Heimark Ronald L, Kaochar Salma, Stamer W Daniel
Department of Surgery, The University of Arizona, Tucson, AZ, USA.
Curr Eye Res. 2002 Nov;25(5):299-308. doi: 10.1076/ceyr.25.5.299.13495.
Purpose. The majority of resistance to outflow of aqueous humor resides at or near the inner wall of Schlemm's canal (SC). Transmembrane proteins that contribute to the generation of resistance to aqueous outflow likely participate in junctional complexes between SC cells. The purpose of the present study was to examine the expression of cadherins in SC cells that play a significant role in adherens junction complexes that control permeability of vascular endothelia. Methods. Identification of cadherin subtype mRNAs was examined by hybridization screening of three different SC cDNA libraries and by polymerase chain reaction analysis with degenerate primers. Expression of endothelial adherens proteins, vascular endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1), was examined by western blot analyses of whole cell lysates prepared from SC and trabecular meshwork cells and by immunofluorescence microscopy of frozen sections of human anterior chambers. As controls, bovine retinal, bovine aortic, human umbilical vein and human iliac vein endothelial cells were examined for VE-cadherin expression. Results. Screens of SC cDNAs revealed abundant expression of N-cadherin and VE-cadherin. Expression of VE-cadherin protein was identified in both inner and outer wall SC cells, appropriately localized to SC intercellular borders and appeared as a single band of approximately 130 kDa by western blot analysis. Specific labeling of PECAM-1 was similar to VE-cadherin and appeared as a single band of approximately 130 kDa by western blot analysis. Conclusions. VE-cadherin and PECAM-1 expression in SC suggests that SC cells are vascular in origin and contain adherens protein likely involved in restricting fluid flow across the inner wall of SC.
目的。房水流出阻力的大部分位于施莱姆管(SC)内壁或其附近。有助于产生房水流出阻力的跨膜蛋白可能参与了SC细胞之间的连接复合体。本研究的目的是检测钙黏蛋白在SC细胞中的表达,这些钙黏蛋白在控制血管内皮通透性的黏附连接复合体中起重要作用。方法。通过对三个不同的SC cDNA文库进行杂交筛选以及使用简并引物进行聚合酶链反应分析,来检测钙黏蛋白亚型mRNA的鉴定。通过对从SC和小梁网细胞制备的全细胞裂解物进行蛋白质免疫印迹分析,以及对人眼前房冰冻切片进行免疫荧光显微镜检查,来检测内皮黏附蛋白、血管内皮(VE)-钙黏蛋白和血小板内皮细胞黏附分子-1(PECAM-1)的表达。作为对照,检测了牛视网膜、牛主动脉、人脐静脉和人髂静脉内皮细胞中VE-钙黏蛋白的表达。结果。对SC cDNA的筛选显示N-钙黏蛋白和VE-钙黏蛋白有丰富表达。在内壁和外壁SC细胞中均鉴定出VE-钙黏蛋白的表达,其在内皮细胞间边界处定位恰当,蛋白质免疫印迹分析显示为一条约130 kDa的单条带。PECAM-1的特异性标记与VE-钙黏蛋白相似,蛋白质免疫印迹分析显示为一条约130 kDa的单条带。结论。SC中VE-钙黏蛋白和PECAM-1的表达表明,SC细胞起源于血管,并且含有可能参与限制房水穿过SC内壁流动的黏附蛋白。