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Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module.

ABSTRACT: Nicotine can induce the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs). We have previously shown that cytoskeletal proteins and RhoGDIA, a negative regulator of the Rho GTPase pathway, are involved in the nicotine-induced dysfunction of VSMCs. Here, we found that nicotine can activate the Rho GTPase pathway and induce the synthesis of the cytoskeletal proteins in VSMCs through the activation of intracellular downstream signaling pathways, including targets such as MYPT1, PAK1 and PI3K/AKT. Upon nicotine treatment, the mRNA level of RhoGDIA is increased but protein level is decreased both in vitro and in vivo, which suggested a mechanism of post-translational regulation. By the dual luciferase reporter assay, we identified the microRNA-200b (miR-200b) as a modulator of the behavioural changes of VSMCs in response to nicotine through targeting RhoGDIA directly. Introducing miR-200b inhibitors into cultured VSMCs significantly attenuated cell proliferation and migration. Additionally, we found that hypomethylation in the CpG island shore region of miR-200b was responsible for the nicotine-induced miR-200b up-regulation in VSMCs. The study demonstrates that nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module through the hypomethylation of miR-200b promoter and suggests that epigenetic modifications may play an important role in the pathological progression.

SUBMITTER: Liang D

PROVIDER: S-EPMC5333142 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module.

Liang Dongli D Wang Zhaoxia Z Yan Zhiqiang Z Hou Shangwei S Xu Wangjie W Wang Lianyun L Shang Meisheng M Qiao Zhongdong Z

Scientific reports 20170302

Nicotine can induce the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs). We have previously shown that cytoskeletal proteins and RhoGDIA, a negative regulator of the Rho GTPase pathway, are involved in the nicotine-induced dysfunction of VSMCs. Here, we found that nicotine can activate the Rho GTPase pathway and induce the synthesis of the cytoskeletal proteins in VSMCs through the activation of intracellular downstream signaling pathways, including targets such as M ...[more]

PMID: 28252009

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Project description:Rationale: During the development of atherosclerosis, macrophages secrete exosomes that regulate vascular smooth muscle cells (VSMCs); however, whether nicotine, a major constituent of cigarettes, can modulate this communication in the context of atherogenesis remains to be further studied. In this study, we hypothesized that nicotine induces macrophages to secrete atherogenic exosomes containing microRNAs (miRNAs) to mediate cell-to-cell crosstalk and encourage proatherogenic phenotypes of VSMCs. Methods: In an in vivo study, nicotine was administered subcutaneously to 8-week-old male ApoE-/- mice fed a high-fat diet (HFD) for 12 weeks. Oil red O and hematoxylin and eosin (HE) were used to stain atherosclerotic lesions. Lesion macrophages, VSMCs and exosomes were stained for CD68, α-smooth muscle actin (α-SMA) and CD9, and plaque exosomes were observed by transmission electron microscopy (TEM). Exosomes derived from control macrophages (M-Exos) and from nicotine-treated macrophages (NM-Exos) were isolated by ultracentrifugation, purified by sucrose density gradient centrifugation and characterized based on specific morphology and surface markers. The IVIS® Spectrum in vivo imaging system showed the biodistribution of NM-Exos and M-Exos in circulation. Chitosan hydrogel-incorporated exosomes were applied to simulate exosome secretion in situ. Scratch wound assay, transwell assay and EdU staining were conducted to assess the effects of NM-Exos on the migration and proliferation of mouse VSMCs. RNA-seq was performed to determine the miRNA profiles of M-Exos and NM-Exos. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of miRNAs and mRNAs. The roles of the candidate miRNA and its target gene were assessed using specific RNA inhibitors, siRNAs and miRNA mimics. Western blotting was used to detect candidate protein expression levels. A dual-luciferase reporting system was utilized to confirm the binding of a specific miRNA to its target gene. Results: Nicotine induced atherosclerotic lesion progression and resulted in plaque exosome retention in vivo. The biodistribution of NM-Exos showed that plaque-resident exosomes might be secreted in situ. VSMCs cocultured in vitro with nicotine-stimulated macrophages presented an increased capacity for migration and proliferation, which was exosome-dependent. In addition, isolated NM-Exos helped promote VSMC migration and proliferation. miRNA profiling showed that miR-21-3p was enriched in NM-Exos, and this miRNA was shown to play a key role in regulating NM-Exos-induced effects by directly targeting phosphatase and tension homologue (PTEN). Conclusion: Exosomal miR-21-3p from nicotine-treated macrophages may accelerate the development of atherosclerosis by increasing VSMC migration and proliferation through its target PTEN.

Dataset Information

Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module.

Publications

Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module.

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