Mitochondria-targeted esculetin mitigates atherosclerosis in the setting of aging via the modulation of SIRT1-mediated vascular cell senescence and mitochondrial function in Apoe-/- mice.

BACKGROUND AND AIMS: Age is a dominant and independent risk factor for the development of atherosclerosis, a major cardiovascular disease, and if left untreated leads to myocardial infarction and death. Mitochondria-targeted anti-oxidants are evolving as a new class of compounds that can alter the pathophysiology of age-related diseases, including atherosclerosis, where mitochondrial dysfunction plays a critical role in disease progression. METHODS: We recently synthesized an alkyl TPP + -tagged esculetin (mitochondria-targeted esculetin or Mito-Esc). Apoe-/- mice were chronically (14 months) administered with Mito-Esc to investigate its efficacy in the mitigation of atherosclerosis in the setting of aging. We monitored BP, and performed various biochemical assays, histopathology, immunohistochemistry, inflammatory factors, qPCR, and Western blotting. Simultaneously, human aortic endothelial cells (HAECs) were used as a model system to study the mechanistic aspects. RESULTS: A chronic low-dose administration of Mito-Esc to Apoe-/- mice greatly prevented alterations in lipid profile, blood pressure, and atherosclerotic plaque formation in the setting of aging. Mito-Esc administration significantly reduced vascular senescence and pro-inflammatory cytokines levels and prevented dysregulation of mitochondrial biogenesis markers in aortic tissue. Further, Mito-Esc treatment prevented replicative and stress-induced premature senescence (SIPS) in HAEC. Importantly, Mito-Esc treatment delayed endothelial cell senescence by increasing human telomerase reverse transcriptase (hTERT) levels via SIRT1 activation. Moreover, Mito-Esc treatment by altering miR-19b and miR-30c via a SIRT1 activation significantly inhibited the increase in PAI-1 levels in HAEC as well as in the serum of Apoe-/- mice. In addition, Mito-Esc treatment improved mitochondrial function in late passage (aged) HAECs by enhancing the oxygen consumption rate (OCR). Furthermore, Mito-Esc administration counteracted the decline in GSH and nitrite levels in Apoe-/- mice and in HAECs. CONCLUSIONS: Overall, Mito-Esc alleviates atherosclerosis in the setting of aging by delaying vascular senescence and pro-inflammatory processes, and by improving mitochondrial biogenesis and function.

Metformin regulates mitochondrial biogenesis and senescence through AMPK mediated H3K79 methylation: Relevance in age-associated vascular dysfunction.

Endothelial senescence in conjunction with mitochondrial dysfunction orchestrates age-associated cardiovascular disorders. In this study we investigated the causal link between these two processes and studied the molecular mechanisms by which metformin acts to coordinate the delay of endothelial senescence via enhancing mitochondrial biogenesis/function. AMPK activators metformin and AICAR delayed endothelial senescence via SIRT1-mediated upregulation of DOT1L, leading to increased trimethylation of H3K79 (H3K79me3). Treatment of cells with either siAMPK or siSIRT1 repressed DOT1L-mediated enhancement of H3K79me3. Moreover, the increase in SIRT3 expression and mitochondrial biogenesis/function by AMPK activators was H3K79me-dependent as H3K79N mutant or siDOT1L abrogated these effects. This was confirmed by the enrichment of H3K79me3 in the SIRT3 promoter with AMPK activation. Intriguingly, enhanced PGC-1α expression by SIRT3 via AMPK activation was responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown caused increased oxidative stress and premature senescence, possibly by depleting hTERT expression. Furthermore, a chronic low dose administration of metformin significantly attenuated vascular aging and inhibited age-associated atherosclerotic plaque formation in ApoE-/- mice. Overall, the results of this study show a novel regulation of mitochondrial biogenesis/function, and cellular senescence by H3K79me acting through SIRT3, thus providing a molecular basis for metformin-mediated age-delaying effects.

Mitochondria-targeted esculetin inhibits PAI-1 levels by modulating STAT3 activation and miR-19b via SIRT3: Role in acute coronary artery syndrome.

In this study we explored the microRNAs responsible for the regulation of PAI-1 during LPS-stimulated inflammation in human aortic endothelial cells and subsequently studied the effect of a newly synthesized mitochondria-targeted esculetin (Mito-Esc) that was shown for its anti-atherosclerotic potential, in modulating PAI-1 levels and its targeted miRs during angiotensin-II-induced atherosclerosis in ApoE-/- mice. LPS-stimulated PAI-1 was accompanied with an upregulation of miR-19b and down-regulation of miR-30c. These effects of LPS on PAI-1 were reversed in the presence of both parent esculetin and Mito-Esc. However, the effect of Mito-Esc was more pronounced in the regulation of PAI-1. In addition, LPS-stimulated PAI-1 expression was significantly decreased in cells treated with Anti-miR-19b, thereby suggesting that miR-19b co-expression plays a key role in PAI-1 regulation. The results also show that incubation of cells with Stattic, an inhibitor of STAT-3, inhibited LPS-stimulated PAI-1 expression. Interestingly, knockdown of SIRT3, a mitochondrial biogenetic marker, enhanced PAI-1 levels via modulation of miR-19b and -30c. Mito-Esc treatment significantly inhibited Ang-II-induced PAI-1, possibly via altering miR-19b and 30c in ApoE-/- mice. The association between PAI-1, miR-19b and -30c were further confirmed in plasma and microparticles isolated from patients suffering from acute coronary syndrome of various degrees. Taken together, LPS-induced PAI-1 involves co-expression of miR-19b and down regulation of miR-30c, and Mito-Esc treatment by modulating miR-19b and miR-30c through SIRT3 activation, inhibits PAI-1 levels that, in part, contribute to its anti-atherosclerotic effects. Moreover, there exists a strong positive correlation between miR-19b and PAI-1 in patients suffering from ST-elevated myocardial infarction.