Background/Purpose: Recent studies demonstrated that the phenotypic transition of endothelial cells (EC) into activated mesenchymal cells, a process known an endothelial-to-mesenchymal transition (EndoMT), may be crucial in the development of tissue and organ fibrosis in fibrotic diseases such as Pulmonary Fibrosis and Systemic Sclerosis (SSc). Furthermore, it was previously demonstrated that TGF-ß induces EndoMT in murine lung EC in vitro. Owing to the important role of caveolin-1 (cav-1) in TGF-ß receptor internalization and TGF-ß signaling the role of cav-1 in induction of EndoMT in murine lung EC was investigated.

Methods: Pulmonary EC were isolated from wild-type (WT) and cav-1 knockout (cav-1 KO) mice employing sequential immunomagnetic selection with anti-CD31 and anti-CD102 antibodies followed by in vitro culture and treatment with TGF-ß1. EndoMT was assessed by immunofluorescence for α-smooth muscle actin (α-SMA) and by Western blot analysis for α-SMA and type I collagen. Induction of the transcriptional repressor, Snail 1, was assessed by real time PCR. The same studies were performed in cav-1 KO pulmonary EC following restoration of functional cav-1 domains employing a cell permeable cav-1 scaffolding domain peptide.

Results: Pulmonary EC from cav-1 KO mice displayed high levels of spontaneous α-SMA and type I collagen expression which increased following TGF-ß treatment. There was a remarkable increase in spontaneous Snail 1 expression. Spontaneous and TGF-ß1-stimulated EndoMT were abrogated by restoration of functional cav-1 domains.

Spontaneous and TGF-ß1 stimulated EndoMT in cav-1 KO primary pulmonary EC.

A. Pulmonary EC from WT and cav-1 KO mice were cultured in media alone (control) or with 10 ng/ml TGF-ß1 for 72h, protein from cell lysates was electrophoresed and probed in Western blots for α-SMA. B.  Culture media from the same samples were analyzed by Western blot for type I collagen.

Conclusion: Cav-1 plays an important role in the regulation of EndoMT by abrogating spontaneous and TGF-ß-induced EndoMT and EndoMT-mediated generation of activated myofibroblasts.  Since reduction of cav-1 expression is an important molecular abnormality present in SSc lung and dermal fibroblasts the results indicate that exaggerated spontaneous and TGF-ß induced EndoMT caused by cav-1 deficiency may play a crucial role in the pathogenesis of SSc tissue fibrosis and vasculopathy.