RESULTS
Cortistatin is downregulated in fibrotic livers. We first analyzed the expression of cortistatin in livers from mice subjected to experimental induction of hepatic fibrosis and from patients with different hepatic fibrosis disorders. The levels of cortistatin gene expression were obtained from the National Cancer for Biotechnology Information Gene Expression Omnibus database based on the gene chips of fibrotic liver tissues associated to hepatitis B virus (HBV-GSE84044, ref. 24), hepatitis C virus-induced hepatocarcinoma (HCV-GSE14323, ref. 25), and nonalcoholic fatty liver disease (NAFLD-GSE49541, ref. 26). We found that cortistatin expression is significantly reduced in fibrotic or cirrhotic livers of these patients compared to normal liver tissue of healthy individuals (Fig. 1A). This inverse correlation between cortistatin expression and human fibrosis was also observed in livers isolated from mice subjected to hepatic fibrosis by intoxication with CCl4 or by cholestasis (Fig. 1B).
Deficiency in cortistatin exacerbates fibrosis caused by cholestatic and toxic-induced hepatic injury. To investigate whether a deficiency in cortistatin predisposes to suffer exacerbated hepatic fibrosis, we induced experimentally chronic liver injury by CCl4-intoxication or by BDL-induced cholestasis in mice that partially (CST+/- ) or totally (CST-/- ) lack the cortistatin gene, and the progression and severity of the fibrotic disease were compared with those observed in wild-type mice (CST+/+ ). We initially observed that in the absence of hepatic insults, cortistatin-deficient mice did not show marked signs of liver fibrosis, although dispersed incipient inflammatory/fibrotic foci were more abundant in livers of sham-operated or vehicle-injectedCST+/- or CST-/- mice than of wild-type animals (fig. S1). Interestingly, whereas BDL did not affect the survival of wild-type mice, it resulted in mortality rates around 40% in cortistatin-deficient mice (Fig. 2A). Histopathological examination of Sirius red-stained liver sections showed thatCST+/- and CST-/- mice had larger areas of fibrosis compared to CST+/+mice (Fig. 2B), which correlated with excessive hepatic damage, as assessed by increased necrotic area (Fig. 2C, fig. S2A) and bilirubin levels (Fig. 2D). Development of severe liver fibrosis in cortistatin-deficient mice subjected to BDL was associated with enhanced presence of αSMA+-activated myofibroblasts in periportal areas and fibrotic portal-to-portal branches (Fig. 2E), excessive collagen content (Fig. 2F) and increased mRNA expression of critical fibrogenic markers, including TGFβ1, collagen I-α2 (Col1a2), connective-tissue growth factor (CTGF) and αSMA (Fig. 2G).
On the other hand, chronic injection of a low dose of CCl4 elevated the mortality rate to 25% in cortistatin-deficient mice (Fig. 3A), which correlated with a significant early and exacerbated progressive liver fibrosis, characterized by increased Sirius red+-areas in most of portal spaces, with occasional portal-to-portal bridging (corresponding to Ishak score of 3) and enlargement of Glisson capsule (Fig. 3B, fig. S2B). Moreover, we observed elevated presence of αSMA+-myofibroblasts and contents of bilirubin and hepatic collagen, and excessive expression of profibrogenic factors inCST+/- and CST-/- mice compared to wild-type mice (Fig. 3C-E). Interestingly, necropsy of cortistatin-deficient mice that died as consequence of chronic CCl4-exposition showed the occurrence of damaged lungs, and histopathological analysis confirmed the presence of extended edematous and hemorrhagic foci with distortion of pulmonary structure (fig. S3).
All together, these findings indicate that cortistatin is a key regulator of pathological fibrosis induced by chronic hepatic injury, acting as a protective anti-fibrotic factor that limits the activation/differentiation of myofibroblasts.
Cortistatin-deficient HSCs show exacerbated fibrotic responses and excessive differentiation capacity to activated myofibroblasts.Numerous evidences demonstrate that pathological fibrosis in liver is driven by differentiated myofibroblasts and that the major contributors of the myofibroblast pool are liver resident activated perisinusoidal HSCs and periportal fibroblasts (PF) (4-6). In order to investigate whether endogenous cortistatin directly regulates these profibrogenic cells, we evaluated the phenotype and fibrogenic responses of the nonparenchymal cell fraction (enriched in HSCs) isolated from livers of wild-type and cortistatin-deficient mice and then cultured as described in Fig. 4A. We first confirmed the expression of cortistatin by mouse HSCs (fig. S4A). After one week in culture, most of the wild-type nonparenchymal cells (>80%) displayed a quiescent/non-activated HSC phenotype (GFAP+/αSMA-), and less than 10% of them corresponded to activated myofibroblasts expressing αSMAhigh-stress fibers (Fig. 4B). However, cortistatin-deficient HSC cultures showed initially a significant elevated percentage (>30%) of activated αSMAhigh-myofibroblasts that progressed rapidly to more than 70% of total cells at the end of culture, a percentage that was largely higher than that observed (<30%) inCST+/+ HSC cultures (Fig. 4B). Moreover,CST-/- HSC cultures showed 1.8-fold cell numbers than CST+/+ HSC cultures. The enhanced fibrogenic/myofibroblastic phenotype observed inCST-/- HSCs correlated with an initial increased expression of profibrotic markers/mediators compared toCST+/+ HSCs (Fig. 4C), suggesting that cortistatin-deficient HSCs are more committed than wild-type HSCs to differentiate to activated myofibroblasts.
To further investigate the genetic profile/signature that could be associated to this commitment, we compared the transcriptomes ofCST+/+ and CST-/- HSCs by using next-generation RNAseq. We observed that 2,289 genes (1,284 upregulated and 1,005 downregulated) displayed significant differential expression (FDR p-value<0.05) in cortistatin-deficient HSCs compared to wild-type HSCs (Fig. 4D). The expression of selected upregulated and downregulated genes of the RNAseq were validated by real-time PCR, showing a high-degree of correlation between both analyses (fig. S4B). The unsupervised hierarchical clustering analysis of the most significant differentially expressed genes (DEGs) revealed two distinct groups with minimal overlap (Fig. 4E). Interestingly, we observed that from the top-25 annotated DEGs that are upregulated inCST-/- HSCs, 22 corresponded to genes related specifically to differentiation/generation, function or markers of muscle cells (Fig. 4F, table S1), and that 53 muscle-related genes were increased at least four-fold in cortistatin-deficient HSCs (fig. S4C). Further, the gene ontology (GO) analysis revealed that the DEGs (mostly upregulated) were mainly associated with biological functions or terms that are related to muscle function, myofibroblast activation/differentiation and extracellular matrix deposition (Fig. 4G, tables S2 to S4). Interestingly, a significant number of downregulated DEGs in CST-/- HSCs are associated with GO terms that are related to defense response, mainly to interferon and viral infection (Fig. 4G, table S3). Moreover, a detailed analysis of DEGs showed that specific markers of activated HSCs and PF were upregulated in nonparenchymal liver cells isolated from cortistatin-deficient mice (fig. S4, D and E). In addition, several inhibitors of HSC senescence were increased inCST-/- HSCs (fig. S4F). These findings suggest that cortistatin could act as an endogenous break of fibroblast and HSC activation and of their differentiation to activated myofibroblasts with potential contractibility. In fact, the addition of exogenous cortistatin to CST-/- HSC cultures reversed significantly this activated myofibroblastic phenotype (Fig. 4H).
To confirm that cortistatin directly downregulates HSC function, we investigated its effect on LX2 cells, a widely used human HSC line to study fibrogenic responses (42). We observed that cortistatin impaired the activation in TGFβ1-stimulated LX2 cells (fig. S5, A to C). Interestingly, the inhibitory effects of cortistatin inLX2 cells were partially blocked by antagonists for sstr and GHSR1, and by inhibiting Gαi and protein kinase A, two signaling factors that are coupled both receptors (fig. S5D). This supports the involvement of sstr and GHSR in the anti-fibrotic activity of cortistatin in HSCs. As occurred in mouse samples, LX2 cells constitutively expressed cortistatin, which was downregulated upon fibrogenic stimulation (fig. S5E). These results are the first insights to potentially translate our observations in preclinical models to humans.
Treatment with cortistatin ameliorates fibrosis induced by toxic and cholestatic liver injury. Our previous results indicate that cortistatin has a critical role in the regulation of hepatic fibrosis and that administration of cortistatin is a potential strategy for the prevention and treatment of liver injury. First, we found that systemic injection of cortistatin reversed the exacerbated fibrosis and hepatic damage observed in cortistatin-deficient mice subject to surgical BDL (Fig. 5) or to chronic injection of low-dose CCl4 (fig. S6). Moreover, treatment with cortistatin protected significantly against severe fibrosis and hepatic damage caused by chronic intoxication with high-dose CCl4 (Fig. 6). Interestingly, the therapeutic effect of cortistatin was observed following both early (from day 5) and delayed (from day 14) regimes, pointing to a wide interventional therapeutic window (Fig. 6). Similarly, cortistatin treatment protected from development of cholestatic-induced hepatic fibrosis (fig. S7). These results suggest that cortistatin-based therapies could avoid the progression to severe hepatic fibrosis in susceptible individuals and attenuate the established liver fibrosis.