Kou-Gi Shyu

Background: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is strongly associated with atrial structural remodeling driven by activated cardiac fibroblasts. Autophagy has been implicated in AF-related atrial remodeling; however, the non-coding RNA mechanisms that govern autophagic activation in atrial fibroblasts under rapid electrical stress remain poorly understood. Methods: Human cardiac fibroblasts from adult atria (HCF-aa) were subjected to rapid electrical stimulation (RES) at 0.5 V/cm and 10 Hz. Expression levels of exosomal metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), cytoplasmic miR-204-5p, and microtubule-associated protein light chain 3B (LC3B) were measured using quantitative real-time PCR and Western blot analyses. Luciferase reporter assays were performed to confirm direct molecular interactions. The functional roles of MALAT1 siRNA, miR-204-5p mimics/antagomirs, rapamycin, and 3-methyladenine (3-MA) on LC3B expression and autophagic activation were assessed by Western blot and immunofluorescence confocal microscopy for LC3B puncta formation. Results: RES significantly induced exosomal MALAT1 expression in a voltage- and time-dependent manner, peaking at 2 h post-stimulation, while cytoplasmic MALAT1 levels remained unchanged. Cytoplasmic miR-204-5p exhibited an initial transient rise followed by a significant decline at 2 h, inversely correlating with peak MALAT1 levels. LC3B mRNA and protein expression subsequently increased, peaking at 6 and 16 h, respectively. Luciferase reporter assays confirmed that miR-204-5p directly binds both the MALAT1 transcript and the 3′-UTR of LC3B mRNA. MALAT1 knockdown augmented miR-204-5p levels and suppressed LC3B expression, while miR-204-5p overexpression attenuated RES-induced LC3B upregulation and LC3B puncta accumulation. Conversely, miR-204-5p inhibition further enhanced autophagic activation, as evidenced by increased LC3B puncta density. Conclusions: In HCF-aa subjected to RES, MALAT1 functions intracellularly as a competing endogenous RNA to putatively sequester miR-204-5p, thereby de-repressing LC3B expression and promoting autophagic activation. Concurrent exosomal secretion of MALAT1 may additionally serve as a paracrine signal to neighboring cells, though this requires future conditioned-media transfer experiments to confirm.

Flavonoid chrysin possesses many biological properties. Autophagy and inflammation interact to cause diabetic cardiovascular diseases. However, whether chrysin affects macrophage autophagy and microRNA 204-5p regulation under hyperglycemic conditions remain unclear. In this study, we examined that the effects of chrysin on the molecular regulatory mechanisms of miR-204-5p in LC3B macrophage autophagy under hyperglycemic stimulation. Macrophages were cultured with 25 mM glucose for hyperglycemia stimulation. A diabetic rat model of carotid artery balloon injury was established. Reverse transcription and real-time quantitative polymerase chain reaction, Western blotting, immunohistochemical staining, and luciferase activity assay were performed. The results indicated that chrysin augmented the macrophage autophagy marker LC3B presentation under high-glucose (HG)-stimulation significantly, however, chrysin attenuated the miR-204-5p level in macrophage cultures. Treatment with chrysin, rapamycin or mutant miR-204-5p increased LC3B presentation through the suppression of miR-204-5p level in macrophage cultures under HG-stimulated condition. By luciferase activity assay, the combination of chrysin with mutant miR-204-5p reduced LC3B binding activity under hyperglycemic stress. Treatment with chrysin (100 mg/kg/day) and wild-type miR-204-5p considerably reduced the neointimal sizes by 46.3% and 36.3%, respectively, at 14 days after carotid artery balloon injury in STZ-induced diabetic rats; ameliorated M1 polarization simultaneously. However, by treating with chrysin suppressed LC3B presentation in diabetic rats. Our findings indicate, under hyperglycemic stress, chrysin regulates the presentation of LC3B autophagy in hyperglycemia-stimulated macrophage cultures and neointimal hyperplasia by mediating the miR-204-5p expression. The results imply that chrysin is potentially effective for preventing hyperglycemia related cardiovascular diseases.

Metastasis-associated lung adenocarcinoma transcript 1(MALAT1) is associated with vascular calcification and diabetes-related complications. However, the effect of exosomal MALAT1 derived from macrophages induced by hyperglycemia on vascular calcification (VC) remains unclear. In this study, we investigated the effect of VC and its regulatory mechanisms in cultured vascular smooth muscle cells (VSMCs) and diabetic rats by exosomal MALAT1 derived from macrophages treated with high levels of glucose. Macrophages and VSMCs were cultured in 25 mM glucose. Macrophages exposed to high glucose exhibited increased expression of exosomal MALAT1. When transferred to VSMCs, exosomal MALAT1 significantly suppressed the expression of miR-143-3p while upregulating Matrix Gla protein (MGP, an inhibitor of VC) mRNA and protein levels. Interventions using MALAT1 siRNA or miR-143-3p mimics effectively reversed this effect. Both MALAT1 siRNA and overexpression of miR-143-3p significantly increased the calcium content in cultured VSMCs and in the carotid artery of diabetic rats following balloon injury. Balloon injury to the carotid artery in diabetic rats treated with macrophage-derived exosomes significantly increased the expression of MALAT1 and MGP while reducing the expression of miR-143-3p in the carotid artery. These findings demonstrate that macrophage-derived exosomal MALAT1 modulates VC via the MALAT1/miR-143-3p/MGP axis under hyperglycemic conditions. The results suggest that targeting exosomal MALAT1 may offer a novel and effective therapeutic approach for mitigating VC in metabolic disorders such as diabetes.

Background and Objectives: Dual antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor is standard care for acute coronary syndrome (ACS). Although ticagrelor showed superiority over clopidogrel in pivotal trials, patients with advanced chronic kidney disease (CKD) or on dialysis were underrepresented and results in Asian populations have been inconsistent. Materials and Methods: We conducted a retrospective cohort study using the Taiwan Society of Cardiology Acute Coronary Syndrome-Diabetes Mellitus (TSOC ACS-DM) registry between 1 October 2013, and 30 September 2016. Eligible patients had type 2 diabetes mellitus and ACS with stage III–V CKD or were on dialysis at index hospitalization and were discharged on aspirin plus either ticagrelor or clopidogrel. The primary endpoint was a composite of cardiovascular (CV) death, CV-related readmission, and repeated revascularization. Cumulative incidence functions were compared using expectation maximization (EM) weighting and propensity score adjustment. Results: After exclusions, 451 patients were analyzed (ticagrelor n = 116; clopidogrel n = 335). Ticagrelor associated with higher myocardial infarction (HR 1.59, 95% CI 1.12–2.28, p = 0.010), CV-related readmission (HR 1.72, 95% CI 1.12–2.65, p = 0.014), repeated revascularization (HR 2.24, 95% CI 1.36–3.68, p = 0.002), and the composite endpoint (HR 1.63, 95% CI 1.06–2.48, p = 0.024) at 2 years. Conclusions: Among real-world Taiwanese patients with type 2 diabetes mellitus, ACS, and CKD, ticagrelor use was linked to increased risks of cardiovascular events compared to clopidogrel. However, these relationships might be affected by potential confounding factors. Randomized controlled trials are necessary to establish the best antiplatelet strategy for this high-risk group.

Autophagy activity is tightly associated with cardiovascular disease development and progression. The effect of exosomal metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) derived from macrophages treated with high levels of glucose on autophagy in cardiomyocytes and diabetic hearts is not known. We investigated the effect of autophagy and its regulatory mechanisms on H9C2 cardiomyocytes and diabetic hearts by macrophage-derived exosomal MALAT1. Mouse macrophages and rat H9C2 cardiomyocytes were cultured, and exosomes were extracted from the culture media. A diabetic model was established through the injection of streptozotocin into adult male Wistar rats. Reverse transcription real-time quantitative polymerase chain reaction, Western blotting, immunohistochemical staining, autophagosome and/or autolysosome fluorescent cell staining, and luciferase activity assays were performed. High glucose significantly enhanced exosomal MALAT1 expression in cultured H9C2 cells and macrophages. Macrophage-derived exosomes significantly increased autophagy-related 7 (ATG7) and decreased miR-204-5p expressions. Silencing MALAT1 by MALAT1 small interference RNA and the overexpression of wild-type miR-204-5p significantly decreased the ATG7 expression induced by macrophage-derived exosomes. MiR-204-5p significantly decreased MALAT1 and ATG7 luciferase activity in cultured H9C2 cells treated with macrophage-derived exosomes. Streptozotocin-induced diabetes mellitus and macrophage-derived exosomes significantly enhanced MALAT1 expression to inhibit miR204-5p expression in the rat hearts. Macrophage-derived exosomes significantly enhanced ATG7 expression in the streptozotocin-induced diabetic hearts. In conclusion, we discovered that exosomal MALAT1 derived from macrophages after high glucose treatment could sequester miR-204-5p, leading to the upregulation of ATG7 expression, and this was linked to the regulation of autophagy in H9C2 cardiomyocytes and streptozotocin-induced diabetic hearts.

Background: Atrial fibrillation (AF) is a common cardiac arrhythmia associated with significant morbidity and mortality. Rapid electrical stimulation (RES) of atrial fibroblasts plays a crucial role in AF pathogenesis, but the underlying molecular mechanisms remain unclear. This study investigates the regulatory axis involving MALAT1, miR-499a-5p, and SOX6 in human cardiac fibroblasts from adult atria (HCF-aa) under RES conditions. Methods: HCF-aa were subjected to RES at 0.5 V/cm and 10 Hz. The expression levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), miR-499a-5p, and SRY-Box Transcription Factor 6 (SOX6) were measured using qPCR and Western blot analyses. Luciferase reporter assays were performed to confirm target relationships. The effects of MALAT1 siRNA, miR-499a-5p mimics/inhibitors, and SOX6 overexpression on gene expression and apoptosis were assessed. Results: RES increased exosomal MALAT1 expression, peaking at 2 h. MiR-499a-5p levels initially increased, then decreased at 2 h, coinciding with peak MALAT1 expression. SOX6 mRNA and protein levels increased, peaking at 4 and 6 h, respectively. Luciferase assays confirmed MALAT1 and SOX6 as miR-499a-5p targets. MALAT1 knockdown increased miR-499a-5p levels and reduced SOX6 expression. MiR-499a-5p overexpression decreased SOX6 levels and inhibited RES-induced apoptosis. Conclusion: In HCF-aa under RES, increased exosomal MALAT1 expression counteracts miR-499-5p’s suppression of SOX6, suggesting that MALAT1-containing exsosomes derived from HCF-aa may offer a novel cell-free therapeutic approach for AF.

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.

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.

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. Graphic abstract 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.