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.