Exosomal MALAT1 from macrophages treated with high levels of glucose upregulates LC3B expression via miR-204-5p downregulation.

BACKGROUND: Metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) plays a critical role in the pathophysiology of diabetes-related complications. However, whether macrophage-derived MALAT1 affects autophagic activity under hyperglycemic conditions is unclear. Therefore, we investigated the molecular regulatory mechanisms of macrophage-derived MALAT1 and autophagy under hyperglycemic conditions. METHODS: Hyperglycemia was induced by culturing macrophages in 25 mM glucose for 1 hour. Exosomes were extracted from the culture media. A rat model of carotid artery balloon injury was established to assess the effect of MALAT1 on vascular injury. Reverse transcription, real-time quantitative polymerase chain reaction, western blotting, immunohistochemical staining, and luciferase activity assays were performed. RESULTS: Stimulation with high levels of glucose significantly enhanced MALAT1 expression in macrophage-derived exosomes. MALAT1 inhibited miR-204-5p expression in macrophage-derived exosomes under hyperglycemic conditions. siRNA-induced silencing of MALAT1 significantly reversed macrophage-derived exosome-induced miR-204-5p expression. Hyperglycemic treatment caused a significant, exosome-induced increase in the expression of the autophagy marker LC3B in macrophages. Silencing MALAT1 and overexpression of miR-204-5p significantly decreased LC3B expression induced by macrophage-derived exosomes. Overexpression of miR-204-5p significantly reduced LC3B luciferase activity induced by macrophage-derived exosomes. Balloon injury to the carotid artery in rats significantly enhanced MALAT1 and LC3B expression, and significantly reduced miR-204-5p expression in carotid artery tissue. Silencing MALAT1 significantly reversed miR-204-5p expression in carotid artery tissue after balloon injury. MALAT1 silencing or miR-204-5p overexpression significantly reduced LC3B expression after balloon injury. CONCLUSION: This study demonstrated that hyperglycemia upregulates MALAT1 . MALAT1 suppresses miR-204-5p expression and counteracts the inhibitory effect of miR-204-5p on LC3B expression in macrophages to promote vascular disease.

Exosomal MALAT1 Derived from High Glucose-Treated Macrophages Up-Regulates Resistin Expression via miR-150-5p Downregulation.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in the pathophysiological process associated with diabetes-related complications. The effect of high glucose levels on macrophage-derived exosomal MALAT1 is unknown. Therefore, we investigated the molecular regulatory mechanisms controlling exosomal MALAT1 in macrophages under high glucose treatment and the therapeutic target of macrophage-derived exosomal MALAT1 using a balloon injury model of vascular disease in diabetic rats. High glucose (25 mM) significantly increased MALAT1 expression in macrophage-derived exosomes. MALAT1 suppressed miR-150-5p expression in macrophage-derived exosomes under high-glucose conditions. Silencing MALAT1 using MALAT1 siRNA significantly reversed miR-150-5p expression induced by macrophage-derived exosomes. Macrophage-derived exosomes under high-glucose treatment significantly increased resistin expression in macrophages. Silencing MALAT1 and overexpression of miR-150-5p significantly decreased resistin expression induced by macrophage-derived exosomes. Overexpression of miR-150-5p significantly decreased resistin luciferase activity induced by macrophage-derived exosomes. Macrophage-derived exosome significantly decreased glucose uptake in macrophages and silencing MALAT1, resistin or overexpression of miR-150-5p significantly reversed glucose uptake. Balloon injury to the carotid artery significantly increased MALAT1 and resistin expression and significantly decreased miR-150-5p expression in arterial tissue. Silencing MALAT1 significantly reversed miR-150-5p expression in arterial tissue after balloon injury. Silencing MALAT1 or overexpression of miR-150-5p significantly reduced resistin expression after balloon injury. In conclusion, high glucose up-regulates MALAT1 to suppress miR-150-5p expression and counteracts the inhibitory effect of miR-150-5p on resistin expression in macrophages to promote vascular disease. Macrophage-derived exosomes containing MALAT1 may serve as a novel cell-free approach for the treatment of vascular disease in diabetes mellitus.

Chrysin boosts KLF2 expression through suppression of endothelial cell-derived exosomal microRNA-92a in the model of atheroprotection.

PURPOSE: Atherosclerosis and its related clinical complications are the leading cause of death. MicroRNA (miR)-92a in the inflammatory endothelial dysfunction leads to atherosclerosis. Krüppel-like factor 2 (KLF2) is required for vascular integrity and endothelial function maintenance. Flavonoids possess many biological properties. This study investigated the vascular protective effects of chrysin in balloon-injured carotid arteries. MATERIALS AND METHODS: Exosomes were extracted from human coronary artery endothelial cell (HCAEC) culture media. Herb flavonoids and chrysin were the treatments in these atheroprotective models. Western blotting and real-time PCRs were performed. In situ hybridization, immunohistochemistry, and immunofluorescence analyses were employed. RESULTS: MiR-92a increased after balloon injury and was present in HCAEC culture media. Chrysin was treated, and significantly attenuated the miR-92a levels after balloon injury, and similar results were obtained in HCAEC cultures in vitro. Balloon injury-induced miR-92a expression, and attenuated KLF2 expression. Chrysin increased the KLF2 but reduced exosomal miR-92a secretion. The addition of chrysin and antagomir-92a, neointimal formation was reduced by 44.8 and 49.0% compared with balloon injury after 14 days, respectively. CONCLUSION: Chrysin upregulated KLF2 expression in atheroprotection and attenuated endothelial cell-derived miR-92a-containing exosomes. The suppressive effect of miR-92a suggests that chrysin plays an atheroprotective role. Proposed pathway for human coronary artery endothelial cell (HCAEC)-derived exosomes induced by chrysin to suppress microRNA (miR)-92a expression and counteract the inhibitory effect of miR-92a on KLF2 expression in HCAECs. This provides an outline of the critical role of the herbal flavonoid chrysin, which may serve as a valuable therapeutic supplement for atheroprotection.

Cyclic stretching boosts microRNA-499 to regulate Bcl-2 via microRNA-208a in atrial fibroblasts.

MicroRNAs that modulate transcription can regulate other microRNAs and are also up-regulated under pathological stress. MicroRNA-499 (miR-499), microRNA-208a (miR-208a) and B-cell lymphoma 2 (Bcl-2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl-2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts-adult atrial (HCF-aa) was investigated. Using negative pressure, HCF-aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up-regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl-2 was up-regulated by longer stretching times. Pre-treatment with antagomir-499 reversed the miR-208a down-regulation, whereas Bcl-2 expression could be suppressed by miR-208a overexpression. In the HCF-aa under stretching for 1 h, miR-499 overexpression decreased pri-miR-208a luciferase activity; this inhibition of pri-miR-208a luciferase activity with stretching was reversed when the miR-499-5p binding site in pri-miR-208a was mutated. The addition of antagomir-208a reversed the Bcl-2-3'UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre-treatment with miR-499 or antagomir-208a inhibited cellular apoptosis in stretched HCF-aa. In hearts with volume overload, miR-499 overexpression inhibited myocardial miR-208a expression, whereas Bcl-2 expression could be suppressed by the addition of miR-208a. In conclusion, miR-208a mediated the regulation of miR-499 on Bcl-2 expression in stretched HCF-aa and hearts with volume overload.

Hyperbaric oxygen-induced long non-coding RNA MALAT1 exosomes suppress MicroRNA-92a expression in a rat model of acute myocardial infarction.

Hyperbaric oxygen (HBO) improves angiogenesis. The effect of HBO on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a pro-angiogenic long non-coding RNA, in cardiac myocyte-derived exosomes and acute myocardial infarction (AMI) is unknown. We aimed to investigate whether MALAT1 is altered in cardiac myocyte-derived exosomes in response to HBO as well as the molecular regulatory mechanisms of MALAT1 in cardiac myocytes treated with HBO. Cardiac myocytes were cultured, and HBO was applied at 2.5 atmosphere absolute in a hyperbaric chamber. Exosomes were extracted from the culture media. A rat model of AMI generated by the ligation of the left anterior descending artery was used. HBO significantly increased MALAT1 expression in cardiac myocytes and HBO-induced MALAT1 and exosomes attenuated miR-92a expression after myocardial infarction. Expression of krüppel-like factor 2 (KLF2) and CD31 was significantly decreased after infarction and HBO-induced exosomes significantly reversed the expression. Silencing of MALAT1 using MALAT1-locked nucleic acid GapmeR significantly attenuated KLF2 and CD31 protein expression after infarction induced by HBO-induced exosomes. HBO-induced exosomes also decreased infarct size significantly. HBO-induced exosomes from cardiac myocytes up-regulate MALAT1 to suppress miR-92a expression and counteract the inhibitory effect of miR-92a on KLF2 and CD31 expression in left ventricular myocardium after myocardial infarction to enhance neovascularization.

(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/ß-catenin.

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/ß-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and ß-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and ß-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/ß-catenin pathways.

Catalpol Ameliorates Neointimal Hyperplasia in Diabetic Rats.

Catalpol, an iridoid glycoside, is an isolated natural product of Rehmannia glutinosa, which has been reported to have antidiabetic properties. This study investigated the vascular protective effects of catalpol in hyperglycemic rats with balloon-injured carotid arteries. Balloon injury stress led to the upregulation of monocyte chemoattractant protein-1 expression in rats with streptozotocin-induced diabetes. Western blotting and real-time PCR were performed. In situ hybridization, immunohistochemistry, and confocal analyses were employed. Monocyte chemoattractant protein-1 levels were increased through streptozotocin induction or balloon injury. After treatment with catalpol, the neointimal hyperplasia area was reduced 2 weeks after balloon injury in hyperglycemic rats. Real-time PCR and immunohistochemical analysis demonstrated reduced levels of monocyte chemoattractant protein-1 2 weeks after the balloon injury. Monocyte chemoattractant protein-1 expression was significantly increased in balloon-injured rats compared with the control groups. Thus, treatment with catalpol affected monocyte chemoattractant protein-1 expression. This study demonstrated that catalpol downregulated monocyte chemoattractant protein-1 expression in carotid arteries and ameliorated neointimal hyperplasia in hyperglycemic rats. The suppressive effect of monocyte chemoattractant protein-1 suggests that it plays a key role in neointimal hyperplasia. The results imply that catalpol is potentially effective for preventing hyperglycemia-related ischemic cardiac diseases.

Hyperbaric oxygen boosts long noncoding RNA MALAT1 exosome secretion to suppress microRNA-92a expression in therapeutic angiogenesis.

BACKGROUND: Hyperbaric oxygen (HBO) could improve wound healing by enhancement of angiogenesis. The effect of HBO on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a proangiogenic long noncoding RNA, and on endothelial cell-derived exosome is unknown. We aim to investigate both whether MALAT1 is altered in human coronary artery endothelial cells (HCAECs)-derived exosomes in response to HBO as well as the molecular regulatory mechanisms of MALAT1 in HCAECs under HBO treatment. METHODS AND RESULTS: HCAECs were cultured and HBO was applied at 2.5 atmosphere absolute (ATA) in a hyperbaric chamber. Exosomes were extracted from culture media. A rat model of hind-limb ischemia was performed by ligation of the right femoral artery. HBO at 2.5 ATA significantly increased MALAT1 expression in HCAECs and HCAECs-derived exosomes. MALAT1 suppressed miR-92a expression in HCAEC-derived exosomes under HBO. Silencing MALAT1 by MALAT1 siRNA significantly inhibited KLF2 mRNA expression induced by HBO, as did MiR-92a. MiR-92a significantly decreased KLF2 luciferase activity in HCAECs under HBO. HBO and HBO-induced exosomes significantly increased cell proliferation and the capillary-like network formation of HCAECs. MALAT1 siRNA and miR-92a overexpression significantly attenuated the cell proliferation and tube formation caused by HBO-induced exosome. HBO and HBO-induced exosomes significantly improved neovascularization in a rat model of hind-limb ischemia. CONCLUSIONS: HBO upregulates MALAT1 to suppress miR-92a expression and counteracts the inhibitory effect of miR-92a on KLF2 expression in HCAECs to enhance neovascularization. HBO-induced derivation of exosomes from HCAECs enhances angiogenesis. Exosomes containing MALAT1 might serve as a valuable therapeutic tool for neovascularization by HBO.

MicroRNA-145 regulates disabled-2 and Wnt3a expression in cardiomyocytes under hyperglycaemia.

AIMS: MicroRNA-145 (miR-145) could protect cardiomyocyte apoptosis against oxidative stress and repair infarcted myocardium. Angiotensin II (Ang II), a pro-inflammatory cytokine could modulate myocardial remodelling. However, the role of hyperglycaemia on miR-145 expression in cardiomyocyte or diabetes is not known. The effect of Ang II on miR-145 expression under hyperglycaemia in cardiomyocytes remains unknown. We sought to investigate the effect of hyperglycaemia and Ang II on miR-145 expression in cardiomyocytes. METHODS: Rat cardiomyocytes were cultured under high glucose concentration (25 mmol/L), and streptozotocin-induced diabetic rats were established. TaqMan® MicroRNA real-time quantitative assay was used to quantitate miR-145. RESULTS: Sustained high glucose concentration (hyperglycaemia) significantly decreased miR-145 expression in cardiomyocytes. Hyperglycaemia significantly increased Ang II mRNA expression and secretion from rat cardiomyocytes. Ang II suppressed miR-145 expression in cardiomyocytes. Hyperglycaemia increased Dab2 and decreased Wnt3a/ß-catenin expression in cardiomyocytes. Repression of miR-145 expression by Ang II resulted in increased Dab2 and decreased Wnt3a and ß-catenin expression under hyperglycaemia. In contrast, overexpression of miR-145 significantly decreased Dab2 mRNA and protein expression, whereas the mRNA and protein levels for Wnt3a and ß-catenin were significantly reduced in left ventricular myocardium from 5 days to 28 days in diabetic rats. The protein expression patterns of Dab2 and Wnt3a/ß-catenin in left ventricular myocardium of diabetic rats could be reversed upon treatment with valsartan. CONCLUSIONS: Ang II downregulates miR-145 to regulate Dab2 and Wnt3a/ß-catenin expression in cardiomyocytes under high glucose concentration. Ang II plays a critical role in the regulation of miR-145 in cardiomyocytes under hyperglycaemic conditions.

Effects of flavonoids on MicroRNA 145 regulation through Klf4 and myocardin in neointimal formation in vitro and in vivo.

MicroRNA 145 (miR-145) is a critical modulator of vascular smooth muscle cell (VSMC) phenotyping and proliferation. Flavonoids have been studied extensively due to their diverse pharmacological properties, including anti-inflammatory effects. The aims of this study is designed to evaluate the atheroprotective effects on angiotensin II (Ang II)-induced miR-145 and Klf4/myocardin expression in vitro and in vivo of flavonoids, including (-)-epigallocatechin gallate (EGCG), chrysin, wogonin, silibinin, and ferulic acid. Ang II significantly reduced the miR-145 compared with the control VSMC groups; all the tested flavonoids increased miR-145 in the 100 nM concentration. Among the test compounds, EGCG showed the strongest augmenting effect on miR-145 and myocardin, however, it also abolished Ang II-induced Klf4. A [3H]-thymidine incorporation proliferation assay demonstrated that EGCG inhibited Ang II-induced VSMC proliferation, and Klf4 siRNA presented with the similar results. Immunohistochemical analysis and confocal microscopy demonstrated increased Klf4 expression and the arterial lumen was narrowed after balloon injury 14 days. With the addition of EGCG (50 mg/kg) and Klf4 siRNA, neointimal formation was reduced by 40.7% and 50.5% compared with balloon injury 14 days; Klf4 expression also was attenuated. This study demonstrated EGCG increased miR-145 and attenuated Klf4, and ameliorated neointimal formation in vitro and in vivo. The novel suppressive effect was mediated through the miR-145 and Klf4/myocardin pathways.

Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor in pressure-overloaded rat heart.

The use of beta-blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1alpha (HIF-1alpha) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1alpha in cardiac hypertrophy due to pressure overload and after treatment with beta-blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1alpha expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 +/- 9 mmHg in the sham group to 94 +/-5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1alpha, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor-beta (NGF-beta) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1alpha and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1alpha, VEGF and BNP increased and mRNA of NGF-beta decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1alpha, VEGF, BNP, and NGF-beta to the baseline values. Increased immunohistochemical labeling of HIF-1alpha, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1alpha, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF-beta mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1alpha, VEGF, BNP, and NGF-beta in the hypertrophic myocardium.