Gallicchio M, Argyriou S, Ianches G, Filonzi E L, Zoellner H, Hamilton J A, McGrath K, Wojta J
Department of Diagnostic Haematology, Royal Melbourne Hospital, Victoria, Australia.
Arterioscler Thromb. 1994 May;14(5):815-23. doi: 10.1161/01.atv.14.5.815.
Regulation of endothelial cell (EC) plasminogen activator inhibitor type-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregulation of secreted and intracellular PAI-1 in human umbilical ECs when cocultured with human smooth muscle cells (SMCs) on amniotic membranes or incubated with SMC conditioned medium (CM) under serum-free conditions as determined by enzyme-linked immunosorbent assay. Cocultured human umbilical vein ECs and SMCs, or human umbilical artery ECs and SMCs, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, since only human aortic, umbilical vein, and umbilical artery SMCs upregulated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonary artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC CM was both time and concentration dependent, with as much as five- and fourfold increases in supernatants and lysates, respectively. PAI-1 synthesis and activity in ECs from other vascular beds were also upregulated by SMC CM. Northern blot analysis paralleled the protein results, showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA expression after incubation with SMC CM for 8 hours. PAI-1 stimulatory activity in SMC CM was completely abolished by boiling or incubation with protamine sulfate and was reduced by transient acidification or heparin-Sepharose pretreatment by 33% or 48%, respectively. The stimulatory factor(s) appeared to have a molecular mass of 23 kD as determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)
内皮细胞（EC）纤溶酶原激活物抑制剂-1（PAI-1）是组织型纤溶酶原激活物（TPA）和尿激酶型纤溶酶原激活物（UPA）的主要生理性抑制剂，其受多种刺激的调控已得到充分表征。我们报告，通过酶联免疫吸附测定法确定，在无血清条件下，人脐静脉内皮细胞与人平滑肌细胞（SMC）在羊膜上共培养或与SMC条件培养基（CM）孵育时，分泌型和细胞内PAI-1会上调。共培养的人脐静脉内皮细胞和平滑肌细胞，或人脐动脉内皮细胞和平滑肌细胞，释放的PAI-1分别增加了73%和68%。SMC衍生的刺激因子释放具有组织特异性，因为只有人主动脉、脐静脉和脐动脉的SMC上调了PAI-1的合成，而来自人乳腺动脉、肺动脉和隐静脉的SMC则没有。SMC CM对EC PAI-1的刺激是时间和浓度依赖性的，上清液和裂解物中分别增加了多达五倍和四倍。其他血管床的EC中PAI-1的合成和活性也被SMC CM上调。Northern印迹分析与蛋白质结果一致，显示与SMC CM孵育8小时后，特异性EC PAI-1 mRNA表达增加了多达2.7倍。SMC CM中的PAI-1刺激活性通过煮沸或与硫酸鱼精蛋白孵育完全消除，通过短暂酸化或肝素-琼脂糖预处理分别降低33%或48%。通过凝胶过滤测定，刺激因子的分子量似乎为23 kD。（摘要截断于250字）
