in the middle of the Saar-Hunsrück Nature Park is the northernmost municipality in Saarland The high altitude in the low mountain range landscape with extensive deciduous and coniferous forests is the prerequisite for the excellent climate many people want to go out into the countryside They find relaxation in activities such as hiking Weiskirchen offers various possibilities to feel the senses and gives you more serenity and a better ability to concentrate Every person has his or her own preferences and interests when it comes to leisure activities the long-distance hiking trail "Saar-Hunsrück Climb" five healing climate trails and a Nordic.Fitness.Park are offered Let's discover nature and replenish our energy reservoir on guided hikes and bike tours there are four Kneipp facilities for refreshing water treatments In the unique natural outdoor pool on the edge of the Weiskirchen forest In the Vitalis bathing centre recharge your batteries and everyday life will look completely different again Whether you want to relax in the massage pool swim a brisk lane or enjoy pure relaxation in the sauna area The Vitalis spa centre's offer is rounded off by the Reha-Vitalis Bridge over the spa park lake in Weiskirchen The spa park with lake A covered footbridge and walkway are part of the spa park path that leads around the lake The lush blossoms of the adjacent perennial garden characterise the ambience from spring to autumn more silhouette-like contours dominate.  The spa park is the starting point for many activities such as hiking The miniature golf course with summer garden is also integrated into the spa park ensemble Weiskirchen offers endless opportunities to spend time and relax Let yourself be inspired and treat yourself to a little breather Colorful blossoms in the perennial garden in Weiskirchen At the Kneipp spa resort in Weiskirchen in Saarland they have mastered the art of combining medical treatments with natural healing remedies Weiskirchen Pure relaxation at Parkhotel Weiskirchen The Parkhotel Weiskirchen offers the perfect oasis for those seeking relaxation and wellness lovers.… Metrics details this drug termed supformin holds promise to be a therapeutic effective in suppressing inflammation-related diseases related to macrophages the exact pathogenesis of inflammation and the contribution of Cu in the activation of macrophages are far from being understood Targeting macrophage-induced inflammation by LCC-12 Macrophage activation is characterized by increased CD44-triggered mitochondrial Cu(II) accumulation and subsequent promotion of NAD(H) cycling Increased activity of α-ketoglutarate and elevated quantities of nuclear iron trigger metabolic changes and epigenetic modifications leading to inflammatory gene expression The drug LCC-12 containing two biguanides linked by 12 methylene groups forms specific highly stable complexes with Cu(II) in mitochondria The sequestering of Cu(II) by LCC-12 attenuates macrophage activation and dampens inflammatory reactions LCC-12 represents a small dimer of metformin composed of two biguanides linked by a stretch of 12 methylene groups This drug forms highly selective Cu(II) complexes but has no affinity for other divalent metal ions The in vivo efficacy of this drug in limiting macrophage activation and inflammation was demonstrated in three mouse models of acute inflammation namely lipopolysaccharide-induced endotoxaemia In all three models that are associated with upregulated CD44 expression and increased cellular Cu LCC-12 showed highly beneficial therapeutic effects as assessed by reduced expression of markers associated with inflammation LCC-12 effectively blocked epithelial-to-mesenchymal transition (EMT) in cancer cells suggesting that the chelatable mitochondrial Cu pool and its associated signaling pathway have biological functions that go far beyond its involvement in regulating the cellular plasticity of macrophages during inflammatory responses LCC-12 is a highly effective super metformin which makes it understandable why the authors renamed LCC-12 to ‘supformin’ the blocking of detrimental mitochondrial Cu by chelating Cu by LCC-12 might also be beneficial in the management of Wilson disease chelating Cu by LCC-12 is a conceivable therapy to cure or mitigate inflammatory lesions associated with activation of macrophages it should be mentioned that macrophages are a highly dynamic cell population that not only play critical roles in the induction but also in the resolution of inflammation it will be of fundamental importance to test the efficacy of LCC-12 in models of chronic inflammation the authors showed that LCC-12 also interfered with the activation of dendritic cells and T lymphocytes that similar to macrophages increase CD44 expression upon exposure to specific biochemical stimuli while not interfering with the activation of neutrophils that is not associated with CD44 upregulation drug carrier and delivery systems designed to selectively target macrophages will be necessary to prevent potential side effects when using this drug in the management of inflammation the fundamental findings of the study of Solier and coworkers might be a significant step forward for the development of anti-inflammatory therapies The finding that targeting the druggable pool of mitochondrial Cu(II) in activated macrophages by application of LCC-12 allows to interfere with the epigenetic fate of macrophages and the progression or outcome of inflammation is of fundamental importance for many research areas It will now be interesting to follow how these basic research findings will be translated into clinical studies and potential new treatments targeting inflammation or other Cu-associated diseases A druggable copper-signalling pathway that drives inflammation Trace metal imaging in diagnostic of hepatic metal disease Is copper a new target to counteract the progression of chronic diseases Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis Mitochondrial copper homeostasis and its derailment in Wilson disease Download references The author is grateful to Sabine Weiskirchen (IFMPEKC RWTH University Hospital Aachen) for drafting the figure for this Research Highlight article The laboratory of RW is supported by the German Research Foundation (grants WE2554/13-1 WE2554/17-1) and the Interdisciplinary Center for Clinical Research within the Faculty of Medicine at the RWTH Aachen University (grant PTD 1-5) None of the funders had any role in the design of this contribution and decision to publish or preparation of this article Open Access funding enabled and organized by Projekt DEAL Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC) RW conceived and wrote the research highlight article All authors have read and approved the article The author declares no competing interests Download citation DOI: https://doi.org/10.1038/s41392-023-01592-4 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Volume 7 - 2016 | https://doi.org/10.3389/fphar.2016.00283 Chronic liver disease (CLD) features constant parenchymal injury and repair together with an increasing hepatic impairment finally leading to fibrosis and cirrhosis and a heightened risk of hepatocellular carcinoma (HCC) the worldwide prevalence of nonalcoholic fatty liver disease has reached an epidemic dimension and is estimated to afflict up to 46% of the general population Up to now there is no effective drug treatment available which is why recommendations encompass both exercise programs and changes in dietary habits Exercise is well-known for unleashing potent anti-inflammatory effects which can principally counteract liver inflammation and chronic low-grade inflammation This review article summarizes the underlying mechanisms responsible for the exercise-mediated anti-inflammatory effects illustrates the application in animal models as well as in humans and highlights the therapeutic value when possible Based on the available results there is no doubt that exercise can even be beneficial in an advanced stage of liver disease and it is the goal of this review article to provide evidence for the therapeutic impact on fibrosis and HCC and to assess whether exercise might be of value as adjuvant therapy in the treatment of CLD all exercise programs carried out in these high-risk patients should be guided and observed by qualified healthcare professionals to guarantee the patients’ safety it is also necessary to additionally determine the optimal amount and intensity of exercise to maximize its value which is why further studies are essential but might likewise be mitigated by reintegrating activities stabilized by evolution The goal of this review is to provide evidence for the therapeutic applicability of exercise in advanced states of CLD and to assess whether exercise might be of value as adjuvant therapy in the treatment of CLD Most of the health-promoting effects of regular exercise are mediated by its anti-inflammatory properties which have been thoroughly investigated and are now well-founded we will outline the most relevant anti-inflammatory mechanisms of exercise on the molecular level Representative health-promoting effects of sports Numerous reports have shown that sporting activity promotes health and functionality of muscles it is reported that doing sports improves sexual function and significantly impacts the composition of the blood Health-promoting effects of physical activity/regular exercise This imbalance in favor of pro-inflammatory adipokines finally results in the development of persistent low-grade inflammation Duration- and intensity-dependent immunomodulatory changes triggered by exercise and sporting activity Exercise increases the number of circulating Tregs stimulates the secretion of adrenaline and cortisol as well as the release of IL-6 and impacts the ratio of M1-type to M2-type macrophages in fat tissue sporting activity reduces expression of TLRs the intrusion of macrophages and monocytes into fat tissue and also the number of circulating pro-inflammatory monocytes these effects inhibit inflammatory reactions within the body In contrast, regular physical activity increases energy expenditure and reduces fat depots, thereby reversing the imbalance in pro- and anti-inflammatory adipokines. As a result, circulating levels of the anti-inflammatory factor adiponectin rise and those of the pro-inflammatory adipokines TNF, IL-6, RBP-4, and leptin decrease (Gleeson et al., 2011) in this way contributing to an overall decline of systemic inflammation and in the long run to a stabilization of this condition In conclusion, IL-6 can be termed exercise factor by definition, because it is synthesized by skeletal muscle as a result of exercise and is then released into the blood circulation (Catoire and Kersten, 2015) SNS-induced secretion of adrenaline and noradrenaline starts within seconds and ACTH-mediated release of cortisol within minutes after the beginning of exercise and the circulating levels of cortisol and adrenaline are proportional to the intensity as well as to the duration of activity (Gleeson et al., 2011) Common to the studies above is the fact that they consistently describe the downregulation of the cell-surface expression of TLR4, a result which becomes more important in view of the fact that TLR4 has been shown to play a key role in the activation of pro-inflammatory signaling induced by FFAs in obesity (Jiao et al., 2009) By comparing obese and lean mice, it was observed that in the latter macrophages within the adipose tissue displayed the M2 phenotype, but with advancing adiposity the M1 cells increased in number and finally constituted the predominant macrophage phenotype in the fat tissue of the animals, a condition characterized by enhanced synthesis of TNF-α, IL-6, and nitric oxide altogether leading to inflammation and insulin resistance (Lumeng et al., 2007) this study exemplifies how diet may influence the development of an inflammatory state and insulin resistance due to phenotype switching in adipose tissue macrophages All of this suggests that members of the miRNA-181 family might be important for both contributing to an anti-inflammatory environment and inducing adaptations in response to exercise Furthermore, characteristic patterns of circulating miRNAs were described, varying from acute exhaustive to sustained aerobic exercise, including miRNAs, like miRNA-146a, which might also be relevant to limit inflammatory reactions (Baggish et al., 2011) these examples illustrate that miRNAs participate in generating the anti-inflammatory effects known to be induced by exercise and one can expect further details in the near future Conclusively, a few remarks should be added. Beside the various anti-inflammatory effects it is noteworthy that exercise also induces multiple antioxidant effects, including modulation of redox-sensitive pathways, such as NF-κB and PGC-1α signaling, downregulation of pro-inflammatory signaling associated with lowered generation of RONS, and promoting repair mechanisms involving, for instance, heat shock proteins and telomerase (Sallam and Laher, 2016) the occurrence of some mechanisms described above is restricted to adipose tissue in obesity such as the intrusion of macrophages/monocytes into fat tissue as well as phenotype switching in macrophages whereas others do arise in response to exercise under normal conditions adrenaline and IL-6 or the rise in the number of circulating Tregs the disease-associated accumulation of toxic products such as physiological metabolites or xenobiotics (e.g. excretion and detoxification within the diseased liver is a hindrance preventing extensive movements the loss of the hepatic gland function (e.g. production of bile acids) and the decreased anabolic activity in the synthesis of proteins and hormones impact the ability to perform intense physical activity patients with liver disease were often incorrectly advised to restrict their physical activity The view that CLD is incompatible with physical exercise has dramatically changed during the last years the previous outcomes imply that the therapeutical effect might rather depend on the intensity of exercise whereas the actual amount of activity might more likely be sufficient for the prevention of NAFLD However, also performing ordinary resistance training exclusively composed of pushups and squats three times a week for 12 weeks was accompanied by a gain in muscle mass, a concomitant drop in fat mass, and improved hepatic steatosis as well as traits of the metabolic syndrome in patients suffering from NAFLD (Takahashi et al., 2015) it is also conceivable that the hepatoprotective effect of exercise via antioxidant mechanisms might similarly contribute to an improved tolerance of drugs by the liver and thus reasonably support drug treatment because - beside lipid peroxidation and involvement of Fas ligands - pro-inflammatory cytokines contribute to cumulative liver injury Up to now there are two studies investigating the effect of exercise on individuals with cirrhosis and both documented exercise-induced enhancements in muscle strength as well as in exercise capacity These results admittedly hint at a positive impact of exercise even in patients with cirrhosis but it is also obvious that further in-depth studies are needed to safely judge the effects in these high-risk patients Interestingly, a further study reported the activation of NK cells induced by one bout of exercise. At least for 24 h after execution of a half marathon, both histone acetylation and the expression of a functional marker of NK cells remained elevated, suggesting that exercise might be helpful to reduce cancer risk as well as recurrence rates (Zimmer et al., 2015) Personalized exercise programs dependent on the severity of hepatic disease might also be beneficial to enhance blood oxygenation or to increase the patient’s mood by stimulating the release of endorphins and modulating neurotransmitter synthesis or activity Despite constant efforts to develop effective drugs which are applicable to the medication of CLD the conventional approach focuses on prevention and early diagnosis whereas at an advanced stage the only remaining possibility often consists in liver transplantation there is always an underlying chronic inflammation in the heart of CLD and dampening the inflammatory state can slow down the progression of the disease thereby preserving and ameliorating liver function H-TS 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) or licensor are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Hans-Theo Schon, dGhlby5zY2hvbkByd3RoLWFhY2hlbi5kZQ== Ralf Weiskirchen, cndlaXNraXJjaGVuQHVrYWFjaGVuLmRl Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details Hepatocellular carcinoma (HCC) is one of the most severe malignancies with increasing incidence and limited treatment options HCC develops during a multistep process involving chronic liver inflammation and liver fibrosis The latter is characterized by the accumulation of extracellular matrix produced by Hepatic Stellate Cells (HSCs) This process involves cell cycle re-entry and proliferation of normally quiescent HSCs in an ordered sequence that is highly regulated by cyclins and associated cyclin-dependent kinases (CDKs) such as the Cyclin E1 (CCNE1)/CDK2 kinase complex we examined the role of Cyclin E1 (Ccne1) and Cdk2 genes in HSCs for liver fibrogenesis and hepatocarcinogenesis we generated conditional knockout mice lacking Ccne1 or Cdk2 specifically in HSCs (Ccne1∆HSC or Cdk2∆HSC) Ccne1∆HSC mice showed significantly reduced liver fibrosis formation and attenuated HSC activation in the carbon tetrachloride (CCl4) model In a combined model of fibrosis-driven hepatocarcinogenesis Ccne1∆HSC mice revealed decreased HSC activation even after long-term observation and substantially reduced tumor load in the liver when compared to wild-type controls the deletion of Cdk2 in HSCs also resulted in attenuated liver fibrosis after chronic CCl4 treatment Single-cell RNA sequencing revealed that only a small fraction of HSCs expressed Ccne1/Cdk2 at a distinct time point after CCl4 treatment we provide evidence that Ccne1 expression in a small population of HSCs is sufficient to trigger extensive liver fibrosis and hepatocarcinogenesis in a Cdk2-dependent manner HSC-specific targeting of Ccne1 or Cdk2 in patients with liver fibrosis and high risk for HCC development could be therapeutically beneficial the major target cell population mediating the pro-fibrotic effect of Ccne1 has not been clearly identified yet While we were able to demonstrate that hepatocytes especially require Cdk2 for hepatocarcinogenesis the effector cells for the oncogenic effects of Ccne1 in the liver have not yet been identified we tried to answer the question of whether the pro-fibrotic effect of Ccne1 is triggered by a function in HSCs and whether this has an effect on downstream hepatocarcinogenesis We provide evidence that the inactivation of Ccne1 only in HSCs is sufficient to substantially reduce liver fibrosis but also HCC development in CCl4-mediated murine injury models which is at least partially dependent on its canonical kinase subunit CDK2 A Experimental setup: Mice with HSC-specific deletion of Cyclin E1 (Ccne1ΔHSC; n = 4) and control littermates (Ccne1f/f; n = 6) were injected with CCl4 three times a week mice were euthanized and examined for markers of liver damage and fibrosis progression Control mice of both genotypes and matching ages were left untreated (ut) B Left: Determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities Right: Liver mass index was calculated as the liver weight (LW): body weight (BW) ratio in percent C Representative Hematoxylin and Eosin (H&E) stained liver paraffin sections from Ccne1f/f and Ccne1ΔHSC mice after 6 weeks of CCl4-treatment D Representative Sirius Red stainings from formalin-fixed paraffin-embedded liver sections E Quantification of liver fibrosis from images in (D) Sirius Red-positive image areas were quantified using ImageJ software and calculated as a percentage of total tissue areas F Determination of Acta2 (encoding alpha-smooth-muscle actin left) and Col1a1 (encoding collagen type 1 alpha 1 right) mRNA expression by quantitative real-time PCR (qPCR) Expression values were normalized to the expression of the housekeeping gene Gapdh and calculated as fold induction in comparison to untreated Ccne1f/f mice of the same genetic background and age A Experimental setup: Ccne1ΔHSC mice (n = 15) and Ccne1f/f littermates (n = 12) were subjected to the DEN/CCl4 HCC model Mice were injected once with DEN on day 14 after birth to induce HCC followed by weekly administration of CCl4 starting at the age of 6 weeks Animals were euthanized at the age of 24 weeks for subsequent analysis B Representative Sirius Red stainings from formalin-fixed paraffin-embedded liver sections C Morphometric quantification of liver fibrosis Sirius Red-stained images were analyzed for the percentage of stained image areas E Acta2 and F Pdfgfb (encoding platelet-derived growth factor receptor beta) by qPCR Gene expression values were normalized to Gapdh expression and calculated as fold induction in comparison to untreated (ut) Ccne1f/f mice of the same genetic background and age G Immunohistochemical staining for alpha-smooth-muscle actin (αSMA) of liver sections from DEN/CCl4 treated Ccne1f/f and Ccne1ΔHSC mice Right: enlarged view of the marked area from left; scale bar: 100 µm H Morphometric quantification of αSMA positive area from the IHC staining shown in (E) using ImageJ software with IHC toolbox these data demonstrate that Ccne1 is a pro-fibrotic key factor in HSCs which is essential for their activation and the onset of liver fibrogenesis in the setting of inflammatory hepatocarcinogenesis Ccne1ΔHSC mice (n = 15, blue bars) and Ccne1f/f littermates (n = 12, gray bars) were subjected to the DEN/CCl4 HCC model as illustrated in Fig. 2A Mice were sacrificed at the age of 24 weeks and analyzed for liver injury A Determination of AST and ALT activities in Units/liter (U/l) B Determination of liver mass index calculated as the liver weight (LW): body weight (BW) ratio in percent C Macroscopic appearance of explanted livers from Ccne1f/f and Ccne1ΔHSC mice Representative liver tumors are highlighted by arrows D Quantification of tumor burden in Ccne1f/f and Ccne1ΔHSC mice by macroscopic evaluation of HCC nodules Left: Number of HCC nodules (tumor number); right: cumulative tumor diameter representing a measure of tumor size E Representative H&E stained liver paraffin sections F Microscopic evaluation of histologic dysplastic areas displayed in (E) Left: The total number of dysplastic lesions per slide for each mouse is shown Right: The proportion of dysplastic lesion areas was calculated as the lesion area/total liver area ratio in percent Expression values were normalized to the expression of Gapdh and calculated as fold induction in comparison to untreated (ut) Ccne1f/f mice our data suggest that the pro-fibrotic function of Ccne1 in HSCs strongly contributes to the process of hepatocarcinogenesis Mice were sacrificed at the age of 24 weeks Explanted livers were analyzed for markers of proliferation and inflammation A Representative immunofluorescence stainings for Ki-67 (green) on liver cryosections The dashed line indicates the border of one representative dysplastic nodule B Quantification of proliferating liver cells shown in (A) in percent D Gene expression of interleukin 6 (Il6) and tumor necrosis factor-α (Tnf) E Gene expression of chemokine (C–C motif) ligand 5 (Ccl5) and Ccl2 F Immunofluorescence staining for CD11b (green); total nuclei are stained with DAPI (blue) G Quantification of CD11b positive cells from the images shown in (F) Values are given as a percentage of total cells H Immune cells were isolated from explanted livers stained with fluorochrome-labeled antibodies and then analyzed via flow cytometry (FACS) Ccne1 seems to be involved in the control of Il6 expression in the fibrotic and tumorous liver independent of Tnf inactivation of Ccne1 in HSCs was associated with a tendency towards reduced mRNA expression of Ccl2 (p = 0.065) compared to controls indicating that Ccne1 could be directly or indirectly involved in immune cell recruitment our data indicate that Ccne1 expression in HSCs affects the myeloid composition of the tumor environment during hepatocarcinogenesis red bars) mice and Cdk2f/f littermates (n = 7 gray bars) at the age of 8 weeks were challenged with CCl4 three times a week for 6 weeks to induce liver fibrosis Mice were euthanized 48 h after the last injection and analyzed for liver histology B Determination of AST and ALT activities in Units/liter (U/l) The liver mass index was calculated as the liver weight (LW): body weight (BW) ratio in percent C Representative H&E-stained liver paraffin sections from Cdk2f/f and Cdk2ΔHSC mice after 6 weeks of CCl4-treatment D Representative Sirius Red stainings of liver sections E Morphometric quantification of liver fibrosis Sirius Red stained images were analyzed for Sirius Red-positive image areas (percentage of total tissue areas) using ImageJ software G Gene expression analysis of F Acta2 and G Col1a1 by qPCR Expression values were normalized to the expression of Gapdh and calculated as fold induction in comparison to untreated Cdk2f/f mice Primary HSCs were isolated from Ccne1ΔHSC and Cdk2ΔHSC mice as well as from respective cre-negative (i.e. Cells were cultivated for up to 10 days (D0–D10) and analyzed at daily intervals B Growth analysis of HSCs derived from Ccne1ΔHSC/Ccne1f/f (left panel) and Cdk2ΔHSC/Cdk2f/f mice (right panel) The same section of each culture plate was imaged daily and subjected to cell counting Relative cell numbers were calculated as percent in relation to the cell number at day 1 of each biological replicate Gene expression analysis of C Acta2 (left) Concomitant inactivation of Ccne1 and Cdk2 in primary HSCs Primary HSCs were isolated from Cdk2f/f and Cdk2ΔHSC mice and cultivated as before HSCs were transfected with lipid nanoparticles (LNPs) loaded with either scrambled (scr.) siRNA or Ccne1 siRNA Cells were harvested after 10 days and characterized by qPCR H Gene expression analysis of Ccne1 in Cdk2f/f and Cdk2ΔHSC mice after siRNA transfection showed approximately 90% knockdown efficiency of Ccne1 siRNA I Growth analysis of Cdk2f/f (left panel) and Cdk2ΔHSC HSCs transfected with either scrambled (scr.) or Ccne1 siRNA Analysis was performed as described for panel (B) J–M Gene expression analysis of J Acta2 (left) All mRNA expression values were normalized to the expression of Gapdh and calculated as fold induction in comparison to either primary Ccne1f/f HSCs (C–F) or Cdk2f/f HSCs treated with scr these experiments demonstrate that Ccne1 and Cdk2 jointly control the expression of several pro-fibrotic genes in HSCs whereas Il6 expression is regulated by Ccne1 in a Cdk2-independent manner Right: Heatmap showing average expression of G1/S-phase genes B t-SNE plots show the relative gene expression strength of selected cell cycle genes and Il6 C Analysis of Ccne1 co-expression with Mki67 and Il6 expression on single cell level Upper panel: Expression of Ccne1 is associated with Mki67 expression in MFB I and MFB III clusters Detection (>0 reads per cell) for Ccne1 and Mki67 were assessed for each cluster Numbers in each tile denote numbers of cells; d: detected; nd: not detected; color shows Chi-Square-residuals of observed versus expected counts P-values represent a Chi-square test of independence between the detection of the two genes Lower panel: Expression of Ccne1 is slightly associated with Il6 expression solely in cluster MFB I Detection (>0 reads per cell) for Ccne1 and Il6 was assessed for each cluster Color code and calculation were performed as above the scRNAseq data suggest that the strong effects of the HSC-specific Ccne1 deletion regarding liver fibrogenesis and HCC development are caused by a small number of actively cycling HSCs and MFBs The data further implicate that the effects of Ccne1 on Il6 expression in HSCs might be indirect as a significant co-expression in the same cells could not be verified our previous data does not exclude any of the main hepatic cell types from being a Ccne1-dependent driver of liver fibrosis or HCC we tested the hypothesis that HSCs are important effector cells for Ccne1-driven liver fibrosis and hepatocarcinogenesis we generated HSC-specific Ccne1 knockout mice and challenged these animals with a pure liver fibrosis model (CCl4) as well as with a model reflecting both liver fibrogenesis and hepatocarcinogenesis (DEN/CCl4) Our study revealed that deletion of Ccne1 only in HSCs substantially reduced liver fibrosis in both experimental settings which was associated with reduced HSC activation the lack of Ccne1 in HSCs also ameliorated HCC initiation and progression we demonstrated that the pro-fibrotic effect of Ccne1 in HSCs is dependent on its associated kinase Cdk2 deletion of Ccne1 in HSCs was associated with reduced expression of genes encoding important signaling molecules such as IL-6 which could be an additional key to explain the pro-fibrotic properties of Ccne1 A Our data demonstrate that Ccne1 and Cdk2 are both required for the differentiation the inactivation of Ccne1 or Cdk2 in HSCs reduces the pro-fibrotic properties of HSCs thereby attenuating fibrosis formation upon chronic injury such as the CCl4 challenge attenuated fibrosis may impact the tissue environment resulting in less liver tumor formation B Ccne1 in HSCs triggers liver fibrogenesis or HCC formation directly through transcriptional control of pro-fibrotic or hepatocarcinogenic signaling molecules C The latter mechanism can be Cdk2-dependent by driving the expression of genes such as Pdgfrb (encoding PDGF-Rβ) and Ccl2 (encoding CCL2) D We demonstrated that Ccne1 is involved in transcriptional control of Il6 in a Cdk2-independent manner which is likely to contribute to HCC formation conclude that CCNE1 alone or in complex with CDK2 can and we postulate that this may also be true for the control of some pro-fibrotic genes although the exact targets of this mechanism have to be evaluated in extensive future studies which further suggests that down-regulation of PDGFR-β transcription by Ccne1 inhibition is mechanistically related to reduced fibrogenesis and hepatocarcinogenesis in our own experimental setting we detected reduced Il6 expression in the fibrotic liver of DEN/CCl4-treated Ccne1ΔHSC mice while ablation of Cdk2 in HSCs had no significant effect on Il6 regulation conclude that the down-regulation of Il6 through Ccne1-depletion in HSCs is significantly involved in the reduced tumor burden of DEN/CCl4-treated Ccne1ΔHSC mice Our scRNAseq analyses detected Il6 gene expression only in a few HSCs following CCl4 treatment the frequency of Il6-expressing HSCs was very similar to the number of HSCs with detectable Ccne1 expression significant co-expression of Ccne1 and Il6 could at a distinct time point was almost undetectable on a single cell level assume that Ccne1 induces Il6 in HSCs either with a time delay or indirectly involving cell-cell communications HSCs are the main effector cells for the pro-fibrotic function of Ccne1 but presumably only one out of several effector cells for the oncogenic function of Ccne1 our data strongly indicate that therapeutic Ccne1 inhibition will be beneficial for the treatment of both liver fibrosis and fibrosis-induced HCC resulting in mice lacking Ccne1 or Cdk2 specifically in HSCs (Ccne1∆HSC we used male mice in a C57BL/6 background; floxed (f/f) cre-negative littermates served as controls 14-day-old pups were injected with DEN (25 mg/kg followed by weekly injections of CCl4 (0.5 ml/kg All animals were euthanized 48 h after the last CCl4 injection by cervical dislocation living cells were plated on 12-well cell+ plates (Greiner Austria) at a density of 30,000 cells per 12-well in DMEM medium (PAA Laboratories GmbH Austria) supplemented with 10% FCS and 100 U/ml penicillin/streptomycin and dead cells were removed by washing with PBS and medium exchange the same area of the plate was imaged using an Axio Imager.Z1 microscope (Carl Zeiss) and analyzed for cell density The used siRNAs were ordered from IDT (Leuven, Belgium) in a lyophilized form and resolved in 0.1 M acetate buffer. Sequences of used siRNAs are listed in Supplementary Table 2 primary HSCs were seeded on Petri dishes immediately after isolation and cultivated for 4 days HSCs were washed once with PBS and supplied with fresh medium containing siRNA at a concentration of 1 µg/ml encapsulated in LNPs as described above Transfected HSCs were then cultivated for another 6 days without further treatment All results were first tested for normal distribution using the Shapiro-Wilk test Comparison of two groups was performed by an unpaired two-tailed t-test in case of a normal distribution or by Mann Whitney test in case of non-normal distributed results Comparison of three or more groups differing in one variable (e.g. genotype) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test In the case of groups differing in two variables (e.g. two-way ANOVA with Tukey’s multiple comparisons test was performed All data were presented as mean ± standard deviation (SD) using combined dot plots with bars to indicate values of individual mice or biological replicates Significances were defined as *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001 Raw expression data of single-cell sequencing datasets used within this study is deposited in the Gene Expression Omnibus (GEO, see https://www.ncbi.nlm.nih.gov/geo/) under accession No The main raw data on which the study is based will be made available by the corresponding author upon reasonable request Cellular and molecular functions of hepatic stellate cells in inflammatory responses and liver immunology beta-PDGF receptor expressed by hepatic stellate cells regulates fibrosis in murine liver injury Molecular and cellular mechanisms of liver fibrosis and its regression The role of cyclin E in the regulation of entry into S phase Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice Targeting CCl4 -induced liver fibrosis by RNA interference-mediated inhibition of cyclin E1 in mice Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation but not for progression of hepatocellular carcinoma An update on the recent advances in antifibrotic therapy The DEN and CCl4-induced mouse model of fibrosis and inflammation-associated hepatocellular carcinoma The PDGF system and its antagonists in liver fibrosis New insights about albumin and liver disease Alpha-fetoprotein expression is a potential prognostic marker in hepatocellular carcinoma CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity The CCR2(+) macrophage subset promotes pathogenic angiogenesis for tumor vascularization in fibrotic livers Targeting of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic strategy against hepatocellular carcinoma Cyclin E1 in murine and human liver cancer: a promising target for therapeutic intervention during tumour progression Single cell RNA sequencing identifies subsets of hepatic stellate cells and myofibroblasts in liver fibrosis Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors Identification of genes periodically expressed in the human cell cycle and their expression in tumors Hepatic stellate cells and hepatocarcinogenesis Cyclin E in normal physiology and disease states Cell cycle kinetics of rat hepatocytes in early putative preneoplastic lesions in hepatocarcinogenesis cyclins and CKIs: roles beyond cell cycle regulation Cdk2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation Cdk2-dependent phosphorylation of the NF-Y transcription factor and its involvement in the p53-p21 signaling pathway Platelet-derived growth factor C induces liver fibrosis Activated hepatic stellate cells induce infiltration and formation of CD163(+) macrophages via CCL2/CCR2 pathway Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production Hepatic stellate cells enhance liver cancer progression by inducing myeloid-derived suppressor cells through interleukin-6 signaling Cyclin E constrains Cdk5 activity to regulate synaptic plasticity and memory formation Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology Lin C, Mostafa A, Jans A, Wolters JC, Mohamed MR, Van der Vorst EPC, et al. Targeting ligand independent tropism of siRNA-LNP by small molecules for directed therapy of liver or myeloid immune cells. Adv Healthc Mater. 2023: e2202670. https://doi.org/10.1002/adhm.202202670 Download references Schwabe for providing us with Lrat-cre mice for the generation of HSC-specific knockout mice We further thank Mariano Barbacid for providing the conditional Cdk2f/f mouse strain and Peter Sicinski for providing conditional Ccne1f/f mice The authors were supported by the German Research Foundation (DFG) received further funding from the DFG project 271777553 (DFG-LI1045/4-2) and from the DFG Research Training Group “Tumor-targeted Drug Delivery,” grant 331065168 (to C.L was funded by the START program of the Medicine Faculty of the RWTH Aachen received financial support from DFG 475 (BA6226/2-1) the COST Action Mye-InfoBank 476 (CA20117) C.Lin was funded by a CSC stipend (ID: 202008320329) These authors contributed equally: Anna Verwaayen Department of Hepatology and Gastroenterology Campus Virchow-Klinikum and Campus Charité Mitte DWI – Leibniz Institute for Interactive Materials Institute of Technical and Macromolecular Chemistry performed the majority of animal experiments and sample analyses with the support of C.P provided and analyzed single-cell sequencing data from fibrotic mice provided essential support for LNP/siRNA experiments contributed technical support for experiments designed and supervised FACS analysis of murine immune cell populations performed data analysis and interpretation All animal experiments were carried out according to German legal requirements and approved by the authority for environment conservation and consumer protection of the state of North Rhine-Westphalia (LANUV) The authors declare no competing interests Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41419-023-06077-4 a shareable link is not currently available for this article Metrics details The molecular mechanisms underlying the transition from nonalcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) are incompletely understood Perilipin 5 (PLIN5) can regulate lipid metabolism by suppressing lipolysis and preventing lipotoxicity Other reports suggest that the lack of PLIN5 decreases hepatic injury indicating a protective role in NAFLD pathology To better understand the role of PLIN5 in liver disease we established mouse models of NAFLD and NAFLD-induced HCC in which wild-type and Plin5 null mice were exposed to a single dose of acetone or 7,12-dimethylbenz[a]anthracene (DMBA) in acetone followed by a 30-week high-fat diet supplemented with glucose/fructose RNA-seq revealed significant changes in genes related to lipid metabolism and immune response pathways such as AMP-activated protein kinase (AMPK) signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) were blunted in Plin5-deficient mice (Plin5−/−) compared to wild-type mice (WT) while Plin5−/− mice were resistant to tumorigenesis only 32 differentially expressed genes associated with NALFD progession were identified in Plin5 null mice The markers of mitochondrial function and immune response such as the peroxisome proliferator‐activated receptor-γ coactivator 1‐α (PGC-1α) and phosphorylated STAT3 Lipidomic analysis revealed differential levels of some sphingomyelins between WT and Plin5−/− mice these changes were not detected in the HCC model indicating a possible shift in the metabolism of sphingomelins during carcinogenesis the molecular mechanisms explaining the transition from NAFLD to NAFLD-HCC remain poorly understood PLIN5 may have important biological functions in the progression from NAFLD to HCC To further elucidate the role of Plin5 during the progression to HCC we performed studies in a NAFLD and a NAFLD-HCC model We demonstrate that the lack of Plin5 prevents liver injury and inhibits liver tumorigenesis by regulating key signaling pathways associated with carcinogenesis A Schematic diagram of the study design To promote the development of NAFLD and hepatic tumors mice were administered with a combination of a single application of either acetone (NAFLD model) or DMBA dissolved in acetone (NAFLD-HCC model) at p4-5 mice were fed with either a ND or WD for 30 weeks before sacrifice Mice were sacrificed 30 weeks after the initiation of the diet B Representative macroscopic appearance of the WT and Plin5−/− livers corresponding to the NAFLD and NAFLD-HCC models WT mice of the NAFLD-HCC model exhibited hepatic tumors indicated by white arrowheads D Representative histopathology of the hepatic tissue and fibrosis scoring of mice subjected to the C NAFLD and D NAFLD-HCC (NT Upper panels show H&E and lower panels show Sirius red staining Collagen deposits are marked by white arrowheads and liver/body weight of the E NAFLD model (n = 6–8 mice per group) and the F NAFLD-HCC model (5–7 mice per group) G Analysis of the CT scans showing liver volume tumor number and total tumor volume of mice used in the NAFLD-HCC model 2D- and 3D-rendering images in the lower panel are representative results obtained from WT and Plin5−/− mice Tumors observed in the 3D rendering are marked by arrowheads Data represent mean ± SD and were analyzed by two-way ANOVA using Tukey’s post-test In the NAFLD-HCC model, only the liver/body weight ratio was significantly reduced in Plin5−/− mice fed with WD (p < 0.05, two-way ANOVA using Tukey’s post-test) (Fig. 1F). CT scans confirmed the higher presence of tumors in WT (Fig. 1G) while most Plin5−/− mice developed no tumors the ones developed had smaller tumors than those found in WT showing that Plin5 deficiency protects against hepatic tumor formation aspartate transaminase (AST) and lactate dehydrogenase (LDH) in serum of mice of the A NAFLD and B NAFLD-HCC groups (n = 5–8 mice per group) D Serum triacylglycerol (TAG) and cholesterol levels in mice of the C NAFLD and D NAFLD-HCC models (n = 5–8 mice per group) F Glucose tolerance test 24 weeks (20 weeks after beginning of the diet) after the beginning of the experiment of the E NAFLD and F NAFLD-HCC models (n = 5–8 mice per group) Data represent mean ± SD except in calculation of AUC where data are represented as mean ± SE All data were analyzed by two-way ANOVA using Tukey’s post-test these results indicate that Plin5 deficiency decreases hepatic injury in NAFLD whereas only a subtle decrease is provoked in the NAFLD-HCC model WD imposed alterations in TAGs and caused impairment in glucose metabolism in both genotypes and both models while Plin5−/− mice fed WD also had lower cholesterol levels in the NAFLD model A Representative Oil Red O staining of liver tissue from mice fed with WD representing the NAFLD and NAFLD-HCC model Images were acquired at 200× magnification (n = 2 biological and 2 technical replicates) C Distribution of lipid droplet size from representative HE stains of mice fed with WD in the B NAFLD or C NAFLD-HCC model (n = 3 for each genotype and 5 representative fields per sample) E RT-qPCR determined expression of genes involved in β-oxidation in the liver of mice of the D NAFLD and E NAFLD-HCC model (n = 5 mice per group F Western blot analysis of whole liver extracts from WT and Plin5−/− mice of the NAFLD and NAFLD-HCC model Images represent the results of at least two independent experiments (left panel) Relative density quantification of Western blots is presented Measurements were normalized to the loading control GAPDH where measurements were normalized to total levels of AMPK (n = 4 mice per sample) (right panel) In the measurement of relative density of protein expression H Representative electron microscopy images of hepatic tissue from WT and Plin5−/− mice fed with a WD from the G NAFLD or D NAFLD-HCC model (n = 3 mice per group) Magnifications are: 6000× and 10,000× as indicated in each panel Abbreviations/symbols used are: LD lipid droplet These data collectively indicate that mitochondrial impairment is more evident in Plin5−/− mice in NAFLD than in NAFLD-induced HCC the composition of liver lipids under WD and the development of HCC have not been previously reported in the respective mice we dissected the lipid profiles by performing a targeted lipidomic analysis B Visual representation of the individual sphingomyelin (SM) species in (A) liver and (B) serum samples of WT and Plin5−/− mice fed with ND or WD from the NAFLD and NAFLD-HCC models Data are given as % of the total amount of SM C Ratio of total SM in liver presented as percentages (n = 4 mice per group) D Ratio of total SM in serum presented as percentages (n = 4 mice per group) Red arrows indicate differences found between WT and Plin5−/− mice data are depicted as Box-Whisker-plots representing the smallest and largest value Statistical significance was determined using multiple t tests and the Holm-Sidak method with α = 0.05 for multiple comparison A Functional analyses of downregulated differentially expressed genes (DEGs) from WT-WD and Plin5−/−-WD of the NAFLD model B Gene set enrichment analysis from WT-WD and Plin5−/−-WD of the NALFD model indicating the reduced enrichment of key inflammatory pathways during NAFLD progression and its corresponding heatmap C Enrichment in FA metabolism process from WT-WD and Plin5−/−-WD acetone-treated mice and its corresponding heatmap D Analysis of transcription factors (TFs) targeting the set of downregulated DEGs from WT-WD and Plin5−/−-WD The average of the fold ratio for all WT and Plin5−/− mice is represented in the heatmaps as average (Av) these results indicate that a lack of Plin5 profoundly affects immunological processes and lipid homeostasis by targeting specific signaling pathways relevant in the pathogenesis of NAFLD Jupiter microtubule-associated homolog 2 (Jpt2) and WD repeat domain 55 (Wdr55) was considered unfavorable for liver cancer prognosis while high expression of glycine N-methyltransferase (Gnmt) was considered favorable These data suggest that depletion of Plin5 in the NAFLD-HCC model has only a small impact on the transcriptional signature A Western blot analysis for detection of effector molecules of various inflammatory signaling pathways (pSTAT3 pAkt) in animals of the A NAFLD and B NAFLD-HCC model Akt) and GAPDH expression served as controls to demonstrate equal protein loading There was no significant difference in the level of ERK phosphorylation between any of the groups due to the high variation the intensities of phosphorylated NF-κB (pNF-κB) dropped after WD feeding there were no differences between WT and Plin5−/− mice Phosphorylation of p38 (pp38) was reduced after 30 weeks of WD in both models compared to ND-fed mice independent of genotype it was found that activation of Akt (pAkt) increased in WD-fed WT animals in both models while WT mice in the NAFLD model had higher levels of pAkt than Plin5−/− mice pAkt levels were comparable in the NAFLD-HCC model the data indicate that loss of Plin5 is associated with reduced levels of important inflammatory markers (pSTAT3 suggesting that deletion of Plin5 protects against inflammation in the pathology of NAFLD and NAFLD-HCC no NAFLD-HCC models have previously been reported to discover the roles of Plin5 we speculate that the addition of glucose/fructose to drinking water blunted the effects caused by the genetic deletion of Plin5 an increase in FAO driven by higher levels of CPT1 is a logical mechanism to compensate for energy requirements These data pinpoint how loss of Plin5 can partially protect against NAFLD by regulating the immune response Although future in vitro experiments would help to elucidate the molecular mechanism involved in this process it is possible to argue that Plin5−/− partially regulates their antifibrotic role by down-regulating Lpl and Lrat PGC-1α peroxisome proliferator‐activated receptor γ coactivator 1α STAT3 signal transducer and activator of transcription 3; VLFA very long chain fatty acids This illustration was created using Procreate Photoshop CS and Microsoft Power Point (version 365) More studies will be necessary to elucidate the molecular mechanisms by which Plin5 promotes the switch from NAFLD to HCC and therapeutic options to target HCC development more studies are needed to explore the mechanistic insights of PLIN5 activity and identify the cell types responsible for mediating these effects All animals were fed ad libitum for 30 weeks The control groups were fed with a normal diet (ND) (V1534 The other four experimental groups were fed with a WD (D17010102 Mice on the WD also had ad libitum access to water supplemented with fructose 55% (w/v) and glucose 45% (w/v) changed every second week After 30 weeks the mice were sacrificed during the light period under inhalation anesthesia using isoflurane (Forene All animals used in this study received humane care All protocols were in full compliance with the guidelines for animal experimentation and were approved by the institutional German Animal Care Committee (LANUV CT scans were performed as previously described [20] The embedded frozen liver tissues were cut into 10-μm sections using a Leica cryostat The slides were washed with phosphate buffered saline and fixed in 4% paraformaldehyde for 2 min The 0.3% Oil-red O solution was filtered before use and applied on each slide for 5 min at room temperature The slides were washed twice with distilled water for 10 min and the slides were washed five times with distilled water The images were acquired with a NIKON Eclipse 80i inverted microscope (Nikon three mice from each group were used and five fields of each image were analyzed At 10 and 20 weeks after the start of the diet the mice were subjected to a glucose tolerance test the mice were fasted for 5 h and baseline glucose levels were measured in tail-tip blood drops with a blood glucose glucometer (Accu-Chek Instant Mice were injected intraperitoneally with D-glucose (G8769 Whole blood was collected from the heart and serum was separated by centrifugation in a Microvette® 500 serum gel extraction tube (Sarstedt and biochemical parameters such as TAGs and cholesterol were measured in serum using standard laboratory techniques Blinded analysis by a technician was performed on the samples The images were acquired at magnifications of 6000× and 10,000× Differentially expressed genes were considered statistically differentially expressed with an adjusted p-value < 0.05 and (log2-fold change) > 1 an online platform for data analysis and visualization membranes were incubated with the listed secondary antibodies coupled to horseradish peroxidase antimouse or antigoat IgG (Invitrogen) and the SuperSignal chemiluminescent substrate (Pierce Germany) using the iBright imaging system (Thermo Fisher Scientific) we first converted the original digitalized images of the Western blots into 8-bit images using the image processing and analysis software ImageJ the images were inverted to black and white and adjusted the gray-scale threshold for the black and white image We then framed the signals within the region of interest and exported the measured signal intensity to a Microsoft Excel spreadsheet we determined the relative quantification of values by calculating the ratio of the net band to the net loading control which could be a house-keeping gene or an unphosphorylated protein Liver samples were homogenized in 500 µl methanol using a Precellys tissue homogenizer (Berlin Technologies Homogenized samples were transferred to a glass vial Lipids were extracted according to the method of Bligh and Dyer using 1:1 chloroform and methanol (1.5 ml each) The samples were mixed for 30 min at 700 rpm and room temperature using a horizontal shaker samples were shaken for 10 min at 700 rpm and room temperature and subsequently centrifuged for 7 min at 3500 rpm and room temperature to separate the lipid-containing organic from the aqueous phase One ml of the lower (organic) phase was withdrawn using a glass syringe (Hamilton) and extraction was repeated by adding 1 ml of chloroform The organic phase was again withdrawn and the organic phases of one sample were combined Ten µl of each sample were automatically sprayed onto a normal phase high performance thin layer chromatography plate Phospholipid classes were separated by chloroform/ethanol/water/triethylamine (30:35:7:35 by vol.) or n-hexane/diethyl ether/glacial acetic acid (80:15:1 Statistical analysis and graphics were performed using GraphPad Prism 8 software (version 8.4.2 The significance of the differences is presented as means ± SD except for the area under the curve (AUC) for the GTT where the results are presented as means ± SE significance values and test performed are indicated in the legends of each figure The probability values given are *p < 0.05 ***p < 0.001 and ****p < 0.0001 respectively we preformed multiple t-tests and corrected for multiple comparisons using the Holm-Sidak methods The figures were assembled using Microsoft Excel (version 2307) and Power Point (version 2307) The data supporting the findings of this study are available from the corresponding authors, AA and RW, upon reasonable request. 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Version 7.5.1. 2022. https://igordot.github.io/msigdbr/ (last accessed 31 August Download references AA and RW received funding from the Wilhelm Sander-Stiftung (project 2020.002.1 / 2020.002.2) AA received further funding from the START program (project 102/20) RW is supported by the German Research Foundation (grants WE2554/13-1 a grant from the Interdisciplinary Center for Clinical Research within the Faculty of Medicine at RWTH Aachen University (IZKF and a grant from the Deutsche Krebshilfe (grant 70115581) TL gratefully acknowledges financial support from the German Research Foundation (SFB 1066) and the IZKF (grant PTD 1–6) PŠ and RK were funded by grants from the Croatian National Science Foundation Project PREDI-COO (project number: IP-2019-04-9308) KME is supported by the German Research Foundation (EN 1279/3-1) These authors contributed equally: Paola Berenice Mass-Sanchez Ralf Weiskirchen & Anastasia Asimakopoulos Institute for Medical Physics and Biophysics Institute for Experimental Molecular Imaging RW and AA conceived and designed the study PB-MS and MK conducted all in vivo experiments and tissue collection PS and RK performed the primary analysis of RNA-seq data KME and JS performed the lipidomic analyses EMB performed transmission electron microscopy JvH assisted with the serum parameters measurements DM and TL performed the CT scans and calculated the liver and tumor volumes SKM helped with the logistics of animal experimentation PB-MS and MK analyzed and interpreted the data AA and RW have organized the funds for this study and requested permission to carry out the outlined animal studies Download citation DOI: https://doi.org/10.1038/s41420-024-01860-4 Estrogens are crucial regulators of ovarian function, mediating their signaling through binding to estrogen receptors. The disruption of the estrogen receptor 1 (Esr1) provokes infertility associated with a hemorrhagic, cystic phenotype similar to that seen in diseased or aged ovaries. Our previous study indicated the possibility of altered iron metabolism in Esr1-deficient ovaries showing massive expression of lipocalin 2, a regulator of iron homeostasis. Therefore, we examined the consequences of depleting Esr1 in mouse ovaries, focusing on iron metabolism. For that reason, we compared ovaries of adult Esr1-deficient animals and age-matched wild type littermates. Volume 15 - 2024 | https://doi.org/10.3389/fendo.2024.1325386 Introduction: Estrogens are crucial regulators of ovarian function mediating their signaling through binding to estrogen receptors The disruption of the estrogen receptor 1 (Esr1) provokes infertility associated with a hemorrhagic cystic phenotype similar to that seen in diseased or aged ovaries Our previous study indicated the possibility of altered iron metabolism in Esr1-deficient ovaries showing massive expression of lipocalin 2 we examined the consequences of depleting Esr1 in mouse ovaries we compared ovaries of adult Esr1-deficient animals and age-matched wild type littermates Results and discussion: We found increased iron accumulation in Esr1-deficient animals by using laser ablation inductively coupled plasma mass spectrometry Western blot analysis and RT-qPCR confirmed that iron overload alters iron transport trivalent iron deposits in form of hemosiderin were detected in Esr1-deficient ovarian stroma The depletion of Esr1 was further associated with an aberrant immune cell landscape characterized by the appearance of macrophage-derived multinucleated giant cells (MNGCs) and increased quantities of macrophages MNGCs in Esr1-deficient ovaries were characterized by iron accumulation and strong autofluorescence deletion of Esr1 led to a significant increase in ovarian mast cells involved in iron-mediated foam cell formation Given that these findings are characteristics of ovarian aging our data suggest that Esr1 deficiency triggers mechanisms similar to those associated with aging Estrogens, especially 17β-Estradiol (E2), play an essential role in a variety of biological processes within the female reproductive system. They control growth and differentiation of uterine tissue and successful ovulation (1). E2 exerts its functions by binding to specific estrogen receptors in the cytoplasm, which are subsequently translocated to the nucleus and initiate signaling via binding to estrogen response elements (2, 3) the exact function of hemosiderin and the consequences of its deposition in various body systems have not yet been conclusively clarified The aim of the present study was to investigate the consequences of depletion of Esr1 on iron homeostasis we analyzed the ovaries of adult Esr1-deficient animals and aged-matched wild type animals with respect to iron regulation we are speculating about the potential link between the depletion of Esr1 and the early indicatiors of ovarian aging The results of the present study highlight that endocrine dysfunction is associated with common signs of ovarian aging the influx of macrophages and the presence of MNGCs which provides additional information such as the specific genotyping protocol and analysis of the animals total body weights For all histological procedures described in the following section The formaldehyde-fixated paraffin-embedded tissue blocks were stored at room temperature (RT) until sectioning 3-µm thick sections were prepared and deparaffinized in xylene and subjected for rehydration to decreasing graded ethanol In order to compare the different histological stains To investigate whether the iron deposits co-localize with lipocalin 2 (LCN2) PPB was performed combined with immunohistochemical staining for LCN2 the tissue sections were first treated as described in Section 2.2 antigen retrieval was done by heating the slices in sodium citrate buffer (10 mM followed by cooling on ice for 20 min) the samples were washed in PBS and PBS supplemented with 0.1% Tween® 20 (PBS-T) USA) was used essentially as described in the manufacturers instructions To block non-specific antibody binding sites tissue sections were incubated in 5% normal rabbit serum (#X0902 Agilent Technologies) in blocking solution (1% BSA 0.05% Tween® 20 in PBS) for 90 min USA) was diluted 1:40 in blocking solution and incubated on slides at 4°C overnight R&D Systems) were used at the same concentration as the primary antibody and served as a negative control endogenous peroxidase was blocked by incubating the tissue slices in 3% hydrogen peroxide (#31642 Tissue was then washed in dH2O and PBS-T and incubated with a biotinylated rabbit polyclonal anti-goat secondary antibody (#E0466 the slices were incubated in ABC-Complex solution (#PK-6100 Vector Laboratories) according to manufacturers instructions for 1 h in RT The chromogen 3,3-diaminobenzidine tetrahydrochloride (DAB Sigma-Aldrich) was used to visualize LCN2 expression the PPB staining kit was used as described above (Section 2.2.2) tissue sections were first incubated for 5 min at 40°C in 5% potassium ferrocyanide (II) and then for 30 min at 40°C in freshly prepared working solution of 5% potassium ferrocyanide (II) in hydrochloric acid solution (1:1) Tissue sections were washed for 5 min in dH2O and counterstained in 0.1% Seed red (nuclear red) for 5 min samples were dehydrated and mounted with DPX as previously described (see Section 2.2.1) The Periodic Acid-Schiff (PAS) reaction is a histochemical staining technique to detect carbohydrate-containing components like glycogen which are typical for macrophages (50) To visualize polysaccharide-enriched phagocytic cells in the female reproductive tract we used a PAS reaction staining kit (#12153 MORPHISTO) according to manufacturers instructions after the rehydration steps (see Section 2.2) tissue sections were incubated in 1% periodic acid for 20 min and the Schiffs reagent was applied on the sections for 10 min the tissue was counterstained with hematoxylin for 2.5 min followed by ‘blueing under running tap water for 3 min The tissue sections were dehydrated and mounted in DPX mounting medium as previously described (see Section 2.2.1) Toluidine blue (TB) is a dye for metachromatic staining of mast cells in tissues, which stains mast cell granules in purple and the background in blue (51) A standard protocol was used with a 1% Toluidine blue (#89640-25G Sigma-Aldrich) stock solution in 70% ethanol sections were stained with 0.1% toluidine blue working solution in 1% sodium chloride for 5 min the tissue sections were dehydrated in ethanol and xylene and mounted in DPX as previously described (see Section 2.2.1) The number of mast cells per mm2 was determined by counting the positive (purple stained) mast cells in the ovary the stained tissue sections were scanned and NDP.view2 software (see Section 2.2.8) was used to determine the areas of the ovaries Autofluorescence of tissues may be due to the accumulation of lipofuscin, an aging pigment that is stored in phagocytic cells (22, 23) For visualization of autofluorescence in reproductive tissue section rehydrated and embedded in aqueous PermaFluor™ mounting medium (#TA-030-FM USA) with or without nuclear counterstaining in 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (#D1306 the sections were incubated for 3 min in the TrueBlack® solution freshly diluted (1:20) in 70% ethanol the tissue sections were washed with PBS and nuclear counterstaining was performed for 30 min using a 200 ng/ml DAPI solution in PBS tissue slices were mounted with aqueous PermaFluor™ mounting medium and stored in the dark at 4°C until fluorescence microscopic evaluation Images were taken with a Nikon Eclipse E80i fluorescence microscope equipped with the NIS-element Vis software (Version 3.22.01 selected tissue slides were scanned using a NanoZoomer (#C13220-04 viewed using NDP.view2 software (version U12388-01 Ovarian tissues of female animals (Wild type: n=5 Esr1–/–: n=5) aged 12-15 weeks were used for laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) measurement Protocols for standardized quantitative LA-ICP-MS analysis with liver tissue were previously established by us (53). In these protocols, it is necessary to flush the tissue prior measurement with saline buffer for detection of proper endogenous iron concentrations. In unflushed liver, the majority of the measured iron concentration results from the blood and thus can lead to massive misinterpretations (54) sacrificed animals were immediately transcardially perfused a 26G needle was inserted from the tip of the heart about 5 mm into the left ventricle and fixed by means of hemostatic forceps (‘Mathieu needle holder) Then a small incision was made in the right atrium with fine scissors and the perfusion with sterile PBS was carefully started and continued until the liver turned pale Afterwards the tissues were carefully dissected and frozen at -80°C For preparing slides for LA-ICP-MS analysis the samples were cut into 30 µm thick slices using a cryomicrotome (#CM3050S in which the temperature of the cryo-chamber was set to -27°C and the object area temperature to -24°C The slices were mounted on StarFrost® self-adhesive microscope slides (B4 0303 Germany) and stored at -80°C until analysis Before the sections were subjected to LA-ICP-MS measurement the cryosections were scanned with a slide scanner (see Section 2.2.8) to obtain a light microscopic overview Microscopic images were viewed and analyzed using the NDP.view2 software Parts of snap-frozen female tissue were placed in RNA lysis buffer with DTT and homogenized as described previously (4). Protocols for RNA extraction and purification including DNase digestion followed by reverse transcription and quantitative real-time PCR (RT-qPCR), and evaluation were previously published (8). A list of all primers used in this study is given in Supplementary Table 1 All calculations were done in Excel v16 (Microsoft Corporation Statistical analysis was performed with GraphPad Prism v.8.0 (GraphPad Software Gaussian distribution was tested with Shapiro-Wilk Tests Students t-test was used if normality could be assumed while otherwise a non-parametric Mann-Whitney Test was applied All data in this study is shown as mean ± standard deviation (SD) Statistical significance between groups was assumed when probability values were below 0.05 (p < 0.05) Significant differences are indicated by asterisks: * p < 0.05 Defined areas of high iron content in the Esr1-deficient ovary are marked by red color The light microscopic analysis further revealed that increased iron content in Esr1-deficient animals is most likely localized to the ovarian stroma Figure 1 Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging of wild type (WT) and Esr1-deficient ovaries LA-ICP-MS imaging was performed on 30 µm-thick cryosections and the distribution of different isotopes was determined Details are described in the Material and Methods section (A) Overview of all analyzed isotopes in ovaries of WT (n=5) and Esr1-deficient (n=5) animals Individual images were generated with the ELAI software tool Light microscopy (LM) images of the cryosections of the individual ovaries are shown on the left side The content of 13C used for normalization is presented in % while other isotope concentrations are shown in μg/g tissue (B) The concentration of 56Fe (in µg/g) is significantly higher in Esr1-deficient ovaries compared to WT controls For statistical analysis a Students t-test was performed The significant difference between both groups is indicated by asterisks: **p<0.01 (C) Images of 56Fe distribution are shown at different concentration-based scales for a representative WT and Esr1-deficient ovary The 56Fe isotope concentration-based scale was lowered from 0-3,000 µg/g to 0-1,000 µg/g to highlight 56Fe accumulation in Esr1-deficient animals we concluded that Esr1-deficient animals had significantly higher amount of iron in the ovaries compared to WT animals these measurements do not allow the determination of exact form of iron present and therefore the iron metabolism of the animals was further investigated there was no change in ferroportin (Fpn1) expression which is an important iron exporter/efflux pump we found significantly higher expression of iron responsive element binding protein 2 (Ireb2) but lower levels of aconitase (Aco1) in Esr1-deficient compared to WT ovaries Figure 2 Analysis of key players in cellular iron metabolism handling in ovarian tissue Wild type (WT) and Esr1-deficient ovarian tissues were either used for mRNA (WT Ftl1 and Tf were measured by RT-qPCR and validated by (B) Western blot analysis Fth1 and Ftl1 were quantified densitometrically and plotted relative to β-actin expression All data (A-C) are displayed as mean ± SD Significant differences between groups are marked with asterisks: *p<0.05 Only the expression of Fpn1 was significantly decreased in Esr1-deficient livers Figure 3 Analysis of cellular iron metabolism in liver tissue Wild type (WT) and Esr1-deficient liver tissues were used for different analysis (A) Formalin-fixated paraffin-embedded liver tissues sections (WT n=4) were stained for iron using Perls Prussian Blue (PPB) The scale bars equal to 50 µm (solid boarder) or 25 µm (dashed border) (B) Liver cryosections were subjected to LA-ICP-MS imaging Concentration of elemental iron isotope (56Fe) (µg/g) is significantly lower in Esr1-deficient livers compared to WTs (left panel) 56Fe distribution is shown for both genotypes at different concentration-based scales for a representative WT and Esr1-deficient liver (left panel) (C) Regulators of cellular iron metabolism (Fth1 (D) Protein expression of transferrin and Fth1 was investigated by Western blot analysis Expression levels were quantified densitometrically and plotted relative to GAPDH expression All data (B–D) are displayed as mean ± SD For statistical analysis a Students t-test was done we found that different players of iron homeostasis are altered in Esr1-deficient ovaries which are associated with iron overload conditions Our data further indicate that there is no systematic iron overload in other organs involved in iron regulation (i.e. Hemosiderin, an iron storage aggregate with poor accessibility, can be formed during iron overload when cells reach their capacity to bind iron to ferritin, such as during hemorrhages (65). This can be easily analyzed in routine hematoxylin and eosin (HE) staining in which hemosiderin appears as golden-brown aggregates (21) the ovaries of Esr1-deficient animals exhibited many dark blue-stained areas that coincided with the deposits occurring in HE staining the ovaries of Esr1-depleted mice also contained light-blue colored cell clusters (see Section 3.5) Figure 4 Hemosiderin deposits in ovarian tissues Formalin-fixated paraffin-embedded ovarian tissues section of wild type (WT n=8) and Esr1-deficient (n=8) animals were used for staining Serial tissue slices of WT (A) and Esr1-deficient (B) ovaries were stained with Hematoxylin-Eosin (HE) or Perls Prussian Blue (PPB) to detect iron in form of hemosiderin Hemosiderin can be seen in HE staining as brown-gold deposits and turns blue with PPB The scale bars equal to 250 µm (solid boarder) or 50 µm (dashed border) Hemosiderin accumulations were found in ovarian stroma of Esr1-deficient animals but not in WTs and were localized around hemorrhagic cysts (C) (D) Immunohistochemical localization of lipocalin 2 (LCN2) was combined with PPB staining to examine whether iron-positive cells co-localize with LCN2-positive cells WT (n=3) and Esr1-deficient (n=4) ovaries were stained showing no co-localization of iron and LCN2 Normal goat IgG was used as a negative control instead of the primary antibody The scale bars equal to 500 µm (solid boarder) or 50 µm (dotted boarder and dashed border) WT ovaries contained only few LCN2-positive cells we found no double-positive cells in WT ovaries Esr1-deficient ovaries showed many cells that were positive for LCN2 and iron Normal goat IgG that were used as a negative control showed no brownish stain that were observed after staining with the LCN2 specific antibody the results show that the vast majority of LCN2-positive cells do not coincide with the iron-loaded cells Figure 5 Macrophages in ovarian tissues (A) Relative mRNA expression of pan-macrophage markers Cd68 and Adgre were detected by RT-qPCR (B) Western blot analysis was used to detect CD68 protein expression which was quantified densitometrically and plotted relative to β-actin expression F4/80 (green) and LCN2 (red) protein expression was visualized by immunofluorescence staining with nuclear DAPI counterstaining in (C) WT and Esr1-deficient (D) ovaries (E) Normal rat and goat IgG were used as a negative control instead of the primary antibodies The scale bars equal to 100 µm (solid boarder) or 50 µm (dashed border) (F) Relative mRNA expression of Nos2 and Il1r1 Cd163 and Mrc (G) were determined by RT-qPCR our results demonstrate a strong increase in phagocytic cells in the Esr1-deficient ovary we found significantly higher expressions of different M2-like macrophage markers in these animals most likely indicating enhanced remodeling and tissue repair activity Figure 6 Multinucleated giant cells (MNGCs) are present in ovarian stroma of Esr1-deficient mice paraffin-embedded serial ovarian tissue sections from Esr1-deficient mice (n=7) were used for different histological stainings (A) Hematoxylin-Eosin (HE) staining shows pale cell clusters with a foamy appearance and multiple nuclei (I-IV) throughout the section of the Esr1-deficient ovary (B) Cell clusters were identified as MNGCs by positive Periodic Acid-Schiff (PAS) reaction (C) Perls Prussian Blue (PPB) staining revealed iron inclusions in the MNGCs (D) PPB-stained MNGCs show strong autofluorescence Scale bar equals 500 µm (solid boarder in (A)) or 50 µm (dashed boarder in (A) and in (B–D)) we detected several clusters of MNGCs in the ovaries of 3-month-old Esr1-deficient mice These findings indicate that disruption of Esr1 leads to signs of reproductive aging confirming altered endocrinology as an underlying cause of MNGCs formation Figure 7 Increased number of mast cells in Esr1-deficient ovaries n=5) and Esr1-deficient mice (Esr1–/– n=5) were dissected and formalin-fixed paraffin-embedded tissue section were prepared Toluidine blue (TB) staining was performed on ovarian tissue sections to detect mast cells (A) WT animals demonstrate lower number of (purple stained) mast cells in the ovary than Esr1–/– animals Scale bar correspond to 250 µm (solid boarder) or 50 µm (dashed boarder) (B) Evaluation of all female mice shows that Esr1–/– animals have significantly more mast cells per mm2 in the ovary compared to WT controls Each dot represents an individual mouse and horizontal lines indicate the mean (± SD) (C) Serial sections either stained with TB or Perls Prussian Blue (PPB) demonstrate that some mast cells in Esr1–/– animals reside in the vicinity of iron-accumulated areas (blue precipitates) degranulated mast cells were found in the Esr1–/– ovary Arrows indicate release of mast cell granules into surrounding ovarian tissue (E) RT-qPCR was performed to evaluate relative mRNA levels of mast cell specific markers (Mcpt6 D) Students t-test was used for statistical analysis Significant differences between groups are marked with asterisks: **p<0.01,***p<0.001 the number of mast cells is increased in Esr1-deficient ovaries compared with WT animals suggesting that ovarian mast cells play an active role in the hemorrhagic cystic Esr1-knockout phenotype and might be involved in the formation of MNGCs For a more detailed analysis of the systemic effects of iron it would be useful to perform a comprehensive serum analysis This analysis should include measuring typical iron-related parameters such as unsaturated iron binding capacity Considering that Slc11a2 mediates the uptake of iron as Fe2+ enhanced Fth1 expression could be essential in protecting against iron-induced oxidative stress These and our results underline that certain conditions which presumably leads to cellular damage in the long term we concluded that the hemosiderin deposition in the ovaries is most likely due to the hemorrhagic phenotype of the Esr1-deficient animals and could probably be cell-damaging we can conclude from our data that defense mechanisms against oxidative stress are activated in the Esr1-deficient animals while we observed no alterations in iron-driven cell death associated with the lack of Esr1 Since there are a large number of parameters associated with iron-induced cell damage it is advisable to comprehensively investigate these in future studies It would be suitable to examine the amount of 4-hydroxynonenal as a marker of lipid peroxidation and analyze the Nrf2-Keap1 axis which is one of the most important signaling pathways in the control of oxidative and electrophilic stress it is important to not only focus on protein expression but also on its localization in the tissue Further studies should investigate systemic levels of inflammatory markers and clearly identify whether these ovarian immune cells are resident or infiltrating preliminary (unpublished) results using Oil Red staining show that the MNGCs in Esr1-deficient ovaries and aged WT ovaries contain numerous lipid droplets which are formed in both the aged WT animals and after Esr1 depletion will shed light on the potential link between aging processes and Esr1 signaling A better understanding of the aging processes in the ovaries could therefore lead to the development of therapies that curb oocyte damage and age-related infertility The raw data supporting the conclusions of this article will be made available by the authors The animal study was approved by internal Review Board of the RWTH University Hospital Aachen The study was conducted in accordance with the local legislation and institutional requirements The author(s) declare financial support was received for the research RW is supported by grants from the German Research Foundation (grants WE2554/13-1 and the Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University (grant PTD 1-5) The funders had no role in the design of this article or in the decision to publish it This work was supported by the Immunohistochemistry Facility a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University Germany) for excellent technical assistance with animal tissue dissection genotyping and staining and to Sabine Weiskirchen (IFMPEGKC Germany) for preparing the cryosections and technical support in using the NanoZoomer SQ digital slide scanner The authors thank Manuela Pinoe-Schmidt (IFMPEGKC Germany) for technical assistance in molecular biology and Sven Thoroe-Boveleth (Institute for Occupational RWTH Aachen University) for performing the LA-ICP-MS measurements The author(s) declared that they were an editorial board member of Frontiers This had no impact on the peer review process and the final decision All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited *Correspondence: Sarah K. Schröder, c2FzY2hyb2VkZXJAdWthYWNoZW4uZGU=; Ralf Weiskirchen, cndlaXNraXJjaGVuQHVrYWFjaGVuLmRl Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish. Volume 15 - 2024 | https://doi.org/10.3389/fendo.2024.1365602 The 25 kDa-sized protein Lipocalin 2 (LCN2) was originally isolated from human neutrophil granulocytes more than 30 years ago LCN2 is an emerging player in innate immune defense as it reduces bacterial growth due to its ability to sequester iron-containing bacterial siderophores LCN2 also serves as a transporter for various hydrophobic substances due to its β-barrel shaped structure LCN2 has been detected in many other cell types including epithelial cells Studies have clearly shown that aberrant expression of LCN2 is associated with a variety of disorders and malignancies including several diseases of the reproductive system LCN2 was proposed as a non-invasive prognostic and/or diagnostic biomarker in this context Although several studies have shed light on the role of LCN2 in various disorders of the female and male reproductive systems a comprehensive understanding of the physiological function of LCN2 in the reproductive tract is still lacking there is evidence that LCN2 is directly related to fertility as global depletion of Lcn2 in mice has a negative effect on their pregnancy rate Since LCN2 expression can be regulated by steroid hormones it is not surprising that its expression fluctuates greatly during remodeling processes in the female reproductive tract Well-founded details about the expression and regulation of LCN2 in a healthy reproductive state and also about possible changes during reproductive aging could contribute to a better understanding of LCN2 as a target in various diseases the present review summarizes current knowledge about LCN2 in the reproductive system and discusses changes in LCN2 expression during pathological events The limited data suggest that LCN2 is expressed and regulated differently in healthy male and female reproductive organs With this review, our goal was to summarize the available knowledge on LCN2 in female and male reproductive organs. The focus was mainly on the female reproductive tract (uterus, vagina, and ovary). However, in the male reproductive tract, we concentrated on the expression of LCN2 in the testes and epididymis, as its expression in healthy and diseased prostates has been previously summarized (10) Although the existence of LCN2 in the uterus has been known for more than 25 years (23, 24), only few studies have addressed the expression, function, and signaling pathways of LCN2 in the female reproductive tract. This is likely due to the fact that so far little is known about the interaction of LCN2 with its various receptors as previously mentioned (14) we summarize the studies on LCN2 expression in the female reproductive tract in chronological order In addition to the small number of studies of LCN2 in mice (Table 1), there are also few studies on its expression in human tissue. In a study analyzing 50 tissues of human origin by RNA dot-blot hybridization, the uterine tissue was positive for LCN2 (39). In their comprehensive study, Friedl and co-workers (40) used immunohistochemistry to examine various healthy human tissues for LCN2 protein expression These results of the study report that LCN2 expression was not found in the female reproductive tract (ovaries not all images of the staining results from the screening are displayed in the publication but the results of the staining have been summarized in tabular form Table 1 Expression pattern of LCN2 in murine female and male reproductive tract Table 2 Summary of LCN2 mRNA and protein expression according to Human Protein Atlas* Figure 1 Schematic representation of LCN2 expression in the main components of the female reproductive system The right side of the figure displays the primary compartments of the mouse female reproductive tract The left side of the figure depicts a scheme of immunohistochemical staining of LCN2 in different parts of the female reproductive system based on previous research and our own findings using the anti-LCN2 antibody AF3508 from R&D Systems Cells shown in shades of pink and purple indicated a lack of LCN2 expression while cells with brown staining indicate positive LCN2 expression The ovarian stroma consists of epithelial cells fibroblast-like cells and smooth muscle cells Growing ovarian follicles contain various cell types including oocytes surrounded by granulosa and theca cells which form from cells remaining in the pre-ovulatory follicle the luminal epithelium displayed positive staining for LCN2 the glandular uterine epithelium showed positive LCN2 expression positive LCN2 staining was observed in several cells of the surface epithelium and stroma (lamina propria) We summarized the current knowledge on the expression of murine LCN2 in different compartments of the female reproductive tract in Figure 1 and included findings of our own (unpublished) immunohistochemical LCN2 stainings Further comprehensive studies are needed to determine the precise cell type expressing LCN2 and vagina and whether they are subject to hormonal fluctuations of the estrus cycle Following birth, the uterus undergoes a process called involution, whose goal is to return the uterus to its prepregnancy state. It has been observed that the postpartum involuting uterus is a major site of LCN2 expression (25) The authors speculate that in this context LCN2 serves as an inducer of neutrophilic apoptosis thus protecting the uterus from oxidative stress and carcinogenesis during the remodeling phase Others studied LCN2 in uterine tissue of rats (45) in context of pregnancy and involution Western blot analysis show that LCN2 was strongly elevated on day 22 of pregnancy and on the first two days after delivery compared to non-pregnant uterine tissue In pregnant and non-pregnant rats (in the proestrus) uterine LCN2 expression was restricted to the uterine luminal and glandular epithelium of the uterine and did not colocalize with Myeloperoxidase (detected via immunostaining) which is also up-regulated during postpartum involution but in different cellular compartments (in the vessel-rich layer and in the endometrial stroma) Several reports implicate LCN2 in the fertilization process (30, 49). A study by Watanabe and colleagues identified LCN2 produced by the female mouse reproductive tract as a sperm-capacitating agent that alters the membrane properties of sperm in preparation for fertilization (30) LCN2 levels reach their peak during estrus at uterotubular junction where sperm maturation is believed to begin forcing the sperm to undergo lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding necessary for fertilization the same study reported that LCN2 has the ability to increase murine in vitro fertilization efficiency making it a great target to study for clinical application the presence of LCN2 reduced the response of sperm to bovine serum albumin (BSA) and calcium by suppressing intracellular pH elevation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm The most important report on the implication of LCN2 in the fertilization process is probably the one that says that Lcn2KO mice show compromised fertility (52) The study reports that although Lcn2 deficient males appear to be fertile Lcn2-deficient females show a significantly (p < 0.05) decreased pregnancy rate compared to WT littermates when crossed to WT or Lcn2-deficient males these data indicate that sperm maturation and fertilization are highly dependent on the LCN2 status of the female individual no further data are available on the physiological expression and function of LCN2 in the male human reproductive tract it was concluded that LCN2 is an important component in spermatozoa processing it was discussed that LCN2 is involved in EMF-induced apoptosis in germ cells Although both publications contain manufacturer information on the antibody used (which was the same) only one of the studies provides information on the catalog number it cannot be completely ruled out that different antibodies were used this discrepancy is not addressed in the study so it is not possible to definitively say where exactly LCN2 is expressed in the rat testes It is important to understand which cells express LCN2 and which putative receptors are essential the molecular mechanisms of its action can be finally uncovered Figure 2 Schematic representation of LCN2 expression in the main components of the male reproductive system The right side of the figure shows the main components involved in sperm maturation and development in the murine male reproductive tract namely the testis (T) and epididymis (E) which can be histologically divided into the caput (Cap) The left side of the figure is a schematic representation of immunohistochemical staining of LCN2 in the different compartments using the anti-LCN2 antibody AF3508 from R&D Systems Cells shown in shades of pink and purple represent cells negative for LCN2 while cells with brown staining are positive for LCN2 Spermatogenesis begins gradually in the germ cells located in the seminiferous tubules of the testis Positive staining for LCN2 was only observed in interstitial located are transported for maturation and storage to the epididymis a high expression of LCN2 is visible in the lumen where sperm maturation occurs A decline in LCN2 expression can be observed when comparing sperm in the lumen of the cauda to the caput epididymis segment The presented article summarizes that there are only limited partially ambiguous studies on the physiological expression and function of LCN2 in the reproductive tract there are several studies that focus on LCN2 in different diseases and pathologies of both male and female reproductive tracts LCN2 is examined in the context of various diseases Further studies should aim to understand the molecular mechanisms which in turn will also allow conclusions to be drawn about the physiological function of LCN2 and tissue are needed to understand the role of LCN2 in PCOS Shortly after the discovery of LCN2, it became obvious that it plays a role in fertilization, implantation, and pregnancy in mice (13, 23, 24, 26) However, LCN2 has also been implicated in pregnancy complications in humans. Meta-analysis comparing women with healthy pregnancy and preeclampsia (PE) detected that high circulating LCN2 is associated with PE, which may be independent of the trimesters for blood sampling and the severity of PE (66). In line, Stepan and colleagues found that maternal LCN2 serum levels increased significantly in PE patients (67) More studies are essential to clarify how LCN2 is related to the initiation and progression of endometriosis It would also be informative to investigate whether and how LCN2 fluctuates during the menstrual cycle which has not been considered in existing studies and could possibly explain the differences seen Gynecological cancers can originate from various sites in the normal tissues of the female reproductive tract (80). LCN2 expression has been shown to be up-regulated in ovarian, cervical tissue, and endometrial cancer, as well as human ovarian cancer cell lines [https://www.proteinatlas.org/ Hao and co-workers found an up-regulated expression of LCN2 in ovarian cancer patients, as well as in different ovarian cancer cell lines compared to normal tissue and cell lines (81). Using human ovarian cancer cell lines, the study provided evidence on the mechanism of LCN2 signaling ovarian cancer (81). LCN2 promotes tumor progression in ovarian cancer cells by activating the ERK/GSK3β/β-catenin signaling pathway (81) the data clearly demonstrate a positive correlation of LCN2 with tumor progression and targeting LCN2 or its signaling pathways could be a therapeutic strategy for targeting ovarian cancer LCN2 expression is speculated to be involved in balance between cell death and proliferation in uterine tissue remodeling and is important in the progression of endometrial cancer All mayor findings on LCN2 expression and role in female reproductive tract cancers have been summarized in a tabular form in Table 3 Table 3 Expression levels of LCN2 in cancers of the human female reproductive tract LCN2 is a known acute phase protein in non-reproductive tissues (52). Interestingly, a study examined Lcn2 in the context of uropathogenic infections (99). In murine cauda epididymis, Lcn2 mRNA was significantly increased after infection with uropathogenic E. coli, highlighting the antibacterial activity of LCN2 in reproductive tissues as seen in female uterine tissue (16, 99) Kessel et al. analyzed LCN2 expression in testes from adult infertile Esr1-deficient mice (31). Interestingly, LCN2 protein expression was strongly increased in these animals compared with age-matched WT animals (31) These studies provide evidence that LCN2 has not only a physiological but also a pathophysiological role in the testes it is mandatory to understand the underlying molecular mechanism which likely involves estrogen receptor signaling Apart from its role in benign prostate diseases and the development and progression of prostate cancer (10, 101), LCN2 has hardly been studied at all in diseases of the male reproductive tract. In 2005, a study analyzed adult male germ cell tumors and found a high increase in LCN2 in teratomas without seminomas but not in other subtypes, classifying it as an suitable predictor (102) it is also important to investigate the expression and localization of LCN2 in healthy human testes and in various diseases such as testicular cancer The presence of LCN2 has been confirmed in both male and female healthy reproductive systems by multiple studies the available studies show that LCN2 has so far been studied much more intensively in a pathological context than in a physiological context we can speculate about the different functions of LCN2 in the female and male reproductive tract Although it appears that in the female reproductive system LCN2 is involved in tissue reorganization during the menstrual cycle and pregnancy the mechanism of its action and the cells responsible for its production have not yet been determined it has been shown to serve as a component of the maturation and 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April 2024 Copyright © 2024 Krizanac, Mass Sanchez, Weiskirchen and Schröder. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Ralf Weiskirchen, cndlaXNraXJjaGVuQHVrYWFjaGVuLmRl; Sarah K. Schröder, c2FzY2hyb2VkZXJAdWthYWNoZW4uZGU= We have identified a phenomenon occurring in the usage of proposed “specific” Mitogen-activated protein kinase (MAPK) inhibitors. We found that especially inhibitors of p38 potentiate the activation of other MAPKs in various cell types. This finding will have tremendous impact on the interpretation of all former studies using MAPK inhibitors. Experimental Pharmacology and Drug Discovery Volume 9 - 2018 | https://doi.org/10.3389/fphar.2018.00098 A commentary has been posted on this article: Commentary: Usage of Mitogen-Activated Protein Kinase Small Molecule Inhibitors: More Than Just Inhibition We have identified a phenomenon occurring in the usage of proposed “specific” Mitogen-activated protein kinase (MAPK) inhibitors We found that especially inhibitors of p38 potentiate the activation of other MAPKs in various cell types This finding will have tremendous impact on the interpretation of all former studies using MAPK inhibitors Reciprocal activation of MAPK signalling by MAPK inhibitors (A) Images of inhibitors used in this study were generated with software Jmol (version 14.2.15) (B) The reporter cell line HSC Col-GFP (left) primary hepatocytes (middle) and (activated) PMF (right) were stimulated for 10 min with PDGF-BB (25 ng/ml) after pre-incubation of cells with the indicated inhibitors (each 10 μM) for 1 h proteins were extracted and subjected to Western blot analysis with the depicted antibodies The inhibition of MAP kinases impacts PDGF responses as PD98059 and UO126 reduce pp42/44 phosphorylation while SB203580 and SB242235 reduce STAT5 phosphorylation (data not shown) (C) Rat PMF were stimulated for 10 min with TGF-β1 (1 ng/ml) or PDGF-BB (25 ng/ml) after pre-incubation of cells with the depicted p38 inhibitors (each 10 μM) for 1 h (D) Deduced impact of inhibitors on MAP kinase activity in cultured HSC Col-GFP Antibodies used are from Santa Cruz (PDGF-Rβ PDGF-BB is a potent mitogen for hepatic stellate cells (HSC) (Borkham-Kamphorst and Weiskirchen, 2016), and stimulation of HSC Col-GFP with PDGF-BB leads to activation of the three major MAP kinases (Figure 1B) the pre-treatment of cells with the MEK1/MEK2 inhibitors resulted in a direct reduction in ERK1/ERK2 MAPK phosphorylation while SB203580 and SP600125 blunted MAPK activity as demonstrated by a reduction in substrate phosphorylation of STAT5 (p38 All experiments were highly reproducible (Supplementary Figure 1) we could show that not only MAPK phosphorylation itself but also substrate phosphorylation is increased which demonstrates a higher activity of non-targeted MAPKs (Supplementary Figure 2) Isolation of primary cells (hepatocytes, PMF) and establishment of cell line HSC Col-GFP were done as described previously (Meurer et al., 2011, 2013; Borkham-Kamphorst et al., 2016). SDS-PAGE and Western blot analysis were done as reported (Borkham-Kamphorst et al., 2016) It is obvious that the mutual “activation by inhibition” is not limited to straight forward MAPK-signaling network Although we don't know if the phenomenon of cross-activation can be generalized when blocking one pathway we think our observations must be critically kept in mind when interpreting experimental results mediated by a “specific” inhibitor Potential mechanisms of MAPK crosstalk and regulation by dual-specificity phosphatases under different conditions are discussed elsewhere (Birkenkamp et al., 2000; Shen et al., 2003; Junttila et al., 2008; Ríos et al., 2014) This study was carried out in accordance with the recommendation of the Landesamt für Umwelt und Naturschutz (LANUV The protocols for isolation of primary cells were approved by the LANUV RW and SM designed the study and drafted manuscript RW is financially supported by the German Research Foundation (projects SFB/TRR57 P13 and Q3) and the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital Aachen (Project O3-1) The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2018.00098/full#supplementary-material The specificities of protein kinase inhibitors: an update an anthrapyrazolone inhibitor of Jun N-terminal kinase The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway Borkham-Kamphorst Adenoviral CCN gene transfers induce in vitro and in vivo endoplasmic reticulum stress and unfolded protein response Borkham-Kamphorst Specificity and mechanism of action of some commonly used protein kinase inhibitors A synthetic inhibitor of the mitogen-activated protein kinase cascade Identification of a novel inhibitor of mitogen-activated protein kinase kinase Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival A protein kinase involved in the regulation of inflammatory cytokine biosynthesis Overexpression of endoglin modulates TGF-β1-signalling pathways in a novel immortalized mouse hepatic stellate cell line Expression and functional analysis of endoglin in isolated liver cells and its involvement in fibrogenic Smad signalling Dual-specificity phosphatases as molecular targets for inhibition in human disease Cross-talk between JNK/SAPK and ERK/MAPK pathways: sustained activation of JNK blocks ERK activation by mitogenic factors a selective inhibitor of p38 mitogen-activated protein kinase Citation: Meurer SK and Weiskirchen R (2018) Usage of Mitogen-Activated Protein Kinase Small Molecule Inhibitors: More Than Just Inhibition Received: 24 November 2017; Accepted: 29 January 2018; Published: 12 February 2018 Copyright © 2018 Meurer and Weiskirchen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited *Correspondence: Steffen K. Meurer, c21ldXJlckB1a2FhY2hlbi5kZQ== Ralf Weiskirchen, cndlaXNraXJjaGVuQHVrYWFjaGVuLmRl Volume 7 - 2016 | https://doi.org/10.3389/fphys.2016.00430 Lipocalin 2 (LCN2) is a secreted protein that belongs to the Lipocalins a group of transporters of small lipophilic molecules such as steroids Two decades after its discovery and after a high variety of published findings LCN2's altered expression has been assigned to critical roles in several pathological organ conditions The significance of this 25-kDa lipocalin molecule has been impressively increased during the last years Data from several studies indicate the role of LCN2 in physiological conditions as well as in response to cellular stress and injury LCN2 in the liver shows a protective role in acute and chronic injury models where its expression is highly elevated LCN2 expression is being considered as a potential strong biomarker for pathological conditions we summarize experimental and clinical findings linking LCN2 to the pathogenesis of liver disease Compilation of data on structure and function of LCN2 The first critical roles given to LCN2 were proposed from its “lipocalin structure” that is most closely related to the structures of the epididymal retinoic acid-binding proteins and the major urinary protein (Goetz et al., 2000). The typical unifying three dimensional fold of lipocalins encompasses an eight-stranded, anti-parallel, symmetrical β-barrel fold with a cylindrical shape (Figure 1) After this very first allocation of LCN2 as a significant factor in guaranteeing immune balance the repertoire of LCN2 functions was further expanded to many biological processes we will summarize some of the major findings of LCN2 in the pathogenesis of organ disease with a special emphasis on inflammatory liver diseases The different reports unanimously show that LCN2 is either increased in the tissue by several resident cell types or alternatively by circulating immune cells that are recruited into the inflamed tissue LCN2 is reported to be expressed in numerous pathological and experimental-induced conditions in many organs and tissues such as liver, kidney, lungs, bone, brain, and heart (Figure 2). Several of the published findings on main organs are listed in Table 2 Representative pathophysiological or malignancies conditions for which elevated LCN2 concentrations in representative organs are indicative are depicted Selected experimental and clinical findings associated with altered LCN2 expression in organ disorders Here we will first focus on some of the most recent reports on LCN2 expression and organ disorders to understand the possible major functions of LCN2 and subsequently highlight the importance of LCN2 in the pathogenesis of hepatic disease In contrast to these findings, another study investigating acute allograft rejection in mice, showed that the treatment with recombinant LCN2 after transplantation reduced the extent of kidney allograft rejection, renal allograft damage, apoptosis of tubular epithelial cells, and induced their proliferation. The proliferation led to an amelioration of damage morphology and improved allograft function (Ashraf et al., 2016) These two studies paradigmatically demonstrate that LCN2 is a versatile biomolecule with outstanding pathophysiological All these data indicate that LCN2 is a very sensitive regulator of cell survival and apoptosis in cardiomyocytes these strong indications of LCN2's correlation to heart disorders makes further elucidation of LCN2 role in heart failure a very interesting and crucial topic to study the precise impact of LCN2 in the brain is still in the very beginning and further studies are required to unravel its roles in the nervous system These studies on LCN2 show that analyzing functional aspects of this lipocalin could also help to understand the pathogenesis or progression of lung diseases Even though LCN2 is suggested to have a role in the early stages of tumor development or as a prognostic marker there is an urgent need for elucidating the role of LCN2 by studying its effects on metastasis behavior the multitude of pathological events (cancer organ failure) and affected organs (kidney muscle) that give rise to alterations in LCN2 serum or urine levels may further require the definition of clear cut-off values that are indicative for a specific disease Figure 3. LCN2 in the pathogenesis of liver diseases. LCN2 is a versatile adiponectin that influences all kinds of liver diseases. For more details on experimental and clinical findings associated with altered LCN2 expression refer to Table 3 Selected experimental and clinical findings associated with altered LCN2 expression in liver alterations in LCN2 expression and functions are reported in many hepatic conditions In the following we will discuss some of the most striking observations that were placed in context with LCN2 in this study it was demonstrated that the measurement of urine LCN2 had a better predictive power than the measurement of serum LCN2 in discriminating acute-on-chronic liver failure (ACLF) patients from cirrhotic patients with acute decompensation The authors suggested that this is due to the fact that the association between urine LCN2 and ACLF was not exclusively related to kidney function and that further urine but not serum LCN2 is independently associated with ACLF it was noted that hepatocytes of mice that lack LCN2 showed lipid droplet accumulation and increased apoptosis giving a slight hint of LCN2 as a modulating factor in lipid metabolism In the same study it was demonstrated that although patients with chronic liver disease exhibited significantly higher concentrations compared to healthy volunteers the levels of LCN2 were not suitable to discriminate between non-cirrhotic and cirrhotic liver-diseased patients suggesting that LCN2 levels provide no correlation to the degree of liver fibrosis but provide a significant positive correlation to inflammation A recent concept suggests that LCN2 is a key factor in controlling intracellular fat metabolisms in hepatocytes by regulating expression of the lipid droplet protein PLIN5/OXPAT (A) The concept is majorly based on the finding that mice lacking LCN2 accumulate more lipids in the liver and show more hepatic damage and inflammation when fed a methionine-choline deficient diet (MCD) representing a nutritional model of NASH it was proposed that LCN2 imports lipids into hepatocytes either via specific receptors (e.g. the LCN2/lipid complexes are first packed into endosomes whose slightly acidified microenvironment causes LCN2 to dissociate from the lipids where they are coated by PLIN5/OXPAT protecting them from intracellular degradation and oxidation PLIN5/OXPAT itself is up-regulated by PPAR-γ that is stimulated by the higher cytoplasmic fat content that is the consequence of facilitated lipid import by LCN2 and PLIN5/OXPAT presents a complex network that affects lipid metabolism intracellular concentrations of free reactive lipid species may be reduced and the overall inflammatory responses suppressed the concentration of free fatty acids within the cytosol is increased predisposing for inflammation and steatosis All these data indicate that LCN2 acts as a paracrine chemoattractant that modulates neutrophil trafficking to the liver and other organs This finding indicates that LCN2 could serve as a biomarker for liver regeneration however further experimentation is still necessary to elucidate the role of LCN2 in liver hepatectomy and regeneration Most popular for quantification are diverse ELISA tests that have been proved to be the most sensitive and quick method to detect LCN2 elevation in early stages and conditions of experimental and clinical liver disease including FLD These contradictory findings indicate that LCN2 might have both protective as well as pathogenic activities These double-edged biological properties may render therapeutic approaches complicated again demonstrating the versatile activities of LCN2 These molecules might be beneficial to target de novo LCN2 expression or its interaction with its cognitive receptors Therapeutic strategies are nowadays tested in cell culture and preclinical models but none of these has been tested in clinical studies so far The lipocalin 2 protein was initially proposed to represent a secretory protein that is involved in the transportation of hydrophobic molecules it has been promoted as a multifunctional siderophore that is involved in innate immunity by sequestering iron that in turn limits bacterial growth A wide variety of studies have shown that the expression of this lipocalin is significantly altered in the pathogenesis of organ damage affecting kidney we have summarized experimental and clinical findings linking LCN2 to liver disorders It is obvious that LCN2 is significantly upregulated during phases of hepatic injury numerous studies correlated LCN2 with general hepatic injury In all these pathological states LCN2 expression seems to be highly elevated underpinning the notion that this lipocalin is a good biomarker candidate for a large variety of hepatic insults Experimental findings have shown that the loss of LCN2 is correlated with a higher susceptibility to bacterial infections and more severe hepatic damage after challenge with appropriate hepatotoxins several diagnostic test systems for detecting and quantification of LCN2 in urine plasma and tissue extracts are on the way to be incorporated into the routine diagnostics the measurement of urine LCN2 could more accurately reflect systemic inflammation or disease progression than serum LCN2 although the knowledge in LCN2 in liver biology has increased dramatically during the last years there is still a mandatory need for studies that further characterized LCN2 and its pathways in the initiation progression and potentially regression of hepatic lesions These studies will potentially lead to novel therapeutic avenues or drugs that might be beneficial to interfere with progression or foster regression of inflammatory hepatic disease All authors agree to be accountable for the content of this work acute kidney injury; ER; endoplasmic reticulum; FLD non-alcoholic steatohepatitis; NF-κB neutrophil gelatinase-associated lipocalin Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart Diet high in fructose leads to an overexpression of lipocalin-2 in rat fatty liver Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis Exogenous Lipocalin 2 ameliorates acute rejection in a mouse model of renal transplantation Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver a “help-me” signal in organ inflammation Lipocalin 2 in the pathogenesis of fatty liver disease and nonalcoholic steatohepatitis CrossRef Full Text | Google Scholar Liver lipocalin 2 expression in severely obese women with non alcoholic fatty liver disease Studies of the release and turnover of a human neutrophil lipocalin Liquid chromatography-mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of nonalcoholic fatty liver disease Neutrophil gelatinase-associated lipocalin immunoexpression in renal tumors: correlation with histotype and histological grade Neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical expression in follicular cell-derived thyroid tumors: a novel diagnostic tool Diagnostic value of neutrophil gelatinase-associated lipocalin (NGAL) immunoexpression in follicular-patterned lesions of the thyroid gland Urinary neutrophil gelatinase-associated lipocalin predicts kidney outcome and death in patients with cirrhosis and bacterial infections Impact of fatty liver disease on health care utilization and costs in a general population: a 5-year observation Urinary biomarkers and progression of AKI in patients with cirrhosis PubMed Abstract | CrossRef Full Text Epidemiology of non-alcoholic fatty liver disease Bläser A sandwich enzyme immunoassay for the determination of neutrophil lipocalin in body fluids Neutrophil gelatinase-associated lipocalin (NGAL) reflects iron status in haemodialysis patients Borkham-Kamphorst Induction of lipocalin-2 expression in acute and chronic experimental liver injury moderated by pro-inflammatory cytokines interleukin-1β through nuclear factor-κB activation Borkham-Kamphorst Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis Induction of neutrophil gelatinase-associated lipocalin in vascular injury via activation of nuclear factor-kappaB Molecular cloning and expression of a cDNA encoding NGAL: a lipocalin expressed in human neutrophils Cabedo Martinez Biochemical and structural characterization of the interaction between the siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal domain of its endocytic receptor SLC22A17 The origin of multiple molecular forms in urine of HNL/NGAL PubMed Abstract | CrossRef Full Text The detrimental role played by Lipocalin-2 in alcoholic fatty liver in mice Comparative mapping of lipocalin genes in human and mouse: the four genes for complement C8 γ chain and progestagen-associated endometrial protein map to HSA9 and MMU2 Lipocalin-2 inhibits autophagy and induces insulin resistance in H9c2 cells Iron metabolism and regulation by neutrophil gelatinase - associated lipocalin in cardiomyopathy Lipocalin 2 is required for pulmonary host defense against Klebsiella infection Tissue- and serum-associated biomarkers of hepatocellular carcinoma Increased plasma levels of lipocalin 2 in mild cognitive impairment Biomarkers for the diagnosis and risk stratification of acute kidney injury: a systematic review Siderocalin/Lcn2/NGAL/24p3 does not drive apoptosis through gentisic acid mediated iron withdrawal in hematopoietic cell lines Altered bone development and turnover in transgenic mice over-expressing lipocalin-2 in bone NGALR is overexpressed and regulated by hypomethylation in esophageal squamous cell carcinoma A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake Neutrophil gelatinase-associated lipocalin and its receptor: independent prognostic factors of oesophageal squamous cell carcinoma Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2 Lipocalin-2 expressed in innate immune cells is an endogenous inhibitor of inflammation in murine nephrotoxic serum nephritis Lipocalin: a novel diagnostic marker for hepatocellular carcinoma in chronic liver disease patients in Egypt Plasma neutrophil gelatinase-associated lipocalin as a marker for the prediction of worsening renal function in children hospitalized for acute heart failure Urinary neutrophil gelatinase-associated lipocalin as biomarker in the differential diagnosis of impairment of kidney function in cirrhosis Fierbinteanu-Braticevici Noninvasive investigations for non alcoholic fatty liver disease and liver fibrosis Nonalcoholic fatty liver disease as a multi-systemic disease Neutrophil gelatinase-associ-ated lipocalin in normal and neoplastic human tissues An apparent autocrine mechanism amplifies the dexamethasone- and retinoic acid-induced expression of mouse lipocalin-encoding gene 24p3 Serum neutrophil gelatinase-associated lipocalin—a sensitive novel marker of renal impairment in liver cirrhosis Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin Lipocalin-2 deficiency impairs thermogenesis and potentiates diet-induced insulin resistance in mice Lcn2 siRNA-encapsulating liposomes are potent anti-angiogenic agents for triple negative breast cancer Immune-induced expression of lipocalin-2 in brain endothelial cells: relationship with interleukin-6 cycloox-ygenase-2 and the febrile response Lipocalin-2 exacerbates psoriasiform skin inflammation by augmenting T-helper 17 response Urinary neutrophil gelatinase-associated lipocalin accurately detects acute allograft rejection among other causes of acute kidney injury in renal allograft recipients Siderocalin [Lcn 2] also binds carboxymycobactins The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake The neutrophil gelatinase-associated lipocalin (NGAL) is a survival factor for thyroid neoplastic cells Could blood Neutrophil Gelatinase-Associated Lipocalin (NGAL) be a diagnostic marker for acute kidney injury in neonates Lipocalin 2 is a selective modulator of peroxisome proliferator-activated receptor-γ activation and function in lipid homeostasis and energy expenditure Increased expression of neutrophil-related genes in patients with early sepsis-induced ARDS Neutrophil gelatinase-associated lipocalin and carbohydrate antigen 19-9 in pancreatic juice: pathobiologic implications in diagnosing benign and malignant disease of the pancreas The role of lipocalin-2 in liver regeneration Increased urinary lipocalin-2 reflects matrix metalloproteinase-9 activity in chronic hepatitis C with hepatic fibrosis Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse Non-alcoholic and alcoholic fatty liver disease - two diseases of affluence associated with the metabolic syndrome and type 2 diabetes: the FIN-D2D survey PubMed Abstract | CrossRef Full Text Clinical evidence for a protective role of lipocalin-2 against MMP-9 autodegradation and the impact for gastric cancer Proteomic profiling in Lipocalin 2 deficient mice under normal and inflammatory conditions Lipocalin-2 deficiency attenuates insulin resistance associated with aging and obesity Urinary neutrophil gelatinase-associated lipocalin as an early predictor of disease severity and mortality in acute pancreatitis Lívero Molecular basis of alcoholic fatty liver disease: from incidence to treatment Neutrophil gelatinase-associated lipocalin is a marker for dysregulated keratinocyte differentiation in human skin Lipocalin 2 is present in the EAE brain and is modulated by natalizumab Plasma and urine neutrophil gelatinase-associated lipocalin in the diagnosis of new onset acute kidney injury in critically ill patients Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages Early diagnosis of pancreatic cancer: neutrophil gelatinase-associated lipocalin as a marker of pancreatic intraepithelial neoplasia Urinary release of 72 and 92 kDa gelatinases N-GAL and conventional prognostic factors in urothelial carcinomas Gelatinase isoforms in urine from bladder cancer patients Circulating biomarkers in hepatocellular carcinoma Noninvasive diagnosis of nonalcoholic fatty liver disease PubMed Abstract | Google Scholar Neutrophil gelatinase-associated lipocalin: a novel inflammatory marker associated with late-life depression Non-Alcoholic steatohepatitis: clinical and translational research Tumour stroma-derived lipocalin-2 promotes breast cancer metastasis PubMed Abstract | CrossRef Full Text Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice Fatty liver disease and fatty acid oxidation 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cirrhosis Up-regulation of miR-138 inhibits hypoxia-induced cardiomyocyte apoptosis via down-regulating lipocalin-2 expression Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation Liver is the major source of elevated serum lipocalin-2 levels after bacterial infection or partial hepatectomy: a critical role for IL-6/STAT3 Lipocalin 2 promotes breast cancer progression Lipocalin-2 mediates non-alcoholic steatohepatitis by promoting neutrophil-macrophage crosstalk via the induction of CXCR2 cathepsin S and chemerin levels and nonalcoholic fatty liver disease Lipocalin-2 test in distinguishing acute lung injury cases from septic mice without acute lung injury Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma: significant correlation with cell differentiation and tumour invasion Neutrophil gelatinase-associated lipocalin worsens ischemia/reperfusion damage of kidney cells by autophagy NGAL and NGALR overexpression in human hepatocellular carcinoma toward a molecular prognostic classification Acute fatty liver of pregnancy: a retrospective analysis of 56 cases Weiskirchen S and Weiskirchen R (2016) Lipocalin 2 (LCN2) Expression in Hepatic Malfunction and Therapy Received: 28 June 2016; Accepted: 09 September 2016; Published: 27 September 2016 Copyright © 2016 Asimakopoulou, Weiskirchen and Weiskirchen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Ralf Weiskirchen, cndlaXNraXJjaGVuQHVrYWFjaGVuLmRl †These authors have contributed equally to this work. Method: Selective literature search in PubMed using the keywords ‘non-alcoholic fatty liver’, ‘fructose’, and ‘fibrosis’ was conducted. Results: The rate of overweight and obesity is significantly higher in both, adult and pediatric NASH patients. The consumption of free sugars is currently three times the maximum recommended amount of 10% of the energy intake. The current literature shows weight gain, negative effects on fat and carbohydrate metabolism and NASH with hypercaloric intake of fructose. Volume 12 - 2021 | https://doi.org/10.3389/fphar.2021.634344 Background: The excessive consumption of free sugars is mainly responsible for the high prevalence of obesity and metabolic syndrome in industrialized countries More and more studies indicate that fructose is involved in the pathophysiology and also in the degree of disease of non-alcoholic fatty liver disease (NAFLD) energy-adjusted higher fructose consumption correlates with NAFLD in overweight adults as an equivalent component of conventional household sugar appears to have negative metabolic effects in particular due to its exclusive hepatic metabolism Liver-related mortality is strictly associated with the degree of fibrosis whereas the most common cause of death in patients suffering from NAFLD and non-alcoholic steatohepatitis (NASH) are still cardiovascular diseases we have summarized the current state of knowledge regarding a relationship between fructose consumption liver fibrosis and life expectancy in NASH Method: Selective literature search in PubMed using the keywords ‘non-alcoholic fatty liver’ and ‘fibrosis’ was conducted Results: The rate of overweight and obesity is significantly higher in both The consumption of free sugars is currently three times the maximum recommended amount of 10% of the energy intake negative effects on fat and carbohydrate metabolism and NASH with hypercaloric intake of fructose Conclusions: Excessive fructose consumption is associated with negative health consequences Whether this is due to an excess of energy or the particular metabolism of fructose remains open with the current study situation The urgently needed reduction in sugar consumption could be achieved through a combination of binding nutritional policy measures including taxation of sugary soft drinks Previous studies suggest that diet-related fructose intake exceeding the amount contained in vegetables and fruits lead to an increase of hepatic lipogenesis further studies to clarify the protective contribution of low-fructose intake to positively influence NAFLD in industrial population are urgently required Fructose-rich diet forms can very quickly lead to almost all basic diseases of the metabolic syndrome. (Hannou et al., 2018). This medical condition is associated with trunk obesity, arterial hypertension, high serum sugar/impaired glucose tolerance (diabetes), elevated serum triglycerides, and reduced high-density lipoproteins (Figure 1) Deleterious effects of a high fructose intake on human health Excessive fructose consumption is a risk factor for several chronic diseases including non-alcoholic fatty liver disease (NAFLD) Histopathology of nonalcoholic steatohepatitis (NASH) histologic features of NASH include steatosis Although not a requirement for the histological diagnosis NASH fibrosis is often paired with the pathological changes (right) The aim of the present review is a compilation of the current data that support the importance of fructose intake for the development of liver fibrosis and life expectancy in NASH A selective literature search in PubMed using the keywords ‘non-alcoholic fatty liver’ and ‘fibrosis’ was conducted at the beginning of December 2020 After excluding pure in vitro trials and animal experiments 35 publications were finally used for this review The extent to which fat as a macronutrient contributes to the development of NAFLD as well as the effects of high carbohydrate intake is the subject of current research prospective multicenter studies analyzing whether marked transformations of hepatic fat amount will influence cholesterol homeostasis are still missing it is indisputable that fructose decreases insulin sensitivity and increases visceral adiposity in overweight/obese adults But how does it work in lean subjects with NAFLD young and healthy humans might at least for a short time period be able to compensate a higher fructose intake Diacylglycerol acyltransferase (DGAT)1 and DGAT2, catalyze the final step of triglyceride synthesis (Harris et al., 2011). A recent study indicates that hepatic DGAT2 deficiency leads to a reduction of diet-induced hepatic steatosis, thus supporting the application of DGAT2 inhibitors as a therapeutic approach to ameliorate NAFLD and associated diseases (Gluchowski et al., 2019) the inhibition of DGAT2 is regarded as a promising therapeutic approach for NAFLD/NASH in humans NAFLD representing a form of chronic hepatitis is one of the most important and rising causes of liver disease worldwide Respective patients have a high frequency of metabolic comorbidities such as obesity impaired insulin resistance and dyslipidemia The current standard of care for NAFLD patients is the limitation and control of risk factors and the advice to follow recommendations for lifestyle changes weight loss provoked by dietary restriction and regular physical exercise can be beneficial in regress of liver disease A Mediterranean diet can significantly reduce liver fat mass even without weight loss and is most recommended there are also a substantial proportion of lean patients with NAFLD and the avoidance of foods enriched in saturated fats are reasonable specific dietary recommendations for NAFLD and NASH patients Sedentary activity patterns contribute as well as ingestion of low amounts of polyunsaturated fatty acid to NAFLD and metabolic syndrome it is very difficult to emphasize the importance of one single nutrient high-quality basic research and well-defined randomized clinical trials assessing dietary interventions that focus on liver-specific endpoints-are urgently needed as a priority All authors listed have made a substantial and intellectual contribution to the work and approved it for publication ER received grants from the German Research Foundation DFG (RO 957/10-1 von-Behring-Roentgen Foundation (#58-0005 and #66-0008 to ER) University Medical Center Giessen and Marburg (UKGM) (#10 and #21_2013 GI to ER) RW is supported by grants from the German Research Foundation (SFB/TRR57 and WE 2554/15-1) and the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the RWTH Aachen University (IZKF Aachen The funders had no role in the design of this review or in the decision to publish it The authors are grateful to Sabine Weiskirchen (IFMPEGKC) who prepared the figures for this review Increased fructose consumption is associated with fibrosis severity in patients with nonalcoholic fatty liver disease PubMed Abstract | CrossRef Full Text | Google Scholar Soft drink consumption is associated with fatty liver disease independent of metabolic syndrome PubMed Abstract | CrossRef Full Text | Google Scholar Soft drink consumption linked with fatty liver in the absence of traditional risk factors PubMed Abstract | CrossRef Full Text | Google Scholar Bagus, T., Roser, S., and Watzl, B. 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Available at: https://www.fda.gov/food/food-additives-petitions/high-fructose-corn-syrup-questions-and-answers Google Scholar Sugar content of popular sweetened beverages based on objective laboratory analysis: focus on fructose content PubMed Abstract | CrossRef Full Text | Google Scholar vegetables or their fiber reduces the risk of type 2 diabetes: a meta-analysis PubMed Abstract | CrossRef Full Text | Google Scholar Challenging the fructose hypothesis: new perspectives on fructose consumption and metabolism PubMed Abstract | CrossRef Full Text | Google Scholar Hepatitis B virus surface proteins accelerate cholestatic injury and tumor progression in Abcb4-knockout mice PubMed Abstract | CrossRef Full Text | Google Scholar The Mediterranean dietary pattern as the diet of choice for non-alcoholic fatty liver disease: evidence and plausible mechanisms PubMed Abstract | CrossRef Full Text | Google Scholar Citation: Roeb E and Weiskirchen R (2021) Fructose and Non-Alcoholic Steatohepatitis Received: 27 November 2020; Accepted: 07 January 2021;Published: 08 February 2021 Copyright © 2021 Roeb and Weiskirchen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use *Correspondence: Elke Roeb, RWxrZS5Sb2ViQGlubmVyZS5tZWQudW5pLWdpZXNzZW4uZGU=; Ralf Weiskirchen, cndlaXNraXJjaGVuQHVrYWFjaGVuLmRl Volume 10 - 2019 | https://doi.org/10.3389/fphar.2019.01289 Montelukast Prevents Mice Against Acetaminophen-Induced Liver Injury A Commentary onMontelukast Prevents Mice Against Acetaminophen-Induced Liver Injury by Pu S, Liu Q, Li Y, Li R, Wu T, Zhang Z, Huang C, Yang X and He J (2019) Front. Pharmacol. 10:1070. doi: 10.3389/fphar.2019.01070 Pu et al. (2019) tested whether the pharmacological inhibition of CysLTR1 by Montelukast impacts acetaminophen (APAP)-induced acute liver failure in C57BL/6J mice. APAP-induced liver injury is a rather complex process in which intracellular and extracellular events are involved including mitochondrial and endoplasmic reticulum oxidative stress, sterile inflammation, microcirculatory dysfunction and liver regeneration (Yan et al., 2018) The authors found that Montelukast-induced CysLTR1 expression and significantly prevented drug-induced liver damage when given at 3 mg/kg body weight by gavage 1 h after APAP administration. In addition, the authors demonstrated that Montelukast upregulated hepatic GSH/GSSH level, provoked expression of Glutathione S-transferase A2 (GSTA2), and prevented liver inflammation, oxidative stress, as well as activation of the c-Jun-N-terminal kinase (JNK) (Figure 1) Montelukast was in vitro effective in blocking cell death and inflammation induced by APAP treatment in mouse primary hepatocytes the authors concluded that the inhibition of CysLTR1 by Montelukast may be a potential treatment strategy in APAP-induced hepatotoxicity Figure 1 Montelukast prevents mice against APAP-induced liver damage The observed beneficial effects of Montelukast in APAP-treated mice are depicted The decrease of pro-inflammatory chemokines or cytokines including Mcp1 and Il18 by Montelukast in primary cultured hepatocytes treated with APAP demonstrates that this drug prevents inflammatory signaling any intervention that blocks NAPQI formation will also prevent downstream effects The data presented by the authors shows that Montelukast impacts NAPQI formation the major and potentially more important mechanism of protection is inhibition of acetaminophen bioactivation not inhibition of JNK signaling or inflammation is likely a secondary effect of the protection The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The author is grateful to Sabine Weiskirchen (Institute of Molecular Pathobiochemistry Experimental Gene Therapy and Clinical Chemistry RWTH University Hospital Aachen) for preparing the figure for this General Commentary Montelukast inhibits inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes Acquired thrombopathia related to montelukast therapy Improvement of hepatic fibrosis by leukotriene inhibition in cholestatic rats Google Scholar doi: 10.7326/0003-4819-140-7-200404060-00042 Acetaminophen-induced liver injury: from animal models to humans The effect of montelukast on liver damage in an experimental obstructive jaundice model Effect of montelukast on nuclear factor kappaB activation and proinflammatory molecules Metabolism and disposition of acetaminophen: recent advances in relation to hepatotoxicity and diagnosis Montelukast prevents mice against acetaminophen-induced liver injury Acute hepatitis associated with montelukast A case of montelukast-induced hepatotoxicity A review on leukotrienes and their receptors with reference to asthma Hepatoprotective and anti-fibrotic agents: it’s time to take the next step New insights into the role and mechanism of c-Jun-N-terminal kinase signaling in the pathobiology of liver diseases Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions Citation: Weiskirchen R (2019) Commentary: Montelukast Prevents Mice Against Acetaminophen-Induced Liver Injury Received: 18 September 2019; Accepted: 08 October 2019;Published: 25 October 2019 Copyright © 2019 Weiskirchen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) The World Heritage Centre is at the forefront of the international community’s efforts to protect and preserve World Heritage partnerships for conservation Ensuring that World Heritage sites sustain their outstanding universal value is an increasingly challenging mission in today’s complex world where sites are vulnerable to the effects of uncontrolled urban development Our Partners Donate Take advantage of the search to browse through the World Heritage Centre information. Miraculously preserved in the beautiful setting of an Alpine valley, the Church of Wies (1745–54), the work of architect Dominikus Zimmermann, is a masterpiece of Bavarian Rococo – exuberant, colourful and joyful. Miraculeusement conservée dans l'écrin d'une vallée des Alpes, l'église de Wies (1745-1754), chef-d'œuvre de l'architecte Dominikus Zimmermann, est probablement l'expression la plus parfaite du rococo bavarois, exubérant, allègre et coloré. ما زالت كنيسة دي فيز (1745-1754) في حالة ممتازة وهي تقع في كنف وادي من وديان الألب. إنها تحفة فنية للمهندس المعماري دومينيكوس زيمرمان وهي على الأرجح أجمل تعبير لنمط الروكوكو البافاري المليء بالحيوية والفرح والألوان. 维斯教堂（1745至1754年）坐落在风景秀美的阿尔卑斯山谷中，保存十分完好，是建筑师多米尼克斯·齐默尔曼(Dominikus Zimmermann)的作品，是巴伐利亚洛可可风格的优秀代表作。整个作品充满了活力，欢快而且色彩丰富。 Церковь в Висе, построенная в 1745-1754 гг., чудесным образом сохранилась в живописной альпийской долине. Это пышный, красочный и преисполненный радости шедевр баварского рококо работы архитектора Доминикуса Циммермана. Conservada milagrosamente en el corazón de un valle de los Alpes, la iglesia de Wies (1745-54), obra del arquitecto Dominikus Zimmermann, es una joya del arte rococó bávaro, exuberante, alegre y lleno de color. The sanctuary of Wies, near Steingaden in Bavaria, is a pilgrimage church extraordinarily well-preserved in the beautiful setting of an Alpine valley, and is a perfect masterpiece of Rococo art and creative genius, as well as an exceptional testimony to a civilization that has disappeared. The church, which is oval in plan, is preceded to the west by a semi-circular narthex. Inside, twin columns placed in front of the walls support the capriciously cut-out cornice and the wooden vaulting with its flattened profile; this defines a second interior volume where the light from the windows and the oculi is cleverly diffused both directly and indirectly. To the east, a long deep choir is surrounded by an upper and a lower gallery. Criterion (i): The sanctuary of Wies, a pilgrimage church constructed in the open countryside, is a perfect masterpiece of Rococo art. Criterion (iii): The Pilgrimage Church of Wies is an exceptional testimony of cultural and religious traditions.  In this sparsely settled area, in complete solitude, it was possible for a religious and architectural idea to be realized unhindered. The site, therefore, contains all elements necessary for Outstanding Universal Value. There are no immediate, adverse effects of development and/or neglect.  The setting is completely untouched. Form and design, material and substance, use and function of the Pilgrimage Church of Wies have remained unchanged.  A core and a buffer zone have been identified to ensure the lasting protection and sustained preservation of the visual and built integrity of the Pilgrimage Church of Wies and its immediate surroundings. The Free State of Bavaria is owner of the Pilgrimage Church of Wies. The property is managed under the responsibility of the Pilgrimage Church Foundation St Joseph. The State Construction Office Weilheim is responsible for construction issues, and the coordination between the stakeholders is organised by the site manager, who is based in the administrative district office of Weilheim. The management system consists of a set of maintenance and conservation measures to ensure the protection of the property and the surrounding cultural landscape by sustainable agriculture. More information on visitor management is provided in the Management Plan and through the Periodic Reporting mechanism. Volume 9 - 2018 | https://doi.org/10.3389/fphar.2018.00426 Lipocalin 2 (LCN2) is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota Human LCN2 contains one N-glycosylation site conserved in other species It was postulated that this post-translational modification could facilitate protein folding is required for proper trafficking from the Golgi apparatus to the cell surface and might be relevant for effective secretion We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types but might be relevant to increase overall solubility these former biochemical studies show that LCN2 is a glycosylated protein data on functional aspects of LCN2 glycosylation is missing Therefore, it is possible that the respective N-glycosylation site is a critical determinant in trafficking of LCN2 to exosomes. In line with this assumption, first evidence for the occurrence of LCN2 in exosomes was demonstrated in urine samples in which the cellular fraction contained lower levels of LCN2 compared with the exosomal fraction (Alvarez et al., 2013) we addressed the question if N-glycosylation of LCN2 is necessary for proper secretion or exosome targeting The inhibition of N-glycosylation by tunicamycin in primary hepatocytes resulted in a slimed core protein that retained its capacity to become secreted and recruited to the exosome fraction thapsigargin leading to endoplasmic reticulum Ca2+ depletion provoking unfolded protein response failed to inhibit LCN2 expression we conclude that the impact of tunicamycin in N-glycosylation of LCN2 is linked to inhibition of the GlcNac phosphotransferase while N-linked glycosylation is not necessary for LCN2 exosome cargo recruitment The prediction of signal peptide cleavage sites were done with the SignalP 4.1 algorithm (Petersen et al., 2011) via a web resource3 The primary sequences of human, mouse, and cheetah LCN2 proteins were analyzed for potential N-glycosylation site using the NetNGlyc N-Glycosylation site predictor with the default parameter settings4 The hydrophobic cluster analysis of the LCN2 protein from different species was done with a web resource for structural Bioinformatics using the default settings5 The stimulation experiments (24 h) were done in cells adapted to advanced DMEM/F12 without FCS and the use of 200 ng/mL LPS (from Salmonella typhimurium Sigma-Aldrich) and/or 50 or 100 ng/mL tunicamycin from Streptomyces sp The cell line NB4 was established from the bone marrow of a 23-year-old female patient with acute promyelocytic leukemia (Lanotte et al., 1991; Duprez et al., 1992) The cells were routinely grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS Experiments and stimulation experiments were done in cells adapted to advanced DMEM/F12 medium under the same conditions described for HL-60 (see section HL-60/dHL-60) For differentiation into granulocytic cells (dNB4) the medium was supplemented with 1 μM ATRA and 0.4% DMSO for 4–5 days LPS and/or tunicamycin stimulation was done as described above (HL-60/dHL-60) This epithelial human prostate cell line (ATCC® CRL-1435™) established from a metastatic site of bone of a 62-year-old male patient suffering from grade IV adenocarcinoma (Kaighn et al., 1979) was obtained from LGC Standards GmbH (Wesel Germany) and routinely cultured in DMEM supplemented with 10% FCS the cells were adapted to advanced DMEM/F12 (+ 10 % FCS Stimulation with 2.5 ng/mL recombinant human IL-1β (IL-1β Germany) and/or 0.5 μg/mL tunicamycin was done for 36 h in advanced DMEM/F12 without FCS This human epithelial carcinoma lung cell line originated from an explant culture derived from a 58-year-old male patient with solid lung carcinoma (Giard et al., 1973) The cells were routinely grown in DMEM supplemented with 10% FCS All experiments and stimulation experiments were done in advanced DMEM/F12 under conditions described for PC-3 cells (see PC-3) In some Western blot experiments depicted, HepG2 cells stimulated with IL-1β were taken as a positive control for LCN2 expression. This human hepatoma derived cell line isolated from liver biopsies of a child with hepatocellular carcinoma (Knowles et al., 1980). When stimulated with IL-1β, the expression of LCN2 is strongly induced (Borkham-Kamphorst et al., 2011) The murine hepatoma cell line TW60 was generated in the laboratory of Christian Liedtke (Department of Internal Medicine III, RWTH Aachen University) as described before (Boaru et al., 2015) this tumorigenic cell line was isolated from hepatocellular carcinoma nodules developed in male mice on C57BL/6 genetic background 40 weeks after single intraperitoneal injection of 25 mg diethylnitrosamine/kg body weight Cell were routinely grown in DMEM containing 1% non-essential amino acids The stimulation experiments were done in advanced DMEM/F12 under conditions described for PC-3 cells (see PC-3) One or two days after initial plating of primary hepatocytes the medium was replaced with fresh medium containing 1–10 μg/mL tunicamycin or 0.025–0.2 μg/mL thapsigargin (#T9033 Sigma-Aldrich) and cultured for additional 24 h the conditioned cell culture media were harvested and cell protein extracts prepared for Western blot analysis Cells cultured for the same time without tunicamycin or thapsigargin or in the presence of vehicle (dimethyl sulfoxide All animal liver specimens used in immunohistochemistry were generated in our laboratory in previous experiments no additional suffering to animals was caused in this study The former experiments were approved by the LANUV (see also comment in Isolation and Culturing of Primary Hepatocytes) The application of lipopolysaccharide (LPS) was performed following a well-standardized protocol previously published (Hamesch et al., 2015) animals received 2.5 mg/kg body weight LPS and were sacrificed 6 h thereafter Control mice received a same volume (100 μl) of a normal saline solution (NSS) A detailed protocol for the application of Concanavalin A (Con A) including preparation of the Con A working solution was previously published (Heymann et al., 2015) Mice received intravenous injection of 25 mg/kg body weight Con A and were sacrificed 8 h thereafter Livers from mice injected with NSS served as control Induction of experimental obstructive cholestasis in mice was induced by bile duct ligation (BDL), essentially following protocols outlined elsewhere (Tag et al., 2015a,b) Animals were sacrificed 4 weeks after the surgery and sham-operated mice served as controls Repeated administration of carbon tetrachloride (CCl4) in mice was performed as outlined in detail elsewhere (Scholten et al., 2015) chronic intoxication with CCl4 was done by intraperitoneal injection of 0.6 μL/g body weight diluted in corn oil The injections were performed twice per week for 8 consecutive weeks Mice receiving equal volumes of corn oil alone served as controls The primary antibodies were visualized using horseradish peroxidase (HRP)-conjugated anti-mouse- or anti-goat IgG (all from Santa Cruz Biotech.) and the SuperSignal chemiluminescent substrate (Pierce Isolation of exosomes from cell culture conditioned media of hepatocytes was carried out following an ultracentrifugation protocol as described by others considering small modifications (Lobb et al., 2015) the conditioned media was harvested from primary murine hepatocytes and centrifuged using a Heraeus Sepatech refrigerated centrifuge at 600 g at 4°C in a BS 4402/A rotor for 15 min to remove detached cells Supernatant was collected and again centrifuged at 3,200 g at 4°C for 30 min to remove cell fragments The second supernatant was collected and filtered slowly through 0.22 μm sterile syringe filters (#431219 Germany) to remove contaminating apoptotic bodies Cleared supernatant was then centrifuged in a Beckman OptimaTM L-70K Ultracentrifuge equipped with a SW 40 Ti rotor at 100,000 g (29,500 rpm; RCFavg 109,895; RCFmax 154,779; k-factor: 252,5) at 4°C for 70 min to pellet exosomes and crude exosome-containing pellets were washed in ice-cold 200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0) and pooled A second round of ultracentrifugation under the same conditions was carried out and the resulting exosome pellet resuspended in 200 μL of 200 mM HEPES buffer (pH 7.0) for electronmicroscopic analysis and NTA measurements or in 200 μl of 50 mM HEPES buffer (pH 7.0) for Western blot analysis For isolation of exosomes from immortalized cell lines or advanced DMEM/F12 supplemented with 10% heat inactivated or normal FCS and PC-3 cells were stimulated for 24 h in serum free advanced DMEM/F12 medium with LPS or IL-1β cell culture media were taken for preparation of exosomes following the protocol given above (see Method for Isolation of Exosomes from Conditioned Media) Particle size and concentration of exosome preparations were measured using the NanoSight NS300 instrument (Malvern Instruments Limited UK) allowing particle concentration to be visualized and measured at 106-109 particles per mL in the 10 nm–2,000 nm diameter range in liquid suspension The high resolution size distributions on a particle-by-particle basis are counted in real-time and the quantities of purified exosome suspensions of each five individual measurements were combined and visualized with the NanoSight NS300 nanoparticle tracking analysis (NTA) v 3.00 software (Malvern Instruments Limited) For details see (Malvern Application note) Unfixed isolated exosomes in 200 mM HEPES buffer (pH 7.0) were allowed to adsorb on glow discharged Formvar/carbon-coated nickel grids (Maxtaform Samples on grids were contrasted by placing on a drop of ready-made pre-packed 0.5% uranyl acetate in aqua dest samples were examined using a TEM LEO 906 (Carl Zeiss operating at an acceleration voltage of 60 kV Representative images of exosomes were taken over a magnification range from 60,000 to 359,700x Liver tissue sections obtained from mice treated with LPS, Con A, CCl4, or subjected to BDL were prepared for immunohistochemical analysis as described previously (Borkham-Kamphorst et al., 2008). Staining for LCN2 and neutrophil marker MPO was performed under conditions given elsewhere (Borkham-Kamphorst et al., 2013; Asimakopoulou et al., 2016a) Immunohistochemistry of LCN2 expression in liver injury (A) Sections were prepared from livers obtained from animals 6 h after LPS injection and stained for LCN2 and MPO Liver sections obtained from normal standard saline (NSS)-injected animals and from Lcn2 deficient mice served as controls (B) Liver sections from animals treated with Con A for 8 h (upper) subjected to bile duct ligation (BDL) for 4 weeks (middle) or from animals receiving CCl4 for 8 weeks (lower) were immunostained for LCN2 or oil-injected mice were taken as controls staining with unspecific goat or rabbit control immune sera served as controls Primary murine hepatocytes isolated from wild type or Lcn2 deficient were incubated for 24 h in the presence of indicated concentrations of tunicamycin (a N-acetylglucosamine transferase inhibitor) or thapsigargin (a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase and ER stressor) Cell extracts were prepared and subjected to Western blot analysis The blots were probed with indicated antibodies Cell extracts from cells without drug treatment and vehicle (DMSO)-treated cells served as controls The probing with a GAPDH specific antibody was performed to demonstrate equal protein loading in each lane Please note the reduced size of LCN2 (LCN2*) in cells that were treated with tunicamycin Western blot analysis of cell extracts and conditioned media (A) Cell extracts or (B) conditioned media of primary hepatocytes cultured for 24 h in the presence of tunicamycin or without any additional supplement were subjected to Western blot analysis and probed for indicated proteins Please note that the cell extracts were probed for CHOP with two different antibodies (CST #2895 and CST #5554) which identified two protein bands with a preference for the upper band supposed to be CHOP The Ponceau S stains were taken to document equal protein loading Please note the lower molecular weight of LCN2* that is due to lack of N-glycosylation the elevated quantities of activated pp65/RelA and the activation of the NF-κB pathway are mechanisms to compensate the lack of LCN2 or are signs of early cellular damage and initiation of apoptotic processes Characterization of isolated exosomes isolated from conditioned medium (A–D) Representative transmission electron microscopic pictures of isolated exosomes negatively stained with 0.5% uranyl acetate Images were taken at original magnifications of (A) 60,000 (E,F) Exosomes were isolated from conditioned media of Ad5-CMV-mLCN2-infected primary hepatocytes cultured for 24 h in the presence of (E) 0.01% DMSO (control) or (F) 0.5 μg/mL tunicamycin Particle size distribution and calculated concentrations as determined by nanoparticle tracking analysis of purified exosome suspensions are depicted Targeting of LCN2 into exosomes of primary hepatocytes Exosomes were prepared from conditioned media isolated from AdEasy1-CMV-mLCN2-infected primary murine hepatocytes treated with tunicamycin (+TUN) or left untreated (–TUN) Fractions of conditioned media as well as supernatant (UC supernatant) and exosome pellet (UC exosomes) after ultracentrifugation were then tested in Western blot for expression of LCN2 (left) or exosome markers CD81 and Alix (right) Ponceau S stain served to document equal transfer of proteins from the SDS gel to the membrane while short and long exposure times for the LCN2 specific Western blot are depicted for better estimation of LCN2 quantities targeted to the exosome fraction Targeting of LCN2 into exosomes of stimulated primary hepatocytes Primary murine hepatocytes were stimulated for 24 h with 400 ng/mL LPS or 2.5 ng/mL IL-1β in the presence or absence of 0.5 μg/ml tunicamycin Protein cell extracts and conditioned media were harvested and exosomes isolated The different fractions were then tested in Western blot for LCN2 expression or exosome markers CD81 and Alix In this set of experiments unstimulated cells served as control Please note that the stimulation with IL-1β resulted in increase of LCN2 in cell extracts and exosomes although both immortalized cell lines expressed the typical myeloid marker MPO Expression and targeting of LCN2 into exosomes in A549 cells A549 cells were stimulated with IL-1β media after ultracentrifugation (UC supernatant) and exosome pellets (UC exosomes) were analyzed for LCN2 expression in Western blot analysis The blots were reprobed with exosome markers Alix and CD81 while GAPDH expression served as control to demonstrate equal gel loading in cell extract fractions the occurrence of two specific Alix protein bands in the A549 cell lysates these bands are found in many human immortalized cell lines including myelogenous leukemia line K-562 and (pro-) monocytic-like lines THP-1 and U-937 when using this mouse monoclonal antibody directed against full length human Alix (Santa Cruz Biotechnology) Expression and targeting of LCN2 into exosomes of PC-3 cells PC-3 cells were treated with tunicamycin (TUN) or left untreated and prepared exosome pellets (UC exosomes) were analyzed for LCN2 expression while GAPDH expression served as loading control Another possibility is that the attached sialic acids in the siderophore LCN2 could act as direct chemoattractants for bacteria. Many bacteria not only use sialic acid as a nutrient, but also incorporate sialic acid in their cell surface, helping them to evade or resist from components of the innate immune response of the host (Severi et al., 2007) Such a biological sugar trap in combination with the affinity of LCN2 to bacterial siderophores might be important in the innate immune response to bacterial infections it is also possible that the N-glycosylated form of LCN2 is more suitable in preserving MPO function by binding tighter to Enterochelin We must admit that there are still several other possibilities how N-glycosylation of LCN2 might fine tune processes counteracting bacterial uptake of iron-loaded siderophores. Some of them had been recently highlighted in a review suggesting the siderophore-binding protein LCN2 acts in a “Tug-of-war” against bacterial siderophores (Wilson et al., 2016) In this scenario the attached sugar tree may be one of the required structural elements in defining the molecular interface necessary to prevent pathogens from acquiring iron through their high-affinity siderophores the direct in vivo application of this drug for blockade of LCN2 glycosylation is too harmful The finding that tunicamycin blocks LCN2 glycosylation will offer a suitable alternate strategy to prepare both LCN2 variant forms for a wealth of applications LCN2 is a secreted protein produced in high quantities in hepatocytes during inflammation It is post-translationally modified at an evolutionarily conserved N-glycosylation site possibly relevant to determine biochemical characteristics of LCN2 The glycosylation of LCN2 can be effectively blocked by application of tunicamycin without preventing its proper secretion or exosome targeting Our study provides a valuable starting point for future work aiming to comparatively analyze the biological characteristics of the glycosylated and unglycosylated LCN2 variants All authors have materially participated in the research of this study and approved the final version of this article RW designed the study and wrote the manuscript RW is supported by grants from the German Research Foundation (DFG and Q3) and the Interdisciplinary Centre for Clinical Research (IZKF) within the Faculty of Medicine at the RWTH Aachen University (E7-6 and O3-1) Lipocalin 2 null mice were a kind gift from Drs The authors thank Steffen Gräber (Helmholtz Institute for Biomedical Engineering RWTH University Hospital Aachen) for technical help in NTA Human cell lines HL-60 and NB4 were kindly provided by Inga Wessels (Institute of Immunology The carcinoma lung cell line A549 was a kind gift of Andreas Ludwig (Institute of Pharmacology The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2018.00426/full#supplementary-material Multiple sequence alignment of LCN2 from various species and prediction of N-glycosylation (A) LCN2 protein sequences from human (HS) and Malayan Pangolin (MJ) were aligned using Clustal Omega An asterisk (*) indicates a position with fully conserved residue a colon (:) indicates a conservation of amino acids with strongly similar properties and a period (•) indicates a conservation of amino acids with weakly similar properties Amino acid positions are given in the right margin Potential N-glycosylation sites determined by the NetNGlyc N-glycosylation site predictor are depicted in reversed type the cysteine residues (Cys 96 and Cys 195 in human LCN2) proposed to be involved in the formation of LCN2 homodimers or intramolecular disulfide bounds are marked in red letters and secretory signal peptides are boxed in yellow (B) The primary LCN2 protein sequences of human (Homo sapiens) mouse (Mus musculus) and cheetah (Acinonyx jubatus) were analyzed for potential N-glycosylation sites using the NetNGlyc N-Glycosylation site predictor The individual graphs illustrate predicted N-glyc sites across the protein chain in which the x-axis represents amino acid positions from N- to C-terminus A position with a potential glycosylation site (vertical line) crossing the threshold (horizontal line at 0.5) is predicted as glycosylated Stimulation of LCN2 expression in TW60 cells Cell extracts and conditioned media prepared from TW60 cells left untreated (control) or stimulated with 400 ng/mL LPS or 2.5 ng/mL IL-1β in the presence or absence of tunicamycin (TUN) were analyzed for expression of LCN2 GAPDH expression served as control to demonstrate equal gel loading in cell extracts (A) Cell extracts and (B) conditioned media of NB4 cells stimulated with LPS LPS and TUN or left untreated were analyzed for expression of LCN2 cells were cultured in medium containing 10% FCS or serum-free medium Ponceau S stain served as control to demonstrate integrity of protein samples Lack of LCN2 expression in cell extracts of dHL-60 and dNB4 cells Cell extracts of differentiated dHL-60 and dNB4 cells stimulated with LPS (200 ng/mL) or LPS and different concentrations of tunicamycin (TUN 50 or 100 μg/mL) were analyzed for expression of LCN2 and MPO A cell extract isolated from IL-1β-stimulated HepG2 cells served as control Equal protein loading was demonstrated by Ponceau S stain and probing with an antibody specific for β-actin although cell extracts were positive for MPO Lack of LCN2 expression in conditioned media of dHL-60 and dNB4 cells Conditioned culture media of differentiated dHL-60 and dNB4 cells stimulated with LPS or LPS and different concentrations of tunicamycin (TUN) were analyzed for expression of LCN2 Equal protein loading was demonstrated by Ponceau S stain (A) Cell extracts and (B) conditioned media of A549 and PC-3 cells left untreated (control) or stimulated with IL-1β IL-1β and TUN were analyzed for expression of LCN2 and cheetah were subjected to a hydrophobic cluster analysis Symbols are used to represent amino acids with peculiar structural properties (red star for proline square and dotted square for threonine and serine The positions of the glycosylated asparagines in the LCN2 of each species are indicated by an arrow that this residue is embedded in a highly hydrophobic surrounding in all species NTA measurement for size determination of exosomes isolated from conditioned media of hepatocytes cultured in the presence of DMSO as vehicle for 24 h Isolation of exosomes was done as described in the Materials and Methods section (see Method for Isolation of Exosomes from Conditioned Media) NTA measurement for size determination of exosomes isolated from conditioned media of hepatocytes cultured in the presence of 0.5 μg/mL tunicamycin for 24 h 1. ^The human protein atlas. 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Department of Bio and Health Informatics. http://www.cbs.dtu.dk/services/SignalP/ (Accessed March 19 4. ^NetNGlyc 1.0 Server. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) Metrics details The Directive 2010/63 EU requires classifying burden and severity in all procedures using laboratory animals This study evaluated the severity of liver fibrosis induction by intraperitoneal carbon tetrachloride (CCl4) injections in mice 29 male C57BL/6N mice were treated three times per week for 4 weeks with an intraperitoneal injection (50 µl) of either 0.6 ml/kg body weight CCl4-vehicle solution germ oil (vehicle-control) or handling only Severity assessment was performed using serum analysis fecal corticosterone metabolite (FCM) measurement The most significant group differences were noticed in the second week of treatment when the highest AST (1463 ± 1404 vs p < 0.0001) and nesting values were measured respective animals showed lower moving distances (4622 ± 1577 vs p < 0.01) and velocity in the Open field identified as main factors in principal component analysis (PCA) a 50% survival rate was observed within the treatment group in which the open field performance was a good tracer parameter for survival this study demonstrates the feasibility of assessing severity in mice using behavioral tests and highlight the open field test as a possible threshold parameter for risk assessment of mortality researchers have to assign different degrees of severity to diverse procedures always according to the behavioral characteristics of the respective species this model is often used in the research of fibrosis development as well as in the investigation of liver repair mechanisms underlining the scientific importance of this model The aim of this research project was to evaluate the severity of induction of liver fibrosis in mice within the periodic administration of CCl4 in a 4-week i.p Fecal corticosterone metabolite (FCM) analysis and Rotarod performance (data not shown) showed no significant differences between the experimental groups and their baseline values during the time of the experiment the animals in the CCl4 group burrowed fewer food pellets than the oil-treated control group (data not shown) due to a lack of showing burrowing behavior in enough animals in the handling group no full statistical analysis between these groups could be performed experimental models of liver fibrosis in mice are of great importance in biomedical research we examined three different rational approaches: (i) the actual stress caused by CCl4 administration taking into account acute toxicity and pain; (ii) chronic effects of cumulative treatment (e.g multiple injections and chronic toxicity); (iii) survival rate and determination of cut-off values in behavioral testing to better estimate the risk of mortality The animals subjected to CCl4 injection showed a significant body weight reduction in the first week compared to the control group animals The development of body weight change in CCl4 treated animals compared to the handling group showed significant differences in week 1 and week 4 This was adjoined with a trend to gain more weight in the handling group while there was no deviation in food intake in all of the three groups detectable weight reduction most likely results from increased metabolism through pain or stress in the first week of treatment when the stress situation caused by the treatment first affects the animals and no adaptive mechanisms can occur Although the animals of the CCl4 group show a slight increase in body weight in the following weeks after the initial weight loss of week 1 the main contributing variables to PCA were further studied by univariate Cox-regression to calculate the individual hazard ratio on animal survival We could demonstrate that the monitoring of the Open field velocity is a suitable measurement parameter for the probability of survival in which a velocity below the defined threshold of 57.8% corresponds with a 4.75-fold higher risk of mortality The severity of the CCl4 model in mice tends to show a moderate severity level it was associated with an increased mortality (33.33%) in our study Although the administration of CCl4 is a long-known and widely used model the publication rate of survival and studies on the mortality of these study models are very rare the severity level of an animal model can most likely also depend on strain Therefore these measures should be strategically investigated and verified for their refinement potential in future studies can be used in estimating severity and animal monitoring The analysis for the survival prediction in this study is not unrestricted The definition of the threshold was based on two criteria the ROC AUC analysis with the value of 0.89 showed that this test procedure is suitable for the assessment of mortality and the definition of a cut-off value for severity assessment this procedure should be validated and refined in further studies the study demonstrates the general feasibility of assessing severity using behavioral tests and could demonstrate that the Open field test is a possible candidate parameter for risk assessment this study also shows that the systematic investigation of severity levels and mortality rates should be investigated to develop sufficient refinement measures for experimental models Humane endpoint criteria were established for all animals These endpoints were applied when > 20% body weight loss Study design with all procedures and treatments training and baseline measurements were performed Body weight was measured initially before allocating the animals to the different treatment groups (CCl4/oil/handling) All animals were allocated randomly according to the average starting weight of both experimental groups the nest was evaluated as follows: score of 1 means that the nestlet is more than 90% untouched and a score of 5 equals a perfect built nest the time until the first contact with the nestlet (latency to start) was determined based on the video analysis Rotarod testing was performed with TSE Rotarod (Rotarod Advanced All animals were trained two times before the test A third rotarod testing was performed to set baseline values for each animal One rotarod test contained three cycles with 120 s/cycle split into two phases of 60 s The frequency in the first 60 s was set to 10 rotations/min and in the second 60 s the frequency increased up to 20 rotations/min the mice were placed in the middle of the Open field and videotaped with a digital video camera (Basler Camera GigE color 1/1.8" disinfected with antifect N liquid (Schülke & Mayr GmbH Germany) and wiped off after each animal testing to avoid chemo-communication via feces or urine and residence time was performed using Noduls Ethovision XT 12.1 software a bottle of counted food pellets (60 pcs) and an empty bottle was placed in the cage The animals were filmed for 16 h until the next morning The weight and the number of moving pellets were evaluated then off-line Blood samples were taken every 2 weeks by retro-bulbar blood sampling under isoflurane anaesthesia after the performance of the behavioral tests exsanguination was performed in terminal anaesthesia by direct cardiac puncture using a 22G needle Serum AST levels were measured with the VITROS 350 Integrated System Analyzer (Ortho-Clinical Diagnostics GmbH the stained areas in the histological samples were measured with the help of the software Image J (National Institutes of Health Prism was used for the statistical evaluation of the histopathological results a two-way analysis of variance (ANOVA) was carried out using the Sidak correction for multiple comparisons For single statistical investigations (e.g planimetry) between the two groups a t-test or non-parametric Mann–Whitney U-test was performed all test results were normalized to their baseline values Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific 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R package version 0.4.0. https://ggplot2.tidyverse.org/ (2020) Kassambara, A., Kosinski, M., & Biecek, P. survminer: Drawing Survival Curves using 'ggplot2'. https://cloud.r-project.org/web/packages/survminer/index.html (2019) Download references This research was funded in part from the German Research Foundation (Deutsche Forschungsgemeinschaft-DFG; FOR-2591) Grant Number TO 542/5-1 Institute for Laboratory Animal Science and Experimental Surgery reviewed and agreed to the published version of the manuscript Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41598-020-72801-1 Sign up for the Nature Briefing newsletter — what matters in science Metrics details The marine environment may be explored as a rich source for novel drugs A number of marine-derived compounds have been isolated and identified and their therapeutic effects and pharmacological profiles are characterized we highlight the recent studies using marine compounds as potential hepatoprotective agents for the treatment of liver fibrotic diseases and discuss the proposed mechanisms of their activities we discuss the significance of similar studies in Oman where the rich marine life provides a potential for the isolation of novel natural bioactive products that display therapeutic effects on liver diseases necessitates effective and cost-efficient treatments The marine environment may be explored as a rich source for novel antifibrotic drugs A number of marine-derived compounds are shown to prevent ROS formation interfere with the TGF-β and PDGF-β pathways most of the compounds have negligible immunogenicity and side effects This review provides an overview of the recent studies using marine compounds as potential hepatoprotective agents for the treatment of fibrotic liver diseases and discusses the proposed mechanisms of their activities (A) The healthy liver is prompted by various triggers (extensive alcohol abuse and drug consumption) to undergo hepatic fibrogenesis resulting in an injured Toxic triggers in the liver promote the activation of quiescent hepatic stellate cells (HSC) that become positive for α-smooth muscle actin (α-SMA) start to proliferate and transdifferentiate into contractile myofibroblasts that have the capacity to synthesize large quantities of extracellular matrix (ECM) Hepatic fibrogenesis is markedly stimulated by platelet-derived growth factor (PDGF) and TGF-β that control collagen expression and cellular proliferation The events are further triggered by other chemokines and cytokines that are secreted by liver residential cells (KC sinusoidal endothelial cells) or infiltrating leukocyte (LC) subsets ECM synthesis is further driven by portal (myo)fibroblast (pMF) and bone marrow derived fibrocytes whether hepatocytes can transit into cells that are capable of ECM synthesis in a process that is commonly known as epithelial-to-mesenchymal-transition (EMT) has to be done for therapeutic applications future clinical trials have to be carried out on these compounds to prove their efficiencies as antifibrotic drugs but again the potential benefits of manipulating this pathway in human liver disease conditions have yet to be fully determined These studies provide basic scientists and clinicians with evidence that novel and antioxidant molecules or compounds isolated from these organisms may assist in the development of novel drugs/means to treat liver diseases A repertoire of marine bioactive molecules has been shown to have profound effects in manipulating human pathophysiological conditions Because liver disease also generally involves inflammation and fibrosis compounds affecting common inflammatory pathways may also have significance in hepatic fibrogenesis Some of the marine compounds affecting these pathways that will be later discussed have the potential to be tested in liver disease or can act as a lead for therapeutic agents Speculation that this molecule or its derivatives will evolve the same therapeutic activity during HSC activation and transdifferentiation is worth consideration The identification of novel marine-derived molecules that exhibit similar effects on the TGF-β pathway or its target gene expression would be very useful While PDGF-A and -B are secreted as bioactive homo- or heterodimers PDGF-C and -D are secreted as latent homodimers that become activated by proteolytic separation of the inhibitory CUB domains a process that is mediated by the activity of the tissue plasminogen activator (tPA) or urokinase plasminogen activator and its receptor system (uPA/uPAR) The PDGF ligands bind to specific tyrosine kinase receptors that lead to the formation of three different dimeric receptor combinations the receptors become phosphorylated at specific tyrosine residues and subsequently trigger the activation of a wide variety of intracellular signaling cascades that leads to altered gene expression the increase of PDGF during liver insult results in increased cell proliferation and actin stress fiber formation the different PDGF isoforms modulate cellular adhesion Based on the outstanding role of the PDGF system in liver fibrosis it will be extremely worthwhile to study the effects of these molecules on the setting of experimental liver disease models Further studies will help to better understand the mechanism of its action and the potential modifications on the saponin backbone may enhance the specificity for individual RTKs It is tempting to speculate that similar technology and scaffolds can be developed that allow the administration of anti-inflammation drugs The novel constituents of fish oil as supplements or drugs might therefore be beneficial in controlling critical pathways in liver pathophysiology while the fate of many other species has not yet been documented Large scale coral reef damage is irreversible to some habitats tremendous efforts must be taken immediately to preserve the marine environment and to explore and characterize novel bioactive molecules from this rich environment Graphical abstract showing a summary of the review article Novel bioactive compounds are isolated from the marine environment and are characterized before in vitro testing The compounds showing in vitro activities will be screened for in vivo activities using animal models and ultimately for their hepatoprotective roles in humans there are presently very few studies focused on their efficacies in hepatic lesions Due to the prevalence of liver diseases and the presence of environmental/other factors contributing to their development more detailed studies related to liver diseases are urgently needed in Oman and other countries The effect of water and food contamination and their impact on liver functionality requires further extensive investigation Because Oman is rich with marine flora and fauna these organisms could provide an important source of novel biomedical products for the treatment of liver disease successful collaborations of basic scientists across different academic disciplines and industrial partners are compulsory and urgently needed to isolate and evaluate these novel drugs There is no potential conflict of interest to declare platelet-derived growth factor β; PPAR-γ receptor peroxisome proliferator-activated receptor-γ; COX-1/COX-2 Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets Ito cells are liver-resident antigen-presenting cells for activating T cell responses CD133+ hepatic stellate cells are progenitor cells Biochem Biophys Res Commun 2007; 352: 410–7 Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition Dominant-negative soluble PDGF-beta receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis Update on hepatic stellate 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Inhibition of inflammatory responses by epitaondiol and other marine natural products a highly oxygenated steroid with the unnatural 14.beta configuration from the marine sponge Petrosia contignata Thiele Effect of contignasterol on histamine release induced by anti-immunoglobulin E from rat peritoneal mast cells Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine sponge Xestospongia bergquistia a novel polyoxygenated 14beta steroid isolated from the New Zealand marine sponge Clathria lissosclera Marine natural products as novel antioxidant prototypes In vivo and in vitro antioxidant activity and hepatoprotective properties of polyphenols from Halimeda opuntia (Linnaeus) Lamouroux Protective effects of Geloina eros extract against carbon tetrachloride-induced hepatotoxicity in rats Analysis of protein composition and antioxidant activity of hydolysates from Paphia undulate Antioxidant and cytotoxic properties of two sea cucumbers Holothuria edulis lesson and Stichopus horrens Selenka Antioxidant peptides isolated from sea cucumber Stichopus Japonicus Four new briarane diterpenoids from Taiwanese gorgonian Junceella fragilis Amino acids from Mytilus galloprovincialis (L) and Rapana venosa molluscs accelerate skin wounds healing via enhancement of dermal and epidermal neoformation Anti-Inflammatory activity of a holothurian (sea cucumber) food supplement in rats and cytotoxic polyketides from the marine sponge Plakortis angulospiculatus collected in Brazil Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes Proteasome and NF- kappa B inhibiting phaeophytins from the green alga Cladophora fascicularis Cacospongionolide B suppresses the expression of inflammatory enzymes and tumour necrosis factor-alpha by inhibiting nuclear factor-kappa B activation Mechanism targeted discovery of antitumor marine natural products Characterization of apoptosis induced by marine natural products in non small cell lung cancer A549 cells Hepatic fibrosis inhibitory effect of peptides isolated from navicula incerta on TGF-β1 Induced activation of LX-2 human hepatic stellate cells Cytotoxic and anti-inflammatory cembranoids from the soft coral Lobophytum crassum anti-inflammatory and antibacterial cembranolides from the soft coral Lobophytum durum Download references The authors thank Mr Claude NOZÈRES, Canadian Register of Marine Species (CaRMS), Government of Canada, Department of Fisheries and Oceans (www.marinespecies.org/carms/photogallery.php) for kindly providing pictures of marine organisms Institute of Clinical Chemistry and Pathobiochemistry Download citation Metrics details Hepatic fibrogenesis is a consequence of hepatic stellate cells that become activated and transdifferentiate into a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components platelet-derived growth factor (PDGF) is the most potent stimulus for hepatic stellate cell proliferation and migration and is overexpressed during active hepatic fibrogenesis This cytokine binds to the PDGF receptor type β activates Ras and sequentially propagates the stimulatory signal sequentially via phosphorylation of Raf-1 MEK and the extracellular-signal regulated kinases ERK1/ERK2 Hepatic injury is associated with both increased autocrine PDGF signaling and upregulation of PDGF receptor we report that a dominant-negative soluble PDGF-β receptor consisting of a chimeric IgG containing the extracellular portion of the PDGF receptor type β blocks HSC activation and attenuates fibrogenesis induced by ligation of the common bile duct in rats In culture-activated hepatic stellate cells the soluble receptor blocks phosphorylation of endogenous PDGF receptor phosphorylation of the ERK1/EKR2 signal and reduces proliferative activities of HSC both the delivery of the purified soluble PDGF antagonist and the administration of adenoviruses expressing the artificial transgene were able to reduce significantly the expression of collagen and α-smooth muscle actin Our results demonstrate that PDGF plays a critical role in the progression and initiation of experimental liver fibrogenesis and suggest that early anti-PDGF intervention should have a therapeutical impact on the treatment of liver fibrogenesis the blots were rehybridized with a cDNA specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5′-d(TCG TGG ATC TGA CGT GCC GCC TG)-3′ and 5′-d(CAC CAC CCT GTT GCT GTA GCC GTA T)-3′ The Netherlands) and electroblotted onto a Protran membrane (Schleicher & Schuell The primary antibodies used were: α-SMA (clone asm-1 obtained from Roche Primary antibodies were visualized using horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz) and the supersignal chemiluminescent substrate (Pierce For quantification of sPDGFRβ in rat serum Germany) were coated overnight at 4°C in 50mM NaHCO3 (pH 9.1) containing 5 μg/ml rabbit anti-human IgG specific for the CH2 domain (A 0089 The plates were washed three times (0.05% Tween 20 154 mM NaCl) and blocked with 200 μl 0.5% BSA (RIA grade Germany) in blocking buffer (50 mM Tris pH 7.7 100 μl diluted rat serum was applied to duplicate wells after being diluted two times with assay buffer (0.05% Tween 20 in blocking buffer) A standard curve was set using 100 μl of the purified sPDGFRβ (0–100 ng/ml) in 50% pooled-rat serum The plates were incubated for 2 h at RT followed by washing and application of 100 μl of 0.5–1 μg/ml polyclonal goat anti-mouse PDGFRβ antibody (AF1042 100 μl of 1:2000 diluted polyclonal horseradish peroxidase-conjugated donkey antigoat IgG antibody (sc-2056 BM blue POD substrate solution containing 3,3′-5,5′-tetramethylbenzidine (Roche) was applied after washing and plates were incubated at RT The reaction was interrupted after 10–30 min with 50 μl of 1 M H2SO4 and absorbance was measured in a Victor Multilabor microplate reader (EG&G Wallac Finland) at 450 nm against a reference wavelength of 690 nm COS-7 cells were infected with Ad5-CMV-sTβRII or Ad5-CMV-sPDGFβ in DMEM supplemented with 10% fetal calf serum (FCS) the medium was changed (0.5% FCS) and cells were cultured for additional 48 h and proteins (sTβRII or sPDGFRβ) were isolated on a Hi Trap G column (Amersham Pharmacia Biotech Germany) according to the instructions of the manufacturer For protein sequencing purified samples were dialyzed extensively against deionized water and five (sPDGFRβ) or six (sTβRII) aminoterminal aminoacids were sequenced by Edman degradation at TOPLAB (Martinsried Sirius Red staining was performed as described previously.31 Briefly liver sections were deparaffinized and the slides were incubated for 1 h in a solution of saturated picric acid containing 0.1% Sirius Red 80 Stained slides were then incubated in 0.01 N HCl for 2 min and washed in running distilled water Tissue sections (1.5 μm) were treated with xylene and blocked against endogenous peroxidase using 3% H2O2 the slides were treated with a monoclonal anti α-SMA (asm-1 Roche) followed by incubation with a biotinylated anti-mouse secondary antibody (BA-9200 the secondary antibodies were complexed with an avidin-conjugated peroxidase (Vectastain ABC-Elite reagent Vector Laboratories) and peroxidase activity was detected with diaminobenzidine (DAKO) For better documentation the tissue sections were briefly counterstained with methyl green Results are presented as the mean of three independent experiments (± s.d.) Statistical analysis was performed with an unpaired Student′s t-test and differences were considered as significant (*) or highly significant (**) at P < 0.05 or < 0.01 Construction and expression of sPDGFRβ in vitro (a) Schematic diagram of the PDGFRβ containing the ECD of PDGF receptor type β and human IgG1 Fc-domain separated by a 379-bp intron splice donor and acceptor sites are underlined (b) Northern blot analysis of Ad5-CMV-sPDGFRβ-infected COS-7 cells Note that the expression of respective mRNAs is dependent on viral doses (c) SDS-PAGE and Western blot analysis of supernatants taken from COS-7 cells Supernatants from cells infected with Ad5-CMV-sPDGFRβ or Ad5-CMV-EGFP (viral control) were separated by SDS-PAGE and subjected to Western blot analysis under nonreducing conditions The infection resulted in strong expression of sPDGFR Also the expression of EGFP was detectable in Coomassie blue staining (*) (d) SDS-PAGE of purified soluble receptors Both sPDGFRβ and sTβRII show redox-dependency and migrate as dimers under nonreducing (−DTT) conditions and as monomers under reducing conditions (+DTT) (a) Proliferation assay of culture-activated HSC Cells were pretreated with indicated amounts of sPDGFRβ and stimulated with 20 ng/ml PDGF-BB (black bars) or left unstimulated (white bars) The experimental design was performed in triplicate and significant differences compared to control values are indicated with P≤ 0.05 (*) or P≤ 0.01 (**) c) Western blot analysis for detection of total (PDGFRβ pp44) of proteins involved in the PDGF signaling pathway HSC were treated with indicated combinations of sPDGFRβ and PDGF-BB and protein extracts were analyzed for expression of pPDGFRβ and PDGFRβ (b) sPDGFRβ decreased collagen expression in BDL model of liver fibrosis (a) Content and deposition of collagen in livers from animals subjected to BDL and injected with saline (non-infected) or Ad5-CMV-sPDGFRβ were analyzed by Sirius red staining Sirius red staining results in dark red staining of collagen fibers (space bar=200 μm) (b) Northern blot analysis of representative liver samples taken from both non-BD ligated-(−BDL) and BD ligated (+BDL) rats after saline (c) The sizes of 18S and 28S rRNAs are indicated in the left margin We next examined the influence of sPDGFRβ on expression of α-smooth muscle actin (α-SMA), another marker of ongoing fibrogenesis and cellular activation. In animals receiving Ad5-CMV-sPDGFRβ, the signals obtained in α-SMA immunostainings were markedly reduced (Figure 5), again confirming that sPDGFRβ is effective in preventing progression of hepatic fibrosis. Reduction of smooth muscle α-actin immunoreactivity in rat livers treated with Ad5-CMV-sPDGFRβ Immunohistological distribution of α-SMA in representative liver sections of BD-ligated rats injected with saline (non-infected) Each liver section is shown at lower (left panel; space bar=200 μm) and higher (right panel; space bar=50 μm) magnification Administration of purified sPDGFRβ protein leads to decreased collagen expression in fibrotic rat liver (a) Sirius red staining of liver sections from BD ligated and rats injected with saline (control) (b) Northern blot analysis of collagen expression in livers obtained from rats with (+BDL) and without (−BDL) receiving injection of saline (control) the reduced fibrosis observed after treatment with sPDGFRβ is not a direct consequence of interfering with the processes involved in controlling the synthesis of fibrogenic markers (α-SMA but may result from blockade of HSC proliferation and inhibition of chemotaxis thereby decreasing the number of cells able to form these destructive components This would suggest that the combined elimination of TGF-β and PDGF could further contribute to a more effective inhibition of hepatic fibrosis The testing of this hypothesis is a major focus of future investigations in our laboratory the data presented herein provide evidence that sPDGFRβ is active as an antifibrogenic protein drug able to reduce the biological effects of PDGF in ongoing fibrogenesis the beneficial effects of this compound make it an attractive therapeutic agent for the treatment of fibrotic liver diseases and possibly other fibrotic organ lesions as well an integrated cellular response to tissue injury PDGF and signal transduction in hepatic stellate cells Induction of β-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis Modular binding 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Germany (BMBF Download citation DOI: https://doi.org/10.1038/labinvest.3700094