마이코 페놀산에 의한 췌도세포주 사멸의 기전

목적: Mycophenolic acid (MPA)는 췌장이식을 포함한 다양한 종류의 장기이식에 사용되는 면역억제제로 inosine monophosphate dehydrogenase (IMPDH)의 선택적이고 비경쟁적인 억제제이나 췌도세포주에서는 세포 사멸을 유도한다고 알려져 있다. 본 연구에서는 인슐린을 분비하는 췌도 세포주인 HIT-T15 세포를 사용하여, MPA가 세포 사멸을 일으키는 기전을 규명하고자 하였다. 방법: 세포주는 American Type Culture Collection에서 구입하였으며 10% fetal bovine serum이 포함된 RPMI-1640을 사용하여 배양하였다. 세포 활성은 methylthiazoletetrazolium (MTT) assay, 세포 사멸은 annexin V와 PI 염색법, mitogen-activated protein kinase (MAPK) 활성화와 caspase-3 분절은 Western blot 분석으로 측정하였다. 결과: MPA 1μM과 10μM을 처리하였을 때 MTT, caspase-3 분절 그리고 annexin V 염색이 24시간에 농도 의존적으로 증가하였으며, 이는 외부에서 함께 투여한 guanosine 500μM에 의하여 부분적으로 회복되었으나 adenosine 500μM 투여에서는 변화가 없었다. 또한 MPA는 extracellular-regulated protein kinase (ERK), p38 MAPK 그리고 c-jun N-terminal protein kinase (JNK)의 활성화를 8시간과 24시간에서 증가시켰고 guanosine 투여는 이를 부분적으로 회복시켰다. ERK의 억제제인 PD98059, p38 MAPK 억제제인 SB203580 그리고 JNK 억제제인 SP600125는 MPA와 함께 처리하였을 때 각 시간에 증가된 MAPK 활성을 감소시켰지만 MTT와 caspase-3 분절을 살펴본 결과 PD98059는 영향이 없었으며 SB203580은 세포사멸을 증가시켰고, SP600125만이 MPA가 일으킨 세포 사멸을 일부 환원시켰다. Pan-caspase 억제제인 Z-VAD-FMK 또한 세포 사멸을 환원시켰다. 결론: MPA는 MAPK 활성을 IMPDH 의존적으로 증가시키지만, ERKl와 p38 MAPK와는 상관없이, JNK 활성화를 통한 caspase-3 증가의 경로로 췌도 세포 사멸을 유도함을 알 수 있었다.ope

Mechanisms involved in the inhibitory effects of mycophenolic acid on the PDGF-induced proliferation of vascular smo

의과학과/박사[한글] 혈관평활근 세포의 증식은 장기이식후 발생하는 혈관경화증이나 동맥경화증의 발생과 진행에 중요한 역할을 한다. Mycophenolic acid (MPA)는 강력한 면역억제제로서 혈관평활근 세포의 증식도 억제한다. 본 연구에서는 흰쥐와 사람 혈관평활근 세포를 이용하여 platelet-derived growth factor (PDGF)로 유도한 세포증식에 대한 MPA의 작용 기전을 연구하였다. 일차배양한 흰쥐와 사람 혈관평활근 세포를 PDGF-BB 10 ng/ml로 자극하였고, MPA를 비롯한 각 신호전달 억제제는 PDGF를 투여하기 1시간 전부터 투여하였다. 세포의 증식은 [H3]-thymidine incorporation, methyl- thiazoletetrazolium (MTT), proliferating cell nuclear antigen (PCNA) 표현, NAD(P)H oxidase subunit의 mRNA 표현은 RT-PCR, dichlorofluorescein (DCF)에 민감한 세포내 활성산소족은 FACS, 과산화수소(H2O2) 농도는 potassium iodide 법, 그리고 PDGF 수용체-β (Tyr 751), rac1, Akt, ERK 1/2 및 p38 MAPK 활성화는 Western blot 분석 방법을 사용하였다. PDGF 10 ng/ml 투여 후 흰쥐 혈관평활근 세포의 증식, PDGF 수용체와 Akt 활성화, 세포내 활성산소족, ERK 1/2와 p38 MAPK 활성화 및 NAD(P)H oxidase subunit 중 rac1의 활성화뿐만 아니라 p22phox와 MOX1의 mRNA 표현이 대조군에 비하여 유의하게 증가하였다. MPA는 PDGF에 의한 세포 증식을 용량의존적으로 억제하였으며, 외부에서 투여한 guanosine 100μM은 MPA에 의한 증식억제 효과를 부분적으로 회복시켰다. 세포내 신호전달계에 대하여 MPA는 PDGF 수용체-β (Tyr 751) 활성화에는 영향을 주지 않았으나, Akt 활성화, rac1의 활성화, p22phox와 MOX1의 mRNA 표현의 상향 조절, 세포내 활성산소족 및 ERK 1/2와 p38 MAPK 활성화를 통계적으로 유의하게 억제하였다. 또한, MPA는 H2O2를 제거하였다. Wortmannin (PI3K 억제제), diphenyleniodonium (NAD(P)H 억제제) 및 NAC와 trolox (항산화제)는 PDGF에 의한 ERK 1/2와 p38 MAPK 활성화와 세포증식을 억제하였다. PD98059 (MEK 억제제)와 p38 I (p38 MAPK 억제제)는 PDGF에 의한 혈관평활근 세포의 증식을 현저하게 억제하였다. 사람의 혈관평활근 세포에서도 MPA는 PDGF에 의한 세포내 활성산소족 및 ERK 1/2와 p38 MAPK의 활성화를 억제하였다. 결론적으로, 본 연구결과는 MPA가 세포내 guanosine 생성 억제, Akt 활성화 억제 및 항산화 효과를 통한 MAPK 활성화 억제를 경유하여 PDGF에 의한 혈관평활근 세포의 증식을 억제함을 시사하였다. MPA의 항산화 효과는 H2O2에 대한 직접적 제거효과와 NAD(P)H oxidase 억제에 기인함을 시사하였다. [영문]Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development and progression of chronic allograft vasculopathy as in atherosclerosis. Mycophenolic acid (MPA), an immunosupressive agent, inhibits VSMC proliferation. In this study, the mechanisms involved in anti-proliferative effect of MPA was examined. Primary rat and human VSMCs were stimulated with platelet-derived growth factor (PDGF)-BB 10 ng/ml in the presence or absence of MPA (10 nM ～ 100 μM). The effect of known inhibitors of signaling molecules involved in VSMC proliferation were compared with that of MPA to confirm the involvement of each signaling molecule. Cell proliferation was assessed by [H3]-thymidine incorporation, methyl- thiazoletetrazolium (MTT), and proliferating cell nuclear antigen (PCNA) expression, NAD(P)H oxidase subunits mRNA expression by reverse transcription-polymerase chain reaction, dichlorofluorescein (DCF)- sensitive cellular reactive oxygen species (ROS) by FACS, hydrogen peroxide (H2O2) concentration by iodometric analysis, and the activation of PDGF receptor-β (Tyr 751), rac1, Akt, ERK 1/2, and p38 MAPK by Western blot analysis. PDGF increased cell proliferation and cellualr ROS, activation of PDGF receptor, Akt, rac1, ERK 1/2, and p38 MAPK, and expression of p22phox and MOX1 mRNA, compared to control. MPA inhibited PDGF-induced VSMC proliferation in a dose-dependent manner. Exogenously administered guanosine partially rescued cell proliferation inhibited by MPA. MPA also inhibited PDGF-induced up-regulation of Akt and rac1 phosphorylation, p22phox and MOX1 mRNA expression, cellualr ROS, and ERK 1/2 and p38 MAPK phosphorylation. However, MPA did not affect PDGF receptor-β (Tyr 751) phosphorylation, suggesting that MPA''s anti-proliferative effect is independent on PDGF receptor activation. MPA directly scavenged H2O2. Wortmannin (PI3K inhibitor), diphenyleneiodonium (DPI, NAD(P)H oxidase inhibitor), N-acetylcysteine and trolox (anti-oxidants) all inhibited PDGF-induced ERK 1/2 and p38 MAPK activation and cell proliferation. PD98059 (MEK inhibitor) and p38 I (p38 MAPK inhibitor) inhibited PDGF-induced cell proliferation. In human VSMCs, MPA also suppressed PDGF-induced cellular ROS and activation of ERK 1/2 and p38 MAPK. In conclusion, these results suggest that MPA inhibits PDGF-induced VSMC proliferation through inhibiting de novo synthesis of guanosine, Akt activation, and cellular ROS leading to ERK 1/2 and p38 MAPK activation. Both direct scavenging and inhibiting NAD(P)H oxidase appear to be involved in anti-oxidative effect of MPA.ope

Mechanisms Involved in the Inhibitory Effects of Mycophenolic Acid on the PDGF-induced Proliferation of Vascular Smooth Muscle Cells

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.ope

An Implementation of an GPGPU-based Digital Channel Selector and a Matched Filter for a Distributed Spectrum Sensing Node

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Effects of Mycophenolic Acid and Rapamycin on PDGF-induced Mesangial Cell Proliferation and Extracellular Matrix Production

Purpose: Excess proliferation and extracellular matrix (ECM) accumulation of mesenchymal cells such as vascular smooth muscle cells (VSMC) and glomerular mesangial cells cause chronic allograft nephropathy showing transplant vascular sclerosis and glomerulosclerosis. Mycophenolic acid (MPA) and rapamycin (RPM) are well known as strong inhibitors of VSMC proliferation, but their effects on the glomerular mesangial cells are not yet clearly understood. This study examined the effects of MPA or RPM on PDGF-induced proliferation and ECM accumulation in rat glomerular mesangial cells. Methods: Mesangial cells isolated from the glomeruli of Sprague-Dawley rats were cultured with DMEM containing 20% fetal bovine serum. Growth arrested and synchronized cells were administered with test drugs (MPA10 nM~10microM , RPM 0.1 nM~1microM) before the addition of PDGF 10 ng/mL. Cell proliferation was assessed by [3H]thymidine incorporation, collagen by [3H]proline incorporation, and fibronectin, ERK, and p38 MAPK by Western blot analysis. Results: PDGF increased mesangial cell proliferation by 4.64-fold. Compared to stimulated control, MPA above 500 nM and RPM above 10 nM showed a significant inhibitory effect in a dose- dependent manner. The IC50 of MPA and RPM against PDGF-induced mesangial cell proliferation were around 500 nM and 100 nM, respectively. The collagen synthesis was also inhibited by MPA and RPM, but the fibronectin secretion was inhibited by MPA alone. The proliferation of mesangial cell correlated with activation of ERK and p38 MAPK. MPA, but not RPM, inhibited ERK and p38 MAPK activation. Conclusion: This study demonstrated that MPA and RPM significantly inhibited PDGF-induced proliferation and ECM production in rat glomerular mesangial cells. The inhibitory effects of MPA, but not RPM, are correlated with ERK and p38 MAPK.ope

Mycophenolic Acid Inhibits Oleic Acid–Induced Vascular Smooth Muscle Cell Activation by Inhibiting Cellular Reactive Oxygen Species

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation and matrix protein accumulation play important roles in the development and progression of chronic allograft vasculopathy. Mycophenolic acid (MPA) inhibits various types of mesenchymal cell proliferation and cellular reactive oxygen species (ROS) are involved in the anti-proliferative effect of MPA. In this study, we investigated the effects of MPA on oleic acid (OA)-induced VSMC proliferation and the role of ROS in this process. METHODS: Primary VSMCs from Sprague-Dawley rats were stimulated with 100 microM OA, with or without MPA (0.1- 10 microM) or 5 mM N-acetylcysteine (NAC) for one hour prior to the addition of OA. Cell proliferation was measured by methylthiazoletetrazolium (MTT) assays, proliferating cell nuclear antigen (PCNA) expression, and fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by fluorescence-activated cell scanning (FACS). RESULTS: OA (100 microM) increased cell proliferation, as measured by MTT (by 1.6-fold), PCNA expression, fibronectin secretion, and cellular ROS (by 1.6-fold). Treatment with MPA dose-dependently inhibited OA-induced VSMC proliferation, fibronectin secretion, and cellular ROS. Treatment with 5 mM NAC also inhibited OA-induced rat VSMC activation. CONCLUSIONS: These results suggest that MPA inhibits OA-induced VSMC proliferation and matrix protein synthesis partially by inhibiting cellular ROS.ope