Circular RNA CircMAP3K5 Acts as a MicroRNA-22-3p Sponge to Promote Resolution of Intimal Hyperplasia Via TET2-Mediated Smooth Muscle Cell Differentiation.

BACKGROUND: Aberrant expression of circular RNA contributes to human diseases. Circular RNAs regulate gene expression by sequestering specific microRNAs. In this study, we investigated whether circMAP3K5 (circular mitogen-activated protein kinase 5) could act as a competing endogenous microRNA-22-3p (miR-22-3p) sponge and regulate neointimal hyperplasia. METHODS: Circular RNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (SMCs) treated with or without platelet-derived growth factor. Expression levels of circMAP3K5 were assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy or coronary heart disease. The role of circMAP3K5 in intimal hyperplasia was further investigated in mice with adeno-associated virus 9-mediated circMAP3K5 transfection. SMC-specific Tet2 (ten-eleven translocation-2) 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 platelet-derived growth factor-BB significantly reduced expression of circMAP3K5 in human coronary artery SMCs. Wire-injured mouse femoral arteries and diseased arteries from patients with coronary heart disease (where platelet-derived growth factor-BB is increased) confirmed in vivo downregulation of circMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of circMAP3K5 inhibited the proliferation of human coronary artery SMCs. In vivo adeno-associated virus 9-mediated transfection of circMap3k5 (mouse circular Map3k5) 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 (human circular MAP3K5) 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 antiproliferative effect of circMap3k5 on vascular SMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the circMap3k5-mediated antiproliferative effect on vascular SMCs. CONCLUSIONS: We identify circMAP3K5 as a master regulator of TET2-mediated vascular SMC 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.

Restoring the Platelet miR-223 by Calpain Inhibition Alleviates the Neointimal Hyperplasia in Diabetes.

Platelet hyperactivity is the hallmark of diabetes, and platelet activation plays a crucial role in diabetic vascular complications. Recent studies have shown that upon activation, platelet-derived miRNAs are incorporated into vascular smooth muscle cells (VSMCs), regulating the phenotypic switch of VSMC. Under diabetes, miRNA deficiency in platelets fails to regulate the VSMC phenotypic switch. Therefore, manipulation of platelet-derived miRNAs expression may provide therapeutic option for diabetic vascular complications. We seek to investigate the effect of calpeptin (calpain inhibitor) on the expression of miRNAs in diabetic platelets, and elucidate the downstream signaling pathway involved in protecting from neointimal formation in diabetic mice with femoral wire injury model. Using human cell and platelet coculture, we demonstrate that diabetic platelet deficient of miR-223 fails to suppress VSMC proliferation, while overexpression of miR-223 in diabetic platelets suppressed the proliferation of VSMC to protect intimal hyperplasia. Mechanistically, miR-223 directly targets the insulin-like growth factor-1 receptor (IGF-1R), which inhibits the phosphorylation of GSK3ß and activates the phosphorylation of AMPK, resulting in reduced VSMC dedifferentiation and proliferation. Using a murine model of vascular injury, we show that calpeptin restores the platelet expression of miR-223 in diabetes, and the horizontal transfer of platelet miR-223 into VSMCs inhibits VSMC proliferation in the injured artery by targeting the expression of IGF-1R. Our data present that the platelet-derived miR-223 suppressed VSMC proliferation via the regulation miR-223/IGF-1R/AMPK signaling pathways, and inhibition of calpain alleviates neointimal formation by restoring the expression of miR-223 in diabetic platelet.

Correction: TRPC6-dependent Ca2+ signaling mediates airway inflammation in response to oxidative stress via ERK pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TRPC6-dependent Ca2+ signaling mediates airway inflammation in response to oxidative stress via ERK pathway.

Ozone (O3) plays an extremely important role in airway inflammation by generating reactive oxygen species (ROS) including hydrogen peroxide, then promoting redox actions and causing oxidative stress. Evidences indicate that TRPC6 (canonical transient receptor potential channel 6) is a redox-regulated Ca2+ permeable nonselective cation channel, but its role in the setting of oxidative stress-related airway inflammation remains unknown. Here, we found that both TRPC6-/- mice and mice pretreated with SAR7334, a potent TRPC6 inhibitor, were protected from O3-induced airway inflammatory responses. In vitro, both knockdown of TRPC6 expression with shRNA and TRPC6 blockage markedly attenuated the release of cytokines IL-6 and IL-8 induced by O3 or H2O2 in 16HBE cells (human bronchial epithelial cell line). Treatment with O3 or H2O2 enhanced TRPC6 protein expression in vivo and vitro. We also observed that TRPC6-dependent increase of intracellular Ca2+ concentration ([Ca2+]i) was triggered by H2O2, which consisted of the release from intracellular calcium store and the influx of extracellular Ca2+ and could be further strengthened by 6-h O3 exposure in both 16HBE cells and HBEpiCs (primary human bronchial epithelial cells). Moreover, we confirmed that the activation of MAPK signals (ERK1/2, p38, JNK) was required for the inflammatory response induced by O3 or H2O2 while only the phosphorylation of ERK pathway was diminished in the TRPC6-knockdown situation. These results demonstrate that oxidative stress regulates TRPC6-mediated Ca2+ cascade, which leads to the activation of ERK pathway and inflammation and could become a potential target to treat oxidative stress-associated airway inflammatory diseases.