Activation of COX‐2/PGE 2 Pathway Is Not Involved in Dedifferentiation of Cardiac Myofibroblasts Induced by Phorbol 12‐myristate 13‐acetate

Abstract

The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGF-β1 is one of the essential molecules that promotes transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. In our previous studies, Phorbol 12-Myristate 13-Acetate (PMA) induces reversal of myofibroblast differentiation via protein kinase C (PKC)-independent mechanism. Prostaglandin E2 (PGE2) has been shown to reserve differentiation of myofibroblasts of fetal and adult lung fibroblasts. The role of PGE2 in cardiac myofibroblast dedifferentiation remains unknown. Human cardiac fibroblasts were cultured in fibroblast medium (FM)-2. TGF-β1 (2ng/mL) was added to FM-2 for 48 hours to convert fibroblasts into myofibroblasts. PMA (50 ng/mL) or PGE2 (500 nM) was added into cultured cells for 48 hours, respectively. Expression of α-smooth muscle action (SMA), a biomarker of myofibroblasts, and fibroblast specific protein 1 (FSP-1), the biomarker of fibroblasts, were detected by using western blotting and immunofluorescence. To explore the involvement of cyclooxygenase 2 (COX-2)/PGE2 pathway in PMA-induced reversal of cardiac myofibroblast differentiation, NS-398, the selective COX-2 inhibitor, and PF-04418948, a selective PGE2 receptor antagonist, were applied. Endogenous levels of PGE2 in cardiac myofibroblasts were detected by using Elisa assay kit. TGF-β1 promoted conversion of cardiac fibroblasts to myofibroblasts as evidenced by increased expression of α-SMA and reduced expression of FSP-1. Treatment with PMA dose-dependently attenuated expression of de novo myofibroblasts. Both NS-398 and PF-04418948 exerted no effects on PMA-induced reversal of cardiac myofibroblasts. Addition of PGE2 into culture medium had no effect on expression of α-SMA from myofibroblasts. PMA dose-dependently enhanced formation of PGE2 levels in cardiac myofibroblasts. In conclusion, PMA-induced reversal of cardiac myofibroblast is independent of activation of COX-2 and PGE2 pathway. The mechanism in PMA-induced reversal of cardiac myofibroblasts remains to be further explored.