Objectives To examine a hypothesis that organic nitrate, widely used to treat vascular contraction, protects endothelial progenitor cells (EPCs) against senescence induced by angiotensinII (AngII); and if so, to investigate the underlying mechanisms involved.
Methods Human-derived EPCs, treated with PBS or nitrate (100 μmol/L), was subsequently exposed to AngII (100 nmol/L) for 24 hours in vitro. The senescence-associated β-galactosidase test and the detection of p16Ink4a/p19Arf expression were used to evaluate the cellular senescence. The miRNAs transcriptome was analysed by microarray and was verified by real-time PCR. The gain- and loss-of-function methods through lentivirus infection were administrated to evaluate the role of miRs in the senescence of EPCs induced by angiotensinII. Additionally, a luciferase reporter assay was performed to confirm associations between miRNAs and their putative targets, which was furthermore verified by small interference RNA (siRNA) and Western-blot.
Results AngII significantly induced EPCs senescence while nitrate not. However, the effect of AngII on cellular senescence could be statistically attenuated by nitrate pre-culture, indicated as the differences in the percentage of senescent EPCs between AngII group and pre-culture group [(72.6 ± 6.92)% vs. (43.7 ± 7.55)%, P<0.05], as well as the expressions of p16Ink4a (3.56 ± 0.76 vs. 1.93 ± 0.42, P<0.05) and p19Arf (4.88 ± 0.72 vs. 2.67 ± 0.54, P<0.05). MiR-210 in pre-culture group was discovered to up-regulate more over 4 times than that in AngII group (P<0.05). Moreover, forced expression of miR-210 in EPCs, compared with scramble over-expression, was evidenced to dramatically reverse cell senescence induced by AngII [(37.6 ± 7.43)% vs. (71.8 ± 6.63)%, P<0.05] and drastically decrease both p16Ink4a and p19Arf expressions (both P<0.05). In contrast, suppression of miR-210 in EPCs, raising putative miR-target SCH9 expression, statistically abolished the protective effect of nitrate on EPCs senescence [anti-miR group vs. null group: (72.6 ± 6.92)% vs. (45.8 ± 8.39)%, P<0.05]. Most importantly, the phenotypic changes were discovered to be recovered by SCH9 knockdown (P<0.05).
Conclusions Nitrate protects EPCs, mediated by miR-210 up-regulation and SCH9 suppression, against cellular senescence induced by AngII, which is of a crucial significance for those rennin-dependent hypertensive patients with vascular injury.