Myoendothelial gap junctional signaling induces differentiation of pulmonary arterial smooth muscle cells

Abstract

Myoendothelial gap junctions are involved in regulating systemic arterial smooth muscle cell phenotype and function, but their role in the regulation of pulmonary arterial smooth muscle cell (PASMC) phenotype is unknown. We therefore investigated in cocultured pulmonary arterial endothelial cells (PAECs) and PASMCs whether myoendothelial gap junctional signaling played a role in PAEC-dependent regulation of PASMC phenotype. Rat PAECs and PASMCs were cocultured on opposite sides of a porous Transwell membrane that permitted formation of heterotypic cell-cell contacts. Immunostaining showed expression of the gap junctional protein connexin 43 (Cx43) on projections extending into the membrane from both cell types. Dye transfer exhibited functional gap junctional communication from PAECs to PASMCs. PASMCs cocultured with PAECs had a more contractile-like phenotype (spindle shape and increased expression of the contractile proteins myosin heavy chain, H1-calponin, and α-smooth muscle cell-actin) than PASMCs cocultured with PASMCs or cocultured without direct contact with PAECs. Transforming growth factor (TGF)-β1 signaling was activated in PASMCs cocultured with PAECs, and the PASMC differentiation was inhibited by TGF-β type I receptor blockade. Inhibition of gap junctional communication pharmacologically or by knock down of Cx43 in PAECs blocked TGF-β signaling and PASMC differentiation. These results implicate myoendothelial gap junctions as a gateway for PAEC-derived signals required for maintaining TGF-β-dependent PASMC differentiation. This study identifies an alternative pathway to paracrine signaling to convey regulatory signals from PAECs to PASMCs and raises the possibility that dysregulation of this direct interaction is involved in the pathogenesis of hypertensive pulmonary vascular remodeling.