Sphingosine 1-phosphate receptor 2 promotes erythrocyte clearance by vascular smooth muscle cells in intraplaque hemorrhage through MFG-E8 production

Intraplaque hemorrhage (IPH) accelerates atherosclerosis progression. To scavenge excessive red blood cells (RBCs), vascular smooth muscle cells (VSMCs) with great plasticity may function as phagocytes. Here, we investigated the erythrophagocytosis function of VSMCs and possible regulations involved. Based on transcriptional microarray analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that genes up-regulated in human carotid atheroma with IPH were enriched in functions of phagocytic activities, while those down-regulated were enriched in VSMCs contraction function. Transcriptional expression of Milk fat globule-epidermal growth factor 8 (MFG-E8) was also down-regulated in atheroma with IPH. In high-fat diet-fed apolipoprotein E-deficient mice, erythrocytes were present in cells expressing VSMC markers aSMA in the brachiocephalic artery, suggesting VSMCs play a role in erythrophagocytosis. Using immunofluorescence and flow cytometry, we also found that eryptotic RBCs were bound to and internalized by VSMCs in a phosphatidylserine/ MFG-E8/integrin alpha(V)beta(3) dependent manner in vitro. Inhibiting S1PR2 signaling with specific inhibitor JTE-013 or siRNA decreased Mfge8 expression and impaired the erythrophagocytosis of VSMCs in vitro. Partial ligation was performed in the left common carotid artery (LCA) followed by intra-intimal injection of isolated erythrocytes to observe their clearance in vivo. Interfering S1PR2 expression in VSMCs with Adeno-associated virus 9 inhibited MFG-E8 expression inside LCA plaques receiving RBCs injection and attenuated erythrocytes clearance. Erythrophagocytosis by VSMCs increased vascular endothelial growth factor-alpha secretion and promoted angiogenesis. The present study revealed that VSMCs act as phagocytes for RBC clearance through S1PR2 activation induced MFG-E8 release.