Therapeutic efficacies of mitochondria-targeted esculetin and metformin in the improvement of age-associated atherosclerosis via regulating AMPK activation.

Atherosclerosis, in general, is an age-associated cardiovascular disease wherein a progressive decline in mitochondrial function due to aging majorly contributes to the disease development. Mitochondria-derived ROS due to dysregulated endothelial cell function accentuates the progression of atherosclerotic plaque formation. To circumvent this, mitochondrially targeted antioxidants are emerging as potential candidates to combat metabolic abnormalities. Recently, we synthesized an alkyl TPP+ tagged esculetin (Mito-Esc), and in the current study, we investigated the therapeutic efficacies of Mito-Esc and metformin, a well-known anti-diabetic drug, in the amelioration of age-associated plaque formation in the aortas of 12 months aged Apoe-/- and 20 months aged C57BL/6 mice, in comparison to young C57BL/6 control mice. Administration of Mito-Esc or metformin significantly reduced age-induced atherosclerotic lesion area, macrophage polarization, vascular inflammation, and senescence. Further, chronic passaging of human aortic endothelial cells (HAEC) with either Mito-Esc or metformin significantly delayed cellular senescence via the activation of the AMPK-SIRT1/SIRT6 axis. Conversely, depletion of either AMPK/SIRT1/SIRT6 caused premature senescence. Consistent with this, Mito-Esc or metformin treatment attenuated NFkB-mediated inflammatory signaling and enhanced ARE-mediated anti-oxidant responses in comparison to late passage control HAECs. Importantly, culturing of HAECs for several passages with either Mito-Esc or metformin significantly improved mitochondrial function. Overall, Mito-Esc and metformin treatments delay age-associated atherosclerosis by regulating vascular senescence via the activation of AMPK-SIRT1/SIRT6 axis.

Mitochondria-targeted esculetin and metformin delay endothelial senescence by promoting fatty acid ß-oxidation: Relevance in age-associated atherosclerosis.

Impaired mitochondrial fatty acid ß-oxidation (FAO) plays a role in the onset of several age-associated diseases, including atherosclerosis. In the current work, we investigated the efficacies of mitochondria-targeted esculetin (Mito-Esc) and metformin in enhancing FAO in human aortic endothelial cells (HAECs), and its relevance in the delay of cellular senescence and age-associated atherosclerotic plaque formation in Apoe-/- mice. Chronic culturing of HAECs with either Mito-Esc or metformin increased oxygen consumption rates (OCR), and caused delay in senescence features. Conversely, etomoxir (CPT1 inhibitor) reversed Mito-Esc- and metformin-induced OCR, and caused premature endothelial senescence. Interestingly, Mito-Esc, unlike metformin, in the presence of etomoxir failed to preserve OCR. Thereby, underscoring Mito-Esc's exclusive reliance on FAO as an energy source. Mechanistically, chronic culturing of HAECs with either Mito-Esc or metformin led to AMPK activation, increased CPT1 activity, and acetyl-CoA levels along with a concomitant reduction in malonyl-CoA levels, and lipid accumulation. Similar results were observed in Apoe-/- mice aorta and liver tissue with a parallel reduction in age-associated atherosclerotic plaque formation and degeneration of liver with either Mito-Esc or metformin administration. Together, Mito-Esc and metformin by potentiating FAO, may have a role in the delay of cellular senescence by modulating mitochondrial function.

Mitochondria targeted esculetin administration improves insulin resistance and hyperglycemia-induced atherosclerosis in db/db mice.

The development and progression of hyperglycemia (HG) and HG-associated atherosclerosis are exacerbated by mitochondrial dysfunction due to dysregulated mitochondria-derived ROS generation. We recently synthesized a novel mitochondria-targeted esculetin (Mito-Esc) and tested its dose-response therapeutic efficacy in mitigating HG-induced atherosclerosis in db/db mice. In comparison to simvastatin and pioglitazone, Mito-Esc administration resulted in a considerable reduction in body weights and improved glucose homeostasis, possibly by reducing hepatic gluconeogenesis, as indicated by a reduction in glycogen content, non-esterified free fatty acids (NEFA) levels, and fructose 1,6-bisphosphatase (FBPase) activity. Interestingly, Mito-Esc treatment, by regulating phospho-IRS and phospho-AKT levels, greatly improved palmitate-induced insulin resistance, resulting in enhanced glucose uptake in adipocytes and HepG2 cells. Also, and importantly, Mito-Esc administration prevented HG-induced atheromatous plaque formation and lipid accumulation in the descending aorta. In addition, Mito-Esc administration inhibited the HG-mediated increase in VACM, ICAM, and MAC3 levels in the aortic tissue, as well as reduced the serum pro-inflammatory cytokines and markers of senescence. In line with this, Mito-Esc significantly inhibited monocyte adherence to human aortic endothelial cells (HAECs) treated with high glucose and reduced high glucose-induced premature senescence in HAECs by activating the AMPK-SIRT1 pathway. In contrast, Mito-Esc failed to regulate high glucose-induced endothelial cell senescence under AMPK/SIRT1-depleted conditions. Together, the therapeutic efficacy of Mito-Esc in the mitigation of hyperglycemia-induced insulin resistance and the associated atherosclerosis is in part mediated by potentiating the AMPK-SIRT1 axis. KEY MESSAGES: Mito-Esc administration significantly mitigates diabetes-induced atherosclerosis. Mito-Esc improves hyperglycemia (HG)-associated insulin resistance. Mito-Esc inhibits HG-induced vascular senescence and inflammation in the aorta. Mito-Esc-mediated activation of the AMPK-SIRT1 axis regulates HG-induced endothelial cell senescence.

DOT1L regulates MTDH-mediated angiogenesis in triple-negative breast cancer: intermediacy of NF-κB-HIF1α axis.

DOT1L, a specific H3K79 methyltransferase, has a tumour-promoting role in various cancers, including triple-negative breast cancer (TNBC). However, the molecular mechanism by which the deregulated DOT1L promotes cancer progression is unclear. Herein, we show that a significantly higher basal level of DOTL1 strongly correlates with MTDH, an oncogene, in clinical TNBC patient cohorts and mediates TNBC progression by enhancing MTDH-induced angiogenesis. In parallel, severe combined immunodeficiency mice-bearing MDA-MB-231 cells with MTDH-Wt or MTDHΔ7 (spliced isoform of MTDH) overexpression constructs showed enhanced blood vessel formations at the tumour site in comparison with control groups. Selective inhibition of DOT1L by EPZ004777, a specific DOT1L inhibitor, or siDOT1L, significantly impaired MTDH-induced proliferation, invasion and angiogenic markers expression in TNBC cells. ChIP assay revealed that Dot1L promotes MTDH-Wt/Δ7 transcription by increasing H3K79me3 levels on its promoter. Dot1L depletion reversed this effect. Mechanistically, DOT1L-induced MTDH caused enhanced nuclear factor kappa B (NF-κB) occupancy on the hypoxia-inducible factor1α (HIF1α) promoter and increased its transcription, leading to elevated levels of proangiogenic mediators in TNBC cells. Moreover, the condition media obtained from MDA-MB-231 cells stably expressing either MTDH-Wt or MTDHΔ7 treated with EPZ004777 or Bay-11-7082 (NF-κB inhibitor) or FM19G11 (HIF1α inhibitor) significantly inhibited MTDH-induced tube formation in human umbilical vein endothelial cells, rat aortic ring sprouting and vessel formations by chick chorioallantoic membrane assay mimicking physiological angiogenic vasculature. Collectively, our findings reveal a novel epigenetic regulation of MTDH by DOTL1, which drives angiogenesis, and that the therapeutic disruption of the DOT1L-MTDH-NF-κB-HIF1α axis may have usefulness in the management of TNBC.

Therapeutic efficacy of mitochondria-targeted esculetin in the improvement of NAFLD-NASH via modulating AMPK-SIRT1 axis.

Mitochondrial dysfunction due to deregulated production of mitochondria-derived ROS is implicated in the development and progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). Recently, we synthesized a novel mitochondria-targeted esculetin (Mito-Esc) and investigated its dose-response therapeutic efficacy in mitigating high-fat diet (HFD)-induced NAFLD and NASH in Apoe-/- mice. Mito-Esc administration, compared to simvastatin and pioglitazone, dose-dependently caused a significant reduction in body weight, improved lipid profile, glucose homeostasis, and pro-inflammatory cytokines level. Mito-Esc administration reduced adipose tissue hypertrophy and lipid accumulation presumably by regulating the levels of CD36, PPAR-γ, EBP-α, and their target genes. Mechanistically, Mito-Esc-induced activation of the AMPK1α-SIRT1 axis inhibited pre-adipocyte differentiation. Conversely, Mito-Esc failed to regulate pre-adipocyte differentiation under AMPK/SIRT1 depleted conditions. In parallel, Mito-Esc administration ameliorated HFD-induced steatosis, fibrosis of the liver, and NAFLD-associated atheromatous plaque formation in the aorta. Importantly, Mito-Esc administration inhibited HFD-induced infiltration of macrophages, a marker of steatohepatitis, in the adipose and liver tissues. The results of the in vitro studies showed that Mito-Esc treatment significantly inhibits TGF-ß-induced hepatic stellate cell differentiation as well as the fibrotic markers. Consistent with the above observations, Mito-Esc treatment by activating the AMPK-SIRT1 pathway markedly reversed palmitate-induced mitochondrial superoxide production, depolarization of mitochondrial membrane potential, and lipid accumulation in HepG2 cells. Together, the therapeutic efficacy of Mito-Esc in the mitigation of HFD-induced lipotoxicity, and the associated NASH is in part, mediated by potentiating the AMPK-SIRT1 axis.

A functional and self-assembling octyl-phosphonium-tagged esculetin as an effective siRNA delivery agent.

Herein, we document a self-assembling octyl-TPP tagged esculetin (Mito-Esc) as functionally active and as a novel small molecule siRNA delivery vector. While Mito-Esc itself induces selective breast cancer cell death, the amphiphilic nature of Mito-Esc delivers therapeutic siRNAs intracellularly without the need for any excipient to exacerbate the anti-proliferative effects.