UC San Diego

Prolonged pulmonary vasoconstriction and vascular remodeling cause idiopathic pulmonary arterial hypertension (IPAH). Pulmonary vasoconstriction is caused by pulmonary artery smooth muscle cell (PASMC) contraction, and enhanced proliferation of PASMC contributes to vascular remodeling. An increase in cytosolic Ca²⁺ concentration ([Ca²⁺ cyt) in PASMC triggers both contraction and proliferation. Inhibition of Ca²⁺ influx completely inhibits prolonged pulmonary vasoconstriction and stops PASMC proliferation. Store-operated Ca²⁺ entry (SOCE) is an important mechanism that mediates Ca²⁺ influx in PASMC and most other cell types. We previously reported that SOCE was significantly enhanced in PASMC exposed to hypoxia and in PASMC from IPAH patients. Stromal interaction molecule (STIM1/2) and Orai proteins (Orai1/2/ 3) were identified recently as essential components that mediate SOCE. We aim to examine whether the expression levels of STIM and Orai are altered in IPAH-PASMC compared to control PASMC, and whether these putative changes in expression level are responsible for the enhanced SOCE and proliferation observed in IPAH-PASMC. The protein expression level of STIM2 was increased in IPAH-PASMC, whereas STIM1 protein expression was decreased. Hypoxia (4% O₂, 5% CO₂, balance N₂) enhanced the protein expression level of STIM2, but not STIM1, in rat PASMC after 48 hours. In IPAH-PASMC knockdown of STIM2 decreased SOCE and proliferation, while knockdown of STIM2 in control PASMC had no effect on either SOCE or proliferation. Overexpression of STIM2 in control PASMC failed to enhance SOCE or proliferation. These data indicate that enhanced protein expression of STIM2 is necessary, but not sufficient, for enhanced SOCE and proliferation of IPAH-PASMC. Orai2 protein expression level was increased in IPAH-PASMC. Orai1 and Orai3 protein expression levels were not statistically significantly altered. Furthermore, 48 hours of hypoxia significantly increased protein expression of Orai1 and Orai2, but not Orai3. Overexpression of Orai2 alone in PASMC did not enhance SOCE. Co-overexpression of both STIM2 and Orai2, however, did augment SOCE in PASMC. In conclusion, upregulated protein expression of STIM2 is necessary, but insufficient, for augmented SOCE and enhanced proliferation in PASMC from IPAH patients. Increased expression of both STIM2 and Orai2 is sufficient to enhance SOCE in normal PASMC