Zamora David O, Riviere Michael, Choi Dongseok, Pan Yuzhen, Planck Stephen R, Rosenbaum James T, David Larry L, Smith Justine R
Casey Eye Institute, Oregon Health and Science University, Portland, OR, USA.
Mol Vis. 2007 Oct 30;13:2058-65.
The ocular vascular endothelium plays a key role in the development of several leading retinal causes of blindness in Western nations. Choroidal endothelial cells are integral to the subretinal neovascular lesions that characterize the exudative form of late age-related macular degeneration (AMD), and retinal endothelial cells participate in the initiation of diabetic retinopathy and posterior uveitis. Vascular endothelial cells at different sites exhibit considerable molecular diversity. This diversity has implications for understanding the pathogenesis of tissue-specific diseases and for the development of targeted therapies to treat these conditions. Previous work from our group has identified significant differences in the gene transcript profiles of human retinal and choroidal endothelial cells. Because the proteome ultimately determines the behavior of any given cell, however, it is critical to determine whether molecular differences exist at the level of protein expression.
Retinal and choroidal endothelial cells were separately isolated from five sets of human eyes by enzymatic digestion with type II collagenase followed by anti-CD31 antibody-conjugated magnetic bead separation. Cells were washed to remove serum peptides in the culture medium, and lysed by sonication in buffer containing 2% sodium dodecyl sulfate. Protein was then precipitated with acetone. Retinal and choroidal endothelial samples from each donor were labeled with Cy3 and Cy5, respectively, mixed with a Cy2-labeled pooled protein sample to facilitate spot matching across gels, and separated by two-dimensional difference gel electrophoresis (2D-DIGE). Following a global normalization, differentially abundant protein spots that were visible in at least four of five donor gels were detected by the significance analysis of microarrays method, with false discovery rate set at 5%. Corresponding spots were excised from additional DIGE-labeled or Coomassie-stained 2D electrophoretic gels. Protein identification was performed by liquid chromatography and tandem mass spectrometry.
Of 123 protein spots detected by 2D-DIGE that qualified for statistical analysis, we found 31 spots that demonstrated a significant difference in abundance between retinal endothelial samples versus choroidal endothelial samples. For 17 proteins, over 50% of the spectral counts could be matched to a single protein in the digested spot. Eleven proteins were more abundant in retinal endothelial cells (i.e., inorganic pyrophosphatase, protein disulfide isomerase A3, calreticulin, peroxiredoxin-4, protein disulfide isomerase, serpin B9, F-actin capping protein subunit beta, coactosin-like protein, vimentin, cathepsin B, and a high molecular weight form of annexin A3). Six proteins were more abundant in choroidal endothelial cells (i.e., glutathione peroxidase 1, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), heat-shock protein beta-1, superoxide dismutase (Cu-Zn), nucleoside diphosphate kinase A, and a low molecular weight form of annexin 3).
Our data indicate that the proteomes of retinal and choroidal vascular endothelial cells are different. Several differentially expressed proteins are implicated in the regulation of angiogenesis; these include cathepsin B and UCH-L1, proteins with transcripts that were also differently expressed according to microarray. Our observations further suggest that angiogenesis within the retina, a component of severe diabetic retinopathy and posterior uveitis, may be controlled by different mechanisms to those regulating choroidal neovascularization, as occur in exudative AMD. Future studies to establish the role of these angiogenic proteins in disease may suggest potential new targets for tissue-specific therapies.
在西方国家，眼部血管内皮在几种主要的导致失明的视网膜疾病的发展过程中起着关键作用。脉络膜内皮细胞是晚期年龄相关性黄斑变性（AMD）渗出型特征性的视网膜下新生血管病变所不可或缺的部分，而视网膜内皮细胞参与糖尿病视网膜病变和后葡萄膜炎的发病起始过程。不同部位的血管内皮细胞表现出相当大的分子多样性。这种多样性对于理解组织特异性疾病的发病机制以及开发针对这些病症的靶向治疗具有重要意义。我们小组之前的工作已经确定了人类视网膜和脉络膜内皮细胞基因转录谱存在显著差异。然而，由于蛋白质组最终决定任何给定细胞的行为，因此确定在蛋白质表达水平上是否存在分子差异至关重要。
通过用II型胶原酶进行酶消化，随后进行抗CD31抗体偶联磁珠分离，从五组人眼中分别分离出视网膜和脉络膜内皮细胞。洗涤细胞以去除培养基中的血清肽，然后在含有2％十二烷基硫酸钠的缓冲液中通过超声处理进行裂解。然后用丙酮沉淀蛋白质。来自每个供体的视网膜和脉络膜内皮样品分别用Cy3和Cy5标记，与Cy2标记的混合蛋白质样品混合以促进凝胶间的斑点匹配，并通过二维差异凝胶电泳（2D-DIGE）进行分离。在进行全局归一化之后，通过微阵列显著性分析方法检测在五个供体凝胶中的至少四个中可见的差异丰富蛋白质斑点，将错误发现率设定为5％。从额外的DIGE标记或考马斯亮蓝染色的二维电泳凝胶中切下相应的斑点。通过液相色谱和串联质谱进行蛋白质鉴定。
在通过2D-DIGE检测到的符合统计分析条件的123个蛋白质斑点中，我们发现31个斑点在视网膜内皮样品与脉络膜内皮样品之间表现出丰度的显著差异。对于17种蛋白质，超过50％的光谱计数可以与消化斑点中的单一蛋白质匹配。11种蛋白质在视网膜内皮细胞中更为丰富（即无机焦磷酸酶、蛋白质二硫键异构酶A3、钙网蛋白、过氧化物酶体增殖物激活受体4、蛋白质二硫键异构酶、丝氨酸蛋白酶抑制剂B9、F-肌动蛋白封端蛋白亚基β、肌动蛋白结合蛋白样蛋白、波形蛋白、组织蛋白酶B和高分子量形式的膜联蛋白A3）。6种蛋白质在脉络膜内皮细胞中更为丰富（即谷胱甘肽过氧化物酶1、泛素羧基末端水解酶同工酶L1（UCH-L1）、热休克蛋白β-1、超氧化物歧化酶（铜锌）、核苷二磷酸激酶A和低分子量形式的膜联蛋白3）。
我们的数据表明视网膜和脉络膜血管内皮细胞的蛋白质组是不同的。几种差异表达的蛋白质与血管生成的调节有关；这些包括组织蛋白酶B和UCH-L1，其转录本根据微阵列分析也有不同表达。我们的观察结果进一步表明，视网膜内的血管生成作为严重糖尿病视网膜病变和后葡萄膜炎的一个组成部分，其调控机制可能与调节脉络膜新生血管形成的机制不同，后者见于渗出型AMD。未来确定这些血管生成蛋白在疾病中的作用的研究可能会提示组织特异性治疗的潜在新靶点。
