GPR4 Signaling is Essential for the Promotion of Acid-mediated Angiogenic Capacity of Endothelial Progenitor Cells by Activating STAT3/VEGFA Pathway in Patients with Coronary Artery Disease

Background: Patients with coronary artery disease (CAD) are characterized by a decline in vascular regeneration, which is related to the dysfunction of endothelial progenitor cells (EPCs). G-protein-coupled receptor 4 (GPR4) is a proton-sensing G-protein-coupled receptor (GPCR) that contributes to neovascularization in acidic microenvironments. However, the role of GPR4 in regulating the angiogenic capacity of EPCs from CAD patients in response to acidity generated in ischemic tissue remains completely unclear.

Methods: The angiogenic capacity of EPCs collected from CAD patients and healthy subjects was evaluated in different pH environments. The GPR4 function of regulating EPC-mediated angiogenesis was analyzed both in vitro and in vivo. The downstream mechanisms were further investigated by genetic overexpression and inhibition.

Results: Acidic environment prestimulation significantly enhanced the angiogenic capacity of EPCs from the non-CAD group both in vivo and in vitro, while the same treatment yielded the opposite result in the CAD group. Among the four canonical proton-sensing GPCRs, GPR4 displays the highest expression in EPCs. The expression of GRP4 was markedly lower in EPCs from CAD patients than in EPCs from non-CAD individuals independent of acid stimulation. The siRNA-mediated knockdown of GPR4 with subsequent decreased phosphorylation of STAT3 mimicked the impaired function of EPCs from CAD patients at pH 6.4 but not at pH 7.4. Elevating GPR4 expression restored the neovessel formation mediated by EPCs from CAD patients in an acidic environment by activating STAT3/VEGFA signaling. Moreover, the beneficial impact of GPR4 upregulation on EPC-mediated angiogenic capacity was abrogated by blockade of the STAT3/VEGFA signaling pathway.

Conclusions: Our present study demonstrated for the first time that loss of GPR4 is responsible for the decline in proton sensing and angiogenic capacity of EPCs from CAD patients. Augmentation of GPR4 expression promotes the neovessel formation of EPCs by activating STAT3/VEGF signaling. This finding implicates GPR4 as a potential therapeutic target for CAD characterized by impaired neovascularization in ischemic tissues.