Voisard Rainer, Viola Sandra, Kaspar Verena, Weber Christian M, von Müller Lutz, Baur Regine, Gastrock-Balitsch Iris, Hombach Vinzenz
Department of Internal Medicine II - Cardiology, Institute of Mikrobiology and Immunology, University of Ulm, Germany.
BMC Cardiovasc Disord. 2005 May 12;5(1):9. doi: 10.1186/1471-2261-5-9.
Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is a rationally designed immunosuppressive drug. The current study investigates the effect of MMF on key pattern of restenosis in a cascade of in vitro and ex vivo models.
Part I of the study investigated in northern blot and cytoflow studies the effect of MMF (50, 100, 150, 200, 250, and 300 microg/mL) on TNF-alpha induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC). Part II of the study applied a human coronary 3D model of leukocyte attack, the 3DLA-model. HCAEC and HCMSMC were cultured on both sides of a polycarbonate filters, mimicking the internal elastic membrane. Leukocyte attack (LA) was carried out by adding human monocytes (MC) on the endothelial side. The effect of MMF (50 microg/mL) on adhesion and chemotaxis (0.5, 1, 2, 3, 4, 6, and 24 h after LA) and the effect on proliferation of co-cultured HCMSMC (24 h after LA) was studied. In part III of the study a porcine coronary organ culture model of restenosis (POC-model) was used. After ex vivo ballooning MMF (50 microg/mL) was added to the cultures for a period of 1, 2, 3, 4, 5, 6, and 7 days. The effect on reactive cell proliferation and neointimal thickening was studied at day 7 and day 28 after ballooning.
Expression of ICAM-1 in northern blot and cytoflow studies was neither clearly inhibited nor stimulated after administration of MMF in the clinical relevant concentration of 50 microg/mL. In the 3DLA-model 50 microg/mL of MMF caused a significant antiproliferative effect (p < 0.001) in co-cultured HCMSMC but had no effect on MC-adhesion and MC-chemotaxis. In the ex vivo POC-model neighter reactive cell proliferation at day 7 nor neointimal hyperplasia at day 28 were significantly inhibited by MMF (50 microg/mL).
Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 microg/mL) in the 3DLA-model. The antiproliferative effect was a direct antiproliferative effect that was not triggered via reduced expression of ICAM-1 or via an inhibition of MC-adhesion and chemotaxis. Probably due to technical limitations (as e.g. the missing of perfusion) the antiproliferative effect of MMF (50 microg/mL) could not be reproduced in the coronary organ culture model. A cascade of focused in vitro and ex vivo models may help to gather informations on drug effects before large experimental studies are initiated.
霉酚酸酯（MMF）是霉酚酸（MPA）的前体药物，是一种经过合理设计的免疫抑制药物。本研究在一系列体外和离体模型中探究了MMF对再狭窄关键模式的影响。
研究的第一部分通过Northern印迹法和细胞流式研究，考察了MMF（50、100、150、200、250和300μg/mL）对肿瘤坏死因子-α诱导人冠状动脉内皮细胞（HCAEC）和人冠状动脉中层平滑肌细胞（HCMSMC）中细胞间黏附分子1（ICAM-1）表达的影响。研究的第二部分应用了人冠状动脉白细胞攻击三维模型，即3DLA模型。将HCAEC和HCMSMC培养在聚碳酸酯滤膜的两侧，模拟内弹性膜。通过在内皮侧加入人单核细胞（MC）进行白细胞攻击（LA）。研究了MMF（50μg/mL）对黏附及趋化作用的影响（LA后0.5、1、2、3、4、6和24小时）以及对共培养的HCMSMC增殖的影响（LA后24小时）。在研究的第三部分中，使用了猪冠状动脉再狭窄器官培养模型（POC模型）。在体外球囊扩张后，将MMF（50μg/mL）加入培养物中，持续1、2、3、4、5、6和7天。在球囊扩张后第7天和第28天研究其对反应性细胞增殖和内膜增厚的影响。
在临床相关浓度50μg/mL的MMF给药后，Northern印迹法和细胞流式研究中ICAM-1的表达既未受到明显抑制也未受到刺激。在3DLA模型中，50μg/mL的MMF对共培养的HCMSMC产生了显著的抗增殖作用（p<0.001），但对MC黏附和MC趋化作用没有影响。在体外POC模型中，MMF（50μg/mL）在第7天对反应性细胞增殖以及在第28天对内膜增生均未产生显著抑制作用。
因此，数据表明临床相关水平的MMF（50μg/mL）在3DLA模型中具有显著的抗增殖作用。该抗增殖作用是一种直接的抗增殖作用，并非通过ICAM-1表达降低或通过抑制MC黏附和趋化作用触发。可能由于技术限制（如缺乏灌注），MMF（50μg/mL）的抗增殖作用在冠状动脉器官培养模型中未能重现。一系列有针对性的体外和离体模型可能有助于在开展大型实验研究之前收集有关药物作用的信息。
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