Aims: Macrophage (MΦ) infiltration during myocardial infarction (MI) amplifies cardiac inflammation and remodeling. We investigated whether activation of the NRLP3 inflammasome by a calcium sensing receptor (CaSR) in MΦ subsets contributes to cardiac remodeling following MI. Methods and Results: Infiltrated MΦ exhibited biphasic activation after MI; M1MΦ peaked at MI 3d and decreased until MI 14d, whereas M2MΦ peaked at MI 7d and decreased at MI 14d as shown via immunohistochemistry. IL-1β co-infiltrated with both M1MΦ and M2MΦ; IL-1β exhibited the same infiltrating tendency as M1MΦ, which was detected by immunohistochemistry. Increasing ventricular fibrosis was confirmed by Masson staining. CaSR and NLRP3 inflammasome in the MI group were upregulated in MΦ subsets in myocardium and peritoneal MΦ (p-MΦ) compared with the sham groups which were detected by immunofluorescence and western blotting. CaSR-activated NLRP3 inflammasome played a role in M1MΦ via PLC-IP₃ but did not play a role in M2MΦ which were polarized by the THP-1 as shown by western blotting and intracellular calcium measurement. CaSR/NLRP3 inflammasome activation in M1MΦ led to the following effects: upregulated α-sma, MMP-2 and MMP-9, and collagen secretion; and downregulated TIMP-2 in cardiac fibroblasts via IL-1β-IL-1RI, which was detected by coculturing M1MΦ and cardiac fibroblasts. Conclusions: We suggest that the CaSR/NLRP3 inflammasome plays an essential role via the PLC-IP₃ pathway in M1MΦ to promote cardiac remodeling post-MI in rats, including accelerated cardiac fibroblast phenotypic transversion, increased collagen and extracellular matrix (ECM) secretion; however, the CaSR/NLRP3 inflammasome does not play a role in this process in M2MΦ.

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-1β 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-1β 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.

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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 withCST 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.

These results indicate that CST inhibits aortic calcification and osteogenic differentiation of VSMCs likely via the GSK3β/β-catenin and PKC signalling pathways, but not JNK signalling pathway.