Acidic/Neutral Amino Acid Residues Substitution in NH2 Terminal of Plasminogen Kringle 5 Exerts Enhanced Effects on Corneal Neovascularization
Acidic/Neutral Amino Acid Residues Substitution in NH2 Terminal of Plasminogen Kringle 5 Exerts Enhanced Effects on Corneal Neovascularization
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纤溶酶原 Kringle 5 NH2 末端酸性/中性氨基酸残基取代对角膜新生血管产生增强作用
DOI：
10.1097/ico.0b013e3182781ec9
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发表时间：
2013-05-01
期刊：
影响因子：
2.8
通讯作者：
Yang, Xia
中科院分区：
文献类型：
--
作者：
Li, Cen;Li, Lei;Yang, Xia
目的：最近的研究表明，纤溶酶原kringle5(K5)被5个丝氨酸残基(突变体K5、Mk5)取代后，对人脐静脉内皮细胞(HUVECs)活力的抑制作用增强。本研究旨在探讨MK5在角膜新生血管(CNV)形成中的抗血管生成作用。方法：用K5和MK5诱导和处理碱烧伤诱导的CNV，共11天。分别用新生血管面积和炎症指数评价新生血管和炎症反应。治疗结束后，取角膜进行末端脱氧核苷酸转移酶dUTP缺口末端标记和免疫组织化学检测。采用四甲基偶氮唑盐比色法、四甲基偶氮唑蓝比色法和流式细胞仪检测MK5和K5对人脐静脉内皮细胞凋亡的影响。Western blotting检测色素上皮衍生因子(PEDF)和血管内皮生长因子(VEGF)的表达水平。结果：在碱诱导的大鼠CNV模型中，MK5局部治疗组与野生型K5组相比，新生血管面积和炎症反应明显减少。同时，MK5和K5在体内外特异性地抑制人脐静脉内皮细胞增殖，诱导血管内皮细胞凋亡，其中MK5具有较高的诱导凋亡作用。在作用机制方面，MK5和K5均显著上调PEDF的表达，轻度下调VEGF的表达。MK5诱导的PEDF/VEGF比值的升高高于K5。结论：MK5对CNV具有比野生型K5更有效的治疗潜力。
Purpose: Recent results showed that plasminogen kringle 5 (K5) has improved inhibitory effect on human umbilical vein endothelial cells (HUVECs) viability when 5 acidic amino acids in NH2 terminal outside kringle domain were replaced by 5 serine residues (mutant K5, mK5). This study was designed to identify the enhanced antiangiogenic activity of mK5 in corneal neovascularization (CNV).Methods: Alkali burn-induced CNV was induced and treated with K5 and mK5 for 11 days. CNV and inflammation were evaluated by the CNV area and the inflammatory index, respectively. At the end of treatment, the corneas were removed for terminal deoxynucleotidyl transferase dUTP nick end labeling detection and immunohistochemistry. The effects of mK5 and K5 on HUVECs apoptosis were tested by MTT, BrdU, and flow cytometry. The expression levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) were detected by Western blot.Results: In a rat model of CNV induced by alkali, topical treatment with mK5 significantly decreased the neovascular area and inflammation compared with the wild-type K5-treated group. Meanwhile, mK5 and K5 specifically inhibited the HUVECs proliferation and induced vascular endothelial cell apoptosis in vitro and in vivo, and mK5 exerted higher apoptosis induction. Toward the mechanism of action, both mK5 and K5 significantly upregulated the expression of PEDF and mildly downregulated the expression of VEGF. The elevation of PEDF/VEGF ratio induced by mK5 was higher than that by K5.Conclusions: These findings suggest that mK5 has more effective therapeutic potential in CNV than wild-type K5.