Deciphering the role of Store Operated Ca2+ entry in post-ischemic angiogenesis

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación

Post-ischemic angiogenesis is essential to improve tissue repair and heart recovery after myocardial infarction (MI). This process is tightly controlled by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which in turn promotes signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recently, we demonstrated that store-operated calcium entry (SOCE) plays a key role in angiogenesis. However, the function of SOCE in post-ischemic angiogenesis remains unknown. The aim of this study is to explore the role of SOCE in this crucial repairing process.

In vitro angiogenesis was studied using Human Umbilical Vein Endothelial Cells (HUVEC) for tube formation assay over Matrigel supplemented with serum from rat model of ischemia/reperfusion (I/R) and from ST elevation MI (STEMI) patients who underwent primary percutaneous coronary intervention (pPCI). We evaluated serum VEGF-A levels using ELISA. Genes and protein expression was determined by RT-qPCR and western blot, respectively. HUVEC migration was assessed by wound healing assay. Moreover, changes in [Ca2+]i were measured using fura-2 AM.

We found that STEMI patients had higher levels of circulating VEGF-A, as compared to controls. The incubation of HUVEC seeded on Matrigel with the serum of these patients promoted vessels-like formation since it enhanced the number of meshes and junctions. Likewise, serum from I/R rats also boosted HUVEC angiogenesis over Matrigel and promoted faster gap closure in wound healing assay, as compared to cells incubated with sham rats’ serum. Furthermore, serum from both rats and patients modulated the expression Hes1 and Hey1, well-known mediators of Notch1 signaling pathway. Moreover, we found that knockdown of Orai1, the pore-forming subunit of the store-operated calcium channel (SOCC), using siRNA impairs VEGF-mediated [Ca2+]i increase, as well as HUVEC tube formation, proliferation, and migration.

Our data show for the first that serum from both STEMI patients and I/R rats promote angiogenesis, involving Orai1 and Notch1 signaling pathways.