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Introduction: The role of cardiac microvascular endothelial cells (CMECs) in diabetic cardiomyopathy is not fully understood. We aimed to investigate whether a glucagon receptor (GCGR) monoclonal antibody (mAb) ameliorated diabetic cardiomyopathy and clarify whether and how CMECs participated in the process. Research Design and Methods: The db/db mice were treated with GCGR mAb or immunoglobulin G (as control) for 4 weeks. Echocardiography was performed to evaluate cardiac function. Immunofluorescent staining was used to determine microvascular density. The proteomic signature in isolated primary CMECs was analyzed by using tandem mass tag‐based quantitative proteomic analysis. Some target proteins were verified by using western blot. Results: Compared with db/m mice, cardiac microvascular density and left ventricular diastolic function were significantly reduced in db/db mice, and this reduction was attenuated by GCGR mAb treatment. A total of 199 differentially expressed proteins were upregulated in db/db mice versus db/m mice and downregulated in GCGR mAb‐treated db/db mice versus db/db mice. The enrichment analysis demonstrated that fatty acid β‐oxidation and mitochondrial fusion were the key pathways. The changes of the related proteins carnitine palmitoyltransferase 1B, optic atrophy type 1, and mitofusin‐1 were further verified by using western blot. The levels of these three proteins were upregulated in db/db mice, whereas this upregulation was attenuated by GCGR mAb treatment. Conclusion: GCGR antagonism has a protective effect on CMECs and cardiac diastolic function in diabetic mice, and this beneficial effect may be mediated via inhibiting fatty acid β‐oxidation and mitochondrial fusion in CMECs. [ABSTRACT FROM AUTHOR]

Diosmetin‐7‐O‐β‐D‐glucopyranoside (Diosmetin‐7‐O‐glucoside) is a natural flavonoid glycoside known to have a therapeutic application for cardiovascular diseases. Cardiac fibrosis is the main pathological change in the end stage of cardiovascular diseases. Endothelial‐mesenchymal transformation (EndMT) induced by endoplasmic reticulum stress (ER stress) via Src pathways is involved in the process of cardiac fibrosis. However, it is unclear whether and how diosmetin‐7‐O‐glucoside regulates EndMT and ER stress to treat cardiac fibrosis. In this study, molecular docking results showed that diosmetin‐7‐O‐glucoside bound well to ER stress and Src pathway markers. Diosmetin‐7‐O‐glucoside suppressed cardiac fibrosis induced by isoprenaline (ISO) and reduced the levels of EndMT, ER stress in mice heart. Primary cardiac microvascular endothelial cells (CMECs) were induced by transforming growth factor‐β1 (TGF‐β1) to perform EndMT. Diosmetin‐7‐O‐glucoside could effectively regulate EndMT and diminish the accumulation of collagen I and collagen III. We also showed that the tube formation in CMECs was restored, and the capacity of migration was partially inhibited. Diosmetin‐7‐O‐glucoside also ameliorated ER stress through the three unfolded protein response branches, as evidenced by organelle structure in transmission electron microscopy images and the expression of protein biomarkers like the glucose‐regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). Further analysis showed that diosmetin‐7‐O‐glucoside could suppress the expression level of Src phosphorylation, then block EndMT with the maintenance of endothelial appearance and endothelial marker expression. These results suggested that the diosmetin‐7‐O‐glucoside can regulate EndMT through ER stress, at least in part via Src‐dependent pathways. [ABSTRACT FROM AUTHOR]

To explore the impact and mechanism of human mesenchymal stem cells (hMSCs) on the angiogenesis of cardiac microvascular endothelial cells (CMECs) after ischemia insult. Exosomes derived from hMSCs (hMSCs-Exo) were identified by Western blotting and labeled by PHK-67. CMECs were isolated from rat myocardial tissues. After hypoxic treatment, CMECs were cultured with hMSCs and exosome inhibitor (GW4869) or transfected with si-COL4A1 + miR-543 inhibitor. CMEC proliferation, migration, invasion, and angiogenesis were examined. Target genes of miR-543 were predicted and then were identified by dual luciferase assay. Myocardial infarction (MI) rat model established by suture occlusion was intravenously injected with hMSCs-Exo. Fluorescence microscope was applied to visualize exosomes in myocardial tissues. Infarction volume and pathologies of myocardial tissues were observed. Ki-67 and miR-543 expressions were detected. The isolated hMSC-Exo expressed TSG101, HSP70, and CD63. Hypoxia-treated CMECs cultured with hMSCs exhibited high proliferation, migration, invasion, and angiogenesis ability, while incubation with exosome inhibitor GW4969 offset the promoting effects of hMSCs on the proliferation, migration, invasion, and angiogenesis of CMECs. hMSCs transfected with miR-543 inhibitor brought CMECs weak viability and angiogenesis ability. CMECs transfected with si-COL4A1 and miR-543 inhibitor showed low proliferation, migration, invasion, and angiogenesis compared to those transfected with si-COL4A1 alone. hMSCs-Exo entered the myocardial tissues of MI rats. Injection of hMSCs-Exo in MI rats diminished infarction size, attenuated MI-induced injuries, and increased Ki-67 expression. hMSCs-Exo facilitates the proliferation, migration, invasion, and angiogenesis of CMECs through transferring miR-543 and downregulating COL4A1 expression. [ABSTRACT FROM AUTHOR]

This study is performed to examine the impacts of microRNA‐218 (miR‐218) on cardiac microvascular endothelial cells (CMECs) injury induced by coronary artery disease (CAD). Reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR) was applied for detecting miR‐218 expression in serum of patients with CAD and healthy controls, and the correlation between miR‐218 expression and the clinical indexes such as creatine kinase, creatine kinase‐myocardial band, cardiac troponin I, and coronary Gensini score was analyzed. CMECs were coincubated with homocysteine for 24 hr for CMECs injury, and the cells were transfected with miR‐218 mimics or miR‐218 inhibitors. Besides, we used oxidized low density lipoprotein as an inducer to incubate with CMECs for 24 hr, and the model of CMECs injury was established to be transfected with miR‐218 mimics. RT‐qPCR and western blot analysis were used to detect miR‐218 and HMGB1 expression in CMECs. A series of experiments were used to determine cell proliferation, apoptosis, migration, and angiogenesis ability of CMECs. Vascular endothelial growth factor expression and inflammatory factor contents were measured. The obtained results suggested that miR‐218 expression in peripheral blood of patients with CAD descended substantially versus that of healthy controls. Low miR‐218 expression was found in CAD‐induced CMECs injury. Overexpressed miR‐218 promoted the proliferation, migration, angiogenesis ability, induced apoptosis, and alleviated the inflammatory injury of CAD‐induced CMECs. miR‐218 may negatively regulate the expression of HMGB1 in CAD. This study demonstrates that upregulation of miR‐218 reduces CMECs injury induced by CAD through the inhibition of HMGB1. [ABSTRACT FROM AUTHOR]

This study is performed to figure out the role of long‐chain noncoding RNA growth‐arrest specific transcript 5 (GAS5) in homocysteine (HCY)‐induced cardiac microvascular endothelial cells (CMECs) injury. CMECs were cultured and the model of CMECs injury was established by coincubation with HCY. To construct stable overexpression of GAS5 cells, the expression of GAS5, microRNA‐33a‐5p (miR‐33a‐5p) and ATP‐binding cassette transporter A1 (ABCA1), and biological characteristics of cells were determined. The messenger RNA (mRNA) level and secretion of vascular endothelial growth factor (VEGF), activity of reactive oxygen species (ROS) and superoxide dismutase (SOD), and the content of malondialdehyde (MDA) were measured. The binding site between GAS5 and miR‐33a‐5p and between miR‐33a‐5p and ABCA1 was verified. CMECs were successfully cultured. Reduction of GAS5 expression and ABCA1 expression together with increased expression of miR‐33a‐5p was found in CMECs induced by HCY. After overexpression of GAS5, there showed increased proliferative activity, decreased cell apoptosis rate and apoptosis index, enhanced cell migration ability, increased number of lumen formation, increased mRNA expression of VEGF in cells and the secretion in the supernatant, decreased activity of ROS and SOD in cells, and decreased content of ROS in cells. miR‐33a‐5p could promote the enrichment of GAS5 and ABCA1 was the direct target gene of miR‐33a‐5p. Our study suggests that the low expression of GAS5 was observed in HCY‐induced CMECs injury, and the upregulation of GAS5 could attenuate HCY‐induced CMECs injury by mediating oxidative stress, and its mechanism is related to the upregulation of ABCA1 expression by competitively binding with miR‐33a‐5p. [ABSTRACT FROM AUTHOR]

Abstract: Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO2, 1% O2, 94% N2) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression. [ABSTRACT FROM AUTHOR]

Extracellular Ub is an immune modulator that plays a role in suppression of inflammation, organ injury, myocyte apoptosis, and fibrosis. The purpose of this study was to investigate the effects of extracellular Ub on the process of cardiac angiogenesis. CMECs and aortic tissue were isolated from rats to measure changes in angiogenic protein levels and to assess angiogenic responses to extracellular Ub. In CMECs, extracellular Ub increased protein levels of VEGF-A and MMP-2, known angiogenesis regulators. CMECs demonstrated enhanced rearrangement of fibrillar actin and migration in response to Ub treatment. Ub-treated CMECs demonstrated an increase in tube network formation which was inhibited by the CXCR4 receptor antagonist, AMD3100. Methylated Ub, unable to form polyubiquitin chains, enhanced tube network formation. Aortic ring sprouting assays demonstrated that Ub increases microvessel sprouting in the Matrigel. The results of our study suggest a novel role for extracellular Ub in cardiac angiogenesis, providing evidence that extracellular Ub, at least in part acting via the CXCR4 receptor, has the potential to facilitate the process of angiogenesis in myocardial endothelial cells. [ABSTRACT FROM AUTHOR]

This study indicates that brain-derived neurotrophic factor ( BDNF) can promote young cardiac microvascular endothelial cells ( CMECs) to migrate via the activation of the BDNF- TrkB- FL- PI3K/Akt pathway, which may benefit angiogenesis after myocardial infarction ( MI). However, the ageing of CMECs led to changes in the expression of receptor Trk isoforms in that among the three isoforms ( TrkB- FL, TrkB- T1 and TrkB- T2), only one of its truncated isoforms, TrkB- T1, continued to be expressed, which leads to the dysfunction of its ligand, a decrease in the migration of CMECs and increased injury in ageing hearts. This shift in receptor isoforms in aged CMECs, together with changes in the ageing microenvironment, might predispose ageing hearts to decreased angiogenic potential and increased cardiac pathology. [ABSTRACT FROM AUTHOR]