A Novel Regulatory Mechanism of Smooth Muscle α-Actin Expression by NRG-1/circACTA2/miR-548f-5p Axis.

RATIONALE: Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor-like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-ß1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown. OBJECTIVE: Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs. METHODS AND RESULTS: Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-ß1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3' untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia. CONCLUSIONS: These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular basis for fine-tuning α-SMA expression and VSMC contraction by transcription factor, circular RNA, and microRNA.

Salvianolic acid B inhibits Ang II-induced VSMC proliferation in vitro and intimal hyperplasia in vivo by downregulating miR-146a expression.

BACKGROUND: Salvianolic acid B (Sal B), a water-soluble compound extracted from Salvia miltiorrhiza that has been widely used to treat cardiovascular diseases for hundreds of years in China, exerts cardiovascular protection by multiple mechanisms. miR-146a is involved in vascular smooth muscle cell (VSMC) phenotypic modulation and proliferation. However, it has yet to be investigated whether the cardiovascular protective effect of Sal B is mediated by miR-146a. PURPOSE: To determine the relationship among the cardiovascular protective effect of Sal B, miR-146a expression, and VSMC proliferation. METHODS: MTS assay and cell counting were performed to evaluate the effect of Ang II, Sal B and miR-146a on VSMC proliferation. The neointima hyperplasia was assessed by hematoxylin/eosin staining. qRT-PCR was used to detect the expression of miR-146a, KLF5, cyclin D1 and PCNA. Western blot analysis was used to detect the expressions of KLF5, cyclin D1 and PCNA after miR-20b-5p was knocked down or overexpressed in VSMC. RESULTS: Sal B suppressed intimal hyperplasia induced by carotid artery ligation and decreased Ang II-induced VSMC proliferation by down-regulating the positive cell-cycle regulators KLF5 and cyclin D1. Further experiments showed that VSMC proliferation and upregulation of KLF5 and cyclin D1 induced by Ang II were accompanied by elevated miR-146a level. Furthermore, overexpression of miR-146a promoted and knockdown of miR-146a reduced Ang II-induced VSMC proliferation and ameliorated intimal hyperplasia induced by carotid artery ligation. Sal B inhibited Ang II-induced VSMC proliferation by suppressing miR-146a expression. CONCLUSION: Sal B inhibited Ang II-induced VSMC proliferation in vitro and intimal hyperplasia in vivo by downregulating miR-146a expression.

Hyperlipidemia-induced apoptosis of hippocampal neurons in apoE(-/-) mice may be associated with increased PCSK9 expression.

Hyperlipidemia is a risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a lipid regulatory gene involved in cell apoptosis. However, the function and mechanism of PCSK9 in neuronal apoptosis following hyperlipidemia remains to be elucidated. The present study established a hyperlipidemic mouse model by feeding a high­fat diet (HFD) to 6­week­old apoE(­/­) mice. Plasma lipid levels, hippocampal lipid accumulation, hippocampal histology, and hippocampal neuronal apoptosis were all monitored for changes. The expression levels of PCSK9, ß­secretase 1 (BACE1), B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), and caspase­3 in hippocampal CA3 and CA1 neurons were also measured. Results demonstrated that a HFD increased the lipid accumulation in the CA3 hippocampus and the levels of plasma lipids, including triglycerides, total cholesterol, low­density lipoprotein, and high­density lipoprotein. In addition, CA3 neurons in the HFD group indicated apparent injuries and increased neuronal apoptosis, which are associated with the expression of Bcl­2, Bax, and caspase­3. A HFD also increased the expression levels of PCSK9 and BACE1. BACE1 promotes cleavage of amyloid precursor proteins to generate ß­amyloid peptide (Aß), which induces neuronal apoptosis. Protein levels of Aß are associated with the observation of amyloid plaques in the hippocampus of the HFD group. The results suggest that hyperlipidemia regulates neuronal apoptosis by increasing PCSK9 and BACE1 expression. Overall, the current study may elucidate the role of lipid metabolism disorder in AD pathogenesis.

Functional regulatory roles of microRNAs in atherosclerosis.

MicroRNAs are a group of endogenously small non-coding RNA molecules that downregulate gene expression at the post-transcriptional level through binding to the 3'UTR of target mRNAs. Recent findings have revealed a key role for microRNAs in the pathophysiological processes of atherosclerosis. As a complex disease, atherosclerosis is influenced by a combination of multiple genes and environmental factors. Both of them play a role in atherogenesis by affecting different types of cells (such as endothelial cell, vascular smooth muscle cell and monocyte/macrophage) function. MicroRNAs control the senescence and dysfunction of endothelial cells, proliferation and migration of vascular smooth muscle cells, and macrophage-driven cytokine production and polarization. By these effects, microRNAs can influence the processes of atherosclerosis and may represent new molecular targets for therapy.