Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells.

BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

A miRNA cassette reprograms smooth muscle cells into endothelial cells.

Cellular reprogramming through targeting microRNAs (miRNAs) holds promise for regenerative therapy due to their profound regulatory effects in proliferation, differentiation, and function. We hypothesized that transdifferentiation of vascular smooth muscle cells (SMCs) into endothelial cells (ECs) using a miRNA cassette may provide a novel approach for use in vascular disease states associated with endothelial injury or dysfunction. miRNA profiling of SMCs and ECs and iterative combinatorial miRNA transfections of human coronary SMCs revealed a 4-miRNA cassette consisting of miR-143-3p and miR-145-5p inhibitors and miR-146a-5p and miR-181b-5p mimics that efficiently produced induced endothelial cells (iECs). Transcriptome profiling, protein expression, and functional studies demonstrated that iECs exhibit high similarity to ECs. Injected iECs restored blood flow recovery even faster than conventional ECs in a murine hindlimb ischemia model. This study demonstrates that a 4-miRNA cassette is sufficient to reprogram SMCs into ECs and shows promise as a novel regenerative strategy for endothelial repair.

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

BACKGROUND: The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. OBJECTIVES: The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. METHODS: Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. RESULTS: This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. CONCLUSION: Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

Lysine demethylase 3A is a positive regulator of cardiac myofibroblast transdifferentiation that increases Smad3 phosphorylation following transforming growth factor beta1 stimulation.

BACKGROUND: The epigenetic modifier molecule lysine demethylase 3A (KDM3A) has been shown to help ameliorate cardiovascular diseases, but its effect on cardiac fibroblasts (CFs) remains unclear. METHODS AND RESULTS: We designed gain- and loss-of-function experiments to investigate the biological functions of KDM3A in CFs. Moreover, we used SIS3-HCl (a specific inhibitor of p-Smad3) to explore the underlying mechanism. Cell viability and migration were verified by CCK-8 and cell migration experiments, respectively, and the degree of fibrosis was measured by Western blot analysis. Our data revealed that KDM3A enhanced the proliferation and migration of CFs and increased the fibroblast-to-myofibroblast transition while enabling the Smad3 phosphorylation response to transforming growth factor beta1 (TGFß1) stimulation. However, these effects were abolished by SIS3-HCl. Furthermore, KDM3A inhibition obviously protected against cardiac myofibroblast transdifferentiation under TGFß1 stimulation. CONCLUSIONS: KDM3A may act as a novel regulator of cardiac myofibroblast transdifferentiation through its ability to modulate the phosphorylation of Smad3 following TGFß1 stimulation.

Exosomal miR-512-3p derived from mesenchymal stem cells inhibits oxidized low-density lipoprotein-induced vascular endothelial cells dysfunction via regulating Keap1.

Atherosclerosis (AS) is a prevalent chronic inflammatory vascular disease. Upregulated oxidized low-density lipoprotein (ox-LDL) in the serum has been found to induce endothelial cells (ECs) apoptosis by increasing oxidative stress and promoting inflammatory response, which are essential mechanisms of AS development. Mesenchymal stem cells (MSCs), which secrete exosomes to transport microRNAs (miRNAs) and regulate cell functions, have become a research focus in recent years. The results of this study manifested that MSCs-derived exosomes were phagocytosed by EC. In addition, miR-512-3p enriched by MSCs- derived exosomes markedly inhibited ox-LDL-mediated EC damage, namely, accelerated EC proliferation, inhibited Caspase-3 activation and cell apoptosis, inhibited the levels of inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6) and oxidative factor MDA, and increased the contents of SOD and GSH-PX. Mechanistically, Keleh-like ECH-associated protein 1 (Keap1) was proved to be a functional target of miR-512-3p. Furthermore, silencing Keap1 limited ox-LDL-mediated EC cell dysfunction, while over-expressing Keap1 mitigated the exosomal miR-512-3p-mediated protective effect in Ox-LDL-induced EC. The above results confirmed that miR-512-3p shuttled by MSCs-derived exosomes protected EC against ox-LDL by targeting Keap1.

LncRNA H19 ameliorates myocardial infarction-induced myocardial injury and maladaptive cardiac remodelling by regulating KDM3A.

Myocardial infarction (MI) remains the leading cause of morbidity and mortality worldwide, and novel therapeutic targets still need to be investigated to alleviate myocardial injury and the ensuing maladaptive cardiac remodelling. Accumulating studies have indicated that lncRNA H19 might exert a crucial regulatory effect on cardiovascular disease. In this study, we aimed to explore the biological function and molecular mechanism of H19 in MI. To investigate the biological functions of H19, miRNA-22-3p and KDM3A, gain- and loss-of-function experiments were performed. In addition, bioinformatics analysis, dual-luciferase reporter assays, RNA immunoprecipitation (RIP) assays, RNA pull-down assays, quantitative RT-PCR and Western blot analyses as well as rescue experiments were conducted to reveal an underlying competitive endogenous RNA (ceRNA) mechanism. We found that H19 was significantly down-regulated after MI. Functionally, enforced H19 expression dramatically reduced infarct size, improved cardiac performance and alleviated cardiac fibrosis by mitigating myocardial apoptosis and decreasing inflammation. However, H19 knockdown resulted in the opposite effects. Bioinformatics analysis and dual-luciferase assays revealed that, mechanistically, miR-22-3p was a direct target of H19, which was also confirmed by RIP and RNA pull-down assays in primary cardiomyocytes. In addition, bioinformatics analysis and dual-luciferase reporter assays also demonstrated that miRNA-22-3p directly targeted the KDM3A gene. Moreover, subsequent rescue experiments further verified that H19 regulated the expression of KDM3A to ameliorate MI-induced myocardial injury in a miR-22-3p-dependent manner. The present study revealed the critical role of the lncRNAH19/miR-22-3p/KDM3A pathway in MI. These findings suggest that H19 may act as a potential biomarker and therapeutic target for MI.

Syringic acid mitigates myocardial ischemia reperfusion injury by activating the PI3K/Akt/GSK-3ß signaling pathway.

Syringic acid is an abundant phenolic acid compound that possesses anti-oxidant, anti-microbial, anti-inflammatory, and anti-endotoxic properties. However, the research of pretreatment with syringic acid against myocardial ischemia reperfusion is still limited. Thus, our research revealed the protective effect of syringic acid in the rat model with myocardial ischemia reperfusion injury. Histological analysis was performed by hematoxylin and eosin (H&E). The myocardial systolic function was detected by echocardiographic. Myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) double staining. The apoptosis index was recorded by Terminal deoxynucleotidyl transferase dUTP nick end labeling staining (TUNEL). The contents of creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH) in the serum were determined by a commercial kit. The expression of the PI3K/Akt/GSK-3ß signaling pathway-related molecules and apoptosis-associated indicators was detected by western blotting or real-time PCR. We found that pretreatment with syringic acid obviously increased the myocardial systolic function (LVEF and LVFS) and decreased the infarct size, the apoptosis index as well as the serum level of CK-MB and LDH. Meanwhile, syringic acid also remarkably augmented the contents of p-PI3K, p-Akt, p-GSK-3ß, Bcl-2 and mitochondria cytochrome c. However, the expression of caspase-3, -9 and Bax significantly reduced. Interestingly, co-treatment with PI3K inhibitor of LY294002 counteracted those effects induced by syringic acid. In conclusion, pretreatment with syringic acid can mitigate myocardial ischemia reperfusion injury by inhibiting mitochondria-induced apoptosis which is regulated by the PI3K/Akt/GSK-3ß signaling pathway.

Silica-coated magnetic nanoparticles labeled endothelial progenitor cells alleviate ischemic myocardial injury and improve long-term cardiac function with magnetic field guidance in rats with myocardial infarction.

Low retention of endothelial progenitor cells (EPCs) in the infarct area has been suggested to be responsible for the poor clinical efficacy of EPC therapy for myocardial infarction (MI). This study aimed to evaluate whether magnetized EPCs guided through an external magnetic field could augment the aggregation of EPCs in an ischemia area, thereby enhancing therapeutic efficacy. EPCs from male rats were isolated and labeled with silica-coated magnetic iron oxide nanoparticles to form magnetized EPCs. Then, the proliferation, migration, vascularization, and cytophenotypic markers of magnetized EPCs were analyzed. Afterward, the magnetized EPCs (1 × 106 ) were transplanted into a female rat model of MI via the tail vein at 7 days after MI with or without the guidance of an external magnet above the infarct area. Cardiac function, myocardial fibrosis, and the apoptosis of cardiomyocytes were observed at 4 weeks after treatment. In addition, EPC retention and the angiogenesis of ischemic myocardium were evaluated. Labeling with magnetic nanoparticles exhibited minimal influence to the biological functions of EPCs. The transplantation of magnetized EPCs guided by an external magnet significantly improved the cardiac function, decreased infarction size, and reduced myocardial apoptosis in MI rats. Moreover, enhanced aggregations of magnetized EPCs in the infarcted border zone were observed in rats with external magnet-guided transplantation, accompanied by the significantly increased density of microvessels and upregulated the expression of proangiogenic factors, when compared with non-external-magnet-guided rats. The magnetic field-guided transplantation of magnetized EPCs was associated with the enhanced aggregation of EPCs in the infarcted border zone, thereby improving the therapeutic efficacy of MI.

Downregulation of microRNA-17-5p improves cardiac function after myocardial infarction via attenuation of apoptosis in endothelial cells.

MicroRNA-17-5p (miR-17-5p) was indicated to suppress the formation of blood vessels, which is associated with cardiac function after myocardial infarction. In this study, the relationship between miR-17-5p and cardiac function was researched. Human umbilical vein endothelial cells were infected with adenoviruses. Apoptosis was determined by Annexin V-7AAD/PI. Real-time RT-PCR was used to evaluate miR-17-5p and ERK levels. Western blotting was used to determine the levels of ERK, the anti-apoptosis protein bcl-2 and apoptosis proteins, including bax, caspase 3, and caspase 9. An in vivo acute myocardial infarction (AMI) model was established in SD male rats. Heart function was evaluated by echocardiography prior to inducing AMI and after 7 and 28 days later. The heart was removed to perform histological examination, real-time RT-PCR, and western blotting, as described above. The result indicated that the ERK pathway was activated by miR-17-5p downregulation and an increase in the level of the anti-apoptosis protein bcl-2; however, the levels of apoptosis proteins (bax/caspase 3/caspase 9) were decreased. The results were completely reversed when miR-17-5p was up-regulated. At 7 and 28 days after the induction of AMI, in the miR-17-5p inhibition group, the infarction areas and collagen fibers were decreased, apoptosis in cardiac tissues was inhibited, and the endothelial growth process was promoted. Therefore, MiR-17-5p silencing protects heart function after AMI through decreasing the rate of apoptosis and repairing vascular injury.

Age-specific cardiovascular disease-related mortality among patients with major gastrointestinal cancers: A SEER population-based study.

BACKGROUND: Studies have reported age as a risk factor for cardiovascular disease (CVD)-related mortality; however, only a few studies have focused on the relationship between age and CVD-related mortality, especially among major gastrointestinal cancers. METHOD: The present retrospective cohort enrolled patients with colorectal, pancreatic, hepatocellular, gastric, and esophageal cancer between 2000 to 2015 from the Surveillance, Epidemiology and End Results Registry (SEER). Standardized mortality ratio (SMR), competing risk regression, and restricted cubic spline (RCS) analyses were used in our study. RESULTS: We analyzed 576,713 patients with major gastrointestinal cancers (327,800 patients with colorectal cancer, 93,310 with pancreatic cancer, 69,757 with hepatocellular cancer, 52,024 with gastric cancer, and 33,822 with esophageal cancer). Overall, CVD-related mortality gradually decreased every year, and the majority were older patients. All cancer patients had a higher CVD-related mortality rate than the general U.S. POPULATION: The adjusted sub-hazard ratios for middle-aged with colorectal cancer, pancreatic cancer, hepatocellular cancer, gastric cancer, and esophageal cancer were 2.55 (95% CI: 2.15-3.03), 1.77 (95% CI: 1.06-2.97), 2.64 (95% CI: 1.60-4.36), 2.15 (95% CI: 1.32-3.51), and 2.28 (95% CI: 1.17-4.44), respectively. The adjusted sub-hazard ratios for older patients with colorectal cancer, pancreatic cancer, hepatocellular cancer, gastric cancer, and esophageal cancer were 11.23 (95% CI: 9.50-13.27), 4.05 (95% CI: 2.46-6.66), 4.47 (95% CI: 2.72-7.35), 7.16 (95% CI: 4.49-11.41), and 4.40 (95% CI: 2.28-8.48), respectively. A non-linear relationship between age at diagnosis and CVD-related mortality was found in colorectal cancer, pancreatic cancer, and esophageal cancer; their reference ages were 67, 69, and 66 years old, respectively. CONCLUSION: This study demonstrated that age was a risk factor for CVD-related mortality among major gastrointestinal cancers.

Corrigendum: KDM3A inhibition ameliorates hyperglycemia-mediated myocardial injury by epigenetic modulation of nuclear factor kappa-B/P65.

[This corrects the article DOI: 10.3389/fcvm.2022.870999.].

KDM3A Inhibition Ameliorates Hyperglycemia-Mediated Myocardial Injury by Epigenetic Modulation of Nuclear Factor Kappa-B/P65.

Objectives: Even after the glucose level returns to normal, hyperglycemia-induced cardiac dysfunction as well as reactive oxygen species (ROS) generation, inflammatory responses, and apoptosis continued deterioration, showing a long-lasting adverse effect on cardiac function and structure. We aimed to unveil the molecular and cellular mechanisms underlying hyperglycemia-induced persistent myocardial injury and cardiac dysfunction. Methods and Results: Recently, the accumulated evidence indicated epigenetic regulation act as a determining factor in hyperglycemia-induced continuous cardiovascular dysfunction. As an important histone demethylase, the expression of lysine-specific demethylase 3A (KDM3A) was continually increased, accompanied by a sustained decline of H3K9me2 levels in diabetic myocardium even if received hypoglycemic therapy. Besides, by utilizing gain- and loss-of-functional approaches, we identified KDM3A as a novel regulator that accelerates hyperglycemia-mediated myocardial injury by promoting ROS generation, aggregating inflammatory reaction, and facilitating cell apoptosis in vitro and in vivo. The KDM3A inhibition could significantly ameliorate the adverse effect of hyperglycemia in both diabetes model and diabetic intensive glycemic control model. Mechanically, our data uncovered that KDM3A could promote the expression and transcriptional activity of nuclear factor kappa-B (NF-κB/P65), and the succedent rescue experiments further verified that KDM3A regulates hyperglycemia-induced myocardial injury in an NF-κB/P65 dependent manner. Conclusion: This study revealed histone-modifying enzymes KDM3A drives persistent oxidative stress, inflammation, apoptosis, and subsequent myocardial injury in the diabetic heart by regulating the transcription of NF-κB/P65.

The histone demthylase KDM3A protects the myocardium from ischemia/reperfusion injury via promotion of ETS1 expression.

Our prior studies have characterized the participation of histone demethylase KDM3A in diabetic vascular remodeling, while its roles in myocardial ischemia/reperfusion (I/R) injury (MIRI) remain to be illustrated. Here we show that KDM3A was significantly downregulated in rat I/R and cellular hypoxia/reoxygenation (H/R) models. Subsequently, gain- and loss-of-function experiments were performed to investigate the effects of KDM3A in the settings of MIRI. KDM3A knockout exacerbated cardiac dysfunction and cardiomyocytes injury both in vivo and in vitro. The deteriorated mitochondrial apoptosis, reactive oxygen species, and inflammation were simultaneously observed. Conversely, KDM3A overexpression developed the ameliorated alternations in MIRI. Mechanistically, the MIRI-alleviating effects of KDM3A were associated with the enhancement of ETS1 expression. ChIP-PCR affirmed that KDM3A bound to the ETS1 promoter and removed dimethylation of histone H3 lysine 9 (H3K9me2), thus promoting ETS1 transcription. Our findings suggest that KDM3A is available for alleviating multi-etiologies of MIRI through the regulation of ETS1.

KDM3A Attenuates Myocardial Ischemic and Reperfusion Injury by Ameliorating Cardiac Microvascular Endothelial Cell Pyroptosis.

Cardiac microvascular endothelial cell ischemia-reperfusion (CMEC I/R) injury occurs in approximately 50% of acute myocardial infarction patients subjected to successful revascularization therapy. This injury leads to cardiac microcirculatory system dysfunctions, which seriously affect cardiac functions and long-term prognostic outcomes. Previously, we elucidated the role of lysine-specific demethylase 3A (KDM3A) in protecting cardiomyocytes from I/R injury; however, its roles in CMEC I/R injuries have yet to be fully established. In this study, hypoxia/reoxygenation (H/R) treatment significantly impaired CMEC functions and induced their pyroptosis, accompanied by KDM3A downregulation. Then, gain- and loss-of-function assays were performed to investigate the roles of KDM3A in CMEC H/R injury in vitro. KDM3A knockout enhanced CMEC malfunctions and accelerated the expressions of pyroptosis-associated proteins, such as NLRP3, cleaved-caspase-1, ASC, IL-1ß, GSDMD-N, and IL-18. Conversely, KDM3A overexpression developed ameliorated alternations in CMEC H/R injury. In vivo, KDM3A knockout resulted in the deterioration of cardiac functions and decreased the no-reflow area as well as capillary density. Mechanistically, KDM3A activated the PI3K/Akt signaling pathway and ameliorated I/R-mediated CMEC pyroptosis. In conclusion, KDM3A is a promising treatment target for alleviating CMEC I/R injury.

KDM3A inhibition modulates macrophage polarization to aggravate post-MI injuries and accelerates adverse ventricular remodeling via an IRF4 signaling pathway.

It has been reported that KDM3A participates in several cardiovascular diseases through epigenetic mechanisms. However, its biological role post myocardial infarction (MI) has not been explored. Excessive and prolonged inflammation period can aggravate post-MI injuries and accelerates left ventricular (LV) remodeling. Previous studies have shown that macrophages play a momentous role in post-MI injuries by regulating the balance between the inflammatory phase. In this study, we aimed to demonstrate whether KDM3A could regulate the polarization of macrophages to affect the inflammatory response after myocardial infarction and whether targeting KDM3A could influence the prognosis of myocardial infarction and adverse LV remodeling. To explore the biological function of KDM3A and the underlying mechanisms, the loss of function experiments were designed in vitro and vivo. we analyzed the function of macrophages by a phagocytosis and migration assay and explored the polarization of macrophages. The expression of macrophage inflammation-related genes in the acute inflammatory phase and surface markers was detected by western blot and immunofluorescence assays. Echocardiography, Masson's trichrome staining and hematoxylin and eosin (H&E) staining were used to detect cardiac ventricular function. Our data showed that KDM3A is essential for the biological function of rat bone marrow macrophages (BMDMs), and KDM3A deficiency decreases the capacity for phagocytosis and migration, promoting M1 but restraining M2 macrophage phenotype polarization in vitro. Furthermore, we constructed MI models of male rats to verify that KDM3A deficiency could regulate macrophage polarization to aggravate the inflammatory response and accelerate LV remodeling in vivo. Among them, we confirmed that IRF4 is a downstream effector of the KDM3A-dependent pathway which could epigenetically influence the transcription of IRF4 by enhancing histone H3 lysine 9 di-methylation(H3K9me2) accumulation on the IRF4 gene proximal promoter region to modulate macrophage polarization. These results demonstrated that KDM3A plays an essential role in the cardiac repair process of post-MI and LV remodeling by modulating the macrophage phenotype, thereby suggesting a promising therapy to treat post MI injuries.

Long noncoding RNA UCA1 inhibits ischaemia/reperfusion injury induced cardiomyocytes apoptosis via suppression of endoplasmic reticulum stress.

BACKGROUND: Ischemia heart disease is one of the major causes of death worldwide which often associated with tissue infarction and limit the recovery of function. Multiple factors involved in the I/R-induced cardiomyocyte dysfunction which were consistent with a role of oxidative stress and altered endothelium-dependent responses. However, the pathogenic mechanisms in I/R injury remain unclear. MATERIALS AND METHODS: The H9C2 cells were in the ischaemia/reperfusion (I/R) condition. After I/R, the cells were transfected with or without adenovirus-urothelial carcinoma associated 1(Ad-UCA1). Then qRT-PCR analysis was performed to quantify mRNA expression of different treatment groups. Cell apoptosis rate was assessed using flow cytometry and ER stress biomarker expression were measured by immunoblotting. Intracellular and mitochondrial ROS generation were assayed by fluorescence microscope after staining with the DCFDA or MitoSOX. RESULTS: I/R conditions trigger lncRNAs UCA1 expression, cellular and mitochondria ROS production, resulting in cell apoptosis through the induction of oxidative and ER stress. Overexpression of UCA1 protects H9C2 cells from I/R-induced ER stress and cell apoptosis. Moreover, UCA1 might be a potential regulator in the protective effect of I/R­induced oxidative stress and mitochondria dysfunction. Subsequently, ER stress inhibitor attenuated the effect of siUCA1 induced injury in H9C2 cells. CONCLUSION: The expression of UCA1 against I/R induced oxidative stress and mitochondria dysfunction via suppression of endoplasmic reticulum stress. UCA1 might be a biomarker to improved diagnosis of I/R injury.

Nobiletin ameliorates myocardial ischemia and reperfusion injury by attenuating endoplasmic reticulum stress-associated apoptosis through regulation of the PI3K/AKT signal pathway.

BACKGROUND: Nobiletin is a natural polymethoxylated flavone that confers antioxidative, anti-inflammatory and anti-apoptotic efficacies. However, the potential benefits of nobiletin preconditioning on myocardial ischemia and reperfusion injury (MIRI) remains largely unknown. METHODS: MIRI was induced by ligation of the left anterior descending coronary artery and reperfusion. Pre-treatment with nobiletin, with or without PI3K/AKT inhibitor LY294002, was performed at the onset of reperfusion. Histological analyses, apoptotic evaluation, plasma biomarkers of myocardial injury, echocardiographic evaluation of cardiac function and myocardial levels of endoplasmic reticulum stress (ERS)-related molecules were observed. RESULTS: Nobiletin pre-treatment significantly deceased the infract size and number of apoptotic cells in the myocardium of MIRI rats, as determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Moreover, the plasma levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) also markedly decreased. In addition, pre-treatment with nobiletin restored the impaired cardiac systolic function, as evidenced by echocardiographic evaluation results. Importantly, pre-treatment with nobiletin significantly downregulated the myocardial mRNA and protein levels of ERS-related signal molecules, including GRP78, CHOP and caspase-12, but upregulated the levels of p-PI3K and p-AKT. Interestingly, co-treatment with LY294002 significantly abolished the benefits of nobiletin pre-treatment on cardiac function, myocardial apoptosis, cardiomyocyte injuries, and changes in myocardial levels of ERS-related signaling molecules. CONCLUSION: Nobiletin pre-treatment may alleviate MIRI probably via the attenuation of PI3K/AKT-mediated ERS-related myocardial apoptosis.

KDM3A inhibition attenuates high concentration insulin­induced vascular smooth muscle cell injury by suppressing MAPK/NF­κB pathways.

Previous studies have indicated that lysine (K)­specific demethylase 3A (KDM3A) is associated with diverse diabetes­associated cardiovascular complications in response to high glucose levels. However, the effects of KDM3A on the pathological progression of cardiovascular injuries in response to high insulin levels remain unknown. The present study aimed to explore whether KDM3A knockdown may attenuate high insulin­induced vascular smooth muscle cell (VSMC) dysfunction, and to further investigate the underlying mechanisms. Primary VSMCs were isolated from the thoracic aorta of Sprague­Dawley rats. Lentiviral vectors encoding control­small interfering (si)RNA or KDM3A­siRNA were transduced into VSMCs for 72 h, and cells were subsequently incubated in medium containing 100 nM insulin for a further 5 days. Cellular proli-feration, migration and apoptosis were measured by Cell Counting kit­8, Transwell chamber assay and flow cytometry, respectively. Reactive oxygen species (ROS) were detected using the dihydroethidium fluorescent probe. The mRNA expression levels of interleukin­6 and monocyte chemotactic protein­1 were measured by reverse transcription­quantitative polymerase chain reaction. Furthermore, the protein expression levels of KDM3A, mitogen­activated protein kinases (MAPKs), nuclear factor (NF)­κB/p65, B­cell lymphoma 2 (Bcl­2)­associated X protein and Bcl­2 were evaluated by west-ern blotting. Lentivirus transduction with KDM3A­siRNA markedly reduced the elevated expression of KDM3A induced by high insulin stimulation in VSMCs. In addition, inhibition of KDM3A significantly ameliorated insulin­induced VSMC proliferation and migration, which was accompanied by decreased ROS levels, cell apoptosis and inflammatory cytokine levels. Furthermore, KDM3A gene silencing mitigated phosphorylation of MAPKs and NF­κB/p65 activation. In conclusion, KDM3A inhibition may exert numerous protective effects on high insulin­stimulated VSMCs, and the underlying mechanisms may be partly associated with inactivation of MAPK/NF­κB signaling pathways.

RP105 ameliorates hypoxia̸reoxygenation injury in cardiac microvascular endothelial cells by suppressing TLR4̸MAPKs̸NF-κB signaling.

The radioprotective 105 kDa protein (RP105) has been implicated in the pathological process of multiple cardiovascular diseases through its functional and physical interactions with Toll­like receptor 4 (TLR4). However, the effects of RP105 on cardiac microvascular endothelial cells (CMECs) in response to hypoxia̸reoxygenation (H̸R) injury have not been extensively investigated. The aim of the present study was to elucidate the potential roles of RP105 in the protection of CMECs against H̸R injury, and investigate the underlying mechanisms. CMECs isolated from Sprague­Dawley rats were transduced with adenoviral vectors encoding RP105 or green fluorescent protein (GFP). At 48 h post­transfection, CMECs were subjected to hypoxia for 4 h and reoxygenation for 2 h (H̸R) to simulate the in vivo ischemia̸reperfusion model. The mRNA and protein levels of RP105 were detected by reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. The effects of RP105 on CMEC proliferation, migration and apoptosis were measured by GFP­8, Transwell chamber and flow cytometry assays, respectively. The secretion of interleukin (IL)­6 and tumor necrosis factor (TNF)­α in the culture medium was measured by ELISA. Moreover, the expression level of TLR4, p38 mitogen­activated protein kinase (MAPK), extracellular-signal-regulated kinase 1̸2, c-Jun N-terminal kinase, nuclear factor (NF)­κB̸p65, IL­6, TNF­α and intercellular adhesion melecule­1 was evaluated by western blot analysis. The results demonstrated that RP105 was minimally expressed in CMECs subjected to H̸R injury. Overexpression of RP105 via adenoviral vectors was able to significantly protect CMECs against H̸R injury, as evidenced by the promotion of cell proliferation and migration, as well as the amelioration of inflammation and apoptosis. These beneficial effects were at least partly mediated through inhibition of TLR4̸MAPKs̸NF­κB signaling. Therefore, RP105 may be a promising candidate for prevention against CMECs­associated H̸R injury.