Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2011;6(6):e21164. doi: 10.1371/journal.pone.0021164. Epub 2011 Jun 17.
Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.
血管通透性增加是许多血管并发症的一个激发事件，包括糖尿病性视网膜病变。我们之前曾报道过，色素上皮衍生因子（PEDF）能够通过一种新型的γ-分泌酶依赖性途径抑制血管内皮生长因子（VEGF）诱导的血管生成。在这项研究中，我们询问了 PEDF 抑制 VEGF 诱导的通透性是否也是通过 γ-分泌酶介导的，并剖析了所涉及的潜在机制。我们通过测量跨内皮电阻和葡聚糖的旁通透性，在体外评估血管通透性，并在存在或不存在 VEGF 和 PEDF 的情况下，通过静脉内 FITC 标记的白蛋白漏入视网膜来评估体内血管通透性。实验在存在或不存在 γ-分泌酶抑制剂的情况下进行。为了评估连接完整性，我们使用培养的细胞和平铺的视网膜进行了黏附连接（AJ）蛋白，VE-钙粘蛋白和β-连环蛋白，以及紧密连接（TJ）蛋白，claudin-5 的免疫组织化学染色。通过免疫沉淀和 Western Blot 分析确定了 AJ 蛋白、VEGF 受体（VEGFRs）和 γ-分泌酶成分之间的蛋白表达和关联。在选定的实验中，还评估了缺氧对连接完整性的影响。PEDF 抑制 VEGF 诱导的通透性，无论是在培养的微血管内皮细胞单层中，还是在小鼠视网膜血管中，都是通过 γ-分泌酶介导的。PEDF 通过以下方式发挥作用：a）防止 AJ 和 TJ 蛋白的解离，b）调节 VEGFR 与 AJ 蛋白的关联，以及随后 AJ 蛋白、VE-钙粘蛋白和 β-连环蛋白的磷酸化。γ-分泌酶与 AJ 蛋白的关联似乎是调节血管通透性的关键。尽管缺氧增加了 VEGFR 的表达，但 VEGFR 与 AJ 蛋白的显著解离。总之，PEDF 通过一种新型的 γ-分泌酶依赖性途径调节 VEGF 诱导的血管通透性，靶向 PEDF 作用的下游效应物可能代表预防或改善血管通透性增加的有前途的治疗策略。
