Okada S S, Golden M A, Raghunath P N, Tomaszewski J E, David M L, Kuo A, Kariko K, Barnathan E S
Georgetown University, Washington, DC, USA.
Thromb Haemost. 1998 Jul;80(1):140-7.
Interaction of proteases with cell surface receptors may modulate cell adhesion, migration, invasion, and matrix degradation. Since the plasminogen activator system has been hypothesized to play a role in intimal thickening after various types of vascular injury, we first studied the expression of urokinase receptor (u-PAR) protein and mRNA by smooth muscle cells (SMC) grown in explant cultures from normal and diseased vessels. Using equilibrium binding studies with radiolabeled 125I-labeled single chain urokinase-type plasminogen activator (scu-PA), we determined that SMC cultured from atherosclerotic arteries expressed a higher maximal number of binding sites/cell (3.6 +/- 0.4 x 10(5) sites/cell vs. 2.1 +/- 0.3 x 10(5), +/- SEM, p < 0.05) with a similar affinity (Kd = 1.5 +/- 0.1 vs. 1.2 +/- 0.2 nM, p = ns). However, SMC subcultured from diseased saphenous vein grafts expressed the highest levels of u-PAR compared to SMC from normal saphenous vein (4.8 +/- 0.6 x 10(5) sites/cell vs. 1.6 +/- 0.9 x 10(5), +/- SEM, p < 0.05). Using binding studies and Northern analysis, we demonstrated a dose and time dependent upregulation of u-PAR protein and mRNA expression respectively in human SMC in response to serum stimulation. Using a rabbit specific u-PAR cDNA probe, we demonstrated a similar upregulation of u-PAR mRNA both in rabbit aortic SMC in culture in response to serum stimulation and up to a 20 fold increase in u-PAR mRNA in rabbit jugular veins in response to implantation as arterial grafts in vivo. Finally, to confirm that u-PAR mRNA is upregulated in human vessels after injury, we performed immunohistochemistry and in situ hybridization studies on coronary arteries, normal saphenous veins and saphenous veins from 10 weeks to 13 years after implantation as grafts. u-PAR mRNA was found mainly in the periadventitial microcirculation in normal veins, but was found to be upregulated in the neointima and media of thickened veins in both macrophages and smooth muscle cells. SMC near the internal elastic laminae in diseased coronary arteries appeared to express increased u-PAR mRNA. These data suggest that this increased expression of u-PAR may contribute to early lesion development.
蛋白酶与细胞表面受体的相互作用可能会调节细胞黏附、迁移、侵袭及基质降解。由于有人提出纤溶酶原激活物系统在各类血管损伤后的内膜增厚过程中发挥作用，我们首先研究了从正常血管和病变血管的外植体培养物中生长的平滑肌细胞（SMC）尿激酶受体（u-PAR）蛋白及mRNA的表达情况。通过对放射性标记的125I标记的单链尿激酶型纤溶酶原激活物（scu-PA）进行平衡结合研究，我们确定从动脉粥样硬化动脉培养的SMC表达的最大结合位点数/细胞更高（3.6±0.4×10⁵个位点/细胞对2.1±0.3×10⁵个位点/细胞，±SEM，p<0.05），亲和力相似（Kd=1.5±0.1对1.2±0.2 nM，p=无显著性差异）。然而，与正常大隐静脉的SMC相比，从病变大隐静脉移植物传代培养的SMC表达的u-PAR水平最高（4.8±0.6×10⁵个位点/细胞对1.6±0.9×10⁵个位点/细胞，±SEM，p<0.05）。通过结合研究和Northern分析，我们证明人SMC中u-PAR蛋白和mRNA表达分别呈剂量和时间依赖性上调，这是对血清刺激的反应。使用兔特异性u-PAR cDNA探针，我们证明在培养的兔主动脉SMC中，对血清刺激的反应u-PAR mRNA有类似上调，并且在体内作为动脉移植物植入后，兔颈静脉中u-PAR mRNA增加了20倍。最后，为了证实损伤后人血管中u-PAR mRNA上调，我们对植入移植物10周至13年后的冠状动脉、正常大隐静脉和大隐静脉进行了免疫组织化学和原位杂交研究。u-PAR mRNA在正常静脉中主要见于外膜周围微循环，但在增厚静脉的新生内膜和中膜中，巨噬细胞和平滑肌细胞中均发现其上调。病变冠状动脉内弹力层附近的SMC似乎表达增加的u-PAR mRNA。这些数据表明u-PAR的这种表达增加可能有助于早期病变的发展。
