Thurman Joshua M, Renner Brandon, Kunchithapautham Kannan, Ferreira Viviana P, Pangburn Michael K, Ablonczy Zsolt, Tomlinson Stephen, Holers V Michael, Rohrer Bärbel
From the Department of Medicine, University of Colorado Denver School of Medicine, Denver, Colorado 80045.
Departments of Ophthalmology and Neurosciences, Division of Research, Charleston, South Carolina 29425.
J Biol Chem. 2009 Jun 19;284(25):16939-16947. doi: 10.1074/jbc.M808166200. Epub 2009 Apr 21.
Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H(2)O(2) (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H(2)O(2) reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H(2)O(2) and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.
补体替代途径的失控激活被认为与年龄相关性黄斑变性（AMD）有关。替代途径在液相中持续被激活，组织表面需要持续的补体抑制以防止自发的自体组织损伤。在此，我们研究了氧化应激对永生化人视网膜色素上皮细胞（ARPE - 19）调节其细胞表面补体激活能力的影响。通过跨上皮电阻（TER）测量发现，用H₂O₂（诱导氧化应激）和补体充足的血清联合处理会破坏稳定的ARPE - 19单层细胞的屏障功能。单独的任何一种处理都没有效果。TER降低与C3在细胞表面沉积增加相关，并且可以通过使用C7缺陷血清（末端补体途径的必需成分）来预防。用H₂O₂处理会降低补体抑制剂DAF、CD55和CD59的表面表达，并损害血清中存在的因子H对细胞表面的调节。用H₂O₂和血清联合处理单层细胞会引发血管内皮生长因子（VEGF）的极化分泌。使用替代途径抑制剂或阻断VEGF受体 - 1/2信号传导都可以减弱VEGF的分泌和TER的降低。综合来看，这些研究表明氧化应激会降低ARPE - 19细胞表面补体的调节，增加补体激活。这种亚溶解激活导致VEGF释放，介导细胞单层的破坏。这些发现将氧化应激、补体激活和顶端VEGF释放联系起来，它们都与AMD的发病机制有关。
