Mycophenolic Acid가 PDGF에 의해 증식 유도된 흰쥐 혈관평활근 세포에서 NAD(P)H Oxidase, 세포내 활성산소족 및 Mitogen-activated Protein Kinases에 미치는 영향 = Mechanisms Involved in the Inhibitory Effects of Mycophenolic Acid on the PDGF-induced Proliferation of Vascular Smooth Muscle Cells

다국어 초록 (Multilingual Abstract)

Background:Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development and progression of chronic allograft vasculopathy as in atherosclerosis. We already reported that mycophenolic acid (MPA) inhibited VSMC proliferation, cellular reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) in human VSMCs. In this study, we examined further molecular mechanisms involved in the anti-proliferative effect of MPA in rat VSMCs.
Methods:Primary rat VSMCs were stimulated with PDGF-BB 10 ng/mL in the presence or absence of MPA and various kinds of cell signaling inhibitors. Cell proliferation was assessed by [H3]- thymidine incorporation, NAD(P)H oxidase subunits mRNA expression by RT-PCR, dichlorofluorescein- sensitive cellular ROS by FACS, and the activation of PDGF receptor-β (Tyr 751), rac1, and MAPK by Western blot analysis.
Results:PDGF increased cell proliferation and cellular ROS, activation of PDGF receptor-β (Tyr 751), rac1, expression of p22phox and MOX1 mRNA, ERK 1/2, and p38 MAPK, compared to control. MPA inhibited up-regulation of rac1 phosphorylation, p22phox and MOX1 mRNA expression, cellular ROS, and phosphorylation of ERK 1/2 and p38 MAPK. However, MPA did not affect PDGF receptor-β (Tyr 751) activation. Wortmannin, diphenyleniodonium (DPI), trolox, and NAC, each inhibited PDGF- induced ERK 1/2 and p38 MAPK activation. PD98059 and p38 MAPK inhibitor also inhibited PDGF-induced cell proliferation.
Conclusion:These results suggest that MPA inhibits PDGF-induced VSMC proliferation through inhibiting NAD(P)H oxidase-dependent cellular ROS leading to ERK 1/2 and p38 MAPK activation.

국문 초록 (Abstract)

배 경:혈관평활근 세포의 증식은 장기이식 후 발생하는 혈관경화증이나 동맥경화증의 발생과 진행에 중요한 역할을 한다. Mycophenolic acid (MPA)는 강력한 면역억제제로서 혈관평활근 세포의 증식도 억제한다. 본 연구는 사람의 혈관평활근 세포에서 MPA가 세포증식에 관여하는 신호전달계인 활성산소족과 mitogen-activated protein kinases (MAPK) 활성화를 억제하는 기존의 연구결과를 바탕으로, PDGF에 의해 증식 유도된 흰쥐 혈관평활근 세포에서 MPA가 세포의 증식을 억제하는 기전을 검색하였다.
방 법:일차 배양한 흰쥐 혈관평활근 세포를 PDGF-BB 10 ng/mL로 자극하였고, MPA를 비롯한 여러 종류의 신호전달 억제제는 PDGF를 투여하기 1시간 전에 투여하였다. 세포증식은 [H3]-thymidine incorporation으로, NAD(P)H oxidase subunit의 mRNA 표현은 RT-PCR 로, dichlorofluorescein에 민감한 세포내 활성산소족은 FACS 방법으로, 그리고 PDGF 수용체-β (Tyr 751), rac1 및 MAPK 활성화는 Western blot으로 각각 분석하였다.
결 과:PDGF는 PDGF 수용체-β (Tyr 751)의 활성화 및 NAD(P)H oxidase subunit 중 rac1의 활성화와 p22phox와 MOX1의 mRNA 표현을 대조군에 비하여 유의하게 증가시켰다. MPA는 PDGF 수용체의 활성화에는 영향을 주지 않았으나, rac1의 활성화, p22phox와 MOX1의 mRNA 표현의 상향 조절, 세포내 활성산소족, 그리고 ERK 1/2와 p38 MAPK의 활성화를 억제하였다. Wortmannin, diphenyleniodonium (DPI) 및 NAC와 trolox는 PDGF에 의한 ERK 1/2와 p38 MAPK 활성화와 세포 증식을 억제하였다. PD98059와 p38 MAPK 억제제는 PDGF에 의한 혈관평활근 세포의 증식을 억제하였다.

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