Heat shock protein 70 attenuates hypoxia-induced apoptosis of pulmonary microvascular endothelial cells isolated from neonatal rats

Pulmonary microvascular endothelial cell (PMVEC) apoptosis is the initial stage of adult pulmonary hypertension (PH), which involves high pulmonary arterial pressure and pulmonary vascular remodeling. However, the mechanism regulating PMVEC apoptosis and its involvement in the early stages of neonatal hypoxic PH (HPH) pathogenesis are currently unclear. The present study aimed to investigate the effects of heat shock protein 70 (HSP70) on hypoxia-induced apoptosis in PMVECs. PMVECs isolated from neonatal Sprague-Dawley rats were transfected with lentivirus with or without HSP70, or treated with the synthetic HSP70 inhibitor N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam under hypoxic conditions (5% O(2)) for 24, 48 or 72 h. PMVEC apoptosis was evaluated by performing flow cytometry and mitochondrial membrane potential (MMP) assays. The expression levels of HSP70, hypoxia-inducible factor-1α (HIF-1α) and apoptosis-associated proteins were determined by conducting reverse transcription-quantitative PCR and western blotting. Following 24, 48 or 72 h of hypoxia, the apoptotic rates of PMVECs were significantly elevated compared with cells under normoxic conditions. The MMP was significantly reduced, whereas the mRNA and protein expression levels of HIF-1α, cytochrome c (cyt C), caspase-3 and HSP70 were enhanced by hypoxia compared with those under normoxic conditions. Additionally, the mRNA and protein expression levels of B-cell lymphoma 2 (Bcl-2) were significantly downregulated in the hypoxia group compared with those in the normoxia group. In hypoxic PMVECs, HSP70 overexpression decreased the apoptotic rate and the expression levels of cyt C, downregulated the expression levels of caspase-3 and HIF-1α, and increased the MMP and the expression levels of Bcl-2. HSP70 inhibition resulted in the opposite outcomes compared with those of HSP70 overexpression. Therefore, the results of the present study suggested that HSP70 may inhibit mitochondrial pathway-mediated apoptosis in isolated neonatal rat PMVECs in early-stage hypoxia, which may be associated with HSP70-mediated HIF-1α downregulation. Overall, HSP70 may be protective against neonatal HPH through the HSP70/HIF-1α pathway.