Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission

Glucagon-Like Peptide-1 Protects Against Cardiac Microvascular Injury in Diabetes via a cAMP/PKA/Rho-Dependent Mechanism

Cardiac microvascular injury often occurs in patients with type 2 diabetes mellitus (T2DM) who develop hyperglycemia and hyperlipidemia. However, besides reported contradictory roles in cardiac diseases, the function of TRPV1 (transient receptor potential vanilloid 1) in cardiac microvessels is not well defined. This study was performed to determine the detailed role of TRPV1 in cardiac microvascular endothelial cells (CMECs) in T2DM. T2DM mice were established by multiple injections of low-dose streptozotocin and high-fat feeding. CMECs were cultured separately in mediums of normal glucose, high glucose (HG), high fatty acid (HF), and HG plus HF (HG-HF). HG-HF inhibited TRPV1 expression in CMECs, reducing cellular Ca2+ content ([Ca2+]i). T2DM impaired cardiac function, disturbed glucose uptake, and damaged microvascular barrier, which were further aggravated by TRPV1-/- Exposure to HG-HF, particularly in TRPV1-/- CMECs, led to a higher level of apoptosis and a lower level of nitric oxide production in viable CMECs. HG-HF markedly enhanced generation of reactive oxygen species and nitrotyrosine, especially in the absence of TRPV1. H2O2 administration reduced TRPV1 expression in CMECs. HG-HF significantly depressed expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) and OPA1 (optic atrophy 1) by reducing [Ca2+]i, whereas OPA1 supplementation partly reversed those detrimental effects induced by TRPV1-/- Furthermore, capsaicin treatment not only attenuated CMECs injury induced by HG-HF but also mitigated cardiac microvascular injury induced by T2DM. Collectively, T2DM leads to cardiac microvascular injury by exacerbating the vicious circle of TRPV1 blockage and reactive oxygen species overload. Long-term capsaicin can protect cardiac microvessels against T2DM via suppressing oxidative/nitrative stress mediated by TRPV1/Ca2+/PGC-1α/OPA1 pathway in CMECs.

Positive regulation of endothelial Tom70 by metformin as a new mechanism against cardiac microvascular injury in diabetes

ObjectivesGlucagon-like peptide-1 (GLP-1) was a hormone predominately synthesised and secreted by intestinal L-cells. Pharmacological modulation of the GLP-1 had emerged as an important treatment target for diabetes mellitus. In addition...

Cardiac microvascular endothelial cells (EC) play an important role in the physiological regulation of coronary blood flow, but their function has not been rigorously examined, because suitable in vitro models have not been available. Cardiac macrovascular and microvascular EC were isolated and cultured from 14-16-week-old Sprague-Dawley rats to examine the pharmacological responses of carbachol-induced nitric oxide (NO) production using a Griess method. Carbachol-induced NO production was only detected in cardiac macrovascular EC, which suggests that endothelial production of NO differs between macrovascular and microvascular EC. Next, cardiac microvascular EC was treated with either vehicle, angiotensin-converting enzyme (ACE) inhibitor (captopril, 10 micromol/L) or angiotensin II type 1 (AT1) receptor antagonist (CV11974, 10 micromol/L) for 4 days. Carbachol-induced NO production was improved by captopril (136+/-45nmol, p<0.01 vs vehicle) and CV11974 (146+/-30nmol, p<0.01 vs vehicle). Angiotensin II concentration in the culture medium and protein expressions of endothelial nitric oxide synthase and AT1 receptor in the EC were similar among the 3 groups. Interestingly, the level of muscarinic subtype 3 (M3) receptor was significantly increased in the EC treated with captopril (214%, p<0.01) and CV11974 (296%, p<0.01). When cardiac microvascular EC were treated with neomycin (non-selective phospholipase C inhibitor), carbachol-induced NO production was also improved (146+/-35nmol, p<0.01, neomycin I mmol/L) together with increased expression of M3 receptor (p<0.01). These data suggest that the upregulation of the M3 receptor by captopril or CV11974 occurs via a phospholipase C-dependent pathway. Cardiac microvascular EC also produced NO constitutively, as did the macrovascular EC, but carbachol-induced NO production was decreased. The present data suggest that the upregulation of the M3 receptor by the ACE inhibitor and AT1 receptor antagonist is a new beneficial effect of these drugs on microvascular endothelial function.

Cardiovascular complications account for a substantial proportion of morbidity and mortality in diabetic patients. Abnormalities of cardiac microvascular endothelial cells (CMECs) lead to impaired cardiac microvascular vessel integrity and subsequent cardiac dysfunction, underlining the importance of coronary microvascular dysfunction. In this study, experimental diabetes models were constructed using Mst1 transgenic, Mst1 knockout and sirt1 knockout mice. Diabetic Mst1 transgenic mice exhibited impaired cardiac microvessel integrity and decreased cardiac function. Mst1 overexpression deceased CMECs autophagy as evidenced by decreased LC3 expression and enhanced protein aggregation when subjected to high glucose culture. Mst1 knockout improved cardiac microvessel integrity and enhanced cardiac functions in diabetic mice. Mst1 knockdown up-regulated autophagy as indicated by more typical autophagosomes and increased LC3 expression in CMECs subjected to high glucose cultures. Mst1 knockdown also promoted autophagic flux in the presence of bafilomycin A1. Mst1 overexpression increased CMECs apoptosis, whereas Mst1 knockout decreased CMECs apoptosis. Sirt1 knockout abolished the effects of Mst1 overexpression in cardiac microvascular injury and cardiac dysfunction. In conclusion, Mst1 knockout preserved cardiac microvessel integrity and improved cardiac functions in diabetic mice. Mst1 decreased sirt1 activity, inhibited autophagy and enhanced apoptosis in CMECs, thus participating in the pathogenesis of diabetic coronary microvascular dysfunction.

BackgroundMicrovascular complications are associated with an overtly increased risk of adverse outcomes in patients with diabetes including coronary microvascular injury which manifested as disruption of adherens junctions between cardiac microvascular endothelial cells (CMECs). However, particular mechanism leading to diabetic coronary microvascular hyperpermeability remains elusive.MethodsExperimental diabetes was induced in mice with adipose tissue-specific Adipsin overexpression (AdipsinLSL/LSL-Cre) and their respective control (AdipsinLSL/LSL). In addition, cultured CMECs were subjected to high glucose/palmitic acid (HG + PA) treatment to simulate diabetes for a mechanistic approach.ResultsThe results showed that Adipsin overexpression significantly reduced cardiac microvascular permeability, preserved coronary microvascular integrity, and increased coronary microvascular density. Adipsin overexpression also attenuated cardiac dysfunction in diabetic mice. E/A ratio, an indicator of cardiac diastolic function, was improved by Adipsin. Adipsin overexpression retarded left ventricular adverse remodeling, enhanced LVEF, and improved cardiac systolic function. Adipsin-enriched exosomes were taken up by CMECs, inhibited CMECs apoptosis, and increased CMECs proliferation under HG + PA treatment. Adipsin-enriched exosomes also accelerated wound healing, rescued cell migration defects, and promoted tube formation in response to HG + PA challenge. Furthermore, Adipsin-enriched exosomes maintained adherens junctions at endothelial cell borders and reversed endothelial hyperpermeability disrupted by HG + PA insult. Mechanistically, Adipsin blocked HG + PA-induced Src phosphorylation (Tyr416), VE-cadherin phosphorylation (Tyr685 and Tyr731), and VE-cadherin internalization, thus maintaining CMECs adherens junctions integrity. LC-MS/MS analysis and co-immunoprecipitation analysis (Co-IP) unveiled Csk as a direct downstream regulator of Adipsin. Csk knockdown increased Src phosphorylation (Tyr416) and VE-cadherin phosphorylation (Tyr685 and Tyr731), while abolishing Adipsin-induced inhibition of VE-cadherin internalization. Furthermore, Csk knockdown counteracted Adipsin-induced protective effects on endothelial hyperpermeability in vitro and endothelial barrier integrity of coronary microvessels in vivo.ConclusionsTogether, these findings favor the vital role of Adipsin in the regulation of CMECs adherens junctions integrity, revealing its promises as a treatment target against diabetic coronary microvascular dysfunction.Graphical Graphical abstract depicting the mechanisms of action behind Adipsin-induced regulation of diabetic coronary microvascular dysfunction.

Naringin attenuates myocardial ischemia-reperfusion injury by promoting mitochondrial translocation of NDUFS1 and suppressing cardiac microvascular endothelial cell ferroptosis.

Empagliflozin attenuates cardiac microvascular ischemia/reperfusion through activating the AMPKα1/ULK1/FUNDC1/mitophagy pathway

Roles of mitochondrial dynamics and mitophagy in diabetic myocardial microvascular injury.

PurposeMitochondria are highly dynamic organelles that continuously undergo fission and fusion, and their dysfunction is associated with various age-related disorders. This study aimed to elucidate the role of mitochondrial fission in the development of choroidal neovascularization (CNV), a hallmark of neovascular age-related macular degeneration (AMD), and to evaluate the therapeutic potential of its pharmacological inhibition.MethodsThe murine CNV model was created by laser photocoagulation using C57BL/6J mice. Expression changes of mitochondrial fission-related protein during CNV development were examined using western blotting and immunofluorescence. To assess the effectiveness of pharmacological inhibition of mitochondrial fission, the effects of mitochondrial division inhibitor-1 (Mdivi-1) and mitochondrial fusion promoter (M1) were evaluated by CNV area measurement, fluorescein angiography, and western blot analysis. The pro-angiogenic mechanisms associated with mitochondrial fission were further investigated in RPE cells cultured under hypoxic condition.ResultsIn a murine laser-induced CNV model, mitochondrial fission-related proteins increased in the retinal pigment epithelium (RPE)–choroid complex, and the high expression of phosphorylated dynamin-related protein 1 (DRP1) was observed in RPE cells surrounding the CNV lesion. Additionally, intravitreal injection of Mdivi-1 or M1 suppressed CNV formation, vascular leakage, and pro-angiogenic factor production. In RPE cells exposed to hypoxia, DRP1-mediated mitochondrial fission was rapidly activated, accompanied by increased mitochondrial reactive oxygen species production. Moreover, inhibition of mitochondrial fission suppressed mitochondrial bioenergetic dysfunction and the upregulation of vascular endothelial growth factor.ConclusionsThese findings support that pharmacological inhibition of activated mitochondrial fission could serve as a potential therapeutic approach for neovascular AMD.

Cardiac microvascular endothelial cells (CMECs) play a critical role in the physiological regulation of coronary blood flow and its dysfunction is associated with myocardium ischemic injury. This study was performed to clarify the effect of microRNA-128 (miR-128) on the CMEC injury in coronary heart disease (CHD) by binding to insulin receptor substrate 1 (IRS1). The rat CMECs were cultured by explant culture method and identified by CD31 immunofluorescence assay. CMECs were treated with homocysteine (Hcy), which underwent stress of CHD, followed by treatment of miR-128 mimics/inhibitors or IRS1 siRNA. Expression of miR-128, IRS1, and vascular endothelial growth factor (VEGF) was determined. The viability, apoptosis, migration ability, and tube formation ability of CMECs were evaluated. The superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS) of CMECs were evaluated, respectively. In rat CMECs, miR-128 was poorly expressed and IRS1 was highly expressed. Notably, miR-128 targeted and negatively regulated IRS1. Additionally, the treatment with Hcy in CMECs led to reduced viability, migration ability, tube formation, VEGF expression, SOD activity as well as increased cell apoptosis, MDA and ROS levels. The experimental results demonstrated that miR-128 mimics and IRS1 siRNA in rat CMECs promoted viability, migration ability, tube formation, VEGF expression, SOD activity, while repressing cell apoptosis, MDA and ROS levels. MiR-128 inhibitors could reverse the tendencies. Collectively, our study provides evidence that miR-128 targeted and negatively regulated IRS1 expression, whereby the functional injury of CMECs induced by Hcy was ameliorated. Furthermore, protection of miR-128 was stimulated by reducing oxidative stress.

Rnd3 Ameliorates Diabetic Cardiac Microvascular Injury via Facilitating Trim40-Mediated Rock1 Ubiquitination.

Tanshinone IIA attenuates cardiac microvascular ischemia-reperfusion injury via regulating the SIRT1-PGC1α-mitochondrial apoptosis pathway.

Introduction: Impaired cardiac microvascular function contributes greatly to diabetes cardiovascular disease. Glucagon-like peptide-1 (GLP-1) was found to have multiple cardioprotective effects besides its glucose lowering properties. Hypothesis: To investigate the protective effects of GLP-1 on cardiac microvessels injury and the underlying mechanism in diabetic rats. Methods: STZ-induced diabetic rats were randomized to 12 weeks of treatment with vehicle, LAF237 or Exenatide. Cardiac function and energetics was examined by echocardiography and 18 F-FDG PET respectively. Lanthanum nitrate perfusion was applied to assess cardiac microvascular barrier function. Cardiac microvascular endothelial cells (CMECs) were cultured in medium alone or medium containing high glucose, GLP-1, high glucose plus GLP-1. Lucigenin-enhanced chemiluminescence assay and DHE staining were used to assess ROS production. The expression of Rho, ROCK, p47 phox and gp91 phox was examined by Western blot. Results: LAF237 or Exetinade treatment improved cardiac function and energetics as well as microvascular barrier function significantly compared with vehicle groups. Compared with vehicle groups, ROS production was markedly decreased in GLP-1 groups. There were also significant reductions in p47 phox and gp91 phox expression. The cAMP/PKA activity was increased and the Rho expression was decreased in high glucose-induced CMECs after treatment with GLP-1, which reproduced the same effects as PKA inhibitor. Conclusions: GLP-1 could protect the cardiac microvessels against oxidative stress injury and the resultant microvascular barrier dysfunction in diabetic rats, which contribute to the improvement of cardiac function. The protective effects of GLP-1 are dependent on downstream inhibition of Rho, which is through cAMP/PKA pathway, resulting in subsequent decreased production of NADPH oxidase.