Ramo Kasmir, Cashman Siobhan M, Kumar-Singh Rajendra
Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 2008 Sep;49(9):4126-36. doi: 10.1167/iovs.08-2025. Epub 2008 May 16.
Complement-mediated damage to the retinal pigment epithelium (RPE), Bruch membrane, and choroid has been associated with pathogenesis in age-related macular degeneration (AMD). The terminal step of complement activation involves lysis of cells by the insertion of the membrane attack complex (MAC) in the plasma membrane. The hypothesis that local overexpression of human CD59 (hCD59) delivered by an adenovirus (Ad) vector to primary murine RPE cells in vitro, RPE in vivo, or cornea ex vivo protects those cells from human MAC deposition and lysis was tested.
A humanized model of MAC deposition on murine cells and murine ocular tissues including RPE and cornea was developed to permit testing of human complement regulators in mice. A recombinant adenovirus-expressing hCD59 was generated, and this virus was injected into the subretinal space of adult mice. Subsequently, eyecups from these mice were exposed to human serum, and the levels of MAC deposition on the RPE were quantified. hCD59 was also expressed on murine cornea ex vivo and in murine hepatocytes, and primary RPE cells in vitro and levels of human MAC deposition and cell lysis were measured.
Adenovirus-mediated delivery of hCD59 to the RPE, cornea, or cells in culture protects those cells from human MAC deposition and MAC-mediated damage and vesiculation.
The humanized model of MAC deposition on murine ocular tissues allows testing of human complement regulators that may have potential in the treatment of AMD or other diseases associated with complement activation.
补体介导的对视网膜色素上皮（RPE）、 Bruch膜和脉络膜的损伤与年龄相关性黄斑变性（AMD）的发病机制有关。补体激活的终末步骤涉及膜攻击复合物（MAC）插入质膜导致细胞溶解。本研究测试了一种假说，即通过腺病毒（Ad）载体将人CD59（hCD59）在体外导入原代小鼠RPE细胞、在体内导入RPE或在体外导入角膜，可保护这些细胞免受人MAC沉积和溶解。
建立了一种人源化的MAC在小鼠细胞和包括RPE及角膜在内的小鼠眼组织上沉积的模型，以允许在小鼠中测试人补体调节因子。构建了一种表达hCD59的重组腺病毒，并将该病毒注入成年小鼠的视网膜下间隙。随后，将这些小鼠的眼杯暴露于人类血清中，并对RPE上的MAC沉积水平进行定量。hCD59也在体外的小鼠角膜、小鼠肝细胞以及体外原代RPE细胞中表达，并检测人MAC沉积水平和细胞溶解情况。
腺病毒介导的将hCD59递送至RPE、角膜或培养中的细胞，可保护这些细胞免受人MAC沉积以及MAC介导的损伤和囊泡形成。
MAC在小鼠眼组织上沉积的人源化模型允许测试可能在治疗AMD或其他与补体激活相关疾病中具有潜力的人补体调节因子。
