Cai Jun, Kehoe Oksana, Smith Gill M, Hykin Philip, Boulton Michael E
Department of Ophthalmology and Visual Sciences, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77550, USA.
Invest Ophthalmol Vis Sci. 2008 May;49(5):2163-71. doi: 10.1167/iovs.07-1206.
The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy.
Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis.
Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2.
These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.
血管生成素（Ang）系统在血管稳定和病理性新生血管形成中起重要作用。本研究的假设是，血管生成素/Tie-2系统除了调节内皮细胞行为外，还调节周细胞凋亡和/或与糖尿病视网膜病变相关的血管成熟。
采用ELISA、蛋白质印迹分析和流式细胞术分析培养的视网膜周细胞中Tie-2的表达。通过蛋白质印迹分析确定周细胞中CD13（氨肽酶N）的表达，并用Tie-2反义处理验证Ang的作用。通过结晶紫摄取监测细胞增殖，并在刮伤伤口中评估周细胞迁移。采用膜联蛋白V-FITC流式细胞术定量周细胞凋亡。
周细胞表达功能性活性Tie-2受体，该受体被Ang-1和Ang-2上调（P<0.05）。在由TNF-α或高糖诱导凋亡的周细胞中，Ang-1提高了存活率（TNF-α处理时P<0.05；高糖处理时P<0.01），而Ang-2增加了凋亡。Ang-1以剂量依赖性方式增强CD13表达（P<0.05），并在合成基质伤口愈合试验中刺激周细胞迁移，这与F-肌动蛋白组织的变化有关。添加Tie-2反义证实血管生成素通过Tie-2发挥作用。
这些发现表明Tie-2在周细胞中具有功能，可能通过调节周细胞丢失并影响周细胞的激活状态和募集，在糖尿病视网膜病变的进展中起重要作用。
