N6-methyladenosine demethylase fat mass and obesity-associated protein suppresses hyperglycemia-induced endothelial cell injury by inhibiting reactive oxygen species formation via autophagy promotion.

INTRODUCTION: Hyperglycemia-induced endothelial cell injury is one of the main causes of diabetic vasculopathy. Fat mass and obesity-associated protein (FTO) was the first RNA N6-methyladenosine (m6A) demethylase identified; it participates in the pathogenesis of diabetes. However, the role of FTO in hyperglycemia-induced vascular endothelial cell injury remains unclear. MATERIALS AND METHODS: The effects of FTO on cellular m6A, autophagy, oxidative stress, proliferation, and cytotoxicity were explored in human umbilical vein endothelial cells (HUVECs) treated with high glucose (33.3 mmol/mL) after overexpression or pharmacological inhibition of FTO. MeRIP-qPCR and RNA stability assays were used to explore the molecular mechanisms by which FTO regulates autophagy. RESULTS: High glucose treatment increased m6A levels and reduced FTO protein expression in HUVECs. Wild-type overexpression of FTO markedly inhibited reactive oxygen species generation by promoting autophagy, increasing endothelial cell proliferation, and decreasing the cytotoxicity of high glucose concentrations. The pharmacological inhibition of FTO showed the opposite results. Mechanistically, we identified Unc-51-like kinase 1 (ULK1), a gene responsible for autophagosome formation, as a downstream target of FTO-mediated m6A modification. FTO overexpression demethylated ULK1 mRNA and inhibited its degradation in an m6A-YTHDF2-dependent manner, leading to autophagy activation. CONCLUSIONS: Our study demonstrates the functional importance of FTO-mediated m6A modification in alleviating endothelial cell injury under high glucose conditions and indicates that FTO may be a novel therapeutic target for diabetic vascular complications.

High-dose remifentanil exacerbates myocardial ischemia-reperfusion injury through activation of calcium-sensing receptor-mediated pyroptosis.

Background: The aim of this study was to investigate whether calcium-sensing receptor (CaSR) was involved in HRF-mediated exacerbation of MI/R injury through NLRP3 inflammasome activation and pyroptosis. Methods: In vivo, a rat MI/R model was established by ligating the left coronary artery, and short-term HRF exposure was induced during reoxygenation. Then, TUNEL, H&E, Masson staining, immunohistochemical (IHC) and serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK), as well as the expression levels of CaSR and pyroptosis-related proteins in heart tissues, were measured. H9c2 cells were cultured to create a hypoxia/reoxygenation (H/R) model and exposed to different concentrations of RF. After pretreatment with the CaSR activator gadolinium chloride (GdCl3) and inhibitor NPS2143 in the H/R model and treatment with HRF, we compared cellular viability, TUNEL, cytosolic [Ca2+]i, the levels of LDH and CK, pyroptosis-related proteins and CaSR in H9c2 cells. We further researched the mechanisms of CaSR-mediated pyroptosis in the H/R+HRF model by CaSR-shRNA, Ac-YVAD-CMK, MCC950 and NAC. Results: We found that HRF significantly increased CaSR expression, rate of cell death, levels of CK and LDH, and exacerbated pyroptosis in MI/R model. In vitro, HRF increased CaSR expression, decreased viability, enhanced cytosolic [Ca2+]i and exacerbated pyroptosis in H/R cells. Pretreated with GdCl3 worsen these changes, and NPS2143, MCC950, Ac-YVAD-CMK, NAC and sh-CaSR can reversed these effects. Conclusion: Exposure to HRF for a short time exacerbates MI/R-induced injury by targeting CaSR to increase cytosolic [Ca2+]i and ROS levels, which mediate the NLRP3 inflammasome and pyroptosis.

PDZK1 improves ventricular remodeling in hypertensive rats by regulating the stability of the Mas receptor.

Ventricular remodeling is one of the main causes of mortality from heart failure due to hypertension. Exploring its mechanism and finding therapeutic targets have become urgent scientific problems to be solved. A number of studies have shown that Mas, as an Ang-(1-7) specific receptor, was significantly reduced in myocardial tissue of rats undergoing hypertensive ventricular remodeling. It has been reported that Mas receptor levels are significantly downregulated in myocardium undergoing ventricular remodeling, but studies focused on intracellular and post-translational modifications of Mas are lacking. The results of this research are as follows: (1) PDZK1 interacts with the carboxyl terminus of Mas through its PDZ1 domain; (2) the expression of PDZK1 and Mas is decreased in rats undergoing hypertensive ventricular remodeling, and PDZK1 upregulation can ameliorate hypertensive myocardial fibrosis and myocardial hypertrophy; (3) PDZK1 enhances the stability of Mas protein through the proteasome pathway, and the proteasome inhibitor MG132 promotes hypertensive ventricular remodeling. PDZK1 improves ventricular remodeling in hypertensive rats by regulating Mas receptor stability. This study provides a scientific basis for the prevention and treatment of ventricular remodeling.

Alcohol Intake Provoked Cardiomyocyte Apoptosis Via Activating Calcium-Sensing Receptor and Increasing Endoplasmic Reticulum Stress and Cytosolic [Ca2+]i.

BACKGROUND: Cardiomyocyte apoptosis plays an important role in alcoholic cardiac injury. However, the association between calcium-sensing receptor (CaSR) and alcohol-induced cardiomyocyte apoptosis remain unclear. Therefore, we investigated the role and its moleculer mechanism of CaSR in rat cardiomyocyte apoptosis induced by alcohol. METHODS: Alcohol-induced cardiomyocyte apoptosis in vivo and in vitro model of rats were applied in this study. The expression of CaSR, endoplasmic reticulum stress markers and apoptosis were tested by immunohistological staining, western blot, TUNEL and flow cytometry, respectively. [Ca2+]i were detected by confocal laser scanning microscopy. RESULTS: Compared with the control group, alcohol intake (AI) led to abnormal arrangements of cardiomyocytes and obvious increase of myocardial apoptosis. Moreover, AI also significantly upregulated protein expression of CaSR, GRP94, caspase-12 and CHOP. Alcohol induced apoptosis of cultured cardiomyocytes of rats in a dose-dependent way. Activation of CaSR markedly enhanced cardiomyocyte apoptosis and ERS induced by alcohol, ERS inducer also significantly increased cardiomyocyte apoptosis without activating CaSR. Furthermore, GdCl3 augmented alcohol-induced increase of [Ca2+]i in cardiomyocytes, which was attenuated by NPS2390 but not 4-PBA pre-treatment. CONCLUSIONS: Alcohol could induce cardiomyocyte apoptosis in rats in vivo and in vitro, which was mediated probably via activating CaSR, and then ERS and the increase of the cytosolic [Ca2+]i. This provides a potential target for preventing cardiomyocyte apoptosis and cardiomyopathy induced by alochol.

Changes in microRNA Expression Profiles in Diabetic Cardiomyopathy Rats Following H3 Relaxin Treatment.

ABSTRACT: MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

Promoting plaque stability by gene silencing of monocyte chemotactic protein-3 or overexpression of tissue factor pathway inhibitor in ApoE-/- mice.

Chemokines may promote the formation and instability of atherosclerotic plaque, which is the most common cause of acute coronary syndrome. The aim of this study was to clarify the function of monocyte chemotactic protein-3 (MCP-3) in the stability of atherosclerotic plaque, to determine the role of tissue factor pathway inhibitor (TFPI) on the development and stability of atherosclerotic plaques, and to further elucidate the anti-atherosclerotic mechanism of TFPI with the emphasis on chemokine MCP-3. We constructed an adenovirus-mediated shRNA against mouse MCP-3 (Ad-MCP-3-shRNA) and an adenovirus-containing TFPI (Ad-TFPI), and tranferred them in a model of vulnerable plaque in ApoE-/- mice respectively. Here, we reported that MCP-3-shRNA and TFPI could both reduce the plaque area and decrease the content of lipids and macrophages, on the contrary, the fibrous cap thickness and content of collagen and smooth muscle cells were increased. In addition, the expression of MCP-3 and CC chemokine receptor 2 (CCR2) was decreased by TFPI transfer. These data provide the first in vivo evidence that MCP-3 is a major contributor to the unstability of atherosclerotic plaque and TFPI may exert its anti-atherosclerotic effects and promote stabilisation of plaque at least partly through inhibiting MCP-3/CCR2 pathway, which may be a new therapeutic method for atherosclerosis.

Calhex231 ameliorates myocardial fibrosis post myocardial infarction in rats through the autophagy-NLRP3 inflammasome pathway in macrophages.

The calcium-sensing receptor (CaSR) is involved in the pathophysiology of many cardiovascular diseases, including myocardial infarction (MI) and hypertension. The role of Calhex231, a specific inhibitor of CaSR, in myocardial fibrosis following MI is still unclear. Using Wistar rats, we investigated whether Calhex231 ameliorates myocardial fibrosis through the autophagy-NLRP3 inflammasome pathway in macrophages post myocardial infarction (MI). The rats were randomly divided into sham, MI and MI + Calhex231 groups. Compared with the sham rats, the MI rats consistently developed severe cardiac function, myocardial fibrosis and infiltration of inflammatory cells including macrophages. Moreover, inflammatory pathway including activation of NLRP3 inflammasome, IL-1ß and autophagy was significantly up-regulated in myocardial tissue, infiltrated cardiac macrophages and peritoneal macrophages of the MI rats. These impacts were reversed by Calhex231. In vitro, studies revealed that calindol and rapamycin exacerbated MI-induced autophagy and NLRP3 inflammasome activation in peritoneal macrophages. Calhex231 and 3-Methyladenine (a specific inhibitor of autophagy) attenuated both autophagy and NLRP3 inflammasome activation; however, the caspase-1 inhibitor Z-YVAD-FMK did not. Our study indicated that Calhex231 improved cardiac function and ameliorated myocardial fibrosis post MI, likely via the inhibition of autophagy-mediated NLRP3 inflammasome activation; this provides a new therapeutic target for ventricular remodelling-related cardiovascular diseases.

Cardioprotection of cortistatin against isoproterenol-induced myocardial injury in rats.

BACKGROUND: The present study was designed to examine whether cortistatin (CORT) could protect rats from myocardial injury induced by subcutaneously injecting isoproterenol (ISO) and to clarify the possible mechanisms. METHODS: Male Sprague-Dawley (SD) rats were placed at random into four groups: the control group, the ISO group, the ISO + CORT 25 µg/(kg·d) group, and the ISO + CORT 50 µg/(kg·d) group. Rat models of myocardial injury were established with the subcutaneous (s.c.) injections of 85 mg/kg ISO for 2 days. In the ISO+ CORT 25 µg/(kg·d) group and ISO+ CORT 50 µg/(kg·d) group, rats were given s.c. injections of CORT 25 µg/(kg·d) and CORT 50 µg/(kg·d) on the day before ISO, 3 days, respectively. Serum malondialdehyde (MDA) content, lactate dehydrogenase (LDH) activity, and creatine kinase isoenzyme (CK-MB) activity were measured by corresponding test kits. Western blot was applied to evaluate the expression of endoplasmic reticulum stress-related protein glucose regulatory protein 78 (GRP78), enhancer-binding protein homologous protein (CHOP), cysteinyl aspartate specific proteinase-12 (caspase-12), LC3-II, Beclin-1, and p62 in the rat myocardium. RESULTS: CORT alleviated the increased enzyme activities of serum LDH and CK-MB, and content of MDA (a typical marker of lipid peroxidation) in rats induced by ISO. CORT also prevented pathological myocardial injury in rats induced by ISO. Moreover, CORT attenuated the increased protein levels of GRP78, CHOP, and caspase-12, and reduced the increase of LC3-II, LC3-II/I, Beclin-1, and p62 in rats induced by ISO. CONCLUSIONS: These data demonstrate that CORT can attenuate ISO-induced acute myocardial injury in rats likely by reducing lipid peroxidation, and inhibiting endoplasmic reticulum stress and autophagy. This supports CORT as a potentially being a new target for preventing and treating myocardial injury and its related disease.

Cortistatin ameliorates Ang II-induced proliferation of vascular smooth muscle cells by inhibiting autophagy through SSTR3 and SSTR5.

AIMS: Vascular smooth muscle cell (VSMC) proliferation plays a significant role in the development of various vascular disorders. However, the effect of cortistatin (CST) on VSMC proliferation remains unclear. Therefore, the purpose of our research aimed to study whether CST protected VSMCs from angiotensin II (Ang II)-induced proliferation and which mechanisms participated in the process. MAIN METHODS: Cultured rat VSMCs were treated with Ang II with or without CST for 24 h. Cell proliferation rate was measured by cell counting kit-8 (CCK8) assay. The expressions of CST and its receptors were assessed by quantitative real-time PCR (qRT-PCR). The protein expression levels were analyzed by western blots. Immunofluorescence and transmission electron microscopy (TEM) were used to observe autophagy. KEY FINDINGS: Our results showed that different concentrations of CST alleviated the Ang II-induced VSMC proliferation. The autophagy and reactive oxygen species (ROS) stimulated by Ang II were attenuated by CST. Furthermore, when the autophagy inhibitor 3-methyladenine (3-MA) was added, it exerted similar inhibition effects like CST, but didn't augment the protective role of CST on Ang II-induced VSMC autophagy and proliferation. Moreover, blocking somatostatin receptor 3 and 5 (SSTR3 and SSTR5) partially abrogated the suppressive effect of CST on Ang II-stimulated VSMC proliferation and autophagy. SIGNIFICANCE: This study indicated that CST could ameliorate Ang II-stimulated VSMC proliferation by inhibiting autophagy partially through its receptors SSTR3 and SSTR5, providing a reasonable evidence for CST as a novel perspective therapeutic target of vascular diseases.

Calcium-Sensing Receptor on Neutrophil Promotes Myocardial Apoptosis and Fibrosis After Acute Myocardial Infarction via NLRP3 Inflammasome Activation.

BACKGROUND: The infiltration of neutrophils aggravates inflammatory response in acute myocardial infarction (AMI), and the role of calcium-sensing receptor (CaSR) in neutrophil-associated inflammation is largely unknown. The aim of this study was to evaluate the regulatory effects of CaSR on nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome in neutrophils and to explore its role in AMI-related ventricular remodelling. METHODS: The expression of CaSR, NLRP3 inflammasome, and interleukin 1ß (IL-1ß) in peripheral blood and infiltrating neutrophils in patients and rats with AMI was detected by western blotting and immunofluorescence. Cardiomyocyte apoptosis was detected by western blotting and transmission electron microscopy. The degree of fibrosis was evaluated by Masson staining and western blotting. RESULTS: We found upregulation of CaSR, NLRP3 inflammasome, Caspase-1, and IL-1ß in peripheral neutrophils from patients with AMI compared with matched healthy controls, peaking on day 1 and decreasing gradually till 7 days. Peripheral and infiltrating neutrophils from rats with AMI showed the same trend. Calindol enhanced NLRP3 inflammasome activation and IL-1ß release in neutrophils from healthy volunteers, which was blocked by inhibitors of the PLC-IP3 pathway and ER-Ca2+ release. Calhex-231 decreased NLRP3 inflammasome activation and IL-1ß release in neutrophils from patients with AMI. The calindol-stimulated neutrophils from healthy rats promoted cardiomyocyte apoptosis and fibrosis of cardiac fibroblasts from healthy rats, which were inhibited by calhex-231. CONCLUSION: The results suggest that CaSR activates NLRP3 inflammasome in neutrophils, contributing to ventricular remodelling after AMI. CaSR inhibition may be a potential therapeutic target for heart failure in AMI.

Cortistatin exerts antiproliferation and antimigration effects in vascular smooth muscle cells stimulated by Ang II through suppressing ERK1/2, p38 MAPK, JNK and ERK5 signaling pathways.

BACKGROUND: Vascular remodeling, that contributes to cardiovascular diseases such as hypertension develops by anomalous proliferation and migration of vascular smooth muscle cells (VSMCs). Cortistatin (CST), a newly discovered biological peptide, has been acknowledged for its protective effects against cardiovascular diseases. Whether CST has an inhibitory regulation role in angiotensin II (Ang II)-induced proliferation and migration of VSMCs and what molecular mechanisms may participate in the CST inhibition process are still unknown. METHODS: VSMCs were divided into control group, Ang II (10-7 M) group, Ang II + PD98059 (5×10-5 M) group, Ang II + SB203580 (10-5 M) group, Ang II + SP600125 (10-5 M) group, Ang II + XMD17-109 (10-6 M) group, Ang II + CST (10-8 M) group and Ang II + CST (10-7 M) group. Cell proliferation was detected by western blotting and cell counting kit-8 (CCK8) analysis. Migration of VSMCs was measured by Transwell assay. RESULTS: Compared with control group, Ang II upregulated the expression levels of proliferating cell nuclear antigen (PCNA) and osteopontin (OPN) and downregulated that of α-smooth muscle actin (α-SMA), increased the proliferation rate as shown by CCK8 and VSMC migration as shown by Transwell assay in cultured VSMCs of the Ang II group. Meanwhile, in Ang II-cultured VSMCs, we found activation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and ERK5 pathways by western blotting at different time points. However, the proliferation and migration stimulated by Ang II were partly reversed by drug inhibitors of the four pathways, namely, PD98059, SB203580, SP600125 and XMD17-109. When Ang II-stimulated VSMCs were cultured with CST pretreatment, we found that proliferation and migration were greatly suppressed as well as that the ERK1/2, p38 MAPK, JNK and ERK5 pathways were deactivated by CST. CONCLUSIONS: The accumulated data suggest that CST may play a protective role in Ang II-promoted proliferation and migration of VSMCs via inhibiting the mitogen-activated protein kinase (MAPK) family pathways, providing a new orientation of CST in protecting against cardiovascular diseases.

H2 relaxin ameliorates angiotensin II-induced endothelial dysfunction through inhibition of excessive mitochondrial fission.

The physiological function of endothelial cells plays an important role in maintaining normal cardiovascular function. Endothelial dysfunction induced by AngII (angiotensin II) is the pathological mechanism of occurrence and development of cardiovascular diseases. Human recombinant relaxin-2 (H2 relaxin), which has protective effect on cardiovascular functions, ameliorates damage to endothelial cells induced by angiotensin II (AngII) treatment. However, the exact mechanisms remain unclear. In this study, we researched the mechanisms of H2 relaxin inhibiting AngII-induced endothelial dysfunction from the protective effect of H2 relaxin on endothelial function though inhibiting excessive mitochondrial fission. Here, we found that H2 relaxin increased eNOS, SOD1 expression, inhibited excessive mitochondrial fission and decreased ROS level in HUVECs treated with AngII. However, overexpression of fission protein 1 (Fis1) prevented H2 relaxin from protecting against AngII-induced low eNOS, SOD1 expression, excessive mitochondrial fission and increased ROS level in HUVECs. Our study indicated that excessive mitochondrial fission could be a target for H2 relaxin to treat endothelial dysfunction in angiocardiopathy.

Nox4-dependent ROS production is involved in CVB3-induced myocardial apoptosis.

Viral myocarditis is a cardiovascular disease that seriously affects human health. Its mechanism is not clear. Coxsackievirus B3 (CVB3) is a member of the picornavirus family and is the leading cause of viral myocarditis. Our group tested the genes in a mouse model of CVB3 virus infection and confirmed that the NADPH oxidase gene had a high expression trend in the acute phase of infection. Whether Nox4, the homologue of NADPH oxidase, participates in the process of viral myocarditis has not been reported. In this study, we found increased expression of Nox4 in viral myocarditis in vivo and in vitro. DPI is a non-specific inhibitor of Nox4 that improved CVB3-induced myocarditis after injection in vivo. DPI also inhibited intracellular ROS release and apoptosis in vitro. Our data indicated that Nox4-dependent ROS production was involved in CVB3-induced myocardial apoptosis.

Cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis.

Cyclosporin A (CsA) is an effective immunosuppressive agent, but its myocardial toxicity limits its widespread and long-term clinical application. In this study, CsA treatment led to damages in myocardial fiber structure, an increase in myocardial fibrosis, and changes in heart size and shape; moreover, the degree of damage was exacerbated with prolonged drug application and increases in dose. However, the mechanism is not clear; therefore, the purpose of this study was to reveal the mechanism of CsA-induced myocardial fibrosis and identify a new target for the prevention and treatment of CsA-induced myocardial injury. Cardiac fibroblasts were treated with CsA (5, 10, or 20 µg/mL) for 24 h. Autophagy was observed by electron microscopy and immunofluorescence. The expression of NRP-2/WDFY-1, autophagy-related proteins (Beclin1 and LC3B), fibrosis-related proteins (MMP2/9), and fibroblast phenotype conversion factor (α-SMA) was evaluated by Western blot. The expression of collagen I was determined by ELISA. Then, we used the gene interference technique to alter WDFY-1 expression with or without CsA or 3-MA treatment for 24 h, and the effects on autophagy and the expression of autophagy-related proteins, fibrosis-associated proteins, IFN-α, TNF-α, and IL-6 were determined. The results showed the following: (1) CsA induced fibrosis-related protein (MMP2/9), fibroblast phenotype conversion factor (α-SMA), and collagen I up-regulation in a dose-dependent manner. (2) CsA induced the formation of autophagosomes and up-regulated the expression of Beclin1, LC3B, and the ERK/MAPK pathway in cardiac fibroblasts. (3) CsA induced NRP-2 down-regulation and WDFY-1 up-regulation. (4) Depletion of WDFY-1 inhibited CsA-induced autophagy, TNF-α and IFN-α up-regulation, and fibrosis. (5) The autophagy inhibitor 3-MA inhibited CsA-induced TNF-α and IFN-α up-regulation and fibrosis. Overall, cyclosporin A induces autophagy in cardiac fibroblasts through the NRP-2/WDFY-1 axis, which promotes the progression of myocardial fibrosis.

Activation of calcium-sensing receptor-mediated autophagy in angiotensinII-induced cardiac fibrosis in vitro.

Cardiac fibrosis is one of the primary mechanisms of ventricular remodeling, and there is no effective method for reversal. Activation of calcium sensing receptor (CaSR) has been reported to be involved in the development of myocardial fibrosis, but the molecular mechanism for CaSR activation has not yet been clarified and needs to be further explored. Here, we found that AngII induces cardiac fibroblast proliferation and phenotypic transformation in a dose-dependent manner with increased CaSR and autophagy related protein (Beclin1, LC3B) expression. CaSR activation results in intracellular calcium release, MEK1/2 pathway phosphorylation, autophagy activation and collagen formation induced by AngII in cardiac fibroblasts. However, pretreating the cells with Calhex231, PD98059 or 3-MA partially blocked AngII-induced cardiac fibrosis. Our data indicate that the activation of CaSR-mediated MEK/ERK and autophagic pathways is involved in AngII-induced cardiac fibrosis in vitro.

H3 Relaxin Protects Against Myocardial Injury in Experimental Diabetic Cardiomyopathy by Inhibiting Myocardial Apoptosis, Fibrosis and Inflammation.

BACKGROUND/AIMS: Apoptosis, fibrosis and NLRP3 inflammasome activation are involved in the development of diabetic cardiomyopathy (DCM). Human recombinant relaxin-3 (H3 relaxin) is a novel bioactive peptide that inhibits cardiac injury; however, whether H3 relaxin prevents cardiac injury in rats with DCM and the underlying mechanisms are unknown. METHODS: To investigate the effect of H3 relaxin on DCM, we performed a study using H3 relaxin treatment in male Sprague-Dawley (SD) rats with streptozotocin (STZ)-induced diabetes (DM). We measured apoptosis, fibrosis and NLRP3 inflammasome markers in the rat hearts four and eight weeks after the rats were injected with STZ (65 mg/kg) by western blot analysis. Subsequently, 2 or 6 weeks after the STZ treatment, the rats were treated with H3 relaxin [2 µg/kg/d (A group) or 0.2 µg/kg/d (B group)] for 2 weeks. Cardiac function was evaluated by echocardiography to determine the extent of myocardial injury in the DM rats. The protein levels of apoptosis, fibrosis and NLRP3 inflammasome markers were used to assess myocardial injury. In addition, we determined the plasma levels of IL-1ß and IL-18 using a Milliplex MAP Rat Cytokine/Chemokine Magnetic Bead Panel kit. RESULTS: The protein expression of cleaved caspase-8, caspase-9 and caspase-3 as well as fibrosis markers increased at 4 and 8 weeks in the STZ-induced diabetic hearts compared with the levels in the control group. Furthermore, the NLRP3 inflammasome was substantially activated in STZ-induced diabetic hearts, leading to increased IL-1ß and IL-18 levels. Compared with the DM group, the A group exhibited substantially better cardiac function. The protein levels of apoptosis markers were attenuated by H3 relaxin, indicating that H3 relaxin inhibited myocardial apoptosis in the hearts of diabetic rats. The protein expression of fibrosis markers was inhibited by H3 relaxin. Additionally, the protein expression and activation of the NLRP3 inflammasome were also effectively attenuated by H3 relaxin. CONCLUSIONS: This study is the first to demonstrate that H3 relaxin plays an anti-apoptotic, anti-fibrotic and anti-inflammatory role in DCM.

Role of the calcium sensing receptor in cardiomyocyte apoptosis via mitochondrial dynamics in compensatory hypertrophied myocardium of spontaneously hypertensive rat.

Calcium sensing receptor (CaSR) mediates pathological cardiac hypertrophy. Mitochondria maintain their function through fission and fusion and disruption of mitochondrial dynamic is linked to various cardiac diseases. This study examined how inhibition of CaSR by the inhibitor Calhex231 affected the mitochondrial dynamics in a hypertensive model in rats. Spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were used in this study. Cardiac function and blood pressure was evaluated at the end of the study. SHRs showed increases in the ratio of heart weight to body weight and the levels of CaSR; all of these increases were suppressed by Calhex231. Additionally, Calhex231 treatment of SHRs changed the expression of proteins involved in mitochondrial dynamics. Our results demonstrated that CaSR activation induced cardiomyocyte apoptosis through the mitochondrial dynamics mediated apoptotic pathway in hypertensive hearts.

Cortistatin inhibits calcification of vascular smooth muscle cells by depressing osteoblastic differentiation and endoplasmic reticulum stress.

Accumulating evidence has indicated that vascular smooth muscular cells (VSMCs) play an important role in the development of vascular calcification (VC). Cortistatin (CST), a novel bio-active peptide, has been shown to exert multiple protective effects on the cardiovascular system. However, the role and possible mechanism of CST in VC remain unclear. Therefore, we used ß-glycerophosphoric acid (ß-GP) to induce calcification in rat and human VSMCs to determine the effects of CST on osteoblastic differentiation and VSMC mineralization in vitro. Compared with the control, ß-GP significantly increased alkaline phosphatase (ALP) activity and calcium content in cultured rat and human VSMCs, as well as multicellular node formation and calcium deposition, as confirmed by von Kossa and Alizarin Red S staining assays. After incubating rat and human VSMCs with ß-GP in the presence of different doses of CST (10-8 or 10-7 mol/L), CST clearly reversed the ß-GP-induced increases in ALP activity and calcium content and formation of pathological calcified nodes of VSMCs in a dose-independent manner. Moreover, 10-8 and 10-7 mol/L CST inhibited the phenotypic transformation of VSMCs into osteoblastic cells by decreasing the osteocalcin protein levels, increasing the SM-α-actin protein levels, and reducing endoplasmic reticulum stress by decreasing the protein expression of glucose-regulated protein 94 and CCAAT/enhancer-binding protein homologous protein. In conclusion, CST directly inhibited ß-GP-induced calcification of VSMCs in vitro, probably by suppressing ERS and phenotypic transformation of VSMCs into osteoblastic cells. These results indicate that CST represents a potential target for the prevention and treatment of VC.

MicroRNA-377 Mediates Cardiomyocyte Apoptosis Induced by Cyclosporin A.

Cyclosporin A (CsA) is a potent immunosuppressant that has wide clinical applications for autoimmune disorders and prevention of rejection in organ transplantation. However, its liver, kidney, and heart toxicity has limited its use. In this study, we investigated the mechanism by which CsA induced cardiomyocyte apoptosis. Through microarray analysis, we found that the expression of microRNA (miR)-377 was regulated by CsA. Ectopic overexpression of miR-377 led to increased apoptosis in cardiomyocytes, as evidenced by an increased number of apoptotic cells, increased levels of proapoptotic proteins, decreased levels of antiapoptotic proteins, and elevated caspase pathway activity. We also found that miR-377 was required for CsA-induced apoptosis, because inhibition of miR-377 expression markedly reduced the ability of CsA to induce cardiomyocyte apoptosis. In addition, we identified XIAP and NRP2 as direct targets for miR-377. The expression levels of these 2 antiapoptotic proteins were negatively regulated by miR-377, as well as by CsA both in vitro and in vivo. Our data suggested that CsA induced cardiomyocyte apoptosis through the miR-377-XIAP/NRP2 axis.

Tissue factor pathway inhibitor gene transfer prevents vascular smooth muscle cell proliferation by interfering with the MCP-3/CCR2 pathway.

Increased vascular smooth muscle cell (VSMC) proliferation substantially contributes to the pathogenesis of atherosclerosis and intimal hyperplasia after vascular injury. The importance of inflammation in VSMC proliferation is now being recognized. Preventing the inflammatory response is one therapeutic strategy that can be used to inhibit atherosclerosis in the clinic. The present study, using RNA interference and gene transfer techniques, was conducted to investigate the effect of monocyte chemotactic protein-3 (MCP-3) on VSMC proliferation that is a result of TNF-α stimulation, and whether overexpression of the tissue factor pathway inhibitor (TFPI) gene could prevent VSMC proliferation by blocking the MCP-3/CC chemokine receptor 2 (CCR2) pathway. Mouse VSMCs were infected in vitro with recombinant adenoviruses containing either mouse MCP-3-shRNA (Ad-MCP-3-shRNA), the TFPI gene (Ad-TFPI), or the negative control, which was shRNA encoding the sequence for EGFP (Ad-EGFP) or DMEM only. The cells were then stimulated with TNF-α for different time periods on the third day after gene transfer. The data show that VSMC proliferation in the Ad-MCP-3-shRNA and Ad-TFPI groups was markedly decreased using BrdU ELISA and MTT assays; MCP-3-shRNA and TFPI inhibited the expression of MCP-3 and CCR2 after long-term stimulation and inhibited the phosphorylation of ERK1/2 and AKT after short-term stimulation, as shown by ELISA and western blot analysis. This study provides convincing evidence that clarifies the effect of the proinflammatory factor MCP-3 in promoting VSMC proliferation. Our data also show, for the first time, that TFPI has an anti-proliferative role in TNF-α stimulated-VSMCs at least partly by interfering with the MCP-3/CCR2 pathway and then via suppression of the ERK1/2 and PI3K/AKT signaling pathways. We conclude that TFPI gene transfer may be a safe and effective therapeutic tool for treating atherosclerosis and intimal hyperplasia.