Hyperoxia Induces Phenotypic Transition of Pulmonary Arterial Smooth Muscle Cells via PI3K/AKT Activation

Background Phenotypic switching of vascular smooth muscle cells (VSMCs) serves as a critical pathological basis for various cardiovascular diseases. This phenotypic transformation enables cells to regain proliferative, secretory, and migratory capabilities, triggering pathological vascular remodeling and subsequent disease development, including pulmonary arterial hypertension (PAH). PAH represents a common complication of congenital heart disease (CHD). Beyond early surgical correction of anatomical defects, effective therapeutic options remain limited. Consequently, there is an urgent need to elucidate the mechanisms underlying PAH pathogenesis and identify novel therapeutic strategies. In CHD, pulmonary arteries are chronically exposed to a high-flow, hyperoxic environment. This study hypothesizes that chronic hyperoxia—a previously overlooked factor—may significantly contribute to phenotypic switching and functional alterations in pulmonary arterial smooth muscle cells (PASMCs). Methods PASMCs were isolated from Sprague-Dawley rats through enzymatic digestion and cultured under hyperoxic conditions to establish an in vitro hyperoxia model(95% O₂/5% CO₂). Cells were divided into two primary groups: Normoxia group (N-group) and Hyperoxia group (H-group). The effects of hyperoxia on phenotypic switching were assessed by examining the expression of contractile markers α-SMA and SM22α, and synthetic marker OPN using qRT-PCR and Western blot analysis. To evaluate changes in cellular secretory capacity, MMP-2 expression was analyzed at both mRNA and protein levels. Cell proliferation and migration capacities were evaluated using CCK-8 assays and scratch wound healing assays, respectively. To investigate the regulatory role of the PI3K/AKT pathway, cells were treated with the PI3K inhibitor LY294002 and divided into four experimental groups: Normoxia (N-group), Hyperoxia (H-group), Hyperoxia + LY294002 (HI-group), Normoxia + LY294002 (NI-group). The phenotypic and functional modifications of PASMCs were assessed using qRT-PCR, Western blot analysis, CCK-8 proliferation assays, and scratch wound healing migration assays to investigate the regulatory involvement of the PI3K/AKT signaling pathway. Results In PASMCs cultured under hyperoxic conditions, a phenotypic transition from contractile to synthetic state was observed. The expression of contractile proteins α-SMA and SM22α was downregulated, while the synthetic protein OPN was upregulated. Concurrently, hyperoxia induced elevated secretion of MMP-2, enhanced cellular viability as demonstrated by CCK-8 assay, and significantly augmented migratory capacity observed in wound healing assays. These alterations exhibited a time-dependent progression. Hyperoxia induced increased phosphorylation of PI3K and AKT, leading to activation of the PI3K/AKT signaling pathway. This activation was associated with enhanced cellular anti-apoptotic capacity and concomitant phenotypic and functional alterations Notably, treatment with the PI3K inhibitor LY294002 effectively abolished the hyperoxia-induced effects on both molecular profiles and cellular behavior. Conclusion The PI3K/AKT signaling pathway mediates hyperoxia-induced phenotypic transition in PASMCs, conferring enhanced proliferative, secretory, and migratory capacities. These findings establish a mechanistic link between chronic hyperoxia and pathological vascular remodeling, providing a novel pathological basis for CHD-associated PAH (CHD-PAH) development.