Hypoxia preconditioning promotes cardiac stem cell survival and cardiogenic differentiation in vitro involving activation of the HIF-1α/apelin/APJ axis

Jingying Hou, Lei Wang, Huibao Long, Hao Wu, Quanhua Wu, Tingting Zhong, Xuxiang Chen, Changqing Zhou, Tianzhu Guo, Tong Wang

Cardiac stem cells (CSCs) transplantation has been regarded as an optimal therapeutic approach for cardiovascular disease. However, inferior survival and low differentiation efficiency of these cells in the local infarct site reduce their therapeutic efficacy. In this study, we investigated the influence of hypoxia preconditioning (HP) on CSCs survival and cardiogenic differentiation in vitro and explored the relevant mechanism. CSCs were obtained from Sprague-Dawley rats and cells of the third passage were cultured in vitro and exposed to hypoxia (1% O2). Cells survival and apoptosis were evaluated by MTS assay and flow cytometry respectively. Cardiogenic differentiation was induced by using 5-azacytidine for another 24 h after the cells experienced HP. Normoxia (20% O2) was used as a negative control during the whole process. Cardiogenic differentiation was assessed 2 weeks after the induction. Relevant molecules were examined after HP and during the differentiation process. Anti-hypoxia-inducible factor-1α (HIF-1α) small interfering RNA (siRNA), anti-apelin siRNA, and anti-putative receptor protein related to the angiotensin receptor AT1 (APJ) siRNA were transfected in order to block their expression, and relevant downstream molecules were detected. Compared with the normoxia group, the hypoxia group presented more rapid growth at time points of 12 and 24 h (p < 0.01). Cells exhibited the highest proliferation rate at the time point of 24 h (p < 0.01). The cell apoptosis rate significantly declined after 24 h of hypoxia exposure (p < 0.01). Expression levels of HIF-1α, apelin, and APJ were all enhanced after HP. The percentage of apelin, α-SA, and cTnT positive cells was greatly increased in the HP group after 2 weeks of induction. The protein level of α-SA and cTnT was also significantly elevated at 7 and 14 days (p < 0.01). HIF-1α, apelin, and APJ were all increased at different time points during the cardiogenic differentiation process (p < 0.01). Knockdown of HIF-1α, apelin or APJ by siRNAs resulted in a significant reduction of α-SA and cTnT. HIF-1α blockage caused a remarkable decrease of apelin and APJ (p < 0.01). Expression levels of apelin and APJ were depressed after the inhibition of apelin (p < 0.01). HP could effectively promote CSCs survival and cardiogenic differentiation in vitro, and this procedure involved activation of the HIF-1α/apelin/APJ axis. This study provided a new perspective for exploring novel strategies to enhance CSCs transplantation efficiency.