**4. Discussion**

Liver injury of various etiologies triggers morphological and functional changes within hepatic sinusoids, resulting in development of fibrosis and portal hypertension [24,43,44]. Disturbances of the intercellular crosstalk within the sinusoids, especially between LSECs and HSCs, contribute to fibrosis development and microvascular dysfunction [24,43,44]. Multiple paracrine signals, such as NO, ET-1, TGF-β, PDGF, and VEGF, as well as various cytokines and chemokines that are being secreted not only from LSECs, hepatocytes, cholangiocytes, immune cells, but also from platelets, modulate LSEC and HSC phenotype under physiological and pathophysiological conditions [44,45]. Gene expression analysis of liver tissue following LCA feeding for 84 h revealed an enrichment in signaling pathways related to inflammation, proliferation, and matrix remodeling, which was in line with histological analysis of the livers. Interestingly, mice deficient for the G protein-coupled BA receptor TGR5 were more susceptible towards LCA-induced liver injury, resulting in elevated AST serum levels, more pronounced bile infarcts, and an elevated portal perfusion pressure. TGR5 is highly expressed in cholangiocytes, where activation of the receptor triggers formation of the bicarbonate umbrella, promotes tight junction integrity, and inhibits apoptosis, thus protecting cholangiocytes from bile acid toxicity [9,11,46,47]. Lack of TGR5 therefore renders mice more susceptible towards bile acid-induced biliary injury, as observed previously, in response to common bile duct ligation (CBDL) [19] and now in the LCA model. Furthermore, TGR5 exerts anti-inflammatory functions in monocytes and macrophages [4,6,13,14,18]. Absence of TGR5 resulted in elevated expression and secretion of chemokines and cytokines in response to lipopolysaccharide injection or common bile duct ligation [18,19]. Following LCA feeding, we observed a significant induction of hepatic mRNA expression of various cytokines, chemokines, and pro-fibrogenic chemokine receptors such as CCR1 and CCR5 [39]. Interestingly, expression of the chemokine receptor CCR5 was already induced 3-fold in TGR5 KO mice under chow-fed conditions, which has been previously reported for white adipose tissue in TGR5-deficient animals [13]. CXCL10 and its receptor CXCR3 have been implicated in fibrosis development in models of congenital hepatic fibrosis and carbon tetrachloride (CCl4)-mediated fibrosis [48,49]. While upregulation in comparison to chow-fed WT animals of CXCL10 was 9-fold in WT and 5-fold in TGR5 KO, respectively, CXCR3 mRNA expression was induced 3-fold in WT and 5-fold in TGR5 KO following LCA feeding. Interestingly, in patients with chronic liver disease, serum CXCL10 levels, but not hepatic CXCL10 mRNA levels, were positively correlated with portal hypertension, fibrosis stage, and disease progression [50,51]. To which extent the CXCL10–CXCR3 signaling pathway contributes to the phenotypes of our LCA-fed mice is unclear. CXCL1, which is secreted from LSECs following mechanical stretching, triggers formation of sinusoidal microthrombi, thereby increasing portal pressure independent of cirrhosis [52]. Hepatic CXCL1 mRNA expression was significantly induced in LCA-fed mice. In comparison to chow-fed WT mice, CXCL1 levels were 14.5- and 9.4-fold higher in LCA-fed WT and TGR5 KO mice, respectively. However, in TGR5 KO mice, CXCL1 levels were low under chow-fed conditions and increased 27-fold in response to LCA. Thus, microthrombosis may also contribute to portal hypertension in LCA-fed TGR5 KO animals.

Moreover, expression of PDGFRα/β, TGF-β1 and ET-1, which are related to HSC activation, development of fibrosis, and portal hypertension, were upregulated in both genotypes in response to LCA. However, mRNA levels were significantly higher in livers of TGR5 KO mice as compared to liver tissue from WT littermates. While periportal fibrosis, as measured by Sirius red staining and hydroxyproline content, was not significantly increased in either genotype as early as 3.5 days after starting the LCA feeding, expression of collagen was significantly upregulated in both genotypes, suggesting initiation of fibrogenesis in line with previous data [21]. Since TGR5 is expressed both in LSECs and activated HSCs, we further explored TGR5-mediated effects in these cell types. Stimulation of TGR5 on LSECs reduced ET-1 expression, as well as its secretion, and reduced the responsiveness of activated HSCs towards ET-1 through internalization of the ETAR. These two mechanisms may act synergistically to reduce ET-1 signaling within the sinusoid (Figure 7B). In rats, treatment with a TGR5 agonist (BAR501) for 6 days prior to cannulation of the portal vein lowered the rise in

portal perfusion pressure in response to norepinephrine [53]. Furthermore, administration of the TGR5 agonist inhibited portal hypertension in mice treated for 9 weeks with CCl4, while it did not affect fibrosis development [53], suggesting an effect on the hepatic microvasculature independent of extracellular matrix deposition. As underlying molecular mechanisms, the authors demonstrated that TGR5 activation induces expression and also non-genomically promotes activation of cystathioneγ-lyase (CSE), resulting in increased production of hydrogen sulfide (H2S), a potent vasodilator [53,54]. Amongst others, we have previously shown that TGR5 may also trigger serine phosphorylation of eNOS, thereby promoting NO generation, which again leads to vasodilation of hepatic sinusoids [1,53]. Taken together, TGR5 agonists promote generation and secretion of vasodilatory agents (H2S and NO) and inhibit expression and secretion of the potent vasoconstrictor ET-1 from LSECs [1,7,46,53,54]. While the function of TGR5 in LSECs has been previously studied, the role of TGR5 for HSC activation and function remained elusive. Interestingly, the rise in hepatic PDGFRα/β expression was more pronounced in TGR5 KO mice as compared to WT littermates after LCA-feeding. PDGF-β, which may be derived from either LSECs or platelets, can signal to activated HSCs through its receptor PDGFRβ and promote cell proliferation, migration, and development of a pro-angiogenic HSC phenotype [55,56]. Furthermore, it was recently demonstrated that PDGF-β plays a role in the trans-differentiation processes of HSCs into an activated phenotype, and that administration of an anti-PDGF-β antibody (MPR8457) inhibits development and progression of biliary fibrosis in Abcb4 KO mice, which serve as a chronic model for sclerosing cholangitis [56,57]. Moreover, cAMP may desensitize activated HSCs towards ET-1 through internalization of the ETAR [42]. Using a non-BA TGR5 ligand, we could demonstrate that activation of the receptor promotes retrieval of ETAR from the plasma membrane, explaining the reduced ET-1-dependent contractility of activated HSCs in the presence of a TGR5 agonist in vitro. In summary, TGR5 activation reduces hepatic vascular resistance through several mechanisms, both in LSECs and in HSCs, that act synergistically. Through modulation of PDGFRβ expression, it may also contribute to the response of HSCs to microvascular thrombosis and platelet-derived signals. A recent study explored the effect of the non-bile acid TGR5 ligand oleanolic acid in a rat model of liver fibrosis development [58]. Treatment with oleanolic acid significantly reduced fibrogenesis in vivo; however, a direct effect on HSCs in vitro was not observed, since the cell lines used did not express TGR5 [58]. It was previously reported that TGR5 expression, which is very low in quiescent HSCs, is upregulated during the activation of HSCs into a myofibroblast-like phenotype [4,26]. Thus, stimulation of TGR5 on activated HSCs may contribute to the beneficial effects of oleanolic acid on fibrosis development in the above model [58]. Furthermore, mice deficient in both TGR5 and the nuclear bile acid receptor FXR were generated and showed an enrichment in gene expression pathways associated with liver fibrosis and inflammation in line with our study [59]. Since FXR agonists convey beneficial effects in preclinical models of liver fibrosis and portal hypertension [60], it is highly anticipated that TGR5/FXR double-KO mice will also develop LSEC and HSC dysfunction and portal hypertension. Thus, targeting of BA signaling becomes an interesting strategy to reverse morphological changes and dysfunction of cell types residing in the hepatic sinusoids, thereby attenuating portal hypertension and fibrosis development.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/11/1467/s1, Figure S1: Expression of TGR5 in rodent HSCs Figure S2: HSC contraction assay Table S1: Differentially upregulated genes LCA vs. chow; Table S2: Differentially downregulated genes LCA vs. chow; Table S3: Enriched GO pathways LCA vs. chow; Table S4: Downregulated GO pathways LCA vs. chow.

**Author Contributions:** C.K. co-designed the study, performed the experiments, interpreted the data, and wrote the manuscript. V.K. designed the study, interpreted the results, and wrote the manuscript. M.R. performed and interpreted the animal experiments. J.S. created the figures and reviewed the manuscript. K.K. performed the gene array experiments and reviewed the manuscript. B.H., J.R., and J.G.H. performed the gene array analysis, designed the figures, and reviewed the manuscript. K.S. provided the transgenic animals, supported the data analysis, and carefully reviewed the manuscript. D.H. provided the stellate cells, interpreted the data, and carefully reviewed the manuscript.

**Funding:** This work was funded by the German Research Foundation (DFG) through the Collaborative Research Centre 974 (SFB 974).

**Acknowledgments:** Expert technical assistance by Stefanie Lindner, Nichole Eichhorst, and Claudia Rupprecht is gratefully acknowledged.

**Conflicts of Interest:** The authors declare that there are no potential conflicts of interest.
