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.

Differential Regulation of Pulmonary Vascular Cell Growth by Hypoxia-Inducible Transcription Factor–1α and Hypoxia-Inducible Transcription Factor–2α

“…In addition, our experiments demonstrate only the autocrine effects of HIF-1a and HIF-2a on pulmonary vascular cells. Because cell-cell interactions contribute to the remodeling process, the paracrine effects of HIFs can also influence this process and warrant further investigation (48)(49)(50). Our present findings, together with previous reports stating that both HIFs play important roles in hypoxia-induced PH (4,5), suggest that targeting both HIFs simultaneously could exert a greater impact in this disorder.…”

Endothelial and Smooth Muscle Cell Interactions in the Pathobiology of Pulmonary Hypertension

“…These effects are prevented by blockers of gap junctions or by knockdown of Cx43 in PAEC. Thus, MEJs appear to act as a gateway for the PAEC-derived signals required for TGF-b-dependent PASMC differentiation (92). The activation of TGF-b signaling and the induction of differentiation of touch-cocultured PASMCs are abolished by the inhibition of serotonin synthesis in PAEC.…”

“…Currently, three different connexins (Cx 37, 40, and 43) are known to be associated with the MEJ that allow the transfer of current and small molecules (,z1.2 kD) between the cytoplasm of these adjacent cells (14). MEJs have been observed in several species and in many vascular beds, including the pulmonary vasculature (13,(90)(91)(92). There are more numerous MEJs in smaller-than in larger-diameter arteries (10,13,93).…”

“…Hence, the interendothelial Cx40-containing gap junctions appear to be critically involved in hypoxic vasoconstriction and the pathobiology of PH in this species (97). In isolated rat pulmonary arteries, sustained constriction, Rho kinase activation, and (92,99), which leads to increased vascular smooth muscle remodeling through the activation of similar mothers against decapentaplegic protein (SMAD) signaling (92,99,135) and phosphorylated TGF-associated kinase 1 (TAK1-p)-MAPK signaling pathways (136). Serotonin may also stimulate the production of superoxide anions by nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) in VSMCs (101,102).…”

“…There is increasing evidence that MEJs are also involved in regulating PASMC phenotype (92,99). When rat pulmonary arterial ECs (PAECs) and PASMCs are touch cocultured on a porous Transwell membrane, MEJs are formed because Cx43 has been found on projections extending into the membrane from both cell types and dye transfers from PAECs to PASMCs, but Cx43 is not present when cells were not touch cocultured.…”