Endoglin and its involvement in transdifferentiation of hepatic stellate cells

Aims: Endoglin, a transmembrane accessory receptor for TGF-beta was recently found to be highly expressed on the surface of hepatic stellate cells. Up to now endoglin was mainly known as a TGF-beta receptor on endothelial cells, in which mutations causes the vascular disorder, hereditary haemorrhagic telangiectasia (HHT1). In the current study we tried to evaluate the role of endoglin in liver fibrosis modulating the classical TGF-beta pathway, which is mainly responsible for an increased extracellular matrix protein (ECM) synthesis during liver fibrosis. Methods: The expression of Endoglin in rat hepatic stellate cells (HSC) was specifically down regulated by siRNA. The knock down efficiency was verified by Western-Blotting. The consequence of downregulating endoglin was evaluated by (CAGA)12MLP-Luc reporter assay and by Western-Blotting using pSmad specific antibodies as well as antibodies against Collagen I, Fibronectin and alpha-sm-actin. Morphological changes in transdifferentiation were monitored by light microscopy. Results: After transfection of 1 day old rat HSC with siRNA against endoglin, the protein level was reduced at day 3 to an extent of approx. 95%. This knock down level was maintained for the following 10 days. Within this time-period we stimulated the cells with TGF-beta1 and analyzed the stimulation by (CAGA)12MLP-Luc reporter assay and Western-Blotting as a read out system. Thus we could demonstrate, that reduction of endoglin expression leads to an increased Smad3 phosphorylation as well as an enhanced (CAGA)12MLP-Luc reporter activity. Furthermore, we were able to show differences in transdifferentiation kinetic by monitoring morphological changes of culture-activated HSC in a time course of 12 days after siRNA transfection. Conclusion: It was possible to show, that endoglin is involved in the TGF-beta pathway in HSC, since reduction of endoglin leads to significant changes in Smad phosphorylation. Furthermore, the reduction of endoglin suppresses trans-differentiation from HSC to myofibroblastic phenotype cells (MFB) while overexpression of endoglin ends up in enhanced transdifferentiation.