ADP에 의해서 활성화된 혈관평활근 세포의 증식에 관여하는 Integrin의 종류와 신호 전달 경로

박찬복,이지현,류종철,차태준,주승재,이재우 고신대학교 의학부 2004 高神大學校 醫學部 論文集 Vol.19 No.1

Background: Adenosine diphosphate(ADP), which is usually secreted from activated platelets activates integrin αvβ3. αvβ5, and α5β1 on human vascular smooth muscle cells. Integrins have an important role in the proliferation of vascular smooth muscle cells. ADP may stimulate the proliferation of vascular smooth muscle dells by the activation of integrins. Integrins mediating ADP-stimulate the proliferation of vascular smooth muscle cells and signal transduction pathway of ADP stimulation were investigated in this study. Methods: MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate ADP or ATP-stimulated proliferation of human aortic smooth muscle cells (HASMC). In some experiments, HASMC were incubated with U73122, an inhibitor of phospholipase C, or monoclonal antibodies (mAb) to integrins after ADP of ATP stimulation. Results: ADP and ATP increased the proliferation of HASMC in a dose-dependent manner. U73122 inhibited ADP or ATP stimulated proliferation of HASMC ADP-stimulated proliferation of HASMC was inhibited either by c7E3, a blocking mAb to integrin β3 or by LM609, a blocking mAb to integrin αvβ3(37% and 26% inhibition, respectively: p<0.05), but neither by P1F5, a blocking mAb to integrin αvβ5 nor by JBSS, a blocking mAb to integrin α5β1. Conclusion: These results indicate that ADP increases the proliferation of human vascular smooth muscle cells through phospholipase C pathway, and only integrin αvβ3 mediates ADP-stimulated proliferation of human vascular smooth muscle cells.

ADP에 의해서 활성화된 혈관평활근 세포의 증식에 관여하는 Integrin의 종류와 신호 전달 경로

박찬복,이지현,류종철,차태준,주승재,이재우 고신대학교(의대) 고신대학교 의과대학 학술지 2004 고신대학교 의과대학 학술지 Vol.19 No.1

Background : Adenosine diphosphate (ADP),which is usually secreted from activated platelets, activates integrin arb3, arb5, and a5b1 on human vascular smooth muscle cells. Integrins have an important role in the proliferation of vascular smooth muscle cells. ADP may stimulate the proliferation of vascular smooth muscle cells by the activation of integrins. Integrins mediating ADP=stimulated proliferation of vascular smooth muscle cells and signal transduction pathway of ADP stimulation were investigated in this study. Methods : MIT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate ADP or ATP-stimulated proliferation of human aortic smooth muscle cells (HASMC). In some experiments, HASMC were incubated with U73122, an inhibitor of phospholipase C, or monoclonal antibodies (mAb) to integrins after ADP or ATP stimulation. Results : ADP and ATP increased the proliferation of HASMC in a dose-dependent manner. U73122 inhibited ADP- or ATP- stimulated proliferation of HASMC. ADP-stimulated proliferation of HASMC was inhibited either by c7E3, a blocking mAb to integrin b3 or by LM609, a blocking mAb to integrin arb3 (37% and 26% inhibition, respectively; p<0.05), but neither by P1F5, a blocking mAb to integrin arb5 nor by JBS5, a blocking mAb to integrin a5b1. Conclusion : These results indicate that ADP increases the proliferation of human vascular smooth muscle cells through phospholipase C pathway, and only integrin arb3 mediates ADP-stimulated proliferation of human vascular smooth muscle cells.

Upregulated thioredoxin and its reductase prevent H₂O₂-induced growth inhibition and death in human pulmonary artery smooth muscle cells

Pergamon 2019 Toxicology in vitro Vol.61 No.-

<P><B>Abstract</B></P> <P>The thioredoxin (Trx) system controls cellular redox in vascular smooth muscle cells. The present study investigated the roles of Trx1 and Trx reductase1 (TrxR1) proteins in regulation of cell growth, death, reactive oxygen species (ROS) and glutathione (GSH) levels in hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>)-treated human pulmonary artery smooth muscle (HPASM) cells. H<SUB>2</SUB>O<SUB>2</SUB> induced growth inhibition and cell death in HPASM cells over 24 h. Overexpression of Trx1 and TrxR1 using adenoviruses significantly weakened cell growth inhibition and cell death caused by H<SUB>2</SUB>O<SUB>2</SUB>. Increases in ROS levels including mitochondrial superoxide anion (O<SUB>2</SUB> <SUP>•−</SUP>) were observed as early as 5–30 min after H<SUB>2</SUB>O<SUB>2</SUB> addition. Administration of adTrxR1 attenuated H<SUB>2</SUB>O<SUB>2</SUB>-induced increases in ROS levels at 30–180 min. adTrx1 and adTrxR1 significantly reduced the increases in O<SUB>2</SUB> <SUP>•−</SUP> level in H<SUB>2</SUB>O<SUB>2</SUB>-treated HPASM cells at 24 h. Furthermore, HPASM cells transfected with Trx1 or TrxR1 siRNA showed increases in ROS levels with or without H<SUB>2</SUB>O<SUB>2</SUB> at 5 min. While H<SUB>2</SUB>O<SUB>2</SUB> transiently decreased GSH level at 5 min, Trx1 and TrxR1 siRNA intensified the decrease in GSH level. In conclusion, upregulation of Trx1 and TrxR1 significantly attenuated cell growth inhibition and death in H<SUB>2</SUB>O<SUB>2</SUB>-treated HPASM cells. As a whole, Trx-related adenoviruses diminished H<SUB>2</SUB>O<SUB>2</SUB>-induced ROS level in HPASM cells whereas Trx-related siRNAs increased ROS levels and decreased GSH level in these cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> H<SUB>2</SUB>O<SUB>2</SUB> induced growth inhibition and cell death in HPASM cells over 24 h. </LI> <LI> Upregulation of Trx1 and TrxR1 significantly attenuated cell growth inhibition and death in H<SUB>2</SUB>O<SUB>2</SUB>-treated HPASM cells. </LI> <LI> Trx-related adenoviruses diminished H<SUB>2</SUB>O<SUB>2</SUB>-induced ROS level in HPASM cells. </LI> <LI> Trx-related siRNAs increased ROS levels and decreased GSH level in HPASM cells. </LI> </UL> </P>

쥐의 폐동맥 평활근 세포에서 저산소에 의한 Vascular Endothelial Growth Factor의 발현

노은석,김여향,현명철,이상범,Nho, Un Seok,Kim, Yeo Hyang,Hyun, Myung Chul,Lee, Sang Bum 대한소아청소년과학회 2003 Clinical and Experimental Pediatrics (CEP) Vol.46 No.2

목 적 : 소아 심장병의 주종을 이루고 있는 선천성 심장병 환아들에서 폐동맥 고혈압은 비교적 흔히 발생하지만 매우 치료하기 어려운 합병증이다. 폐동맥 고혈압의 원인과 치료 및 예방에 대해서는 아직 많이 알려지지 않은 실정이므로 이의 원인을 산소결핍이라는 전형을 이용하여 VEGF란 유전인자의 차원에서 규명하고, 나아가서는 폐동맥 고혈압의 치료 및 예방책을 마련하기 위하여 이 연구를 시행하였다. 방 법 : 폐동맥 평활근 세포는 생후 6주 Fischer rat의 주폐동맥을 적출하여 작은 조각으로 잘라 20% fetal bovine serum을 첨가한 DMEM 배지를 사용하여 5% 이산화탄소 배양기에서 배양하였다. 배양된 세포는 평활근 세포에만 선택적으로 염색되는 평활근 myosin과 ${\alpha}$-actin 항체를 이용하여 염색함으로써 순수 평활근 세포임을 확인하였다. 5% 이산화탄소 배양기에서 배양한 대조군 세포와 1 또는 3% $O_2$ tension에서 배양한 실험군 세포에서의 VEGF 발현 차이와 starvation한 군과 하지 않은 군에서의 VEGF 발현 차이를 RT-PCR과 northern blotting을 이용하여 비교하였다. 결 과 : 대조군과 저산소 조건에서 배양한 실험군에서 VEGF 발현 정도는 차이가 없었다. 결 론 : 아직 국내에서는 유전인자 차원에서의 폐동맥고혈압의 원인규명이나 이에 따른 치료에 대한 연구가 전혀 없는 상태이며, 이 연구에 이어 신생쥐와 성숙쥐와의 차이점 및 나아가서 사람과 쥐의 폐동맥 평활근 세포의 차이점 등을 규명할 예정이며, 이번 연구 결과를 바탕으로 폐동맥 고혈압의 원인기전 규명, 치료 및 예방방법 개발에 기여하고자 한다. Purpose : Pulmonary vascular hypertension is a common problem in congenital heart disease, the most common cardiac condition in childhood. However, the mechanisms responsible for this pathologic change, treatment, and prevention are poorly understood. Therefore, we studied the gene expression of vascular endothelial growth factor(VEGF) by using a hypoxic model of the pulmonary artery smooth muscle cells. Methods : The main pulmonary artery and its proximal branches of a 6 wk old Fischer rat were excised. They were cut into multiple small pieces and suspended in DMEM medium supplemented with 20% fetal bovine serum and incubated in 5% $CO_2$-95% air atmosphere. The smooth muscle cells were confirmed by immunostaining with smooth muscle myosin and ${\alpha}$-smooth muscle actin antibodies. The VEGF gene expression in the hypoxic group was compared with the one in control the group as well as the one in the starved group by RT-PCR and Northern blot hybridization. Results : There was no statistically significant difference among the control, hypoxic and starved groups. Conclusion : There are few studies of pulmonary vascular hypertension at the molecular level in Korea. Therefore, we studied the expression of VEGF gene in hypoxic pulmonary vascular smooth muscle cells. Further studies will be needed to find the difference between newly born and adult rats, or human and rat pulmonary vascular smooth muscle cells in gene expression. We hope that the study will lead to a better understanding of pulmonary vascular hypertension.

생체에서 분리된 혈관조직에서 아데노바이러스벡터를 이용한 특정 단백질의 발현

허양훈,김학림 대한약학회 2013 약학회지 Vol.57 No.4

Treatments of vascular disease via modulating the expression of specific proteins by gene transfer have been attempted in various studies over the past few years. Among several methods to deliver genes, adenovirus currently has been used because of a number of positive aspects. In this study, we test adenoviral vector as a potential mediator in the treatment of vascular disease by using freshly isolated vascular tissues not cultured vascular cells. Freshly isolated vascular tissues were directly exposed to adenoviral vector pAd5CMVmcsIRESeGFPpA to check the possibility of GFP expression in different layer of vascular tissues. We found that the GFP expression by using adenoviral vector experiments is mainly focused on the adventitia and failed to detect GFP expression at endothelial layer or vascular smooth muscle layer in vas- cular tissues. However, we also found that several integrin receptors are robustly expressed in vascular smooth muscle, thus the limited expression of protein in vascular smooth muscle are not likely the lack of integrin receptors. In conclusion, adenovirus could not be a good tool for a specific protein expression in vascular smooth muscle cell. Thus, the application of adenovirus as a tool for gene therapy of vascular smooth muscle cells in clinical therapeutic trial need to be optimized further.

Retroviral vector를 이용하여 쥐 과립구 집락자극인자 유전자를 형질이입시킨 혈관평활근세포 이식 후 백혈구계의 변화

박국태,김승택,김영규,오태근,김동운 大韓血液學會 1998 大韓血液學會誌 Vol.33 No.2

Porphyromonas gingivalis Lipopolysaccharide Regulates Migration of Vascular Smooth Muscle Cells

Yeon Kim,So-Jeong Kim,Mi-Kyoung Kim,Hyun-Joo Park,Hyung Joon Kim,Soo-Kyung Bae,Moon-Kyoung Bae 대한구강생물학회 2016 International Journal of Oral Biology Vol.41 No.4

Porphyromonas gingivalis, a foremost periodontal pathogen, has been known to cause periodontal diseases. Epidemiologic evidences have indicated the involvement of P. gingivalis in the development of cardiovascular diseases. In this study, we show that the P. gingivalis lipopolysaccharide increases the mRNA expression and protein secretion of interleukin-6 in vascular smooth muscle cells. We demonstrate that P. gingivalis LPS activates the extracellular signalregulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt, which mediate the IL-6 expression in vascular smooth muscle cells. Also, P. gingivalis LPS stimulates the vascular smooth muscle cell migration, which is a critical step for the progression of atherosclerosis. Moreover, neutralization of the IL-6 function inhibits the migration of vascular smooth muscle cells induced by P. gingivalis LPS. Taken together, these results indicate that P. gingivalis LPS promotes the expression of IL-6, which in turn increases the migration of vascular smooth muscle cells.

Functional expression of smooth muscle-specific ion channels in TGF-β₁-treated human adipose-derived mesenchymal stem cells

Park, Won Sun,Heo, Soon Chul,Jeon, Eun Su,Hong, Da Hye,Son, Youn Kyoung,Ko, Jae-Hong,Kim, Hyoung Kyu,Lee, Sun Young,Kim, Jae Ho,Han, Jin American Physiological Society 2013 American journal of physiology. Cell physiology Vol.305 No.4

<P>Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β<SUB>1</SUB> (TGF-β<SUB>1</SUB>)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β<SUB>1</SUB> dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and <I>h</I>-caldesmon. We observed Ca<SUP>2+</SUP>, big-conductance Ca<SUP>2+</SUP>-activated K<SUP>+</SUP> (BK<SUB>Ca</SUB>), and voltage-dependent K<SUP>+</SUP> (K<SUB>v</SUB>) currents in TGF-β<SUB>1</SUB>-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Ca<SUB>v</SUB>1.2) and T-type (Ca<SUB>v</SUB>3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Ca<SUB>v</SUB>β<SUB>1</SUB> and Ca<SUB>v</SUB>β<SUB>3</SUB> subtypes in TGF-β<SUB>1</SUB>-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BK<SUB>Ca</SUB> channels varied, the major α-subtype BK<SUB>Ca</SUB> channel (K<SUB>Ca</SUB>1.1) clearly increased in the TGF-β<SUB>1</SUB>-induced, differentiated hASCs. Most of the K<SUB>v</SUB> subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β<SUB>1</SUB>-induced, differentiated hASCs. Our results suggest that TGF-β<SUB>1</SUB> induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.</P>

The Expression of Adiponectin Receptors and the Effects of Adiponectin and Leptin on Airway Smooth Muscle Cells

신주화,김정호,이원영,심정연 연세대학교의과대학 2008 Yonsei medical journal Vol.49 No.5

Purpose: Obesity is a major risk factor for asthma and it influences airway smooth muscle function and responsiveness. Adiponectin is inversely associated with obesity and its action is mediated through at least 2 cell membrane receptors (AdipoR1 and AdipoR2). Leptin is positively associated with obesity. We investigated whether human airway smooth muscle (ASM) cells express adiponectin receptors and whether adiponectin and leptin regulate human ASM cell proliferation and vascular endothelial growth factor (VEGF) release. Materials and Methods: Human ASM cells were growth- arrested in serum-deprived medium for 48 hours and then stimulated with PDGF, adiponectin and leptin. After 48 hours of stimulation, proliferation was determined using a cell proliferation ELISA kit. Human AdipoR1 and -R2 mRNA expressions were determined by RT-PCR using human- specific AdipoR1 and -R2 primers. Concentrations of VEGF, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha in cell culture supernatant were determined by ELISA. Results: Both AdipoR1 and AdipoR2 mRNA were expressed in the cultured human ASM cells. However, adiponectin did not suppress PDGF-enhanced ASM cell proliferation, nor did leptin promote ASM cell proliferation. Leptin promoted VEGF release by human ASM cells, while adiponectin did not influence VEGF release. Neither leptin nor adiponectin influenced MCP-1 secretion from human ASM cells. Adiponectin and MIP-1alpha were not secreted by human ASM cells. Conclusion: Human ASM cells expressed adiponectin receptors. However, adiponectin did not regulate human ASM cell proliferation or VEGF release, while leptin stimulated VEGF release by human ASM cells.

Interleukin-24 Suppresses the Growth of Vascular Smooth Muscle Cells by Inhibiting H₂O₂-Induced Reactive Oxygen Species Production

Lee, Ki-Mo,Kang, Haeng-A.,Park, Min,Lee, Hwa-Youn,Song, Min-Ji,Ko, Kisung,Oh, Jae-Wook,Kang, Hyung-Sik S. Karger AG 2012 Pharmacology Vol. No.

<P>Abstract</P><P><B><I>Background/Aim:</I></B> The abnormal growth of vascular smooth muscle cells (VSMCs) induced by reactive oxygen species (ROS) is considered a major pathogenic process in vascular diseases. Interleukin (IL)-24 specifically inhibits cancer cell growth through the induction of cell cycle arrest and apoptosis. However, the role of IL-24 in ROS-induced VSMC growth has not yet been investigated. <B><I>Methods:</I></B> An MTT assay, gene expression analysis, flow cytometry and a scratch wound healing assay were performed to determine the anti-growth effects of IL-24 in H<SUB>2</SUB>O<SUB>2</SUB>-treated mouse vascular aortic smooth muscle (MOVAS) cells. To elucidate the effect of IL-24 on ROS-induced signaling, Western blot analysis was employed. <B><I>Results:</I></B> IL-24 inhibited the growth of normal MOVAS cells treated with H<SUB>2</SUB>O<SUB>2</SUB> by inducing a cell cycle arrest at the G<SUB>0</SUB>/G<SUB>1</SUB> phase through the regulation of p21 and cyclin D1. Furthermore, IL-24 suppressed mRNA expression of vascular endothelial growth factor and platelet-derived growth factor and subsequently decreased the level of cell migration in response to H<SUB>2</SUB>O<SUB>2</SUB>. Interestingly, IL-24 attenuated the H<SUB>2</SUB>O<SUB>2</SUB>-induced ROS production by reducing the mitochondrial H<SUB>2</SUB>O<SUB>2</SUB> production and enhancing the expression of antioxidant enzymes. We also showed that the ability of H<SUB>2</SUB>O<SUB>2</SUB> to induce the PI3K/Akt and Erk signaling pathways was blocked by IL-24. <B><I>Conclusion:</I></B> These findings suggest a novel mechanism in which IL-24 suppresses the growth of normal VSMCs by inhibiting H<SUB>2</SUB>O<SUB>2</SUB>-induced ROS production through the regulation of mitochondrial ROS production and expression of antioxidant enzymes.</P><P>Copyright © 2012 S. Karger AG, Basel</P>