• Journal of Nutrition and Health
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• v.53 no.6
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• pp.563-569
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• 2020

Purpose: The vascular smooth muscle cells (VSMCs) in mature animals have implicated to play a major role in the progression of cardiovascular diseases such as atherosclerosis. This study aimed at optimizing the protocol in culturing primary VSMCs (pVSMCs) from rat thoracic aorta and investigating the effect of cellular zinc (Zn) deficiency on cell proliferation of the isolated pVSMCs. Methods: The thoracic aorta from 7-month-old Sprague Dawley rats was isolated, minced and digested by the enzymatic process of collagenase I and elastase, and then inoculated with the culture Dulbecco Modified Eagle Medium (DMEM) at 37℃ in an incubator. The primary cell culture morphology was observed using phase-contrast microscopy and cellular Zn was depleted using Chelex-100 resin (extracellular zinc depletion only) or 3 µM N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) (extracellular and intracellular zinc depletion). Western blot analysis was used for the detection of SM22α and calponin as smooth muscle cell marker proteins and von Willebrand factor as endothelial cell marker protein to detect the culture purity. Cell proliferation by Zn depletion (1 day) was measured by MTT assay. Results: A primary culture protocol for pVSMCs from rat thoracic aorta was developed and optimized. Isolated cultures exhibited hill and valley morphology as the major characteristics of pVSMCs and expressed the smooth muscle cell protein markers, SM22α and calponin, while the endothelial marker von Willebrand factor was hardly detected. Zn deprivation for 1 day culture decreased rat primary vascular smooth muscle cell proliferation and this pattern was more prominent under severe Zn depletion (3 µM TPEN), while less prominent under mild Zn depletion (Chelexing). Conclusion: Our results suggest that cellular Zn deprivation decreased pVSMC proliferation and this may be involved in phenotypic modulation of pVSMC in the aorta.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.1
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• pp.39-48
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• 2024

Wogonin, extracted from the roots of Scutellaria baicalensis Georgi, has been shown to suppress collagen deposition in spontaneously hypertensive rats (SHRs). This study was performed to investigate the role and mechanism of wogonin underlying vascular remodeling in SHRs. After injection of SHRs with 40 mg/kg of wogonin, blood pressure in rats was measured once a week. Masson's trichrome staining was conducted to observe the changes in aortas and mesenteric arteries. Vascular smooth muscle cells (VSMCs) isolated from rat thoracic aortas were treated with Angiotensin II (Ang II; 100 nM) in the presence or absence of varying concentrations of wogonin. The viability and proliferation of VSMCs were examined using Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine assay, respectively. The migration of VSMCs was examined using wound healing assay and transwell assay. We found that wogonin administration alleviated hypertension, increased lumen diameter, and reduced the thickness of the arterial media in SHRs. Ang II treatment enhanced the viability of VSMCs, which was inhibited by wogonin in a concentration-dependent manner. Wogonin reversed Ang II-induced increases in the viability, proliferation, and migration of VSMCs. Moreover, wogonin inhibited Ang II-induced activation of mitogen-activated protein kinase (MAPK) signaling in VSMCs. Overall, wogonin repressed the proliferative and migratory capacity of VSMCs by regulating the MAPK signaling pathway, thereby attenuating vascular remodeling in hypertensive rats, indicating that wogonin might be a therapeutic agent for the treatment of vascular diseases.

• IMMUNE NETWORK
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• v.9 no.2
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• pp.64-73
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• 2009

Background: 15d-$PGJ_2$ has been known to act as an anti-inflammatory agent and has anti-hypertensive effects. As a result of these properties, we examined the effect of 15d-$PGJ_2$ on the LPS-induced IL-8/CXCL8 mRNA expression in VSMCs from SHR. Methods: Effect and action mechanism of 15d-$PGJ_2$ on the expression of LPS-induced IL-8/CXCL8 mRNA in VSMCs from SHR and WKY were examined by using real-time polymerase chain reaction, electrophoretic mobility shift assay for NF-${\kappa}B$ avtivity, Western blotting analysis for ERK and p38 phosphorylation and flow cytometry for NAD(P)H oxidase activity. Results: 15d-$PGJ_2$ decreased the expression of LPS-induced IL-8/CXCL8 mRNA in WKY VSMCs, but increased the expression of LPS-induced IL-8/CXCL8 mRNA in SHR VSMCs. The upregulatory effect of 15d-$PGJ_2$ in SHR VSMCs was mediated through PPAR${\gamma}$, and dependent on NF-${\kappa}B$ activation and ERK phosphorylation. However, inhibition of the p38 signaling pathway augmented the upregulatory effect of 15d-$PGJ_2$ on LPS-induced IL-8/CXCL8 mRNA. A NAD(P)H oxidase inhibitor inhibited the upregulatory effect of 15d-$PGJ_2$ on LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs, and an increase in NAD(P)H oxidase activity was detected in SHR VSMCs treated with 15d-$PGJ_2$/LPS. Conclusion: Our results indicate that the upregulatory effect of 15d-$PGJ_2$ on LPS-induced IL-8/CXCL8 expression in SHR VSMCs is mediated through the PPAR${\gamma}$ and ERK pathway, and may be related to NAD(P)H oxidase activity. However, p38 inactivation may also play an important role in 15d-$PGJ_2$/LPS-induced IL-8/CXCL8 expression in SHR VSMCs.

• Food Science of Animal Resources
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• v.40 no.1
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• pp.106-117
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• 2020

Cellular senescence is associated with age-related vascular disorders and has been implicated in vascular dysfunctions. Here, we show that duck oil-loaded nanoemulsion (DO-NE) attenuates premature senescence of vascular smooth muscle cells (VSMCs) triggered by angiotensin II (Ang II). Compared with control nanoemulsion (NE), DO-NE significantly inhibited the activity of senescence-associated β-galactosidase, which is a biomarker of cellular senescence, in Ang II-treated VSMCs. SIRT1 protein expression was dose- and time-dependently induced in VSMCs exposed to DO-NE, but not in those exposed to NE, and SIRT1 promoter activity was also elevated. Consistently, DO-NE also dose-dependently rescued Ang II-induced repression of SIRT1 expression, indicating that SIRT1 is linked to the anti-senescence action of DO-NE in VSMCs treated with Ang II. Furthermore, the SIRT1 agonist resveratrol potentiated the effects of DO-NE on VSMCs exposed to Ang II, whereas the SIRT1 inhibitor sirtinol elicited the opposite effect. These findings indicate that DO-NE inhibits senescence by upregulating SIRT1 and thereby impedes vascular aging triggered by Ang II.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.5
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• pp.299-304
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• 2010

Losartan is a selective angiotensin II (Ang II) type 1 ($AT_1$) receptor antagonist which inhibits vascular smooth muscle cells (VSMCs) contraction and proliferation. We hypothesized that losartan may prevent cell proliferation by activating AMP-activated protein kinase (AMPK) in VSMCs. VSMCs were treated with various concentrations of losartan. AMPK activation was measured by Western blot analysis and cell proliferation was measured by MTT assay and flowcytometry. Losartan dose- and time-dependently increased the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in VSMCs. Losartan also significantly decreased the Ang II- or 15% FBS-induced VSMC proliferation by inhibiting the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. Compound C, a specific inhibitor of AMPK, or AMPK siRNA blocked the losartan-induced inhibition of cell proliferation and the $G_0/G_1$ cell cycle arrest. These data suggest that losartan-induced AMPK activation might attenuate Ang II-induced VSMC proliferation through the inhibition of cell cycle progression.

• Archives of Pharmacal Research
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• v.29 no.1
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• pp.67-72
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• 2006

The abnormal proliferation of aortic vascular smooth muscle cells (VSMCs) plays a central role in the pathogenesis of atherosclerosis and restenosis after angioplasty and possibly also in the development of hypertension. The present study was designed to examine the inhibitory effects and the mechanism of luteolin 7-glucoside (L7G) on the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs. L7G significantly inhibited the PDGF-BB-induced proliferation and the DNA synthesis of the VSMCs in a concentration-dependent manner. Pre-incubation of the VSMCs with L7G significantly inhibited the PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and the phospholipase C $(PLC)-{\gamma}1$ activation. However, L7G had almost no affect on the phosphorylation of $PDGF-{\beta}$ receptor tyrosine kinase, which was induced by PDGF-BB. These results suggest that L7G inhibits the PDGF-BB-induced proliferation of VSMCs via the blocking of $(PLC)-{\gamma}1$, Akt, and ERK1/2 phosphorylation.

• BMB Reports
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• v.50 no.12
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• pp.628-633
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• 2017

Gastrin-releasing peptide (GRP) has been reported to be implicated in the pathogenesis of inflammatory disorders. The migration and proliferation of vascular smooth muscle cells (VSMCs) are key components of vascular inflammation that leads to the development of atherosclerosis. The present study aimed to investigate the molecular effect of GRP on VSMC proliferation and migration. We report that GRP significantly enhanced the proliferation and migration of rat VSMCs. GRP increased mRNA and protein expression of matrix metalloproteinase-2 and -9 (MMP-2/9) in VSMCs. The induction of MMP-2/9 by GRP was regulated by the activation of the signal transducer and activator of transcription-3 (STAT3). In addition, STAT3-knockdown of VSMCs by siRNA or blockade of the GRP receptor inhibited GRP-induced migration of VSMCs. Taken together, our findings indicate that GRP promotes the migration of VSMCs through upregulation of MMP-2/9 via STAT3 activation.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.4
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• pp.307-314
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• 2013

Connective tissue growth factor (CTGF) is a potent pro-fibrotic factor, which is implicated in fibrosis through extracellular matrix (ECM) induction in diabetic cardiovascular complications. It is an important downstream mediator in the fibrotic action of transforming growth factor ${\beta}$ ($TGF{\beta}$) and is potentially induced by hyperglycemia in human vascular smooth muscle cells (VSMCs). Therefore, the goal of this study is to identify the signaling pathways of CTGF effects on ECM accumulation and cell proliferation in VSMCs under hyperglycemia. We found that high glucose stimulated the levels of CTGF mRNA and protein and followed by VSMC proliferation and ECM components accumulation such as collagen type 1, collagen type 3 and fibronectin. By depleting endogenous CTGF we showed that CTGF is indispensable for the cell proliferation and ECM components accumulation in high glucose-stimulated VSMCs. In addition, pretreatment with the MEK1/2 specific inhibitors, PD98059 or U0126 potently inhibited the CTGF production and ECM components accumulation in high glucose-stimulated VSMCs. Furthermore, knockdown with ERK1/2 MAPK siRNA resulted in significantly down regulated of CTGF production, ECM components accumulation and cell proliferation in high glucose-stimulated VSMCs. Finally, ERK1/2 signaling regulated Egr-1 protein expression and treatment with recombinant CTGF reversed the Egr-1 expression in high glucose-induced VSMCs. It is conceivable that ERK1/2 MAPK signaling pathway plays an important role in regulating CTGF expression and suggests that blockade of CTGF through ERK1/2 MAPK signaling may be beneficial for therapeutic target of diabetic cardiovascular complication such as atherosclerosis.

• Journal of Acupuncture Research
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• v.23 no.2
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• pp.139-157
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• 2006

In the present study, I have investigated the bee venom (BV) and melittin (a major component of BV) -mediated anti-proliferative effects, and defined its mechanisms of action in cultured rat aortic vascular smooth muscle cells (VSMCs). BV and melittin $(0.4{\sim}0.8\;{\mu}g/ml)$ effectively inhibited 50 ng/ml platelet derived growth factor BB (PDGF-BB)-induced VSMCs proliferations. The regulation of apoptosis has attracted much attention as a possible means of eliminating excessively proliferating VSMCs. In the present study, the treatment of BV and melittin strongly induced apoptosis of VSMCs. I examined the effects on $NF-{\kappa}B$ activation to investigate a possible mechanism for anti-proliferative effects of BV and melittin, the PDGF-BB-induced $I{\kappa}B{\alpha}$ phosphorylation and its degradation were potently inhibited by melittin, and DNA binding activity and nuclear translocation of $NF-{\kappa}B$ p50 subunit in response to the action of PDGF-BB were potently attenuated by melittin. In further investigations, melittin markedly inhibited the PDGF-BB-induced phosphorylation of Akt but not ERK1/2, upstream signals of $NF-{\kappa}B$. Treatment of melittin also potently induced pro-apoptotic protein p53, Bax, and caspase-3 expression, but decreased anti-apoptotic protein Bcl-2 expression. These results suggest that the anti-proliferative effects of BV and melittin in VSMCs through induction of apoptosis via suppressions of $NF-{\kappa}B$ and Akt activation, and enhancement of apoptotic signal pathway. Based on these results, BV acupuncture can be a candidate as a therapeutic method for restenosis and atherosclerosis.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.3
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• pp.349-360
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• 2018

Autophagy has been studied as a therapeutic strategy for cardiovascular diseases. However, insufficient studies have been reported concerning the influence of vascular smooth muscle cells (VSMCs) through autophagy regulation. The aim of the present study was to determine the effects of VSMCs on the regulation of autophagy under in vitro conditions similar to vascular status of the equipped micro-tubule target agent-eluting stent and increased release of platelet-derived growth factor-BB (PDGF-BB). Cell viability and proliferation were measured using MTT and cell counting assays. Immunofluorescence using an $anti-{\alpha}-tubulin$ antibody was performed to determine microtubule dynamic formation. Cell apoptosis was measured by cleavage of caspase-3 using western blot analysis, and by nuclear fragmentation using a fluorescence assay. Autophagy activity was assessed by microtubule-associated protein light chain 3-II (LC-II) using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured using $H_2DCFDA$. The proliferation and viability of VSMCs were inhibited by microtubule regulation. Additionally, microtubule-regulated and PDGF-BB-stimulated VSMCs increased the cleavage of caspase-3 more than only the microtubule-regulated condition, similar to that of LC3-II, implying autophagy. Inhibitory autophagy of microtubule-regulated and PDGF-BB-stimulated VSMCs resulted in low viability. However, enhancement of autophagy maintained survival through the reduction of ROS. These results suggest that the apoptosis of conditioned VSMCs is decreased by the blocking generation of ROS via the promotion of autophagy, and proliferation is also inhibited. Thus, promoting autophagy as a therapeutic target for vascular restenosis and atherosclerosis may be a good strategy.