SIRT1 promotes doxorubicin-induced breast cancer drug resistance and tumor angiogenesis via regulating GSH-mediated redox homeostasis.

Sirtuin 1 (SIRT1), a member of histone deacetylase III family, plays a pivotal role in mediating chemoresistance in several cancers, including breast cancer. However, the molecular mechanism by which the deregulated SIRT1 promotes doxorubicin (Dox) resistance is still elusive. Here, we showed that the cell proliferation rates and invasive properties of MDA-MB-231 breast cancer cells were increased from low- to high-Dox-resistant cells. In agreement, severe combined immunodeficiency disease (SCID) mice bearing labeled MDA-MB-231high Dox-Res cells showed significantly higher tumor growth, angiogenesis, and metastatic ability than parental MDA-MB-231 cells. Interestingly, the levels of SIRT1 and glutathione (GSH) were positively correlated with the degree of Dox-resistance. Dox-induced SIRT1 promoted NRF2 nuclear translocation with an accompanying increase in the antioxidant response element promotor activity and GSH levels. In contrast, inhibition of SIRT1 by EX527 greatly reversed these events. More so, Dox-resistance-induced pro-proliferative, proangiogenic, and invasive effects were obviated with depletion of either SIRT1 or GSH. Together, Dox-induced SIRT1 promotes dysregulation of redox homeostasis leading to breast cancer chemoresistance, tumor aggressiveness, angiogenesis, and metastasis.

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.

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.

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.

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.

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.

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.

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.

sp3 -Rich Glycyrrhetinic Acid Analogues Using Late-Stage Functionalization as Potential Breast Tumor Regressing Agents.

Late-stage functionalization (LSF) aids drug discovery efforts by introducing functional groups onto C-H bonds on pre-existing skeletons. We adopted the LSF strategy to synthesize analogues of the abundantly available triterpenoid, glycyrrhetinic acid (GA), by introducing aryl groups in the A-ring, expanding the A-ring and selectively activating one methyl group of the gem-dimethyl groups. Intriguingly, two compounds were found to preferentially accumulate in the mitochondrial compartment of MDA-MB-231 breast cancer cells, to cause depolarization of mitochondrial membrane potential and to induce antiproliferative and anti-invasive effects through enhanced mitochondrial superoxide production with parallel depletion of GSH levels. Furthermore, intraperitoneal administration of these two compounds, in comparison with GA, greatly regressed breast tumor growth and metastasis in a SCID mouse model bearing labeled MDA-MB-231 cells.

N-end rule pathway inhibitor sensitizes cancer cells to antineoplastic agents by regulating XIAP and RAD21 protein expression.

Anticancer drugs exert their effects on cancer cells by deregulating many pathways linked to cell cycle, apoptosis, etc. but cancer cells gradually become resistive against anticancer drugs, thereby necessitating the development of newer generation anticancer molecules. N-end rule pathway has been shown to be involved in the degradation of many cell cycle and apoptosis-related proteins. However, the involvements of this pathway in cancer are not well established. Recently, we developed a non-peptide-based N-end rule pathway inhibitor, RF-C11 for type 1 and 2 recognition domains of E3 ubiquitin ligases. The inhibitor significantly increased the half-life of potential N-degrons leading to significant physiological changes in vivo. We hypothesized RF-C11 may be used to decipher the N-end rule pathway's role in cancer towards the development of anticancer therapeutics. In this study, we showed that RF-C11, barring noncancer cells, significantly sensitizes cancer cells towards different anticancer agents tested. We further find that the profound cellular sensitization to anticancer drugs was affected by (a) downregulation of X-linked inhibitor of apoptosis protein, an antiapoptotic protein and (b) by stabilization of RAD21, and thereby inhibiting metaphase to anaphase promotion. The study shows that RF-C11 or its analogs may be used as a novel additive in combination therapy against cancer.

A novel metadherinΔ7 splice variant enhances triple negative breast cancer aggressiveness by modulating mitochondrial function via NFĸB-SIRT3 axis.

Metadherin (MTDH) expression inversely correlates with prognosis of several cancers including mammary carcinomas. In this work, we identified a novel splice variant of MTDH with exon7 skipping (MTDHΔ7) and its levels were found significantly high in triple negative breast cancer (TNBC) cells and in patients diagnosed with TNBC. Selective overexpression of MTDHΔ7 in MDA-MB-231 and BT-549 cells enhanced proliferation, invasion, and epithelial-to-mesenchymal (EMT) transition markers in comparison to its wildtype counterpart. In contrast, knockdown of MTDHΔ7 induced antiproliferative/antiinvasive effects. Mechanistically, MTDH-NFĸB-p65 complex activated SIRT3 transcription by binding to its promoter that in turn enhanced MnSOD levels and promoted EMT in TNBC cells. Intriguingly, mitochondrial OCR through Complex-I and -IV, and glycolytic rate (ECAR) were significantly high in MDA-MB-231 cells stably expressing MTDHΔ7. While depletion of SIRT3 inhibited MTDH-Wt/Δ7-induced OCR and ECAR, knockdown of MnSOD inhibited only ECAR. In addition, MTDH-Wt/Δ7-mediated pro-proliferative/-invasive effects were greatly obviated with either siSIRT3 or siMnSOD in these cells. The functional relevance of MTDHΔ7 was further proved under in vivo conditions in an orthotopic mouse model of breast cancer. Mice bearing labeled MDA-MB-231 cells stably expressing MTDHΔ7 showed significantly more tumor growth and metastatic ability to various organs in comparison to MTDH-Wt bearing mice. Taken together, MTDHΔ7 promotes TNBC aggressiveness through enhanced mitochondrial biogenesis/function, which perhaps serves as a biomarker.

Doxorubicin induces prostate cancer drug resistance by upregulation of ABCG4 through GSH depletion and CREB activation: Relevance of statins in chemosensitization.

Multidrug resistance mediated by ATP-binding cassette (ABC) transporters remains a major impediment to cancer chemotherapy. In the present study, we documented that doxorubicin (Dox) or cisplatin-induced prostate cancer (PCa) chemoresistance is predominantly mediated by the induction of ABCG4 in androgen-independent PCa cells. Treatment of DU-145 or PC-3 cells with Dox significantly enhanced the expression of ABCG4 that resulted in the efflux of intracellular Dox. However, incubation of cells with ABCG4 short hairpin RNA resulted in a significant accumulation of Dox and sensitized cells to Dox-induced cytotoxicity. Interestingly, simvastatin synergistically potentiated Dox-induced cytotoxicity by inhibiting ABCG4 in DU-145 and DU-145 Doxres cells. Mechanistically, ABCG4 expression was regulated redox-dependently by intracellular glutathione (GSH) levels. Treatment of cells with N-acetylcysteine or simvastatin restored Dox-induced depletion of GSH levels that in turn inhibited ABCG4 levels. In addition, a reduction in GSH levels by Dox caused a nuclear factor-κB dependent enhancement of c-Myc expression, which led to cAMP-regulatory element-binding protein (CREB) activation. Furthermore, chromatin immunoprecipitation experiments revealed that Dox-induced CREB activation transcriptionally upregulates ABCG4 expression. These results were further confirmed in an in vivo PCa xenograft mice model. Combination of simvastatin and Dox significantly regressed the tumor growth and size with no noticeable Dox-induced cardiotoxic side effects. Intriguingly, DU-145 cells with stably depleted ABCG4 levels not only significantly delayed the development of the tumor but also greatly sensitized the tumor to a low dose of Dox that resulted in complete tumor regression. Collectively, this data reinforces a novel function of ABCG4 in Dox-mediated chemoresistance, and as a potential therapeutic target in drug-induced PCa chemoresistance.

High Affinity Neutral Bodipy Fluorophores for Mitochondrial Tracking.

We report the first high affinity neutral Bodipy fluorophores for selective imaging of mitochondria with notable sensitivity (∼100 nM) and insignificant cytotoxicity even at very high concentration (∼100 µM), when tested against HeLa cells. Further, these fluorophores are chemically robust and require no special conditions for storage.

Recent trends in electrochemical biosensors of superoxide dismutases.

Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.

Metformin treatment prevents SREBP2-mediated cholesterol uptake and improves lipid homeostasis during oxidative stress-induced atherosclerosis.

Lipids are responsible for the atheromatous plaque formation during atherosclerosis by their deposition in the subendothelial intima of the aorta, leading to infarction. Sterol regulatory element-binding protein 2 (SREBP2), regulating cholesterol homeostasis, is suggested to play a pivotal role during the early incidence of atherosclerosis through dysregulation of lipid homeostasis. Here we demonstrate that oxidative stress stimulates SREBP2-mediated cholesterol uptake via low density lipoprotein receptor (LDLR), rather than cholesterol synthesis, in mouse vascular aortic smooth muscle cells (MOVAS) and THP-1 monocytes. The enhancement of mature form of SREBP2 (SREBP2-M) during oxidative stress was associated with the inhibition of AMP-activated protein kinase (AMPK) activation. In contrast, inhibition of either SREBP2 by fatostatin or LDLR by siLDLR resulted in decreased cholesterol levels during oxidative stress. Thereby confirming the role of SREBP2 in cholesterol regulation via LDLR. Metformin-mediated activation of AMPK was able to significantly abrogate cholesterol uptake by inhibiting SREBP2-M. Interestingly, although metformin administration attenuated angiotensin (Ang)-II-impaired lipid homeostasis in both aorta and liver tissues of ApoE-/- mice, the results indicate that SREBP2 through LDLR regulates lipid homeostasis in aorta but not in liver tissue. Taken together, AMPK activation inhibits oxidative stress-mediated SREBP2-dependent cholesterol uptake, and moreover, metformin-induced prevention of atheromatic events are in part due to its ability to regulate the SREBP2-LDLR axis.

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.

Resveratrol attenuates monocyte-to-macrophage differentiation and associated inflammation via modulation of intracellular GSH homeostasis: Relevance in atherosclerosis.

Monocyte-to-macrophage differentiation promotes an inflammatory environment within the arterial vessel wall that causes a mal-adaptive immune response, which contributes to the progression of atheromatous plaque formation. In the current study, we show that resveratrol, a well-known antioxidant, dose-dependently attenuated phorbol myristate acetate (PMA)-induced monocyte-to-macrophage differentiation, as measured by cell adhesion, increase in cell size, and scavenger receptor expression in THP-1 monocytes. Also, resveratrol significantly inhibited PMA-induced pro-inflammatory cytokine/chemokine and matrix metalloprotease (MMP-9) production. This inhibitory effect of resveratrol on monocyte differentiation results from its ability to restore intracellular glutathione (GSH) status, as resveratrol in the presence of buthionine sulfoximine (BSO) failed to affect monocyte differentiation. Furthermore, PMA-induced monocyte differentiation and inflammation was greatly inhibited when cells were co-treated with N-Acetyl-l-cysteine (NAC), a GSH precursor, while the presence of BSO aggravated these processes. These results also show that resveratrol mediated up-regulation of GSH is due to AMP-activated protein kinase (AMPK)-α activation, as compound C (AMPK inhibitor) treatment drastically depleted intracellular GSH and exacerbated PMA-induced monocyte differentiation and pro-inflammatory cytokine production. More importantly, chronic administration of resveratrol efficiently prevented monocyte infiltration and markedly diminished angiotensin (Ang)-II-induced atheromatous plaque formation in apolipoprotein-E knockout (ApoE(-/-)) mice. We conclude that, intracellular GSH status plays a critical role in regulating monocyte-to-macrophage differentiation and inflammation and resveratrol, by restoring GSH levels, inhibits these processes. Taken together, these results suggest that resveratrol can attenuate atherosclerosis, at least, in part, by inhibiting monocyte differentiation and pro-inflammatory cytokines production.

Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis.

Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE(-/-) mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE(-/-) mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects.