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Title: Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation

Abstract

Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K{sup +} channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.

Authors:
; ; ; ; ;
Publication Date:
OSTI Identifier:
22606187
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 4; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACTIN; ARTERIOSCLEROSIS; CELL PROLIFERATION; CORONARIES; GROWTH FACTORS; INJURIES; KAONS PLUS; MUSCLES; PHENOTYPE; POTASSIUM IONS; RATS; RECTIFIERS

Citation Formats

Qiao, Yong, Tang, Chengchun, E-mail: tangchengchun@medmail.com.cn, Wang, Qingjie, Wang, Dong, Yan, Gaoliang, and Zhu, Boqian. Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.134.
Qiao, Yong, Tang, Chengchun, E-mail: tangchengchun@medmail.com.cn, Wang, Qingjie, Wang, Dong, Yan, Gaoliang, & Zhu, Boqian. Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation. United States. doi:10.1016/J.BBRC.2016.06.134.
Qiao, Yong, Tang, Chengchun, E-mail: tangchengchun@medmail.com.cn, Wang, Qingjie, Wang, Dong, Yan, Gaoliang, and Zhu, Boqian. 2016. "Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation". United States. doi:10.1016/J.BBRC.2016.06.134.
@article{osti_22606187,
title = {Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation},
author = {Qiao, Yong and Tang, Chengchun, E-mail: tangchengchun@medmail.com.cn and Wang, Qingjie and Wang, Dong and Yan, Gaoliang and Zhu, Boqian},
abstractNote = {Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K{sup +} channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.},
doi = {10.1016/J.BBRC.2016.06.134},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 477,
place = {United States},
year = 2016,
month = 9
}
  • Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCsmore » induced a marked increase in rat aortic VSMC proliferation and [{sup 3}H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.« less
  • Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK andmore » DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.« less
  • miR-140-5p is down-regulated in patients with pulmonary arterial hypertension (PAH) and experimental models of PAH, and inhibits hypoxia-mediated pulmonary artery smooth muscle cell (PASMC) proliferation in vitro. Delivery of synthetic miR-140-5p prevents and treats established, experimental PAH. DNA methyltransferase 1 (Dnmt1) is up-regulated in PAH associated human PASMCs (HPASMCs), which promotes the development of PAH by hypermethylation of CpG islands within the promoter for superoxide dismutase 2 (SOD2) and down-regulating SOD2 expression. We searched for miR-140-5p targets using TargetScan, PicTar and MiRanda tools, and found that Dnmt1 is a potential target of miR-140-5p. Based on these findings, we speculated that miR-140-5pmore » might target Dnmt1 and regulate SOD2 expression to regulate hypoxia-mediated HPASMC proliferation, apoptosis and differentiation. We detected the expression of miR-140-5p, Dnmt1 and SOD2 by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays, respectively, and found down-regulation of miR-140-5p and SOD2 and up-regulation of Dnmt1 exist in PAH tissues and hypoxia-mediated HPASMCs. Cell proliferation, apoptosis and differentiation detection showed that miR-140-5p inhibits proliferation and promotes apoptosis and differentiation of HPASMCs in hypoxia, while the effect of Dnmt1 on hypoxia-mediated HPASMCs is reversed. Luciferase assay confirmed that miR-140-5p targets Dnmt1 directly. An inverse correlation is also found between miR-140-5p and Dnmt1 in HPASMCs. In addition, we further investigated whether miR-140-5p and Dnmt1 regulate HPASMC proliferation, apoptosis and differentiation by regulating SOD2 expression, and the results confirmed our speculation. Taken together, these results indicated that miR-140-5p at least partly targets Dnmt1 and regulates SOD2 expression to inhibit proliferation and promote apoptosis and differentiation of HPASMCs in hypoxia. - Highlights: • miR-140-5p and SOD2 are down-regulated in PAH tissues and hypoxia-mediated HPASMCs. • Dnmt1 is up-regulated in PAH tissues and hypoxia-mediated HPASMCs. • miR-140-5p regulates HPASMC proliferation, apoptosis and differentiation. • Dnmt1 and SOD2 regulates HPASMC proliferation, apoptosis and differentiation. • miR-140-5p targets Dnmt1 and regulates SOD2 expression.« less
  • The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba{sup 2+}-sensitive inward rectifier K{sup +} current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level butmore » not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 2+}. On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba{sup 2+}. We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca{sup 2+} entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia. •Kir2.1 up-regulation is responsible for hypoxia-enhanced BEC proliferation.« less
  • Purpose: The aim of the study was to examine the effects of meclofenamic acid on proliferation, clonogenic activity,migratory ability, cell cycle distribution and p44/42 MAPK (mitogen activated protein kinase) expression in serum-stimulated human aortic smooth muscle cells (haSMCs). Methods: haSMCs were treated with meclofenamic acid in three different concentrations (10mM, 100 mM, 200 mM) for 4 days. Then meclofenamic acid-free culture medium was supplemented until day 20. Growth kinetics were assessed. Cell cycle analysis was performed by flow cytometry.Clonogenic activity was evaluated with colony formation assays.Migratory ability was investigated by stimulation with platelet-derived growth factor (PDGF-BB) in 24-well plates withmore » 8 mm pores membrane inserts. p44/42 MAPK was detected by Western blot technique. Results: Meclofenamic acid inhibited the proliferation,clonogenic activity and migratory ability of haSMCs in a dose-dependent manner. Cell cycle analysis revealed a G2/M-phase block. The p44/42MAPK was significantly reduced. Conclusion: Meclofenamic acid inhibits the proliferation and migration of haSMCs. If a sufficient dose of meclofenamic acid can be applied systemically or by local drug delivery it could be a valuable substance to prevent restenosis after angioplasty.« less