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. 2022 Jan 14;23(2):867. doi: 10.3390/ijms23020867

Figure 1.

Figure 1

Expression and release of CXCL12 by SMCs and EPCs. (A) Analysis of CXLC12 release using ELISA. Cell supernatants from monocultured SMCs and EPCs, treated as indicated, were collected 24 h after cultivation. * p < 0.05 vs. untreated cells; n = 6. (B) Real-time RT-PCR analysis of CXCL12 expression SMCs and EPCs treated as indicated. Results were normalized to CXCL12 expression in SMCs. * p < 0.05 vs. untreated cells; n = 5. (C) Detection of CXCL12 in MVs derived from monocultured SMCs and EPCs. Isolated MVs were lysed in RIPA buffer and CXCL12 levels were determined using ELISA. * p < 0.05 vs. non-injured SMCs, # p < 0.05 vs. SMC-MV; n = 5. (D) Enumeration of MVs in the supernatant of EPCs, non-injured SMCs and injured SMCs using flow cytometry with calibrated microbeads. * p < 0.05 vs. non-injured SMCs; n = 4. (E) Evaluation of the effect of EPC-SMC co-cultivation and engagement of CXCR4 on the release of CXCL12. Supernatants from monocultured SMCs, monocultured EPCs and EPCs co-cultured with SMCs, each in the presence or absence of a blocking CXCR4 Ab, were analyzed for CXCL12 concentration using ELISA. * p < 0.05 vs. SMCs, # p < 0.05 vs. EPC-SMC co-culture in the absence of anti-CXCR4; n = 5. (F) Real-time RT-PCR analysis of CXCL12 expression to test the impact of EPC-SMC co-cultivation and involvement of CXCR4. CXCL12 transcripts were determined in SMCs, EPCs and EPC-SMC co-cultures in the presence or absence of a blocking CXCR4 Ab. * p < 0.05 vs. SMCs, # p < 0.05 vs. EPC-SMC co-culture in the absence of anti-CXCR4; n = 5. (G) Real-time RT-PCR analysis of CXCL12 expression in SMCs treated with various doses of rCXCL12, CM-EPC or EPC-MV in the presence or absence of an anti-CXCR4 Ab. * p < 0.05 vs. untreated SMCs (control), # p < 0.05 vs. respective treatment in the absence of anti-CXCR4; n = 5. (H) Adhesion of EPCs to SMCs under flow conditions in vitro. EPCs pretreated with/without an anti-CXCR4 Ab were perfused in a parallel flow chamber and the number of cells EPCs adherent to the SMC monolayer was determined and expressed as adherent cells per 1 mm². For some experiments, the SMC monolayer was wounded by a linear scratch before perfusion of EPCs. * p < 0.05 vs. untreated and non-scratched SMCs (control), # p < 0.05 vs. respective treatment in the absence of anti-CXCR4; n = 4 to 6.