Department of Vascular Biology and Tumor Angiogenesis, Center for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), Heidelberg, Germany.
5th Medical Department, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
Invest Ophthalmol Vis Sci. 2014 Jun 10;55(7):4295-303. doi: 10.1167/iovs.13-13619.
Corneal neovascularization can cause loss of vision. The introduction of anti-VEGF therapy has been a major improvement in therapeutic options. Recently, we established Kelch-like Ect2-interacting protein (KLEIP/KLHL20) knockout mice as a model of spontaneous corneal neovascular dystrophy. The aim of the present study was to characterize corneal neovascularization in progressive corneal dystrophy in KLEIP(-/-) mice, to evaluate the efficacy of anti-VEGF therapy, and to identify novel molecular regulators in this experimental model.
Corneal neovascularization was assessed by immunohistochemistry. Vascular endothelial growth factor signaling was inhibited by injection of a blocking antibody. Microarrays were used to measure expression of mRNA and microRNA (miRNA) in dystrophic corneae. Results were validated by immunohistochemistry and Western blotting.
Blood vessels and lymphatics grew from the limbus toward the dystrophic epithelium in corneae of KLEIP(-/-) mice. Blocking VEGF signaling did not reduce phenotype progression. Correspondingly, microarray analysis revealed no upregulation of canonical vascular growth factors in late dystrophy. During phenotype progression, angiopoietin-1 expression increased while miR-204 expression decreased. Bioinformatic analysis identified a binding site for miR-204 in the angiopoietin-1 gene. Validation by in vitro experiments confirmed regulation of angiopoietin-1 by miR-204.
Vascular endothelial growth factor does not act as a major player in corneal neovascularization in KLEIP(-/-) mice. However, the proangiogenic factor angiopoietin-1 was strongly upregulated in late-stage phenotype, correlating with loss of miR-204 expression. Correspondingly, we identified miR-204 as a novel regulator of angiopoietin-1 in vitro. These findings may explain the incomplete efficacy of anti-VEGF therapy in the clinic and may provide new candidates for pharmaceutical intervention.
角膜新生血管可导致视力丧失。抗血管内皮生长因子（VEGF）治疗的出现极大地改善了治疗选择。最近，我们建立了 Kelch 样 Ect2 相互作用蛋白（KLEIP）/KLHL20 基因敲除小鼠作为自发性角膜新生血管营养不良的模型。本研究旨在描述 KLEIP 基因敲除（-/-）小鼠进行性角膜营养不良中的角膜新生血管形成，评估抗 VEGF 治疗的疗效，并在该实验模型中鉴定新的分子调节因子。
通过免疫组织化学评估角膜新生血管形成。通过注射阻断抗体抑制血管内皮生长因子信号通路。使用微阵列测量营养不良角膜中的信使 RNA（mRNA）和 microRNA（miRNA）的表达。通过免疫组织化学和 Western blot 验证结果。
在 KLEIP（-/-）小鼠的角膜中，血管和淋巴管从角膜缘向营养不良的上皮生长。阻断 VEGF 信号通路不能减缓表型进展。相应地，微阵列分析显示在晚期营养不良中没有经典血管生长因子的上调。在表型进展过程中，血管生成素 1 的表达增加，而 miR-204 的表达减少。生物信息学分析确定了 miR-204 在血管生成素 1 基因中的结合位点。通过体外实验验证了 miR-204 对血管生成素 1 的调控作用。
VEGF 不是 KLEIP（-/-）小鼠角膜新生血管形成的主要因素。然而，在晚期表型中，促血管生成因子血管生成素 1 强烈上调，与 miR-204 表达的丧失相关。相应地，我们在体外确定了 miR-204 是血管生成素 1 的新调节因子。这些发现可能解释了抗 VEGF 治疗在临床上的不完全疗效，并可能为药物干预提供新的候选药物。
