Singh Nirbhai, Jani Pooja D, Suthar Tushar, Amin Shivan, Ambati Balamurali K
Department of Ophthalmology, Medical College of Georgia, 1120 15th Street, Atlanta, GA 30912, USA.
Invest Ophthalmol Vis Sci. 2006 Nov;47(11):4787-93. doi: 10.1167/iovs.06-0419.
To determine whether Flt24K, a recombinant construct of domains 2 to 4 of VEGFR-1 (Flt) coupled with an endoplasmic reticulum retention signal (KDEL) can bind VEGFR-2 and induce apoptosis, unfolded protein response (UPR), and regression of injury-induced corneal neovascularization.
Human microvascular endothelial cells were transfected with pCMV.Flt24K and subjected to hypoxia. Cell lysates underwent Western blot analysis with anti-XBP-1 antibody and RT-PCR for CHOP. Human malignant melanoma cells (which express VEGFR-2 but not Flt), were transfected with pCMV.Flt24K, and lysates underwent immunoprecipitation with anti-FLT antibody, and Western blot analysis for VEGF and VEGFR-2. Mouse corneas sustained injury induced by topical NaOH and mechanical scraping and were injected with pCMV.Flt24K 2 weeks later. Corneas were harvested 2 days later for Western blot analysis for XBP-1 and caspase-3 or 1 week later for quantification of neovascularization and TUNEL staining. Saline and empty pCMV vector were used in control experiments.
The mean percentage area of corneal neovascularization in mice 3 weeks after corneal injury and 1 week after intrastromal injection of empty pCMV vector or pCMV.Flt24K was 55.4% +/- 2.7% vs. 19.3% +/- 6.1%, respectively (P < 0.001). Flt24K was found to bind VEGFR-2 and upregulate activated XBP-1 and CHOP in vitro. In vivo, pCMV.Flt24K upregulated activated XBP-1 and caspase-3. Apoptosis was observed in corneal neovascular endothelium in corneas treated with pCMV.Flt24K but not in the control.
The Flt24K intraceptor can bind VEGFR-2 within cells, induce the unfolded protein response in vitro and in vivo, elicit apoptosis of vascular endothelial cells in vivo, and induce regression of corneal neovascularization in vivo.
确定Flt24K（一种将血管内皮生长因子受体-1（Flt）的第2至4结构域与内质网滞留信号（KDEL）偶联的重组构建体）是否能结合血管内皮生长因子受体-2并诱导细胞凋亡、未折叠蛋白反应（UPR）以及损伤诱导的角膜新生血管消退。
用pCMV.Flt24K转染人微血管内皮细胞并使其处于缺氧状态。细胞裂解物用抗XBP-1抗体进行蛋白质印迹分析，并用CHOP进行逆转录聚合酶链反应。将人恶性黑色素瘤细胞（表达血管内皮生长因子受体-2但不表达Flt）用pCMV.Flt24K转染，裂解物用抗FLT抗体进行免疫沉淀，并用血管内皮生长因子和血管内皮生长因子受体-2进行蛋白质印迹分析。用局部氢氧化钠和机械刮擦诱导小鼠角膜损伤，2周后注射pCMV.Flt24K。2天后收获角膜用于XBP-1和半胱天冬酶-3的蛋白质印迹分析，或1周后用于新生血管定量和末端脱氧核苷酸转移酶介导的缺口末端标记染色。在对照实验中使用生理盐水和空的pCMV载体。
角膜损伤后3周及基质内注射空的pCMV载体或pCMV.Flt24K后1周，小鼠角膜新生血管的平均面积百分比分别为55.4%±2.7%和19.3%±6.1%（P<0.001）。发现Flt24K在体外能结合血管内皮生长因子受体-2并上调活化的XBP-1和CHOP。在体内，pCMV.Flt24K上调活化的XBP-1和半胱天冬酶-3。在用pCMV.Flt24K处理的角膜新生血管内皮中观察到细胞凋亡，而在对照中未观察到。
Flt24K细胞内受体可在细胞内结合血管内皮生长因子受体-2，在体外和体内诱导未折叠蛋白反应，在体内引发血管内皮细胞凋亡，并在体内诱导角膜新生血管消退。
