Oncofetal Protein CRIPTO Is Involved in Wound Healing and Fibrogenesis in the Regenerating Liver and Is Associated with the Initial Stages of Cardiac Fibrosis

Abstract

Oncofetal protein, CRIPTO, is silenced during homeostatic postnatal life and often re-expressed in different neoplastic processes, such as hepatocellular carcinoma. Given the reactivation of CRIPTO in pathological conditions reported in various adult tissues, the aim of this study was to explore whether CRIPTO is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of CRIPTO expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesis. Circulating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and 1 year after LT. The expression of CRIPTO and fibrotic markers (αSMA, collagen type I) was determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure, and in mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4), were evaluated for CRIPTO expression and fibrotic markers. CRIPTO was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for CRIPTO and fibrotic markers in three models of heart injury following myocardial infarction, pressure overload, and ex vivo induced fibrosis. Patients with end-stage liver cirrhosis showed elevated CRIPTO levels in plasma, which decreased 1 year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of CRIPTO in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. CRIPTO expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, CRIPTO-positive cells were observed in damaged liver areas around the central vein, which preceded the expression of αSMA-positive stellate cells, i.e., mediators of fibrosis. In the chronic mouse models, the fibrosis and CRIPTO expression were still present after 11 weeks, whereas in the acute model the liver regenerated and the fibrosis and CRIPTO expression resolved. In vivo overexpression of CRIPTO in this model led to an increase in fibrotic markers, while blockage of CRIPTO secreted function inhibited the extent of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. CRIPTO expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction CRIPTO is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, CRIPTO expression decreased concomitantly with the αSMA expression. Temporal expression of CRIPTO in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, CRIPTO expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis. Our results are indicative for a functional role of CRIPTO in the induction of fibrogenesis as well as a potential target in the antifibrotic treatments and stimulation of tissue regeneration.