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Effects of homocysteine on endothelial progenitor cells in vitro Homocysteine (Hcy) is an independent cardiovascular risk factor linked to the induction of endothelial dysfunction (Eikelboom et al., 1999; McCully et al,. 1975). Circulating endothelial progenitor cells (EPC) have been shown to play an important role in the maintenance and repair of the endothelium (Asahara et al., 1997) and may therefore be a potential target for Hcy mediated toxicity. We investigated the effects of Hcy on EPC in vitro. EPC were isolated from the peripheral blood of healthy volunteers (n = 10) by density gradient centrifugation. The cells were cultured in EBM-2 + EGM-2-MV-Single Quots medium containing 10% foetal calf serum (FCS) and plated on fibronectin (10μg/ml) coated coverslips for 3 days, prior to overnight treatment with Hcy (3 - 100 μM). EPC were characterised by dual-positive staining for AcLDL-DiI uptake and lectin binding. The senescence of EPC was assessed by senescence-associated β-galactosidase staining. Early and late apoptosis of EPC was measured by an annexin V / propidium iodide assay and 4’-6-diamidino-2-phenylindole (DAPI) staining respectively. Data are presented as mean ± SEM and comparisons were made using one-way ANOVA with Dunnett’s post hoc test or by paired Student’s t-test; p < 0.05 was considered to be significant. IMAGE Figure 1: Effect of Hcy on A) total EPC number and B) % senescence. Data are mean ± SEM (n= 7). Hcy produced a significant concentration-dependent decrease in EPC (Figure 1A.) and a concentration-dependent increase in EPC senescence (Figure 1B.). The percentage of early and late apoptotic EPC was significantly increased by Hcy (10 μmol/L) from 21.3 ± 3.0 to 30.0 ± 3.4 (p = 0.04; n=6), and 14.0 ± 1.2 to 23.9 ± 3.7 (p=0.02; n=6) respectively. Hcy increases senescence and apoptosis of EPC. This effect may contribute to endothelial injury and cardiovascular disease (CVD) caused by Hcy.
Asahara et al., (1997) Science, 275 , 964-967. |
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