Zhang Xu, Chaudhry Aisha, Chintala Shravan K
Eye Research Institute, Oakland University, Rochester, MI 48309, USA.
Mol Vis. 2003 Jun 12;9:238-48.
The mechanisms that trigger ganglion cell death in ischemic retinal diseases are not clearly understood. Using a mouse optic nerve ligation model, the objective of this study was to test the hypothesis that extracellular matrix (ECM) modulating plasminogen activators (PAs) potentiate ganglion cell loss.
Optic nerve ligation was performed to initiate ganglion cell loss in the retina. Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity in retinal extracts was determined by plasminogen/fibrinogen zymography. Immunostaining and western blot analysis was performed to detect uPA and tPA proteins. Plasmin activity was determined by casein gel-zymography. Plasminogen and plasmin proteins were detected and quantified by western blotting. Morphology was assessed using hematoxylin and eosin stained retinal cross sections, and programmed cell death was monitored by an apoptotic assay. Laminin degradation in retinal extracts was assessed by western blot analysis.
Optic nerve ligation led to a transient increase in uPA and plasmin proteolytic activity in the retina. Urokinase inhibitor, amiloride, blocked uPA activity in retinal extracts. We found a correlation between the increased uPA activity, and conversion of zymogen plasminogen to active plasmin in retinal extracts with laminin degradation in the retina and apoptosis of ganglion cells. We found that by adding exogenous plasmin, in vitro, laminin present in control retinal extracts could be degraded in similar fashion. In addition, uPA or tPA failed to degrade laminin in control retinal extracts unless plasminogen was added, indicating that plasminogen activation is necessary for laminin degradation, in vitro. After intravitreal injection of plasmin inhibitor, alpha-2 antiplasmin, we found a significant protection against optic nerve ligation-induced ganglion cell loss.
Optic nerve ligation-induced plasmin(ogen) activation that precedes ganglion cell loss suggest that specific targeting of plasmin activity may have therapeutic potential in preventing ganglion cell loss in retinal diseases.
缺血性视网膜疾病中触发神经节细胞死亡的机制尚不清楚。本研究利用小鼠视神经结扎模型，旨在验证细胞外基质（ECM）调节纤溶酶原激活剂（PAs）会加剧神经节细胞丢失这一假说。
进行视神经结扎以引发视网膜中的神经节细胞丢失。通过纤溶酶原/纤维蛋白原酶谱法测定视网膜提取物中尿激酶型纤溶酶原激活剂（uPA）和组织型纤溶酶原激活剂（tPA）的活性。进行免疫染色和蛋白质印迹分析以检测uPA和tPA蛋白。通过酪蛋白凝胶酶谱法测定纤溶酶活性。通过蛋白质印迹法检测并定量纤溶酶原和纤溶酶蛋白。使用苏木精和伊红染色的视网膜横切片评估形态，并通过凋亡检测监测程序性细胞死亡。通过蛋白质印迹分析评估视网膜提取物中的层粘连蛋白降解情况。
视神经结扎导致视网膜中uPA和纤溶酶蛋白水解活性短暂增加。尿激酶抑制剂阿米洛利可阻断视网膜提取物中的uPA活性。我们发现视网膜提取物中uPA活性增加、酶原纤溶酶原向活性纤溶酶的转化与视网膜中层粘连蛋白降解以及神经节细胞凋亡之间存在相关性。我们发现，在体外添加外源性纤溶酶后，对照视网膜提取物中存在的层粘连蛋白可被以类似方式降解。此外，除非添加纤溶酶原，uPA或tPA无法降解对照视网膜提取物中的层粘连蛋白，这表明在体外纤溶酶原激活对于层粘连蛋白降解是必要的。玻璃体内注射纤溶酶抑制剂α-2抗纤溶酶后，我们发现对视神经结扎诱导的神经节细胞丢失有显著保护作用。
视神经结扎诱导的神经节细胞丢失之前的纤溶酶（原）激活表明，特异性靶向纤溶酶活性可能在预防视网膜疾病中的神经节细胞丢失方面具有治疗潜力。
