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.

Singuru G ，Pulipaka S ，Shaikh A ，Sahoo S ，Jangam A ，Thennati R ，Kotamraju S ... -  《-》

SGLT2 inhibitor downregulates ANGPTL4 to mitigate pathological aging of cardiomyocytes induced by type 2 diabetes.

Senescence is recognized as a principal risk factor for cardiovascular diseases, with a significant association between the senescence of cardiomyocytes and inferior cardiac function. Furthermore, type 2 diabetes exacerbates this aging process. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) has well-established cardiovascular benefits and, in recent years, has been posited to possess anti-aging properties. However, there are no reported data on their improvement of cardiomyocytes function through the alleviation of aging. Consequently, our study aims to investigate the mechanism by which SGLT2i exerts anti-aging and protective effects at the cardiac level through its action on the FOXO1-ANGPTL4 pathway. To elucidate the underlying functions and mechanisms, we established both in vivo and in vitro disease models, utilizing mice with diabetic cardiomyopathy (DCM) induced by type 2 diabetes mellitus (T2DM) through high-fat diet combined with streptozotocin (STZ) administration, and AC16 human cardiomyocyte cell subjected to stimulation with high glucose (HG) and palmitic acid (PA). These models were employed to assess the changes in the senescence phenotype of cardiomyocytes and cardiac function following treatment with SGLT2i. Concurrently, we identified ANGPTL4, a key factor contributing to senescence in DCM, using RNA sequencing (RNA-seq) technology and bioinformatics methods. We further clarified ANGPTL4 role in promoting pathological aging of cardiomyocytes induced by hyperglycemia and hyperlipidemia through knockdown and overexpression of the factor, as well as analyzed the impact of SGLT2i intervention on ANGPTL4 expression. Additionally, we utilized chromatin immunoprecipitation followed by quantitative real-time PCR (ChIP-qPCR) to confirm that FOXO1 is essential for the transcriptional activation of ANGPTL4. The therapeutic intervention with SGLT2i alleviated the senescence phenotype in cardiomyocytes of the DCM mouse model constructed by high-fat feeding combined with STZ, as well as in the AC16 model stimulated by HG and PA, while also improving cardiac function in DCM mice. We observed that the knockdown of ANGPTL4, a key senescence-promoting factor in DCM identified through RNA-seq technology and bioinformatics, mitigated the senescence of cardiomyocytes, whereas overexpression of ANGPTL4 exacerbated it. Moreover, SGLT2i improved the senescence phenotype by suppressing the overexpression of ANGPTL4. In fact, we discovered that SGLT2i exert their effects by regulating the upstream transcription factor FOXO1 of ANGPTL4. Under conditions of hyperglycemia and hyperlipidemia, compared to the control group without FOXO1, the overexpression of FOXO1 in conjunction with SGLT2i intervention significantly reduced both ANGPTL4 mRNA and protein levels. This suggests that the FOXO1-ANGPTL4 axis may be a potential target for the cardioprotective effects of SGLT2i. Collectively, our study demonstrates that SGLT2i ameliorate the pathological aging of cardiomyocytes induced by a high glucose and high fat metabolic milieu by regulating the interaction between FOXO1 and ANGPTL4, thereby suppressing the transcriptional synthesis of the latter, and consequently restoring cardiac function.

Wen Y ，Zhang X ，Liu H ，Ye H ，Wang R ，Ma C ，Duo T ，Wang J ，Yang X ，Yu M ，Wang Y ，Wu L ，Zhao Y ，Wang L ... -  《Cardiovascular Diabetology》

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-β-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.

Singuru G ，Pulipaka S ，Shaikh A ，Balaji Andugulapati S ，Thennati R ，Kotamraju S ... -  《-》

Circulating mitochondria carrying cGAS promote endothelial Secreted group IIA phospholipase A2-mediated neuroinflammation through activating astroglial/microglial Integrin-alphavbeta3 in subfornical organ to augment central sympathetic overdrive in heart

Sympathoexcitation, a manifestation of heart-brain axis dysregulation, contributes to the progression of heart failure (HF). Our recent study revealed that circulating mitochondria (C-Mito), a newly identified mediator of multi-organ communication, promote sympathoexcitation in HF by aggravating endothelial cell (EC)-derived neuroinflammation in the subfornical organ (SFO), the cardiovascular autonomic neural center. The precise molecular mechanism by which C-Mito promotes SFO-induced endothelial neuroinflammation has not been fully elucidated. C-Mito carrying cGAS promote sympathoexcitation by targeting PLA2G2A in ECs of the SFO in HF rats. Male Sprague-Dawley (SD) rats received a subcutaneous injection of isoprenaline (ISO) at a dosage of 5 mg/kg/day for seven consecutive days to establish a HF model. C-Mito were isolated from HF rats and evaluated. The level of cGAS, a dsDNA sensor recently discovered to be directly localized on the outer membrane of mitochondria, was detected in C-Mito. C-Mito from HF rats (C-MitoHF) or control rats (C-MitoCtrl) were intravenously infused into HF rats. The accumulation of C-Mito in the ECs in the SFO was detected via double immunofluorescence staining. The SFO was processed for RNA sequencing (RNA-Seq) analysis. Secreted group IIA phospholipase A2 (PLA2G2A), the key gene involved in C-MitoHF-associated SFO dysfunction, was identified via bioinformatics analysis. Upregulation of PLA2G2A in the SFO ECs was assessed via immunofluorescence staining and immunoblotting, and PLA2G2A activity was evaluated. The interaction between cGAS and PLA2G2A was detected via co-immunoprecipitation. The dowstream molecular mechanisms of which PLA2G2A induced astroglial/microglial activation were also investigated. AAV9-TIE-shRNA (PLA2G2A) was introduced into the SFO to specifically knockdown endothelial PLA2G2A. Neuronal activation and glial proinflammatory polarization in the SFO were also evaluated. Renal sympathetic nerve activity (RSNA) was measured to evaluate central sympathetic output. Cardiac sympathetic hyperinnervation, myocardial remodeling, and left ventricular systolic function were assessed in C-Mito-treated HF rats. Respiratory functional incompetence and oxidative damage were observed in C-MitoHF compared with C-MitoCtrl. Surprisingly, cGAS protein levels in C-MitoHF were significantly higher than those in C-MitoCtrl, while blocking cGAS with its specific inhibitor, RU.521, mitigated respiratory dysfunction and oxidative injury in C-MitoHF. C-Mito entered the ECs of the SFO in HF rats. RNA sequencing revealed that PLA2G2A is a key molecule for the induction of SFO dysfunction by C-MitoHF. The immunoblotting and immunofluorescence results confirmed that, compared with C-MitoCtrl, C-MitoHF increased endothelial PLA2G2A expression in the SFO of HF rats, which could be alleviated by attenuating C-MitoHF-localized cGAS. Furthermore, we found that cGAS directly interacts with PLA2G2A, increased the activity of PLA2AG2, which produced arachidonic acid, and also promoted PLA2G2A secretion in brain ECs. In addition, the inhibition of PLA2G2A in brain ECs significantly mitigated the proinflammatory effect of conditioned cell culture medium from C-MitoHF-treated ECs on astroglia and microglia. Also, we found that PLA2G2A secreted from ECs insulted by C-Mito induced neuroinflammation through activating astriglial/microglial Integrin-alphavbeta3 in the SFO, which further promote central sympathetic overdrive in HF rats. Specific knockdown of endothelial PLA2G2A in the SFO mitigated C-MitoHF-induced presympathetic neuronal sensitization, cardiac sympathetic hyperinnervation, RSNA activation, myocardial remodeling, and systolic dysfunction in HF rats. C-Mito carrying cGAS promoted cardiac sympathoexcitation by directly targeting PLA2G2A in the ECs of the SFO in HF rats. Secreted PLA2G2A derived from ECs insulted by C-Mito induced neuroinflammation through activating astriglial/microglial Integrin-alphavbeta3 in the SFO, which further promote central sympathetic overdrive in HF rats. Our study indicated that inhibiting cGAS in C-Mito might be a potential treatment for central sympathetic overdrive in HF.

Zhang S ，Huang Y ，Han C ，Chen M ，Yang Z ，Wang C ... -  《-》

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.

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. 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. 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. 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.

Karnewar S ，Pulipaka S ，Katta S ，Panuganti D ，Neeli PK ，Thennati R ，Jerald MK ，Kotamraju S ... -  《-》