호산구의 혈관내피세포 접착에 대한 IL - 13의 영향

송소향(So Hyang Song),김관형(Kwan Hyoun Kim),문화식(Hwa Sik Moon),송정섭(Jeong Sup Song),박성학(Sung Hak Park) 대한천식알레르기학회 1999 천식 및 알레르기 Vol.19 No.1

Background: Infiltration of eosinophils and activated T cells into the airway is a charac- teristic feature of allergic inflammation such as asthma. IL-4 has been shown to mediate adhesion of eosinophils and T cells to endothelial cells by inducing VCAM-1 expression on endothelial surface. IL-13 shares a number of biologic properties with IL-4. Objective: We aimed to investigate the effects of IL-13 on the adhesion of eosinophils to human umbilical vein endothelial cells (HUVEC) and on the expression of VCAM-1 in HUVEC. Method: HUVEC was incubated for 24h with IL-13 (10ng/ml), IL-4 (10ng/ml) and TNF-a (10ng/ml). Surface expression of VCAM-1 in HUVEC was detected using irnmuno-cytochemical stain and reverse transcription-polymearse chain reaction (RT-PCR), and the adhesion of eosinophils to HUVEC was quantitated using eosinophil peroxidase (EPO) assay. Results: The VCAM-1 expression on IL-13-treated HUVEC increased more than in the expression on medium-treated HUVEC (p <0.05). The adhesion of eosinophil to IL-13- treated HUVEC also increased more than in the adhesion to medium-treated HUVEC (p < 0.05). The VCAM-1 expression was synergistically induced by TNF-a and IL-13 (p (0. 05). IL-13 induced VCAM-1 expression and adhesion of eosinophils to HUVEC, similar to IL-4. IL-13 also induced VCAM-1 mRNA expression, with greater expression than with medium and TNF-a(p <0.05). IL-13-induced surface VCAM-1 was associated with expres- sion of mRNA transcripts and adhesion of eosinophils to HUVEC(r=0.89, r=0.93, p <0. 05). Conclusion: These findings demonstrate that IL-13 stimulates HUVEC to express surface VCAM-1 and has a possible role in promoting VCAM-1/VLA-4 dependent accumulation of eosinophils during allergic and other inflammatory responses.

재대정맥 내피세포의 증식에 미치는 글루타민 및 혈청 결핍의 영향

정진우(Jin-Woo Jeong),이혜현(Hye Hyeon Lee),박철(Cheol Park),김원재(Wun-Jae Kim),최영현(Yung Hyun Choi) 한국생명과학회 2013 생명과학회지 Vol.23 No.7

글루타민과 혈청은 세포의 생존과 증식에 기본적으로 요구되지만, 그들의 양적 변화에 따른 내피세포 반응에 관한 신호전달 관련 연구는 거의 이루어지지 않았다. 본 연구에서는 인체 재대정맥 내피세포(human umbilical vein endothelial cells, HUVECs)의 증식에 미치는 글루타민과 혈청의 결핍에 관한 영향을 조사하였다. 본 연구의 결과에 의하면 글루타민 및 혈청이 결핍된 조건에서 배양된 HUVECs의 증식 억제는 apoptosis 유발과 연관성이 있었음을 DAPI staining에 의한 핵의 형태 변화와 유세포 분석을 통하여 확인하였다. 비록 혈청이 결핍된 조건보다 글루타민 결핍에 의한 apoptosis 유발 정도가 더 높게 나타났으나, 두 현상에 의한 apoptosis의 유발은 anti-apoptotic Bcl-2 및 Bcl-xL의 발현 저하와 pro-apoptotic Bax의 발현 증가, IAP family 단백질의 발현 감소, caspase의 활성 증가에 따른 PARP 단백질의 단편화와 연관성이 있었다. 또한 이러한 조건에서 HUVECs의 Bid 발현의 감소 또는 tBid 발현의 증가 현상이 관찰되어, 글루타민 또는 혈청 결핍에 의한 HUVECs의 apoptosis 유발은 세포막 수용체 및 미토콘드리아 활성 경로를 동시에 통하여 이루어지고 있음을 알 수 있었다. 그러나 글루타민과 혈청이 동시에 결핍된 조건에서 배양된 HUVECs의 증식 억제 현상은 각각의 조건에 비하여 증가되었으나 apoptosis는 유발되지 않았다. Glutamine and serum are essential for cell survival and proliferation in vitro, yet the signaling pathways that sense glutamine and serum levels in endothelial cells remain uninvestigated. In this study, we examined the effects of glutamine deprivation and serum starvation on the fate of endothelial cells using a human umbilical vein endothelial cell (HUVEC) model. Our data indicated that glutamine deprivation and serum starvation trigger a progressive reduction in cell viability through apoptosis induction in HUVECs as determined by DAPI staining and flow cytometry analysis. Although the apoptotic effects were more predominant in the glutamine deprivation condition, both apoptotic actions were associated with an increase in the Bax/Bcl-2 (or Bcl-xL) ratio, down-regulation of the inhibitor of apoptosis protein (IAP) family proteins, activation of caspase activities, and concomitant degradation of poly (ADP-ribose) polymerases. Moreover, down-regulation of the expression of Bid or up-regulation of truncated Bid (tBid) were observed in cells grown under the same conditions, indicating that glutamine deprivation and serum starvation induce the apoptosis of HUVECs through a signaling cascade involving death-receptor-mediated extrinsic pathways, as well as mitochondria- mediated intrinsic caspase pathways. However, apoptosis was not induced in cells grown in glutamine- and serum-free media when compared with cells exposed to glutamine deprivation or serum starvation alone. Taken together, our data indicate that glutamine deprivation and serum starvation suppress cell viability without apoptosis induction in HUVECs.

황산마그네슘이 인간제대정맥내피세포의 VEGF와 Caspase-3에 미치는 영향

이귀세라 ( Gui Se Ra Lee ),김사진 ( Sa Jin Kim ),김소영 ( So Young Kim ),이영 ( Young Lee ),박상희 ( Sang Hi Park ),신종철 ( Jong Chul Shin ),유영옥 ( Young Oak Lew ),김수평 ( Soo Pyung Kim ) 대한주산의학회 2004 大韓周産醫學會雜誌 Vol.15 No.3

목적 : 황산마그네슘이 혈관 내피 세포의 caspase-3와 VEGF의 농도에 어떠한 영향을 미치는지를 알아보기 위하여 본 연구를 하였다. 방법 : 임신 말기에 계획된 제왕절개를 시행한 태아의 제대로부터 정맥내피세포(HUVECs)를 추출하여 TNF-α로 처리한 군과 처리하지 않은 두 군으로 나누어 황산마그네슘을 각 농도 별(0, 4, 8, 12 mM)로 가하여 배양한 후 배양 상청액으로 VEGF에 대한 ELISA assay를, caspase-3에 대하여 colorimeter assay를 시행하여 그 농도를 측정하였다. 결과 : HUVECs에서의 VEGF의 발현 농도는 TNF-α로 처리한 경우가 처리하지 않은 경우에 비하여 유의적으로 증가하였다. TNF-α를 가한 경우는 황산마그네슘의 농도에 따른 VEGF의 농도에 유의적인 차이가 없으나 TNF-α를 가하지 않은 경우 황산마그네슘의 농도가 4 mM 및 8 mM인 경우에 가장 낮은 농도로 발현되었다. caspase-3의 농도는 TNF-α와 황산마그네슘의 농도에 따른 유의적인 차이가 없었다. 결론 : HUVECs에서의 VEGF의 발현은 TNF-α에 의하여 증가되나 황산마그네슘의 농도 변화에 따른 영향은 없고, TNF-α에 의한 자극이 없는 경우는 황산마그네슘의 치료적 농도에서 그 발현은 감소되는 반면 caspase-3의 발현은 TNF-α와 황산마그네슘의 영향을 받지 않는 것 같다. Objectives : To determine the effects of magnesium sulfate on Caspase-3 and Vascular endothelial growth factor (VEGF) of human umbilical vein endothelial cells (HUVECs) under presence or absence of Tumor necrosis factor (TNF-α). Methods : HUVECs were isolated from normal term umbilical cords and cultured in several physiolo-gically relevant concentrations of magnesium sulfate with or without exposure of TNF-α. The concentrations of VEGF and caspase-3 were estimated by colorimetric assay and ELISA assay, respectively. Results : The concentration of VEGF in HUVECs significantly increased in the presence of TNF-α compared with in the absence of TNF-α. However, the concentration of VEGF did not show significant difference in several concentrations of magnesium sulfate concentrations with addition of TNF-α and it showed the lowest concentration under 4 mM and 8 mM of magnesium sulfate concentration without addition of TNF-α. The concentration of caspase-3 in HUVECs did not show statistically significant difference with the addition of TNF-α and magnesium sulfate. Conclusion: TNF-α induce HUVECs to stimulate the VEGF, and magnesium sulfate might not have the effects on the expression of VEGF with addition of TNF-α. However, the concentration for treatment of magnesium sulfate inhibits the expression of VEGF without addition of TNF-α. Magnesium sulfate might not have an effect of the expression of caspase-3 with or without addition of TNF-α.

청국장 에탄올 추출물의 혈관내피세포 증식과 이동 촉진효과

황재성,성대일,이환명,정영신,김한복,Hwang, Jae Sung,Sung, Dae Il,Lee, Whan Myung,Chung, Young Shin,Kim, Han Bok 한국미생물학회 2014 미생물학회지 Vol.50 No.3

청국장은 대두발효식품으로 대두 단백질이 발효 중 분해되면서 다양한 생리활성물질이 만들어진다. 혈관내피세포는 혈관의 기능은 물론 신생혈관 생성을 주도하는 세포이다. 뇌졸중이나 심근경색, 뇌경색 등의 혈관관련 질병들은 신생혈관 생성을 촉진하는 치료법이 필요하다. Vascular Endothelial Growth Factor (VEGF)는 새로운 혈관 형성을 촉진하는 역할을 한다. 본 연구에서는 청국장 에탄올추출물(CEE)이 HUVEC (혈관 내피세포) 증식에 미치는 영향을 조사하여 보았다. 청국장 추출물(100, $1000{\mu}g/ml$)을 HUVEC에 처리했을 때, VEGF (10 ng/ml)를 처리한 대조군과 같은 정도로 세포를 증식시켰다. 열처리한 청국장추출물을 혈관 내피세포에 처리해도 세포 증식효과는 마찬가지였다. 청국장이 세포 증식뿐 아니라 HUVEC이동에도 영향을 주는지 sprout 분석법으로 확인하였다. 청국장 추출물($100{\mu}g/ml$)을 처리했을 때, VEGF (10 ng/ml)와 비슷할 정도로 HUVEC 이동이 일어났다. 청국장 추출물에서 HUVEC 증식과 이동에 영향을 미치는 특정 peptide의 분리가 필요할 것이다. In the fermented soybean product known as "chungkookjang", diverse bioactive compounds are produced when the soybean proteins are degraded during fermentation. Vascular endothelial cells (EC) are crucial in vein function and the formation of new vessels. A treatment to stimulate formation of new blood vessels is needed in cerebrovascular diseases that lead to ischaemic stroke and heart attack, as well as for diabetic ulcers. VEGF (Vascular Endothelial Growth Factor) simulates EC formation. The effect of Chungkookjang ethanol extract (CEE) on the proliferation of EC was studied. CEE (100, $1000{\mu}g/ml$) and boiled CEE were as effective as VEGF (10 ng/ml) for the proliferation of human umbilical vascular endothelial cells (HUVEC). The effect of CEE on the migration of HUVEC was investigated using sprout analysis. CEE ($100{\mu}g/ml$) was as effective as VEGF (10 ng/ml) for the migration of HUVEC. Isolation of specific peptides influencing the growth and migration of EC is needed.

NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat) induces NOXA-dependent apoptosis through up-regulation of ATF-4

Liu, Xiaojun,Jiang, Yanan,Wu, Jianfu,Zhang, Wenjuan,Liang, Yupei,Jia, Lijun,Yu, Jinha,Jeong, L.S.,Li, Lihui Elsevier 2017 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>It has been reported that MLN4924 can inhibit cell growth and metastasis in various kinds of cancer. We have reported that MLN4924 is able to inhibit angiogenesis through the induction of cell apoptosis both in vitro and in vivo models. Moreover, Neddylation inhibition using MLN4924 triggered the accumulation of pro-apoptotic protein NOXA in Human umbilical vein endothelial cells (HUVECs). However, the mechanism of MLN4924-induced NOXA up-regulation has not been addressed in HUVECs yet. In this study, we investigated how MLN4924 induced NOXA expression and cellular apoptosis in HUVECs treated with MLN4924 at indicated concentrations. MLN4924-induced apoptosis was evaluated by Annexin V-FITC/PI analysis and expression of genes associated with apoptosis was assessed by Quantitative RT-PCR and western blotting. As a result, MLN4924 triggered NOXA-dependent apoptosis in a dose-dependent manner in HUVECs. Mechanistically, inactivation of Neddylation pathway caused up-regulation of activating transcription factor 4 (ATF-4), a substrate of Cullin-Ring E3 ubiquitin ligases (CRL). NOXA was subsequently transactivated by ATF-4 and further induced apoptosis. More importantly, knockdown of ATF-4 by siRNA significantly decreased NOXA expression and apoptotic induction in HUVECs. In summary, our study reveals a new mechanism underlying MLN4924-induced NOXA accumulation in HUVECs, which may help extend further study of MLN4924 for angiogenesis inhibition treatment.</P> <P><B>Highlights</B></P> <P> <UL> <LI> MLN4924 triggered NOXA-dependent apoptosis in a dose-dependent manner in HUVECs. </LI> <LI> Inactivation of neddylation caused up-regulation of ATF4, a substrate of CRL. </LI> <LI> NOXA was transactivated by ATF4 and further induced apoptosis. </LI> <LI> Knockdown of ATF-4 significantly decreased NOXA expression and apoptotic induction. </LI> <LI> We revealed a new mechanism underlying MLN4924-induced NOXA accumulation in HUVECs. </LI> </UL> </P>

사람제대정맥 내피세포의 한탄바이러스 감염

유석희 ( Yu Seog Hui ),강응택 ( Kang Eung Taeg ),정상인 ( Jeong Sang In ),양용태 ( Yang Yong Tae ),이병직 ( Lee Byeong Jig ) 대한내과학회 1993 대한내과학회지 Vol.44 No.5

Backgrounds: The pathogenesis of korean hemorrhagic fever (Hemorrhagic fever with renal syndromes) is not elucidated until today. The basic mechaism of disease is vascular injury which increases its permeability. This study took aim to demonstrate the tropism of Hataan virus to human umbilical vein endothelial cells (HUVEC) and the infectivity of virus porliferated in HUVEC. Methods: Cultrued HUVEC were inoculated with Hantaan virus (starin 76-118). Replication of Hantaan virus in HUVEC was demonstrated by immunofluorescence and immunohistochemistry using mouse anti·Hantaan virus monoclonal antibody. The infectivity of replicated virus in was determined with Vero E-6 cells. Results: The infectivity of Hantaan virus was 10⁴. After 3 days after virus inoculation, specific fine granular fluorecsence was found in the cytoplasm of HUVEC. After 7 days nearly all of cells showed specific fluorescence. Infected HUVEC could infect Vero E-6 cells. Conclusions: We demonstrated the tropism of Hantaan virus to cultured HUVEC and infectivity of virus in infected HUVEC.

혈관내피세포에서 고농도 포도당으로 유도된 산화스트레스에 대한 조릿대잎 추출물의 보호효과

황지영(Ji-Young Hwang),한지숙(Ji-Sook Han) 한국식품영양과학회 2010 한국식품영양과학회지 Vol.39 No.12

혈관내피세포는 만성적인 고혈당 상태에 노출되면, 반응성 산소기의 급격한 증가로 인해 산화스트레스를 일으키게된다. 이를 보호하기 위해 사용되는 항산화제는 혈관내피세포의 생존율을 높이며 2차적인 질병의 유발을 감소 및 완화시켜서 당뇨합병증에 도움이 되는 것으로 알려져 있는데, 본 연구에서는 항산화 효과가 있는 것으로 알려진 조릿대잎추출물을 이용하여 고농도의 포도당으로 유도된 산화스트레스에 대한 혈관내피세포의 보호효과를 조사하고자 하였다. 즉, 30 mM 포도당에 노출되어 산화적 스트레스가 유발된 HUVECs에 조릿대잎 추출물 ethyl acetate(ESLE) 층을 분주한 결과, 농도 의존적으로 세포 생존율이 증가하였고, 활성산소종 수준과 지질과산화물 가는 ESLE 첨가에 의해 유의적으로 감소하는 것을 확인할 수 있었다. 또한 고농도 포도당으로 GSH 함량과 SOD, GSH-px, catalase 등과 같은 항산화효소의 활성이 감소된 HUVECs에 ESLE를 분주하였을 때 이들의 함량 및 활성이 유의적으로 증가되었으며, western blotting을 통해 ESLE 첨가가 SOD와 catalase 발현을 증가시킴을 확인할 수 있었다. 따라서 ESLE는 혈관내 피세포에서 고농도 포도당으로 인해 유발된 산화적 스트레스에 대해 세포를 보호하는 효과가 있는 것으로 나타났으며, 이는 ESLE가 세포내 활성산소종을 감소시키고 항산화효소계의 활성을 증가시킴으로써 산화스트레스를 감소시킨 결과에 기인하는 것으로 사료되었다. This study was designed to investigate the protective effects of Sasa borealis leaves on high glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). Freeze-dried Sasa borealis leaves were extracted with 70% methanol and followed by a sequential fractionation with dicholoromethan, ethyl acetate, butanol and water. The ethyl acetate fraction from Sasa borealis leaves extract (ESLE) was used in this study because it possessed the strongest antioxidant activity among the various solvent fractions. Exposure of HUVECs to 30 mM high glucose for 48 hr resulted in a significant (p<0.05) decrease in cell viability, glutathion (GSH) concentration, activities of antioxidant enzymes including superoxide dimutase (SOD), glutathion peroxidase (GSH-px) and catalase, and a significant (p<0.05) increase in intracellular ROS and lipid peroxidation formation in comparison to the cells treated with 5.5 mM glucose. ESLE treatment decreased intracellular ROS and lipid peroxidation formation and increased cell viability, GSH concentration and expressions of SOD and catalase in HUVECs. These results suggest that ESLE may be able to protect HUVECs from high glucose-induced oxidative stress, partially through the antioxidative defense systems.

Thrombin and activated protein C inhibit the expression of secretory group IIA phospholipase A₂ in the TNF-@?-activated endothelial cells by EPCR and PAR-1 dependent mechanisms

Bae, J.S.,Rezaie, A.R. Pergamon Press ; Elsevier Science Ltd 2010 Thrombosis research Vol.125 No.1

Introduction: Thrombin and tumor necrosis factor (TNF)-α up-regulate the expression of proinflammatory molecules in human umbilical vein endothelial cells (HUVECs). However, activated protein C (APC) down-regulates the expression of the same molecules. The expression level of secretory group IIA phospholipase A<SUB>2</SUB> (sPLA<SUB>2</SUB>-IIA) is known to be elevated in inflammatory disorders including in sepsis. Here, we investigated the effects of APC and thrombin on the expression of sPLA<SUB>2</SUB>-IIA and extracellular signal-regulated kinase (ERK) in HUVECs. Materials and methods: The expression level of sPLA<SUB>2</SUB>-IIA was quantitatively measured by an enzyme-linked-immunosorbent-assay following stimulation of HUVECs with either thrombin or TNF-α in the absence and presence of the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 and the cholesterol-depleting drug methyl-β-cyclodextrin (MβCD). Results and conclusions: Thrombin had no effect on the expression of sPLA<SUB>2</SUB>-IIA in HUVECs, however, TNF-α potently induced its expression. The prior treatment of cells with APC inhibited expression of sPLA<SUB>2</SUB>-IIA through the EPCR-dependent cleavage of PAR-1. Further studies revealed that if HUVECs were pretreated with the zymogen protein C to occupy EPCR, thrombin also inhibited the TNF-α-mediated expression of sPLA<SUB>2</SUB>-IIA through the cleavage of PAR-1. The EPCR-dependent cleavage of PAR-1 by both APC and thrombin increased the phosphorylation of ERK ½. Pretreatment of cells with either LY294002 or MβCD abolished the inhibitory activity of both APC and thrombin against sPLA<SUB>2</SUB>-IIA expression, suggesting that the protein C occupancy of EPCR confers a PI3-kinase dependent protective activity for thrombin such that its cleavage of the lipid-raft localized PAR-1 inhibits the TNF-α-mediated expression of sPLA<SUB>2</SUB>-IIA in HUVECs.

葛根黃蓮黃芩湯과 疎風活血湯이 HUVEC 내에 MCP-1, ICAM-1, VCAM-1 and eNOS의 유전자 발현량에 대해 미치는 영향

정현진,전상윤,장혜연,김민욱 대한한방내과학회 2020 大韓韓方內科學會誌 Vol.41 No.4

Objectives: The aim of this study was to compare the effects of Galkunhwanglyeonhwanggum-tang (GGT), and Sopunghwalhyeol-tang (SPT) on gene expression of MCP-1, ICAM-1, VCAM-1, and eNOS in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were treated with GGT and SPT at concentrations of 50, 100, and 200 μg/mL. Gene expression of MCP-1, ICAM-1 ,VCAM-1, and eNOS in HUVECs was analyzed by the polymerase chain reaction (PCR), and electrophoresis was performed to verify the gene expression level. Results: 1. MCP-1 gene expression was more strongly decreased by SPT than by GGT. 2. ICAM-1 and VCAM-1 gene expressions were more strongly decreased by SPT than by GGT 3. GGT significantly increased eNOS gene expression, but SPT did not. Conclusions: These findings suggest that GGT and SPT regulate gene expression related to anti-inflammatory effects in HUVECs. Clinical application of these Korean medicines to diseases related to dyslipidemia, such as cardiovascular disease, will require additional in vivo experiments to verify the anti-inflammatory effects of GGT and SPT.

Effects of Astragalus membranaceus on Angiogenesis

Seo Dong-Min,Choi Do-Young,Lee Jae-Dong 대한침구의학회 2007 대한침구의학회지 Vol.24 No.2

Objectives : The aim of this study is to identify and characterize whether Astragalus membranaceus (AM) could induce angiogenesis in vitro and in vivo. Therefore, this study may allow better understanding of the relationship between angiogenesis and wound healing, and ischemia control by AM in oriental medicines and be the basement of developing herbal acupuncture for treatment of wound healing, ischemia and bone fracture. Methods : This study investigated the effects on angeogenesis of AM using human umbilical vein endothelial cells (HUVECs) and Matrigel angiogenesis model. Results : AM significantly increased HUVECs proliferation in a dose-dependent manner. In addition, AM increased migration and tube-like formation in HUVECs. The expression of basic fibroblast growth factor (bFGF), an angiogenesis-stimulating growth factor, was dose-dependently increased by AM. The angiogenic activity of AM was confirmed using an in vivo Matrigel angiogenesis model, showing promotion of blood vessel formation. 목적 : 황기가 혈관 신생 작용이 있는지에 관하여 관찰한다. 황기는 상처의 치유나 허혈성 질환에 효과를 나타내는 것으로 알려져 있다. 이러한 효과가 황기의 혈관 신생 작용과의 관련성을 이해하며 향후 임상에 쓰일 수 있는 황기 약침액 개발을 위한 기초 자료를 목표로 한다. 방법 : 황기의 혈관 신생 작용의 관찰을 위하여 human umbilical vein endothelial cells (HUVECs)와 Matrigel angiogenesis model 을 이용하여 연구하였다. 결과 : 황기는 용량에 따라서 HUVECs의 증식을 나타내었다. 또한 혈관 내피 세포의 이동과 관형 형성을 보였다. 혈관 신생 물질인 basic fibroblast growth factor (bFGF) 가 황기에 의해 증가하였다. Matrigel angiogenesis model에서 황기는 조직학적으로 혈관 형성을 촉진하였으며, 헤모글로빈의 증가를 나타내었다.