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1 Antioxidant effect of Estrogen on Bovine aortic endothelial cells 가톨릭대학교의과대학서울성모병원산부인과김미란 Journal of Steroid Biochemistry and Molecular Biology 117 (2009) 74-80

2 Background Objectives Materials & Methods Results & Discussion Limitation Conclusion

3 폐경이행기 (Menopausal transition) Declining estrogen levels -> multifarious metabolic and physiologic changes ->strongly associated with increased incidence of hypertension and susceptibility to CVD

4 Menopause and CVD

5 Menopause and CVD Mainly due to Estrogen deficiency - Change in body composition : android type fat distribution - Metabolic syndrome - More atherogenic lipoprotein profile - Impaired fibrinolytic potential

6 Observational Studies of CVD Risk: ET Compared With HT Rosenberg et al, 1993 Mann et al, 1994 Psaty et al, 1994 Sidney et al, 1997 Grodstein et al, 1999 Swedish cohort Grodstein et al, 2000 Nurses Health Study (NHS) Varas-Lorenzo et al, 2000 ET HT Relative Risk (95% CI)

7 Women s Health Initiative (WHI) Study Outline end in March 2005 F/U : 8.5 yrs <Outcomes> AGE (mean 63) Hysterctomy - Primary outcome Coronary heart diseases (CHD) Yes (63.6 세 ) No(63.2 세 ) non-fatal myocardial infarction and CHD death N=10739 N= Secondary outcome Stroke, Venous Thromboembolism, Colorectal Cancer, Osteoporosis-related fractures, CEE(0.625mg/d) Placebo CEE(0.625mg/d) Placebo stopped in Feb F/U : 6.8 yrs all-cause Mortality -Primary adverse outcome Invasive breast cancer + MPA(2.5mg/d) stopped in May, 2002 F/U : 5.2 yrs

8 WHI clinical outcome Event CHD event Nonfatal MI CHD death Invasive breast cancer Stroke Pulmonary embolism Colorectal cancer Endometrial cancer Hip fracture Vertebral fracture Death due to causes other than events above Global index Relative Risk CEE/MPA vs. placebo (Hazard ratio) 1.29 ( )* 1.32 ( )* 1.18 ( ) 1.26 ( ) 1.41 ( )* 2.13 ( )* 0.63 ( )* 0.83 ( ) 0.66 ( )* 0.66 ( )* 0.92 ( ) Relative Risk CEE vs. placebo (Hazard ratio) 0.91 ( ) 0.94 ( ) 0.84 ( ) 0.77 ( ) 1.39 ( )* 1.34 ( ) 1.03 ( ) ( )* 0.62 ( )* 1.01 ( ) * statistical significance

9 Estrogen as antioxidant Antioxidant protection of LDL by physiological concentrations of 17 beta-estradiol. Shwaery GT et al. Circulation Mar 18;95(6): The inhibition of low-density lipoprotein oxidation by 17-beta estradiol. Rifici VA et al. Metabolism Oct;41(10): Effects of oral estrogen on aortic ROS-generating and - scavenging enzymes and atherosclerosis in apoe-deficient mice. Wing LY et al. Exp Biol Med (Maywood) Sep;234(9): Epub 2009 Jun 22. Long-term oral estrogen treatment reduces ROS levels and atherosclerosis progression in apoe(-/-) mice. Oral estrogen alters ROS-generating and -scavenging enzyme expression, suggesting that anti-oxidative actions in the vessel wall contribute to atheroprotective effects of estrogen.

10 Objectives 대동맥혈관내피세포에 Hydrogen peroxide (H 2 O 2 ) 를이용한직접적인산화자극에대한 Estrogen의작용을알아보고그작용기전에대하여알아보는데목적이있다.

11 DNA damage Oxidative stress (H2O2) Lipid peroxidation Seperation of cytochrome C from mitochondria Cell death

12 Materials & Methods 1mM of H2O2 bovine Aortic Endothelial Cells (baec) 24 hrs 3 hrs 1uM of 17β-estradiol (E2) 1mM of H2O2 bovine Aortic Endothelial Cells (baec) Cell survival MTT assay Reactive oxidative stress Cellular apoptosis Intracellular mechanism 2,7-dichlorofluorescin diacetate (DCF-DA) Hoechst staining Fluorescence activated cell sorter (FACS) Western blotting for phospho-p38, p38, and Bcl-2

13 Materials & Methods (contin.) 1. 세포배양및전처치 소혈관내피세포주인 baec(bovine Aortic Endothelial Cell), DMEM (Dulbecco's Modified Eagle Medium, Gibco/BRL, MD, U.S.A.) 대조군은산화자극을가하기위하여 30% 의 H 2 O 2 (Sigma Chemicals, St. Louis, MO, U.S.A.) 를단독처리하였으며, 에스트로겐처리군은 1 μm 의 17β-estradiol(E 2 ) (Sigma Chemicals, St. Louis, MO, U.S.A.) 을산화자극을가하기 24 시간전에투여하여전배양 2. 산화자극과 MTT 분석 96 well plate 에 1 x 10 4 / ml개의 baec 세포를배양시켜대조군은 30% 의 H 2 O 2 (Sigma Chemicals) 를인산완충용액 (PBS) 에 500 μm 로희석하여 15, 30, 60 분동안산화자극을가하였으며, 에스트로겐처리군은 1 μm 의 17β-estradiol(E 2 ) 을 24 시간전에처리하여전배양후 15, 30, 60 분동안산화자극을가했다. 세포의산화제에대한감수성평가는 tetrazolium based colorimetric assay 방법을변형한 MTT[3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyl-tetrazolium bromide] (Colorimetric assay kit, Chemicon Inc. CA, U.S.A.) 측정방식을이용하여 450 nm에서흡광도를측정했다.

14 Materials & Methods (contin.) 3. 세포내 reactive oxygen species (ROS) 측정 6 well tissue culture dish 에 5 x 10 4 / ml개의 baec 세포를배양하고 1 mm 농도의 H 2 O 2 에 3 시간노출시켰다. 에스트로겐처리군은 1 μm 의 17βestradiol 을산화자극을가하기 24 시간전에투여하여전배양한후에같은방법으로산화자극. 이후에 PBS 완충용액으로 2 회세척, 30 μm 의 2,7-dichlorofluo- rescin diacetate (DCF-DA (Sigma Chemicals, St. Louis, MO, U.S. A.) 를첨가하여 1 시간동안 CO 2 를 5% 함유한 37 incubation - 세척후 flow cytometer (FACScan, Becton-Dickinson, Mountain View, CA, U.S.A.) 로분석. 4. 세포자멸사의형태학적관찰 4 well chamber slide 에 1 x 10 4 / ml개의 baec 세포를배양하고 1 mm 농도의 H 2 O 2 에 3 시간노출. 에스트로겐처리군은방법 2 와같이전처치후역시 H 2 O 2 에 3 시간노출. 인산염완충식염수로 2 회세척하고 3.7% formaldehyde 로세포를고정시키고, 다시인산염완충식염수로 3 회씻어낸후 10 μg / ml의 Hoechst (Sigma Chemicals, St. Louis, MO, U.S.A.) 를첨가하여 1 시간동안실온의암실에서반응시키고세척후형광현미경으로관찰했다.

15 Materials & Methods (contin.) 5. Western blotting 2 x 10 5 / ml개의 baec 세포를 100 mm2 dish 에배양시켜 30% 의 H 2 O 2 를인산완충용액 (PBS) 에 1 mm 로희석하여 3 시간동안산화자극을가했다. 에스트로겐처리군은역시위와동일한방법으로전처치후 3 시간동안산화자극을가했다. phospho-p38 의활성억제를확인하고자대조군과에스트로겐처리군에서 SB203580(Sigma Chemicals) 를 20 μm 농도로 1 시간전처치한후산화자극을가하였다. 일차항체는 phospho-p38, p38 MAP kinase (Thr180/Tyr182) (Cell signaling Technology, Beverly, MA, U.S.A. ), Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) 항체

16 Materials & Methods (contin.) 6. 형광표지세포분리기 (fluorescence activated cell sorter, FACS) 를이용한세포자멸사분석 인산염완충식염수로세척한후 100 μl의 Annexin Ⅴ binding buffer(140 mm NaCl, 10 mm Hepes, ph 7.4, 25 mm CaCl 2 ) 로재부유시켰다. 5 μl의 Annexin Ⅴ-FITC conjugate 와 5 μl의 propidium iodide (PI) 를넣어암실에서 15 분간반응시켰다. 400 μl의 Annexin Ⅴ binding buffer 를재첨가하여 FACScan (Becton-Dickinson) 으로분석. 7. 통계분석 (Statistical analysis) 같은실험을 3 회에걸쳐서반복하여결과를얻으며, 모든자료는평균 ± 표준편차로표시하고, 각군들사이의비교는 SPSS (SPSS Inc, Chicago, U.S.A) 를사용하며 t-test 로분석. 통계학적유의수준은 P < 0.05 로정의.

17 Results cell survival by MTT assay 17β-estradiol inhibited H2O2 induced baec death. 55.7± 3.0 % 39.1 ± 3.7 % 64.3 ± 3.9 % 66.0 ± 4.8 %

Results cellular apoptosis by Hoechst 33342 staining

18 Results cellular apoptosis by Hoechst staining 17β-estradiol reduced apoptotic bodies and nuclear chromatin condensation.

19 2`,7`-dichlorofluoroscein diacetate ( DCF - DA ) Results Reactive oxygen species by DCF-DA 17β-estradiol inhibited intracellular ROS production ± 68.9 % P < ± 3.9 %

22 Results intracellular mechanism of apoptosis after oxidative stress by western blotting for phospho-p38, p38, and Bcl-2

23 Results cellular apoptosis by FACS with Annexin V and propidium iodide (PI) ± 2.4 % 3.5 ± 2.4 % 23.6 ± 7.1 % 48.8 ± 2.4 % 23.6 ± 7.1 % 3.5 ± 2.4 %

25 Limitations Bovine aortic endothelial cells In vitro experiment

26 Conclusion 소의대동맥내피세포에서 E2 의전처치는 H 2 O 2 에대한직접적인산화자극에대해서 혈관내피세포를보호하는작용을하였다. E2 는세포내의 Reactive oxygen species 를줄이고, antiapoptotic effect 를보였다. 이러한보호작용에대한기전으로는세포내 p38 MAP Kinase를활성화시키는것으로생각되며, 향후이러한기전에대한연구가더필요하다. 따라서, 여성호르몬치료가폐경여성에서산화자극에대하여 cardiovascular integrity 유지에중요한역할을할것이라는과학적근거로제시될수있겠다.

27 Thank you for your attention!!

Treatment and Role of Hormaonal Replaement Therapy

Treatment and Role of Hormaonal Replaement Therapy Treatment and Role of Hormone Replacement Therapy Chung-Ang University Hospital Chee Jeong Kim 7.2 5.3 6.4 5.6 7.3 5.5 1 1 2 3 4 10 11 7 34 49 122 123 1.0 - - 10. 2001 1998 1998 Improvement of postmenopausal