Endothelial progenitor cells (EPCs) play an important role in accelerating endothelial repair after vascular injury. The proliferation and migration of EPCs is a critical first step in restoring endothelial. However, mechanisms for modulating EPC proliferation and migration are still being elucidated. Our previous study found that transient receptor potential canonical-1 (TRPC1) is involved in regulating store-operated Ca 2 + entry in EPCs through stromal interaction molecule 1. Therefore, in the present study, we sought to further investigate the regulation of proliferation and migration of EPCs by TRPC1. We found that the silencing of TRPC1 by 2 different RNA interference methods suppressed the proliferation and migration of EPCs. In addition, knockdown of TRPC1 significantly reduced of the amplitude of store-operated Ca 2 + entry and caused arrest of the EPC cell cycle in G1 phase. Analysis of the expression of 84 cell cycle genes by microarray showed that 9 genes were upregulated and 4 were downregulated by > 2-fold in EPCs following TRPC1 silencing. The genes with expression changes were Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, Prm1, Bcl2, Mki67, Pmp22, and Ppp2r3a. Finally, we found that a Schlafen 1-blocking peptide partially reversed the abnormal cell cycle distribution and proliferation induced by TRPC1 knockdown, suggesting that Schlafen 1 is downstream of TRPC1 silencing in regulating EPC proliferation. In summary, these findings provide a new mechanism for modulating the biological properties of EPCs and suggest that TRPC1 may be a new target for inducing vascular repair by EPCs.

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.

Cardiac stem cells (CSCs) can home to the infarcted area and regenerate myocardium. Stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) axis is pivotal in inducing CSCs migration. However, the mechanisms remain unclear. This study set out to detect if SDF-1α promotes migration and engraftment of CSCs through the CXCR4/PI3K (phosphatidylinositol 3-kinase) pathway. In the in vitro experiment, c-kit+ cells were isolated from neonatal mouse heart fragment culture by magnetic cell sorting. Fluorescence-activated cell sorting results demonstrated that a few c-kit+ cells expressed CD45 (4.54%) and Sca-1 (2.58%), the hematopoietic stem cell marker. Conditioned culture could induce c-kit+ cells multipotent differentiation, which was confirmed by cardiac troponin I (cTn-I), α-smooth muscle actin (α-SMA), and von Willebrand factor (vWF) staining. In vitro chemotaxis assays were performed using Transwell cell chambers to detect CSCs migration. The results showed that the cardiomyocytes infected with rAAV1-SDF-1α-eGFP significantly increased SDF-1α concentration, 5-fold more in supernatant than that in the control group, and subsequently attracted more CSCs migration. This effect was diminished by administration of AMD3100 (10 µg/ml, CXCR4 antagonist) or LY294002 (20 µmol/L, PI3K inhibitor). In myocardial infarction mice, overexpression of SDF-1α in the infarcted area by rAAV1-SDF-1α-eGFP infection resulted in more CSCs retention to the infarcted myocardium, a higher percentage of proliferation, and reduced infarcted area which was attenuated by AMD3100 or ly294002 pretreatment. These results indicated that overexpression of SDF-1α enhanced CSCs migration in vitro and engraftment of transplanted CSCs and reduced infarcted size via CXCR4/PI3K pathway.

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