DISCUSSION
Although wound-healing is a physiological process aimed at restoring
normal tissue structure and function after an injury, it can be more
damaging than the insult itself if becomes uncontrolled and excessive
(1). In the liver, a dysregulated fibrotic response to tissue injury of
various etiologies, including viral infections, toxics, biliary
obstruction and nonalcoholic steatohepatitis, occurs and is associated
to poor prognosis in several of the chronic hepatic disorders with
elevated incidence, morbidity and mortality worldwide (2,3). Evidence
indicates that a precise balance between fibrogenic and anti-fibrotic
factors must exist to tune adequately the wound-healing response (43).
By using two well-characterized experimental models of chronic liver
fibrosis, we here recognize cortistatin as an endogenous protective
factor. We found that a deficiency in cortistatin predisposes for
developing exacerbated fibrotic responses in injured livers after
exposition to hepatotoxic compounds, even at low doses, or after
cholestatic damage, and to suffer subsequently more severe clinical
signs, hepatic damage and increased mortality. Hallmarks of the
exacerbated fibrogenic responses observed in injured livers of
cortistatin-deficient mice included the excessive occurrence of
portal-to-portal fibrous scars, ECM-deposition and activated
myofibroblasts.
Our data indicate that cortistatin could acts as an endogenous negative
regulator in the activation and/or differentiation of myofibroblast, a
major player in the development of pathological hepatic fibrosis (44).
Non-parenchymal cells isolated from livers of cortistatin-deficient mice
showed excessive presence of cells compatible with an activated
myofibroblast phenotype (45,46) characterized by enhanced expression of
intracellular αSMA+-stress fibers and production of
fibrogenic markers. In agreement with this commitment to myofibroblastic
differentiation, the genetic signature of the cortistatin-deficient
hepatic non-parenchymal cells displayed increased expression of a
significant number genes related to collagen-containing ECM secretion,
fiber formation and focal adhesion, but mainly, linked to function and
development of muscle, actin cytoskeleton and contractile cellular
fibers, including many components of muscular myosin complexes.
Moreover, whereas genes, such as myogenin, myomarker (Mymk) and myogenic
differentiation 1 (Myod1) that are involved in muscle
differentiation/formation, are highly upregulated in
cortistatin-deficient cells, the expression of myostatin, a gene that
blocks myogenesis, was significantly decreased. This muscle-like
phenotype is supported by the fact that cortistatin-deficient cells
expressed genes encoding for delta and gamma subunits of
cholinergic/nicotinic-receptors, which are solely expressed in muscle
cells, which are almost 200-fold upregulated, but did not differentially
express other cholinergic-receptor subunits that are present in other
cells. Similarly, the gene encoding the muscle-specific carbonic
anhydrase isoform Car3, but not other isoforms, was differentially
increased (40-fold) in cortistatin-deficient cells. These findings
suggest that lack of cortistatin in non-parenchymal hepatic cells favors
the generation of myofibroblasts with contractile functions, and
treatment with cortistatin impairs their differentiation. This entails
important pathological consequences, because contractile myofibroblasts
are abundant in the advanced phases of liver fibrotic disorders and are
highly resistant to reversion/resolution of the fibrogenic response
(47,48).
Numerous evidences support that HSCs are the major sources for activated
myofibroblasts (4-6,49). However, several studies demonstrated that
other fibrogenic cells could also contribute to myofibroblast generation
depending of liver damage etiology (4-6). Whereas fibrosis in
hepatotoxic liver injury is attributed to activated HSCs, activated PFs
are implicated in liver fibrosis caused by cholestatic liver injury
(4-6,49). Our and other studies showed that cortistatin and its
receptors are expressed in HSCs and PFs, and therefore, it could act in
both fibrogenic cells in an autocrine/paracrine manner (14,18,20). In
fact, treatment with cortistatin reversed the activated myofibroblastic
phenotype observed in cortistatin-deficient hepatic cells, and impaired
the activation of human cell line of HSCs, pointing to these cells as
major targets for the anti-fibrotic effect of cortistatin. Moreover, we
observed that non-parenchymal liver cells lacking cortistatin showed
significant increased expression of genes that are specifically
associated to activated HSCs. However, deficiency in cortistatin also
enhanced the levels of genes that are mostly expressed in activated PFs.
Therefore, cortistatin could act as an endogenous break of activated
HSCs and PFs and of their differentiation to activated myofibroblasts,
and consequently, as a critical protective factor for developing severe
liver fibrosis, independently of the hepatic injury type. Despite this
direct effect on fibrogenic hepatic cells, we cannot fully discard the
anti-inflammatory activity of cortistatin as an indirect additional
mechanism involved in its anti-fibrotic effect in vivo, because
inflammation is a major driver of fibrosis in many organs, including
liver. However, the fact that cortistatin treatment efficiently reduced
hepatic fibrosis when initiated once that the inflammatory response was
fully established also supports the capacity of cortistatin to directly
limit fibrogenic responses in injured livers.
Our findings have several clinical implications both from diagnostic and
therapeutic points of view. The fact that a simple partial deficiency in
cortistatin could predispose for developing exacerbated fibrotic
responses could be used to anticipate the diagnostic of more severe
forms of chronic hepatic disorders. Indeed, we found an inverse
correlation between hepatic cortistatin levels and fibrosis/cirrhosis in
patients and animals with different types of liver damage, and it will
be intriguing corroborating these findings in plasma of patients to
consider cortistatin as a potential biomarker of disease prognosis and
susceptibility. The deficiency in cortistatin, and therefore the
susceptibility to suffer exacerbated fibrosis, could be circumstantial
and more or less transitory (i.e., chronic stress, sleep-deprivation) or
permanent (i.e., individuals with 1p36 monosomy, the most common
subtelomeric terminal-deletion syndrome, which are heterozygous for
cortistatin) (50,51). In any case, our study demonstrates that a
systemic cortistatin-based treatment would correct easily this
deficiency and improve disease progression. Noteworthy is that
cortistatin-treatment has a favorable safety profile in humans and
demonstrated clinical efficacy in Cushing’s disease (52), and that the
interest of pharmaceutical industry in developing cortistatin-based
analogues with improved half-life in serum and clinical efficiency has
increased lately (53). Previous studies described the therapeutic effect
on fibrogenic responses by various agonists that signal through
receptors that are recognized by cortistatin, including sstr and GHSR
(13-20), suggesting that binding of cortistatin to both receptor-classes
could allow a kind of synergic anti-fibrotic effect in liver, a
hypothesis that is confirmed here in activated LX2 cells.
In summary, this study provides new insights into the protective
function of cortistatin in liver fibrosis, acting as an endogenous break
of activation/differentiation of myofibroblasts. Beside as a potential
biomarker of disease susceptibility/protection, cortistatin emerges as
an attractive candidate for designing anti-fibrotic therapies to treat
chronic hepatic disorders of different etiologies.