Background: Aging increases cardiovascular morbidity. Aging in vascular smooth muscle cells (VSMC) induces cell senescence, leading to impaired vasodilation with decreased vascular compliance, increased inflammation and oxidative stress. MicroRNA-34a (miR-34a) has been reported as a biomarker of aging.
Methods and Results: After screening for a group of miRs, there was a > 2 fold of miR-34a in aortic SMCs isolated from transgenic mice overexpressing Nox1 in SMCs (Nox-1 SMCs). Therefore, we hypothesized that miR34 contributes to VSMC aging. TaqMan miR assay confirmed an increased miR-34a by 2.1 fold in young NOX-1 SMCs (passage 3-7) with an increase by 5.25 fold in activity of senescence-associated β-galactosidase (SA-β-Gal ) compared to WT. miR-34a in old NOX-1 and WT SMCs (passage 14-16) was increased by 3.8 fold and 2.1 fold, respectively, compared to young SMCs (both P< 0.01 ). There was 1.8 fold more miR-34a detected in old NOX-1 vs. old WT SMCs. SA-β-Gal positive cells accounted for 0.8±1% and 4±1.3% in young WT and NOX-1 SMCs (P<0.01), and 5±1.5% and 32±5% in old WT and NOX-1 SMCs (P <0.005). After transfection with miR-34a inhibitor, mature miR-34a was decreased by 75% and SA-β-Gal activity by 58% in old NOX-1 cells with parallel changes in the protein expression for Gamma-H2AX( a DNA damage marker) and p21( an activator for cell senescence). Moreover, phosphatase nuclear targeting subunit (PNUTS, a recently identified anti-age protein and direct miR-34a target) was not changed in young NOX-1 SMCs but reduced by 78% in old NOX-1 SMCs. Interestingly, nuclear factor (erythroid-derived 2)-like 2 (Nrf2, a key transcription factor for antioxidant genes) mRNA and protein were decreased by 55% and 32% in old NOX-1 SMCs (P<0.01 and 0.05). Nrf2 activator tert-butylhydroquinone (tBHQ) reduced miR-34a from 2.9±0.4 to 1.8±0.2 fold in those cells.
Conclusion: Increased NOX-1 in SMCs induces miR-34a that accelerates SMC senescence. The pathway may involve downregulation of Nrf2 and increased P21 through P53 pathway. This provides potential targets for protecting against vascular aging.