Angiotensin II 와 shear stress 상호작용이혈관염증에미치는영향 측면에서혈관내피세포의기능조절에밀접한연관성을나타낸다. 1) 고혈압과같은혈관의병적인상태는레닌안지오텐신시스템의비정상적인활성으로조직내안지오텐신 II가과발현된다. 이는세포벽에분포하는안지오텐신 II

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1 Original Articles Vol. 17, No. 1, March 2011 ISSN doi: /jksh Copyright c The Korean Society of Hypertension 안지오텐신 II 와 Shear stress 상호작용이혈관염증에미치는영향 최승현 1, 박은혜 1, 오승한 3, 백상홍 1,2 1 가톨릭대학교의과대학심혈관연구실, 2 가톨릭대학교의과대학서울성모병원심혈관센터 3 Department Biology, University of Miami College of Arts and Sciences,Coral Gables, FL, USA Effects of Angiotensin II and Shear Stress Interaction on Vascular Inflammation Sung Hyun Choi, MS 1, Eun Hye Park, MS 1, Yujin Oh, BSc 3, Sang Hong Baek, MD 1,2 1 Laboratory of Cardiovascular Research, The Catholic University of Korea School of Medicine; 2 Division of Cardiovascular Medicine, St. Seoul Mary s Hospital, The Catholic University of Korea School of Medicine, Seoul, Korea; 3 Department Biology, University of Miami College of Arts and Sciences,Coral Gables, FL, USA ABSTRACT Background: Angiotensin II (AngII) and abnormal oscillatory shear stress are highly associated with vascular inflammation including atherosclerosis. However, it is poorly understood how interactions between AngII and shear stress in human aortic endothelial cells (HAEC) are involved in mechanisms by which cellular adhesion molecules are expressed. The purpose of this study was to improve that understanding. Methods: AngII (10-7 M for 6 hr) and two-types of shear stress treatments were used: laminar shear stress (LS: unidirectional, 12 dynes/cm 2 ) and oscillatory shear stress (OS: bi-directional, 5 dynes/cm 2, 1 Hz) in HAEC. Immunoblotting was used to detect expression of cellular adhesion molecules markers such as vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1). Results: AngII significantly increased VCAM1 and ICAM1 expression in HAEC that had been reduced due to pretreatment with telmisartan. AngII-LS co-stimulation and AngII-OS co-stimulation significantly increased VCAM1 and ICAM1 expression in HAEC. The expression levels of VCAM1 and ICAM1 were also, significantly reduced when pretreated with telmisartan. However, VCAM1 and ICAM1 expression were significantly reduced under LS and OS stimulation. Conclusions: Telmisartan may modulate the expressions of VCAM1 and ICAM1 via different types of shear stress in HAEC that are activated by AngII. (J Korean Soc Hypertens 2011;17(1):17-27) Key Words: Vascular cell adhesion molecule 1, Intercellular adhesion molecule 1, Angiotensin II, Shear stress, Human aortic endothelial cell 서론 논문접수일 : , 심사완료일 : , 게재승인일 : 교신저자 : 백상홍주소 : 서울시서초구반포동 505 가톨릭대학교의과대학서울성모병원심혈관센터 Tel: 02) , Fax: 02) whitesh@catholic.ac.kr 본연구의연구비지원은 2007 년대한고혈압학회산학협동연구비수혜로이루어짐. 안지오텐신 II는수용체자극을통해혈관의염증반응을조장하며, shear stress는종류와정도에따라동맥경화 의촉진또는항동맥경화작용을하는등의혈류역학적 J Korean Soc Hypertens 2011;17(1):

2 Angiotensin II 와 shear stress 상호작용이혈관염증에미치는영향 측면에서혈관내피세포의기능조절에밀접한연관성을나타낸다. 1) 고혈압과같은혈관의병적인상태는레닌안지오텐신시스템의비정상적인활성으로조직내안지오텐신 II가과발현된다. 이는세포벽에분포하는안지오텐신 II 수용체의자극을통해 NADPH oxidase 와같은효소의활성화를증가시켜조직내과도한활성산소종의생성을초래한다. 2,3) 활성산소종은조직의직접적인산화로표적기관의손상을초래하기도하며, 수산화라디칼과같은강력한산화물질로전환되어지질과산화및단백질산화와같은이차적으로간접적인표적기관의손상을야기한다. 4,5) 혈관내활성산소종증가와더불어염증반응의증가는혈관내피세포에 vascular cell adhesion molecule 1 (VCAM1) 및 intercellular adhesion molecule 1 (ICAM1) 과같은세포부착분자들의발현을증가시키고, 이와같은현상은단핵구들의혈관내피세포로의부착을유도하여동맥경화를비롯한염증성혈관질환을유발한다. 6,7) Shear stress는혈관벽에작용하는혈류흐름으로정상적인혈류흐름과유사한 laminar shear stress (LS) 와병적상태의와류와유사한 oscillatory shear stress (OS) 로구분한다. 최근보고된연구에따르면, 혈관의병적상태로비정상적인와류가형성되는 OS는세포에서안지오텐신 II 의자극과유사하게 VCAM1, ICAM1 및 E-selectin과같은세포부착단백질의발현을증가시키고, 이로인한동맥경화의진행을촉진시킨다고알려져있다. 8) 이와반대로 LS의자극은혈관내피세포와평활근세포의증식을억제하여이를통한항동맥경화작용이나타난다고알려져있다. 9) 고혈압으로인한혈관내피세포의기능적상실에안지오텐신 II 제1형수용체차단제는세포벽에분포한안지오텐신 II 수용체에가해지는안지오텐신 II의자극을차단하여하위신호전달체계의불활성화를통해고혈압에서유발되는혈관내피세포및평활근세포의증식억제와산화스트레스를감소시켜혈관의염증반응을감소시킬수있다 ) 또한, 본연구팀에서는선천성고혈압백서에 telmisartan을통한고혈압치료후, 심혈관조직에서활성산소종의생성이억제되고, 이로인한심혈관조직 의지질과산화가감소됨을확인하여 telmisartan을통한심혈관조직의항산화효과를보고한바있다. 13) 그러나, 지금까지보고된연구결과들은혈관내피세포에서비정상적인 shear stress인 OS의자극이나안지오텐신 II의각각의세포자극이세포부착분자의발현증가를통해동맥경화유발에각각중요한역할을담당하고있음을시사하고있지만, 안지오텐신 II와 OS의병합처치가인체대동맥내피세포에서세포부착분자들의발현에미치는영향에대한기전연구는미미한실정이다 ) 이에, 본연구는인체대동맥내피세포에서안지오텐신 II와서로다른 shear stress의병합처치가세포부착분자들의발현조절에미치는영향을분석하고, 안지오텐신 II 수용체차단제인 telmisartan을통한안지오텐신 II 제1형수용체차단효과가대동맥내피세포의세포부착분자들의발현에미치는영향을비교분석하고자수행되었다. 대상및방법 1. 인체대동맥내피세포 (HAEC) 배양 Human aortic endothelial cells (HAEC) 은 EGM-2 ( 포함 : 2% FBS, hydrocortison, hfgf-b, VEGF, R3-IGF-1, ascorbic acid, heparin, hegf, amphotericin B; gentamicine, Lonza, Wokingham, UK) 로 37, 5% CO 2 의조건에서배양하였다. 사용된세포는계대배양횟수가 3회이상이나, 7회가초과되지않은세포를사용하였다. 2. 안지오텐신 II에따른 VCAM1 의발현분석 1) 안지오텐신 II에의한 HAEC 의분화능력분석안지오텐신 II의농도에따른 HAEC의분화능력을분석하기위해 CellTiter 95 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI, USA) 을이용하였다. HAEC에서로다른농도의 (10-8 M, 10-7 M, 및 10-6 M) 안지오텐신 II (Sigma-Aldrich, St Louis, MO, USA) 를 18시간동안처리한후, 3-(4,5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-Sulfopheynyl)-2H-tetrazolium (MTS) 18 The Korean Society of Hypertension

3 최승현 박은혜 오승한 백상홍 와 phenazine methosulfate (PMS) 를혼합하여, 20 μl씩분주했다. MTS/ PMS 용액이분주된시료들은 37, 5% CO 2 습윤배양기에서 3시간동안배양하였으며, 배양이완료된시료들은 490 nm에서흡광도를측정하였다. 는 0.5 의각을가지고있는회전이가능한 Teflon cone을이용했다. 19,20) Laminsar shear stress (LS) 는 12 dynes/cm 2 의세기로단방향으로회전시켜자극을주었으며, oscillatory shear stress (OS) 의자극은 5 dynes/cm 2 의세기와 1 Hz 의회전 2) 안지오텐신 II의농도에따른 VCAM1 의발현안지오텐신 II의농도를결정하기위해 6-well plate에배양된 HAEC에서로다른농도의 (10-8 M, 10-7 M, 및 10-6 M) 안지오텐신 II를 18시간동안처리하였다. 안지오텐신 II 처리후, 각세포들은 PRO-PREB (intron, Seoul, Korea) 으로총단백질을추출했고, 추출된단백질은 Bradford 분석법을이용해농도를정량하였다. 각시료의총단백질양을 μg으로전기영동한후, VCAM1 일차항체 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) 를통한면역점적법으로단백질의발현정도를분석하였다. 3) 안지오텐신 II의처리시간에따른 VCAM1 의발현 HAEC에안지오텐신 II를각기다른처리시간으로 (3, 6, 12, 18, 및 24 시간 ) 처리한후, PRO-PREB 을이용해총단백질을추출하였다. 추출된총단백질은전기영동후, VCAM1 항체를이용해단백질의발현을분석하였다. 3. Telmisartan 에의한세포부착분자의발현조절분석 Telmisartan으로안지오텐신 II 제1형수용체자극의차단이세포부착분자들의발현에미치는영향을분석하기위해 10-7 M 농도의 telmisartan (Sigma-Aldrich) 을 HAEC 에 30분간전처리한후안지오텐신 II (10-7 M) 을 6시간동안처리하였다. 그후, 총단백질을추출하여 VCAM1 과 ICAM1의항체 (Santa-Cruz) 를이용해면역점적법으로세포부착분자의단백질발현을분석하였다. 율로양방향회전시켜자극을주었다. 자극이진행되는동안 HAEC은 37, 5% CO 2 조건을유지했다. 2) LS 자극을통한세포배양후, 안지오텐신 II가세포부착분자의발현에미치는영향분석안지오텐신 II와 LS의병합처치가세포부착분자들의발현에미치는영향을분석하기위하여, HAEC을 37, 5% CO 2 조건에서 12 dynes/cm 2 의 LS로 18시간동안자극후, 안지오텐신 II (10-7 M) 를 6시간동안처리하였으며, telmisartan (10-7 M) 은 30분동안안지오텐신 II 처리전처치하였다. HACE에스트레스자극의종료후 PRO-PREB 으로총단백질을추출하였으며, VCAM1과 ICAM1의특이항체를이용하여면역점적법으로단백질의발현을분석하였다. 3) OS 자극을통한세포배양후, 안지오텐신 II가세포부착분자의발현에미치는영향분석안지오텐신 II와 OS의병합처치가세포부착분자들의발현에미치는영향을분석하기위하여, HAEC을 37, 5% CO 2 조건에서 5 dynes/cm 2, 1 Hz의 OS로 18시간동안자극후, 안지오텐신 II (10-7 M) 를 6시간동안처리하였으며, telmisartan (10-7 M) 은 30분동안안지오텐신 II 처리전에처치하였다. HACE에스트레스자극의종료후 PRO-PREB으로총단백질을추출하였으며, VCAM1 과 ICAM1의특이항체를이용하여면역점적법으로단백질발현을분석하였다. 4. 안지오텐신 II와 shear stress의병합처치에따른세포부착분자의발현분석 1) Shear stress 배양된 HAEC에서로다른 shear stress의자극은 cone과 plate 혼합 shear 기계를사용했으며, 사용된 cone과 plate 5. 통계처리본연구의모든실험결과는 평균 ± 표준편차 로기술하였으며, 통계처리는 SPSS ver (SPSS Inc., Chicago, IL, USA) 을이용했다. 본연구의유의성판단은각실험군간 t-test를이용한비교분석에 p값이 0.05인경우에만통계 J Korean Soc Hypertens 2011;17(1):

4 Angiotensin II 와 shear stress 상호작용이혈관염증에미치는영향 적의의를두었으며, 모든실험은 3회씩분리하여실험을진행하여통계처리하였다. 결과 1. 안지오텐신 II의농도에따른 HAEC의증식능력 10-8 M에서 10-6 M의안지오텐신 II 처리결과, 10-6 M의안지오텐신 II 처리군에서대조군에비하여 HAEC의증식능력이유의하게감소되었다 (AngII [0 M] vs. AngII [10-6 M], p < 0.05) (Fig. 1). 2. 안지오텐신 II 의농도에따른 VCAM1 의발현 HAEC에서 10-7 M의안지오텐신 II 처리군은대조군에비하여 VCAM1의발현이유의하게증가되었으나 (AngII [0 M] vs. AngII [10-7 M], p < 0.05) (Fig. 2.), 다른농도의안지오텐신 II 처리군 (10-6 M과 10-8 M) 에서는 VCAM1의발현변화가통계적으로유의하지않았다 (AngII [0 M] vs. AngII [10-8 M], AngII [0 M] vs. AngII [10-6 M], p > 0.05) (Fig. 2). Fig. 2. Analysis of VCAM1 expression according to different concentrations of AngII. The expression of VCAM1 was measured by western blot (A), and quantitative results that normalized with actin are shown (B). AngII was incubated for 18 hours with EBM-2. Data were obtained in at least 3 independent experiments, obtained with different sets of cells. VCAM1, vascular cell adhesion molecule 1; AngII, angiotensin II. HAEC viability assay Fig. 1. MTS (HAEC viability) assay. HAEC viability was significantly decreased in 10-6 M AngII treated group as compared to the control. OD (optical density) ratio. AngII was incubated for 18 hours with EBM-2. *p<0.01 compared to the control. Data were obtained in at least 3 independent experiments, obtained with different sets of cells. MTS, 3-(4,5-demethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-Sulfopheynyl)-2H-tetrazolium; HAEC, human aortic endothelial cells. Fig. 3. Analysis of the VCAM1 expression according to different exposure durations of Ang II. The expression of VCAM1 was measured by western blot (A) and quantitative results that normalized by actin are shown (B). AngII treated concentration was 10-7 M cotreated with EBM-2. Data were obtained in at least 3 independent experiments, obtained with different sets of cells. VCAM1, vascular cell adhesion molecule 1; AngII, angiotensin II. 20 The Korean Society of Hypertension

5 최승현 박은혜 오승한 백상홍 3. 안지오텐신 II 의처리시간에따른 VCAM1 발현분석 HAEC에 10-7 M의안지오텐신 II를시간대별로 (3, 6, 12, 18, 24시간 ) 처리한결과안지오텐신 II의처리 6시간후대조군에비하여 HAEC에서 VCAM1의발현이증가했으며, 24시간후에는 HAEC에서대조군에비하여 VCAM1의발현이감소하는경향을보였으나통계적으로유의하지는않았다 (0시간 vs. 24시간, p > 0.05) (Fig. 3). 위의결과들을바탕으로본연구에서처리된안지오텐신 II의농도는 10-7 M, 처리시간은 6시간을기준으로하여실험을진행하였다. Fig. 5. Representative HAEC morphology after laminar shear stress and oscillatory shear stress. Cell morphologyunder light microscopy of the control group (A), treatment with unidirectional laminar shear stress (12 dynes/cm 2 ) in HAEC (B), and treatment with bidirectional oscillatory shear stress (5 dynes/cm 2, 1 Hz) in HAEC (C). Shear stresses were treated for 18 to 19 hours. HAEC, human aortic endothelial cells. 4. Telmisartan 에의한세포부착분자발현조절 HAEC에서안지오텐신 II 처리군은대조군에비하여 VCAM1의발현이약 1.6배증가하였으나 (Con vs. AngII, p < 0.01) (Fig. 4.), ICAM1의발현은대조군에비하여약 7.1배증가하였다 (Con vs. AngII, p < 0.01) (Fig. 4). Fig. 4. Analysis of the VCAM1 and ICAM1 expression after AngII with and without pre-treatment of ARB (telmisartan). The expression of VCAM1 and ICAM1 was analyzed by western blot (A), and quantitative results that normalized by actin are shown (B, C). VCAM1 and ICAM1 expressions were increased by treatment with AngII as compared to the control, and their expression was significantly reduced after pre-treatment of ARB. The degree of reduction in ICAM1 was larger than VCAM1. AngII: 10-7 M, for 18 hours in EBM-2, ARB: telmisartan, 10-7 M, for 30 minutes before treatment with AngII in EBM-2. Data were obtained in at least 3 independent experiments, obtained with different sets of cells. VCAM1, vascular cell adhesion molecule 1; AngII, angiotensin II. Fig. 6. Analysis of VCAM1 and ICAM1 expressions by interaction between laminar shear stress (LS) and Ang II. The expression levels of VCAM1 and ICAM1 were detected by western blot (A), and quantitative result that normalized by actin are shown (B). LS or LS and AngII costimulation increased VCAM1 and ICAM1 expression levels as compared to the control. VCAM1 expression was significantly reduced by pre-treatment of ARB. LS (force: 12 dynes/cm 2, incubation times: 18 hr), AngII (concentration: 10-7 M, incubation times: 6hr), ARB (telmisartan, concentration: 10-7 M, incubation times: 30 min). *p < 0.01, compared to control, **p < 0.05, compared to control, p< 0.01, compared to LS group. Data were obtainedin at least 3 independent experiments, obtained with different sets of cells. VCAM1, vascular cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1; AngII, angiotensin II. J Korean Soc Hypertens 2011;17(1):

6 Angiotensin II 와 shear stress 상호작용이혈관염증에미치는영향 Table 1. The expression levels of VCAM1 and ICAM1 in LS and OS treatment groups as compared to control group Compared with Control Group (%) LS OS - Ang II TERT TERT+Ang II - Ang II TERT TERT+Ang II VCAM NS NS ICAM LS, lanimar shear stress; OS, oscillatory shear stress; Ang II, angiotensin II; TERT, telmisartan; NS, not significant. VCAM, vascular cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1. Table 2. The expression levels of VCAM1 and ICAM1 in LS and OS treatment groups as compared to each groups control (LS only and OS only) Compared with control group (%) LS OS Ang II TERT TERT+Ang II Ang II TERT TERT+Ang II VCAM1 NS NS NS ICAM NS NS LS, lanimar shear stress; OS, oscillatory shear stress; Ang II, angiotensin II; TERT, telmisartan; NS, not significant. VCAM, vascular cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1. Telmisartan의전처리를통한 HAEC에서안지오텐신 II 제1형수용체차단 (AngII+ARB) 은안지오텐신 II 처리군보다 VCAM1을약 2.1배감소시켰으나 (AngII vs. AngII + ARB, p < 0.01) (Fig. 4), ICAM1 의발현은약 6.4배감소했다 (AngII vs. AngII+ARB, p < 0.01) (Fig. 4). 5. LS 자극을통한세포배양후, 안지오텐신 II가세포부착분자의발현에미치는영향 1) LS의자극이세포부착자의발현에미치는영향 HAEC에서 LS의자극은대조군에비하여 VCAM1 의발현이유의하게증가하였으며 (Con vs. LS, p < 0.01) (Figs. 5, 6), 또한 ICAM1의발현도유의하게증가하였다 (Con vs. LS, p < 0.01) (Fig. 6). 그러나, HAEC에서 LS의자극은 VCAM1의발현이 ICAM1 에비하여더높았다 (Con vs. LS, VCAM1; +Δ2.7배, ICAM1; +Δ1.5배 ) (Table 1). 증가시켰다 (Con vs. AngII+LS, p < 0.01) (Fig. 6). 그러나 VCAM1 의발현에비하여 ICAM1 의발현이더높았다 (Con vs. AngII+LS, VCAM1; +Δ2.3배, ICAM1; +Δ3.1배 ) (Table 1). LS와안지오텐신 II의병합처치군은 LS의단일처치군에비하여 VCAM1의발현에는차이가없었으나 (LS vs. AngII + LS, p > 0.05) (Table 2, Fig. 6), ICAM1의경우약 2배정도더증가되었다 (LS vs. AngII+LS, p < 0.01) (Table 2, Fig. 6). 3) Telmisartan이 LS와안지오텐신 II의병합처치에의한세포부착분자의발현조절에미치는영향 HAEC에서 LS와안지오텐신 II의병합처치와함께 telmisartan을투여하여안지오텐신 II 제 1형수용체의자극을차단한경우, 대조군과비교하여 VCAM1의발현에는변화가없었으나 (Con vs. LS+AngII+ARB, p > 0.05) (Table 1, Fig. 6), ICAM1의발현은약 4.7배증가되었다 (Con vs. LS+AngII+ARB, p < 0.01) (Table 1, Fig. 6). 2) LS 자극을통한세포배양후, 안지오텐신 II가세포부착분자의발현에미치는영향 HAEC에서 LS와안지오텐신 II의병합처치는대조군에비하여 VCAM1의발현을유의하게증가시켰고 (Con vs. AngII+LS, p < 0.01) (Fig. 6), ICAM1 의발현또한유의하게 LS+ARB+AngII군은 LS와안지오텐신 II의병합처치군에비하여 VCAM1의발현이약 2배감소했으나 (LS+AngII vs. LS+AngII+ARB, p < 0.01) (Table 2, Fig. 6), ICAM1의발현은약 1.5배증가하였다 (LS+AngII vs. LS+AngII+ARB, p < 0.01) (Table 2, Fig. 6). 22 The Korean Society of Hypertension

7 최승현 박은혜 오승한 백상홍 2) OS 자극을통한세포배양후, 안지오텐신 II가세포부착분자의발현에미치는영향 HAEC에서 OS와안지오텐신 II 병합처치는대조군에비하여 VCAM1의발현을유의하게증가시켰으며 (Con vs. AngII+OS, p < 0.01) (Fig. 7), 대조군에비하여 ICAM1의발현도유의하게증가시켰다 (Con vs. AngII+OS, p < 0.01) (Fig. 7). HAEC에서 OS와안지오텐신 II의병합처치는 OS의단일처치군과같이 ICAM1 의발현증가가 VCAM1의발현증가에비하여높은결과를보였다 (Con vs. AngII+OS, VCAM1; +Δ1.7배, ICAM1; +Δ6.4배, Table 1). 그러나, HAEC에서 OS와안지오텐신 II의병합처치는 OS 단일처치군에비하여 VCAM1 과 ICAM1 의발현에차이가없었다.(OS vs. AngII+OS, VCAM1; p > 0.05, ICAM1; p > 0.05) (Table 2, Fig. 7) Fig. 7. Analysis of VCAM1 and ICAM1 expressions by interaction between oscillatory shear stress (OS) and Ang II. The expression levels of VCAM1 and ICAM1 were detected by western blot (A) and quantitative result that normalized by actin are shown (B) OS or OS and AngII costimulation increased VCAM1 and ICAM1 expression levels as compared to the control. ICAM1 expression was significantly reduced by pre-treatment of ARB. LS (force: 12 dynes/cm 2, incubation times: 18 hr), AngII (concentration: 10-7 M, incubation times: 6hr), ARB (telmisartan, concentration: 10-7 M, incubation times: 30 min). *p < 0.01, compared to control, **p < 0.05, compared to control, p < 0.01, compared to LS group. VCAM1, vascular cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1; LS, laminar shear stress. 6. OS 자극을통한세포배양후, 안지오텐신 II가세포부착분자의발현에미치는영향 1) OS 의자극이세포부착분자의발현에미치는영향 HAEC 에서 OS 의처치는대조군에비하여 VCAM1 의 3) Telmisartan 이 OS와안지오텐신 II의병합처치에의한세포부착분자의발현조절에미치는영향 HAEC에서 OS와안지오텐신 II의병합처치와함께 telmisartan을투여하여안지오텐신 II 제 1형수용체의자극을차단한경우 (OS+ARB+AngII) 대조군에비하여 VCAM1은약 2.8배 (Con vs. OS+ARB+AngII, p < 0.01) (Table 2, Fig. 7), ICAM1 은약 4.5배발현이증가했다 (Con vs. OS+ARB+AngII, p < 0.01) (Table 2, Fig. 7). HAEC에서 OS+ARB+AngII 처치군은 OS와안지오텐신 II의병합처치군에비하여 VCAM1의발현이약 1.7배증가했으나 (OS+AngII vs. OS+ARB+AngII, p < 0.01) (Table 2, Fig. 7), ICAM1의발현은약 0.7배감소했다 (OS+AngII vs. OS+ARB+AngII, p < 0.01) (Table 2, Fig. 7). 고찰 발현을유의하게증가시켰으며 (Con vs. OS, p < 0.01) (Figs. 5, 7), 또한 ICAM1의발현도유의하게증가시켰다 (Con vs. OS, p < 0.01) (Fig. 7). 그러나, HAEC에서 OS 의처치는 VCAM1의발현이 ICAM1에비해더낮게나타났다 (Con vs. OS, VCAM1; +Δ2.3배, ICAM1; +Δ7.2 배, Table 1). 본연구는 HAEC에서안지오텐신 II와서로다른종류의 shear stress의병합처치가세포부착분자인 VCAM1 과 ICAM1의발현은증가시켰지만, 안지오텐신 II 제1형수용체자극차단제인 telmisartan은이들 VCAM1과 ICAM1의발현억제에다른결과를보였다. J Korean Soc Hypertens 2011;17(1):

8 Angiotensin II 와 shear stress 상호작용이혈관염증에미치는영향 혈관내피세포에서세포부착분자의발현증가는단핵구의부착을증가시켜 foam 세포형성을촉진하며, 이는혈관벽의죽상반형성에핵심적인작용을나타낸다. 21,22) 고혈압은동맥경화의발병에중요한위험인자로조직내안지오텐신 II가비정상적으로높게발현되어있다. 이들은세포벽에분포하는수용체자극을통해직접적으로혈관내피세포의기능부전을야기할뿐만아니라, 17,23,24) 산화스트레스의증가와염증반응을매개로한 VCAM1 및 ICAM1과같은세포부착분자의발현증가로혈관의동맥경화초기병변발생을촉진시킨다 ) 본연구결과에서안지오텐신 II에의한 HAEC의자극은 VCAM1과 ICAM1의발현을유의하게증가시켰으며, telmisartan의전처리를통해두분자의발현이유의하게감소되는것을확인하였다 (Fig. 4). 이는 HAEC에서안지오텐신 II에의한 VCAM1과 ICAM1의발현은안지오텐신 II 제1형수용체의신호전달을통해발현이조절됨을강력하게시사한다 ) 또한, HAEC에서안지오텐신 II의자극은세포부착분자중 VCAM1의발현증가폭에비해 ICAM1의발현이더두드러지게증가되었으며 (Fig. 4), 이는최근에보고된몇몇다른연구들과유사한경향성을보인다. 32,33) ICAM1은다양한세포들에서염증성사이토카인들에의해강력하게발현되지만, 이와반대로 VCAM1은제한적으로발현이조절되는것으로알려져있다. 32,33) VCAM1과 ICAM1 은막투과성당단백질 (transmembrane glycoprotein) 로백혈구의 β-integrin과상호작용을나타내며, 특히 ICAM1 은혈관내피세포에발현되어대부분의백혈구의 β2-integrin과작용하여세포부착을조절한다. 24) 하지만, VCAM1 은단핵구의 β1-integrin에선택적이면서도제한적인작용을나타낸다. 29,30,32,33) 이러한결과는 VCAM1 이 ICAM1 에비하여제한적인발현을나타내는것으로보이나, 안지오텐신 II나 TNF-α와같은염증성사이토카인에의한자극을통해 ICAM1 과동력학적으로유사한활성을나타내는것으로생각된다. HAEC에서로다른 shear stress (LS 및 OS) 의자극은정상적인 HAEC에비해세포부착분자의발현을유의하게증가시켰고 (Figs. 6, 7), HAEC에안지오텐신 II와 LS 또는, OS의병용처치도대조군보다세포부착분자들의발현을유의하게증가시켰다 (Figs. 6, 7). 혈류의흐름을통해혈관벽에작용하는 shear stress는크게두가지로분류할수있는데, 정상적인생리적형태의혈관에작용하는 LS는세포부착분자들의발현을감소시켜항동맥경화효과를나타낸다. 34) 반면에, 동맥경화에서 foam 세포의침착으로인한동맥경화의초기병변의발생은정상적인혈류의흐름을방해하여와류를형성시키는데이를 OS라하며, 이와같은비정상적인혈류의흐름은동맥경화의진행을촉진시키는것으로알려져있다. 35,36) 그러나, 상기연구결과에서는 HAEC에서 LS의자극을통한항동맥경화효과를확인할수없었다 (Fig. 6). 이러한결과는상대적으로낮은유속을가지고있는 LS의경우에도병적인상태의 OS와같이혈류의속도에따라혈관내피세포에서세포부착분자들의발현을증가시켜동맥경화의유발을촉진시킬수있을것으로보인다. 37) 더불어 HAEC에서로다른 shear stress의처리결과, LS의처치는 VCAM1의발현이우세하였지만 OS의처치는 ICAM1이우세한다른결과를확인하였다. 이와같이 HAEC에서각각의 shear stress에의한처치에따라세포부착분자의발현양상이다르게나타나는이유는아직명확하게밝혀지지는않았지만, 혈관내피세포를비롯한세포에서세포부착분자의발현은 NFkB의활성에의해조절되는데본연구에서는 LS와 OS의자극이 HAEC에서 NFkB의활성에각기다르게작용함에따른것으로생각된다 ) 안지오텐신제1형수용체차단제인 telmisartan은 LS나 OS와안지오텐신 II의병합처치에따른 HAEC의세포부착분자의발현증가를유의하게감소시켰으나 (Figs. 6, 7), Shear stress의처치종류에따라감소효과가상이하게나타났다 (Table 2). 위의결과는안지오텐신 II와 LS 및 OS의자극이 HAEC 에서안지오텐신 II 제1형수용체의자극으로 VCAM1과 ICAM1의발현을서로다른기전을통해조절함을강력하게사사하나, 이에대한기전설명은아직명확하지않은실정이므로추가적인연구가수반되어야할것으로생각된다. 24 The Korean Society of Hypertension

9 최승현 박은혜 오승한 백상홍 안지오텐신 II는제1형수용체의직접적인자극으로하위신호전달체계를통해 NFkB의활성을촉진하고이를통한세포부착분자들의발현을조절할수있다. 35) Shear stress와같은기계적자극은 enos의상, 하위신호전달체계인 PKA 36) 와 PKG 37) 를통해안지오텐신 II 제1형수용체의발현을조절한다. 이로인한 shear stress의자극후 telmisartan을통한안지오텐신 II 제1형수용체의차단은 HAEC에서 NFkB의활성을통해 VCAM1과 ICAM1 과같은세포부착분자들의발현을조절하는것으로생각된다. 38) 또한, 이들의세포부착분자들의발현조절은 DNA의결합능력이나전사인자의활성도의차이를나타내며, 이로인해본연구에서는안지오텐신 II와함께서로다른 shear stress 처치가 telmisartan에의한제1형수용체차단이후 VCAM1이나 ICAM1의발현조절에차이를보이는것으로생각되나 38,39) 안지오텐신 II와 shear stress 의병용처치가안지오텐신 II 제1형수용체차단제를통한 NFkB의발현조절에미치는 DNA의결합능력이나, 전사인자의활성도에보이는정확한차이를분석한추가연구가병행되야할것이다. 본연구의제한점으로 HAEC에서안지오텐신 II에의한자극이 shear stress보다먼저작용한경우에세포부착분자들의발현조절에미치는영향의차이에대한연구가진행되지않아안지오텐신 II와 shear stress의병합처치에대한세포부착분자들의발현에미치는영향을완벽하게입증하는데제한이있다. 또한 shear stress의세기에따른세포부착분자들의발현과다양한혈관내피세포들에서부착분자들의발현양상에대한추가연구가필요하며, 상기의결과를바탕으로한 in vivo 실험이병행되어야할것으로생각된다. 상기연구결과를통한결론은안지오텐신 II와서로다른 shear stress의병합처치는 HAEC에서세포부착분자들인 VCAM1과 ICAM1의단백질발현을증가시키고, 안지오텐신 II 수용체차단제인 telmisartan은서로다른물리적화학적자극따라 VCAM1과 ICAM1의발현의조절을통해항동맥경화효과를나타낼수있을것으로보인다. 요약 연구배경 : 안지오텐신 II 와병적상태의 oscillatory shear stress 는혈관염증반응촉진과세포부착분자발현의증가 로인한동맥경화발병에높은연관성을보인다. 그러나 안지오텐신 II 와서로다른종류의 shear stress 의병합처 치가세포부착분자의발현에미치는영향에대한연구는 미미한실정이다. 이에인체대동맥내피세포 (HAEC) 에서 안지오텐신 II 와서로다른종류의 shear stress 의병합처 치와함께안지오텐신 II 제 1 형수용체차단이세포부착분 자의발현에미치는영향을비교분석하고자한다. 방법 : 안지오텐신 II (10-7 M, 6 시간 ) 와서로다른종류의 shear stress (laminar shear stress; LS, 12 dynes/cm 2, oscillatory shear stress; OS, 5 dynes/cm 2, 1 Hz) 의자극이 HAEC 에서세포부착분자인 VCAM1 과 ICAM1 의발현에 미치는영향을면역점적법 (immunoblotting) 을이용하여 분석했다. 결과 : 안지오텐신 II 처리는 HAEC 에서 VCAM1 과 ICAM1 의단백질발현을증가시켰으며, telmisartan 의전 처리후세포부착분자들의발현이유의하게감소되었다. 안지오텐신 II 와 LS 및 OS 의병합처치에의한자극에 의해서도세포부착분자의발현이증가하였으나, telmisartan 의전처리후 LS 처리군에서는 VCAM1 의발현이유의하게 감소되었고, OS 처리군에서는 ICAM1 의발현이유의하게 감소되는상반되는양상을보였다. 결론 : 서로다른종류의 shear stress 와안지오텐신 II 의 병합처치는 HAEC 에서세포부착분자의발현을증가시 켰지만, telmisartan 은스트레스의종류에따라서로다른 기전을통해 HAEC 의 VCAM1 과 ICAM1 의발현을조절 하는것으로보인다. 중심단어 : VCAM1, ICAM1, AngII, Shear stress, 인체 대동맥내피세포 References 1. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002;105: J Korean Soc Hypertens 2011;17(1):

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