Phorbol 12‐myristate 13‐acetate Dedifferentiates Human Cardiac Myofibroblasts to Fibroblasts

Fibroblast plays an important role in keeping heart shape and elasticity as well as maintaining normal cardiac function. The differentiation of fibroblast to myofibroblast is generally considered as an irreversible conversion which represents a critical step in pathogenesis of fibrosis. Transforming growth factor (TGF)‐β1 is a major cytokine that mediates the conversion of fibroblast to myofibroblast, defined by the overly expression of α‐smooth muscle actin (α‐SMA)‐ a biomarker of myofibroblast with little to no appearance of fibroblast specific protein 1 (FSP‐1) – a biomarker of fibroblast. The cellular and molecular mechanisms of myofibroblast differentiation from fibroblasts have been studied extensively. Reversal of myofibroblast differentiation to fibroblasts remains unclear and incompletely understood. Phorbol 12‐myristate 13 acetate (PMA) is involved in multiple cellular functions such as cell growth, differentiation, programmed cell death via protein kinase C (PKC) signaling pathways. To investigate whether PMA dedifferentiates the formed myofibroblasts, NIH 3T3 fibroblasts and human cardiac fibroblast (HCF), cultured in DMEM and fibroblast medium (FM)‐2 respectively, were induced to convert into myofibroblasts in the presence of 2 ng/ml of TGF‐β1 for 24‐ or 48‐hour incubations. Expression of α‐SMA and FSP‐1 in both cell lines was detected by using western blotting and immunofluorescence. Collagen gel contraction induced by cardiac fibroblasts was determined as well. After incubation with TGF‐β1, morphology changes in the shape and the size of NIH 3T3 and HCF cells were observed by the presence of large nuclei and cytoplasm. The levels of expression of α‐SMA were significantly increased whereas expression of FSP‐1 was reduced after 48‐hour incubation with TGF‐β1. NIH 3T3 and HCF cells were then treated with 50 ng/ml of DMSO which used as control groups and with various PMA concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) for additional 24‐ and 48‐ hour incubations. Chemiluminescence detection of Western blot confirmed the reduction in expression of α‐SMA. The shape and the size of the both cell lines were recovered under the presence of PMA. The results indicated that the reduction in expression of α‐SMA was directly proportional to the concentration of PMA. As PMA concentration increased, the expression of α‐SMA remarkably decreased. PMA also reduced TGF‐β1‐induced collagen gel contraction. These data unambiguously elucidate the reversal of myofibroblast differentiation induced by PMA. Under basic condition, TGF‐β1 induces myofibroblast conversion from fibroblast, and myofibroblast de‐differentiates back into fibroblast in the presence of PMA. Although the mechanism remains to be identified, the novel findings of this study shed light on future development of novel agents to treat fibrotic diseases. Support or Funding Information This project was supported by the Seed Grant from California Northstate University, College of Pharmacy.