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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 1β (IL-1β) 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-1β 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-1β 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-1β 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.

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.

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.

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.

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.

High concentrations of glucose induce cardiomyocyte apoptosis, and contribute to diabetic cardiomyopathy. Relaxin-2 and relaxin-3 are two members of the relaxin peptide family that are cardioprotective. However, it remains unknown whether relaxin-2 or relaxin-3 can regulate apoptosis in high glucose treated-neonatal rat ventricular myocytes (NRVMs). In cultured NRVMs, 33 mmol/l high glucose (HG) increased apoptosis in a time-dependent manner. HG-increased the protein expression of cleaved caspase-8 and -9, two initiators of the extrinsic and intrinsic pathways of apoptosis, Caspase-3 was attenuated by human recombinant relaxin-2 (H2 relaxin) or relaxin-3 (H3 relaxin), indicating that H2 and H3 relaxin inhibited HG-induced apoptosis. Furthermore, endoplasmic reticulum stress (ERS) markers CHOP and caspase-12 were markedly increased in HG-treated NRVMs, leading to apoptosis; this effect was also effectively attenuated by H2 relaxin or H3 relaxin. Treatment of NRVMs with HG reduced autophagy which cannot be adjusted by H2 relaxin or H3 relaxin. In conclusion, HG-induced apoptosis in NRVMs was mediated, in part, by the activation of the extrinsic and intrinsic pathways of apoptosis and ERS, all inhibited by H2 relaxin or H3 relaxin.

In our previous study, we have demonstrated that tissue factor pathway inhibitor (TFPI) gene could induce vascular smooth muscle cell (VSMC) apoptosis. This study was conducted to investigate whether the overexpression of the TFPI gene can induce VSMC apoptosis by inhibiting JAK-2/STAT-3 pathway phosphorylation and thereby inhibiting the expression of such downstream targets as the apoptotic protein Bcl-2 and cell cycle protein cyclin D1. The effect of TFPI on the expression of survivin, a central molecule in cell survival, was also investigated.Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. TUNEL staining and electron microscope were carried out to determine the apoptosis of VSMCs. The expression levels of JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1, Bcl-2 and survivin were examined by western blot analysis.TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th days after gene transfer, the apoptotic rates by TUNEL assay in the TFPI group were 10.91 ± 1.66%, 13.46 ± 1.28% and 17.04 ± 1.95%, respectively, whereas those in the LacZ group were 3.28 ± 0.89%, 4.01 ± 0.72% and 4.89 ± 1.17%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. JAK-2, p-JAK-2, STAT-3, p-STAT-3, cyclin D1 and Bcl-2, which are all involved in the JAK-2/STAT-3 pathway, were detected in the VSMCs on the 3rd, 5th and 7th days after gene transfer, which is consistent with previously demonstrated time points when VSMCs apoptosis occurred. The expression levels of p-JAK-2, p-STAT-3, cyclin D1 and Bcl-2 were significantly decreased over time in the TFPI group (each P0.05) but not in the Ad-LacZ and DMEM groups. However, this attenuation of expression was not observed for JAK-2 and STAT-3 in any of the groups at any time points after gene transfer (each P0.05). The expression level of survivin in the TFPI group also weakened significantly over time compared with the levels in the Ad-LacZ and DMEM groups (each P0.05) at the 3rd, 5th and 7th days after gene transfer.The results demonstrated that TFPI played an apoptosis-inducing role in VSMCs in a manner that involves both the suppression of JAK-2/STAT-3 pathway phosphorylation and the down-regulation of survivin. Our data show for the first time that targeting the JAK-2/STAT-3 pathway and survivin by overexpressing TFPI may be a new avenue for the treatment of restenosis.