Axel D I, Frigge A, Dittmann J, Runge H, Spyridopoulos I, Riessen R, Viebahn R, Karsch K R
Medical Clinic III, Department of Cardiology, University of Tübingen, Otfried-Müller St. 10, D-72076, Tübingen, Germany.
Cardiovasc Res. 2001 Mar;49(4):851-62. doi: 10.1016/s0008-6363(00)00312-6.
The vitamin-A derivative all-trans retinoic acid (atRA) is a potent regulator of cell growth, differentiation, and matrix formation of various cell types and plays an important role in embryogenesis. However, sparse data are available about its effects on human vessel diseases. Thus, we studied the effects of atRA on human arterial smooth muscle cell (haSMC) and endothelial cell (haEC) proliferation, migration, differentiation and extracellular matrix (ECM) turnover in mono- and transfilter cocultures.
Effects of atRA on human arterial cells in monocultures were determined using cell counting assays, BrdU-ELISA and MTT-tests. In transfilter cocultures haSMC-growth was studied under the stimulatory effect of proliferating haEC. Using Northern blot analysis, effects of atRA on mRNA expression of ECM-proteins were examined while protein expression and activity of matrix metalloproteinases were determined by Western blotting and zymography.
atRA caused a dose dependent inhibition of haSMC-growth in monocultures (IC(50) at 0.022 microM) whereas haEC-growth was inhibited less potently (IC(50) at 97 microM). In addition, proliferation and migration of haSMC through a porous membrane were inhibited dose dependently by micromolar atRA-doses after non-stop and single dose application of atRA on the endothelial side of the complex transfilter coculture system. Immunostainings and Northern blotting demonstrated an enhanced alpha-smooth muscle actin and heavy chain myosin expression in haSMC after atRA-treatment. Whereas mRNA-expression of the glycoproteins thrombospondin-1 and fibronectin were decreased, collagen-1 mRNA expression was even slightly stimulated. Transcription of biglycan and TGF-beta1 were not influenced in a specific manner. Finally, protein expression and activity of the matrix metalloproteinases MMP-2 and MMP-9 were inhibited significantly by atRA.
atRA was found to be a potent inhibitor of both haSMC-proliferation and -migration, even in coculture with haEC releasing growth factors. In addition, redifferentiation, ECM synthesis and ECM degradation were regulated by atRA which also influence haSMC migration and intima formation. Thus, atRA-treatment seems to be a promising strategy for the inhibition of processes involved both in atherosclerosis and restenosis.
维生素A衍生物全反式维甲酸（atRA）是多种细胞类型的细胞生长、分化及基质形成的有效调节剂，在胚胎发育中起重要作用。然而，关于其对人类血管疾病影响的数据却很稀少。因此，我们研究了atRA在单培养和跨滤共培养中对人动脉平滑肌细胞（haSMC）和内皮细胞（haEC）增殖、迁移、分化及细胞外基质（ECM）周转的影响。
使用细胞计数法、BrdU-ELISA和MTT试验确定atRA对单培养中人类动脉细胞的影响。在跨滤共培养中，研究了在增殖的haEC刺激作用下haSMC的生长情况。采用Northern印迹分析检测atRA对ECM蛋白mRNA表达的影响，同时通过蛋白质印迹和酶谱法测定基质金属蛋白酶的蛋白表达和活性。
在单培养中，atRA对haSMC的生长具有剂量依赖性抑制作用（IC50为0.022微摩尔），而对haEC生长的抑制作用较弱（IC50为97微摩尔）。此外，在复合跨滤共培养系统的内皮侧连续或单次给予微摩尔剂量的atRA后，haSMC通过多孔膜的增殖和迁移受到剂量依赖性抑制。免疫染色和Northern印迹显示，atRA处理后haSMC中α-平滑肌肌动蛋白和肌球蛋白重链表达增强。而糖蛋白血小板反应蛋白-1和纤连蛋白的mRNA表达降低，I型胶原mRNA表达甚至略有增加。双糖链蛋白聚糖和转化生长因子-β1的转录未受到特异性影响。最后，atRA显著抑制了基质金属蛋白酶MMP-2和MMP-9的蛋白表达和活性。
发现atRA即使在与释放生长因子的haEC共培养时，也是haSMC增殖和迁移的有效抑制剂。此外，并由atRA调节再分化、ECM合成和ECM降解，这也影响haSMC迁移和内膜形成。因此，atRA治疗似乎是抑制动脉粥样硬化和再狭窄相关过程的一种有前景的策略。
