• 생명과학회지
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• 제26권2호
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• pp.226-233
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• 2016

혈관평활근세포의 세포자멸사는 죽상경화증을 비롯한 여러 혈관질환에서 일어나며, 죽상판의 불안정화에 중요한 역할을 한다. Oxysterol은 혈관평활근세포의 세포자멸사를 야기하는데, 죽상경화 병변에는 비효소적으로 생성되는 주된 oxysterol인 7-ketocholesterol (7KC)이 대량 존재한다고 알려져 있다. 그러나 7KC에 의한 혈관평활근세포의 세포자멸사에 관하여 밝혀진 자세한 기전은 아직 미흡하다. 본 연구는 7KC가 혈관평활근세포의 세포자멸사를 일으키는 기전을 구명하고자 하였다. Sprague-Dawley계 숫쥐의 대동맥 절편으로부터 배양한 혈관평활근세포에 7KC를 처리하여 혈관평활근세포의 생존력의 변동과 세포자멸사를 각각 methylthiazole tetrazolium bromide 분석법 및 trypan blue 분석법 그리고 유세포 분석법, 면역 형광 측정법, 면역침전법 및 웨스턴블럿 등으로 측정하였다. 7KC는 혈관평활근세포의 생존력을 시간- 및 농도-의존적으로 감소시켰고, 혈관평활근세포내에 지질과산화 최종산물인 4-hydroxynonenal (HNE) 생성을 증가시켰으며, HNE 단독 처리 또한 혈관평활근세포의 생존력을 농도-의존적으로 감소시켰다. 7KC 또는 HNE에 의하여 감소되었던 혈관평활근세포의 생존력은 HNE 생성 억제제인 2,4-dinitrophenyl hydrazine 전처치에 의하여 회복되었다. 더욱이 세포생존 매개인자로 잘 알려져 있는 Akt의 발현이 7KC와 HNE에 의하여 농도-의존적으로 감소되었고, 2,4-dinitrophenyl hydrazine 또는 N-acetylcysteine 전처치에 의하여 회복되었다. 단백질분해효소복합체 억제제인 lactacystin은 7KC에 의한 세포자멸사와 Akt 감소는 억제하였지만 7KC에 의한 HNE 생성은 억제하지 못하였다. HNE에 결합된 Akt의 양은 7KC와 HNE에 의하여 현저히 증가하였으며 단백질분해효소복합체 억제제인 lactacystin 전처치 시에도 유의하게 증가하였다. 이상의 결과로 보아 죽상경화 병변에서 7KC는 혈관평활근세포내 HNE 생성을 증가시키고, 이 HNE에 결합된 Akt는 단백질분해효소복합체에 의하여 분해되는 것으로 보이며, 이와 같은 기전에 의하여 죽상판의 불안정화가 촉진되는 것으로 생각된다.

• 대한한의학회지
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• 제21권2호
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• pp.60-67
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• 2000

Objectives : Radix Angelicae Gigantis(RAG) and Resina Ferulae(RF) have been used in oriental medicine or folk medicine to increase stamina. The aim of this study was the characterization of the mechanism of action of RAG and RF on smooth muscle and macrophages in rats to find new substances for the treatment of erectile dysfunction, cardiovascular diseases and immune dysfunction. Methods : We investigated the effects of the water extracts of RAG and RF on phenylephrine or KCl-contracted rat endothelium-denuded aorta, the production of NO in vascular smooth muscle cell (VSMC) and the production of NO and induction of iNOS in the $IFN-{\gamma}-primed$ RAW 264.7 cells. Results : The water extracts of the RAG and RF showed significant concentration-dependent relaxation effects on phenylephrine or KCl-contracted rat endothelium-denuded aorta. It also reduced the tension of the rat endothelium denuded aorta which was contracted in $Ca^{2+}-free$ media. On the other hand, it increased production of NO in VSMC which was stimulated with $IL-{\beta}$ or $IL-{\beta}$ plus $IFN-{\gamma}$. The water extracts of RAG and RF increased production of NO and induction of iNOS in the $IFN-{\gamma}-primed$ RAW 264.7 cells. Conclusions : According to the above results, the water extracts of RAG and RF relaxed the smooth muscle effectively and increased the production of NO in VSMC and macrophages. So, these herbs can be applied to erectile dysfunction, hypertension, angina pectoris, artherosclerosis and a defense defect for virus or microbe.

• The Korean Journal of Physiology and Pharmacology
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• 제19권5호
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• pp.421-426
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• 2015

The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through $G_0/G_1$ to S phase of the cell cycle, as measured by [$^3H$]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at $G_0/G_1$ phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis.

• 동의생리병리학회지
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• 제23권5호
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• pp.974-979
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• 2009

Proliferation of vascular smooth muscle cell(VSMC) is one of the key features in onset of atherosclerosis and restenosis after vascular surgery such as stent implant. Atherosclerotic plaques are usually composed of collagen, elatsin and smooth muscle cells. Release of matrix metalloproteinases(MMPs) is considered to have correlation with development of atherosclerotic plaques. Based on the hypothesis that MMP inhibition would be helpful in the treatment of atherosclerosis, we investigated inhibition of MMP activity and migration of TNF-$\alpha$-induced human aortic smooth muscle cell(HASMC) by Cinnamomi Ramulus(CC). The result from gelatin zymography showed that CC inhibited MMP-9 activity in a dose-dependent manner. In addition, CC considerably inhibited the migration of HASMC induced by TNF-$\alpha$, while it showed little cytotoxic effect on HASMC. These results suggest that CC can be a potential anti-atherosclerotic agent through inhibition of MMP-9 activity and SMC migration.

• Environmental Analysis Health and Toxicology
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• 제19권1호
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• pp.109-117
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• 2004

The purpose of the present study was to examine the effect of diesel exhaust particles on human aortic vascular smooth muscle cells (VSMCs). DNA synthesis, cell viability and morphology of VSMCs after treatment of diesel exhaust particles (DEP) and fine particulate matter (PM$_{2.5}$) were assayed. PM$_{2.5}$ inhibited the DNA synthesis of VSMCs in a concentration -dependent manner, whereat DEP did not affect VSMCs up to 50$\mu\textrm{g}$/mL. These results were confirmed by morphological examination of VSMCs. PM$_{2.5}$ showed a dose-dependent cytotoxicity of VSMCs by MTT assay. Fraction 4 (organic acids) and fraction 8 (moderately polar compounds) showed the most potent inhibition of DNA synthesis of VSMCs, and fraction 7 (slightly polar compounds), fraction 9 (higher polar compounds), and fraction 6 (aromatic compounds) were next order. These results were confirmed by morphological examination of VSMCs. These results suggest that PM$_{2.5}$ inhibits the DNA synthesis of VSMCs through the cytotoxicity.oxicity.

• The Korean Journal of Physiology and Pharmacology
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• 제9권2호
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• pp.117-123
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• 2005

A cumulative evidence indicates that consumption of tea catechin, flavan-3-ol derived from green tea leaves, lowers the risk of cardiovascular diseases. However, a precise mechanism for this cardiovascular action has not yet been fully understood. In the present study, we investigated the effects of different green tea catechins, such as epigallocatechin-3 gallate (EGCG), epigallocatechin (EGC), epicatechin-3 gallate (ECG), and epicatechin (EC), on angiotensin II (Ang II)-induced hypertrophy in primary cultured rat aortic vascular smooth muscle cell (VSMC). [$^3H$]-leucine incorporation was used to assess VSMC hypertrophy, protein kinase assay, and western blot analysis were used to assess mitogen-activated protein kinase (MAPK) activity, and RT-PCR was used to assess c-jun or c-fos transcription. Ang II increased [$^3H$]-leucine incorporation into VSMC. However, EGCG and ECG, but not EGC or EC, inhibited [$^3H$]-leucine incorporation increased by Ang II. Ang II increased phosphorylation of c-Jun, extracellular-signal regulated kinase (ERK) 1/2 and p38 MAPK in VSMC, however, EGCG and ECG , but not EGC or EC, attenuated c-Jun phosphorylation increased by Ang II. ERK 1/2 and p38 MAPK phosphorylation induced by Ang II were not affected by any catechins. Ang II increased c-jun and c-fos mRNA expression in VSMC, however, EGCG inhibited c-jun but not c-fos mRNA expression induced by Ang II. ECG, EGC and EC did not affect c-jun or c-fos mRNA expression induced by Ang II. Our findings indicate that the galloyl group in the position 3 of the catechin structure of EGCG or ECG is essential for inhibiting VSMC hypertrophy induced by Ang II via the specific inhibition of JNK signaling pathway, which may explain the beneficial effects of green tea catechin on the pathogenesis of cardiovascular diseases observed in several epidemiological studies.

• 생명과학회지
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• 제22권12호
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• pp.1595-1599
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• 2012

혈관 증식 질환에서 세포주기 활성화와 진행은 중요한 치료 목적으로 사용된다. Luteolin는 glycosylated 형태로 샐러리, 후추, 들깨 잎 그리고 카밀레 차에 존재하며 항돌연변이, 항종양, 항산화 그리고 항염증을 나타낸다. 본 연구에서는 흰쥐 동맥으로부터 분리한 혈관평활근세포를 배양하여 소태아혈청으로 유도된 증식에서 luteolin 효과에 대해 조사했다. Luteolin이 5% 소태아혈청으로 유도된 흰쥐의 혈관평활근세포 증식과 DNA 합성을 5, 20 그리고 $50{\mu}M$에서 억제했다. 혈관평활근세포 증식을 각각 29.6, 50.8 그리고 83.1% 억제했고 DNA 합성은 각각 25.8, 57.6 그리고 81.0% 억제했다. 게다가, 유세포분석 결과 소태아혈청으로 유도된 혈관평활근세포의 세포주기는 luteolin에 의해 차단되었다. 이러한 결과는 세포독성에 의해서도 나타날 수 있기 때문에 WST-1 분석으로 세포독성을 확인한 결과 세포독성 없이 세포주기를 차단하는 효과임을 확인했다. 이상의 결과들은 luteolin이 혈관스텐트와 동맥경화의 치료를 위한 의미있는 항증식 물질임을 보여준다.

• The Korean Journal of Physiology and Pharmacology
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• 제10권3호
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• pp.161-165
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• 2006

NO and cyclooxygenase-2 (COX-2) are contributes to vascular inflammation induced by various stimulation. The mechanism, which explains a linkage between NO and COX-2, could be of importance in promoting pathophysiological conditions of vessel. We investigated the effects of NO donors on the COX-l and COX-2 mRNA/protein expression, as well as the nitrite production in culture medium of vascular smooth muscle cell (VSMC). VSMC was primarily cultured from thoracic aorta of rat. In this experiments, COX-l and COX-2 mRNA/protein expressions were analysed and nitrite productions were investigated using Griess reagent. VSMC did not express COX-2 protein in basal condition (Nonlipopolysaccharide (LPS) stimulated). In LPS-stimulated experiments, after 3 hours of NO donor pretreatment, LPS $10{\mu}g/ml$ was treated for 24 hours. COX-l protein expressions were unchanged by SNP and NOR-3. NOR-3 significantly increased COX-2 mRNA/protein expression under LPS stimulation. In contrast, SNP did not increase COX-2 mRNA/protein expression under LPS stimulation. Nitrite production was higher in NOR-3 treatment than SNP treatment under LPS stimulation. These results suggest that the expression of COX-2 in VSMC is regulated by NOR-3, COX-2 expressions were depending on the types of NO donor and LPS stimulation in VSMC.

• The Korean Journal of Physiology and Pharmacology
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• 제17권4호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제15권1호
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• pp.31-36
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• 2011

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express $PSY_1$, $PSY_6$, and $PSY_{11}$ receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to $NAD^+$, an agonist of the human $PSY_{11}$ receptor, and $NADP^+$ as well as ATP, an agonist for $PSY_1$ and $PSY_{11}$ receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by $NAD^+$ and $NADP^+$ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. $NAD^+$ and $NADP^+$ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. $NAD^+$- and $NADP^+$-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.