Background: Aberrant expression of circular RNA (CircRNA) contributes to human diseases. CircRNAs regulate gene expression by sequestering specific microRNAs (miRNAs). In this study, we investigated whether CircMAP3K5 could act as a competing endogenous miR-22-3p sponge and regulate neointimal hyperplasia. Methods: CircRNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (HCASMCs) treated with or without PDGF. Expression levels of circular MAP3K5 (CircMAP3K5) was assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy (DCM) or coronary heart disease (CHD). The role of CircMAP3K5 in intimal hyperplasia was further investigated in mice with AAV9-mediated CircMAP3K5 transfection. SMC-specific Tet2 knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which CircMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. Results: RNA sequencing demonstrated that treatment with PDGF-BB significantly reduced expression of CircMAP3K5 in HCASMCs. Wire-injured mouse femoral arteries and diseased arteries from CHD patients (where PDGF-BB is increased) confirmed in vivo downregulation of CircMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of CircMAP3K5 inhibited the proliferation of HCASMCs. In vivo AAV9-mediated transfection of CircMap3k5 specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, CircMAP3K5 was found to sequester miR-22-3p, which in turn inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the anti-proliferative effect of CircMap3k5 on VSMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the CircMap3k5-mediated anti-proliferative effect on VSMCs. Conclusions: We identify CircMAP3K5 as a master regulator of TET2-mediated VSMC differentiation. Targeting the CircMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia including restenosis and atherosclerosis.

Previous genetic studies in mice have shown that functional loss of activin receptor-like kinase 7 (ALK7), a type I transforming growth factor-β receptor, increases lipolysis to resist fat accumulation in adipocytes. Although growth/differentiation factor 3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess conditions, it is unknown how GDF3 production is regulated. Here, we show that a physiologically low level of insulin converts CD11c adipose tissue macrophages (ATMs) into GDF3-producing CD11c macrophages ex vivo and directs ALK7-dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and adipocytes represents a previously unrecognized mechanism by which insulin regulates both fat metabolism and mass.

Platelets have emerged as key inflammatory cells implicated in the pathology of sepsis, but their contributions to rapid clinical deterioration and dysregulated inflammation have not been defined. Here, we show that the incidence of thrombocytopathy and inflammatory cytokine release was significantly increased in patients with severe sepsis. Platelet proteomic analysis revealed significant upregulation of gasdermin D (GSDMD). Using platelet-specific Gsdmd -deficient mice, we demonstrated a requirement for GSDMD in triggering platelet pyroptosis in cecal ligation and puncture (CLP)-induced sepsis. GSDMD-dependent platelet pyroptosis was induced by high levels of S100A8/A9 targeting toll-like receptor 4 (TLR4). Pyroptotic platelet-derived oxidized mitochondrial DNA (ox-mtDNA) potentially promoted neutrophil extracellular trap (NET) formation, which contributed to platelet pyroptosis by releasing S100A8/A9, forming a positive feedback loop that led to the excessive release of inflammatory cytokines. Both pharmacological inhibition using Paquinimod and genetic ablation of the S100A8/A9–TLR4 signaling axis improved survival in mice with CLP-induced sepsis by suppressing platelet pyroptosis.

Activin receptor-like kinase 7 (ALK7) is a type I receptor in the transforming growth factor-β superfamily preferentially expressed in adipose tissue and associated with lipid metabolism.Inactivation of ALK7 signaling in mice results in increased lipolysis and resistance to both genetic and diet-induced obesity. Human genetic studies have recently revealed an association between ALK7 variants and both reduced waist-to-hip ratios and resistance to development of diabetes. The present study found that treatment with a neutralizing monoclonal antibody against ALK7 causes a substantial loss (40-60%) of adipose mass and improves glucose intolerance and insulin resistance in both genetic and diet-induced mouse obesity models. The enhanced lipolysis increased fatty acid supply from adipocytes to promote fatty acid oxidation in muscle and O2 consumption at the whole-body level. The treatment temporarily increased hepatic triglyceride levels, which resolved with long-term antibody treatment. Blocking of ALK7 signals also decreased production of its ligand, growth differentiation factor 3, by downregulating S100A8/A9 release from adipocytes and subsequently interleukin-1β release from adipose tissue macrophages. These findings support the feasibility of potential therapeutics targeting ALK7 as a treatment for obesity and diabetes.

BackgroundKawasaki disease (KD) is an acute vasculitis that may result in permanent coronary artery damage with unknown etiology. Endothelial cell (EC) dysfunction and platelet hyperactivity are the hallmarks of KD. Platelets are involved in the development of endothelial dysfunction. MiR-223 transferred by platelet microparticles (PMPs) has been found to involve in the functional regulation of endothelial cells in sepsis. However, the role of platelet-derived miR-223 in endothelial dysfunction has not yet been investigated in KD.ObjectivesWe seek to investigate the role of platelet-derived miR-223 in endothelial dysfunction of KD vasculopathy.Methods and resultsForty-five acute KD patients and 45 matched controls were randomly recruited in the study. When co-cultured with human coronary artery endothelial cells (HCAECs), KD platelets with higher levels of miR-223 were incorporated into HCAECs, resulting in the horizontal transfer of miR-223. Using KD platelets, PMPs, and platelet-releasate from the same amount of blood co-cultured with HCAECs, we found the increased expression of miR-223 in HCAECs was primarily derived from KD platelets, rather than PMPs or free miRNAs from platelet- releasate. KD platelet-derived miR-223 attenuated TNF-α induced intercellular cell adhesion molecule-1 (ICAM-1) expression in HCAECs. KD platelet-derived miR-223 also suppressed the monocyte adhesion to HCAECs. In vivo, platelet-specific miR-223 knockout (PF4-cre: miR-223flox/flox) C57BL/6 mice and miR-223flox/flox C57BL/6 mice were used. Using Lactobacillus casei cell wall extract (LCWE) to establish KD murine model, we showed that in LCWE-injected PF4-cre: miR-223flox/flox mice, deficiency of platelet-miR-223 exacerbates the medial thickening of the abdominal aorta, increased ICAM-1 expression with concomitant CD45+ inflammatory cells infiltration into the endothelium compared to LCWE-injected miR-223flox/flox mice.ConclusionsThe horizontal transfer of platelet-derived miR-223 suppresses the expression of ICAM-1 in HCAECs, which at least in part attenuates leukocyte adhesion, thereby reducing endothelial damage in KD vasculitis