학술대회안내사전등록초록등록안내초록등록/관리숙박 및 교통
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FKHRL1 induces endothelial cell apoptosis via suppression of endothelial cell-extracellular matrix interaction by matrix metalloproteinase activation
서울대학교 의과대학 내과학교실; 서울대학교병원 임상의학연구소 심혈관 연구실; 서울대학교병원 심혈관센터¹ : 서울대학교병원 임상의학연구소 심혈관 연구실 ² : 분당서울대학교병원 심혈관센터³
유현정², 이해영¹ , 정재웅² , 윤석원² , 김효수¹ , 채인호 ¹ , 손대원¹ , 오병희¹ , 이명묵¹ , 박영배¹ , 최윤식¹
Background & Aim: The interaction with the extracelluar matrix (ECM) is important in endothelial cell (EC) survival, and the forkhead transcription factors are known to induce EC apoptosis. To investigate the role of forkhead transcription factor in EC-ECM interaction, we evaluated the response of matrix metalloproteinases (MMPs) following FKHRL1 overexpression.
Methods: FKHRL1-triple mutant (TM), FKHRL1-dominant negative (DN), or the green fluorescent protein (GFP) genes were transferred to HUVECs by adenoviral vector and the full range of effects were evaluated by oligonucleotide microarray at 16, 24 and 40 hours post-transfection. To evaluate EC-ECM interaction, HUVECs were cultured on fibronectin-coated plates. At 24 hours after transfection, ECs were fixed, incubated with fibronectin monoclonal antibody, and observed with immunofluorescence microscopy.
Results: 1) Morphologically, FKHRL1-TM transfected ECs showed increased dehiscence from the plate and remarkable cytotoxicity compared to GFP group. The FACS analysis showed that the major mechanism of the cytotoxicity was apoptosis. 2) In microarray, MMP-3 and MMP-7 mRNA expressions increased while TIMP-1 mRNA expression decreased. By real-time PCR, mRNA expressions of MMP-3 and MMP-7 were increased compared to GFP group and Western blot analysis confirmed that these mRNA changes corresponded to enzyme synthesis, and casein zymography showed MMP activity was also increased. 3) FKHRL1-TM transfected ECs degraded fibronectin, and they had less adhesive capacity. Moreover, VE-cadherin and β-catenin at cell periphery were decreased and the discontinuity in alignment was observed with immunofluorescence microscopy. 4) On the contrary, FKHRL1-DN transfected ECs produced less MMPs compared to GFP group and no cytotoxicity was observed. FKHRL1-TM group showed less apoptosis when treated with an MMP inhibitor, GM6001.
Conclusion: These data show that FKHRL1 suppresses EC-ECM interaction by the degradation of the ECM via MMP activation. The finding that endothelial cell apoptosis was reduced by MMP inhibition suggests an important role of MMPs in forkhead induced apoptosis of ECs.


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