CD151调控VE-cadherin的囊泡运输维持血管稳定性的机制研究

Maintaining the stability of new blood vessels is a key step in angiogenesis, and the regulation of VE-cadherin vesicle trafficking is considered to be one of the most important mechanisms to maintain the vascular stability and the endothelial cell barrier integrity. Our group and other studies have demonstrated that the high expression of CD151 can promote angiogenesis in vivo and in vitro, but the mechanism involved is not clear. As we known, it was reported that the CD151 C-terminal cytoplamic tail contains a YXXΦ trafficking motif and the mutation of CD151 YXXφ sequence leads to impaired vesicle trafficking of CD151. Our previous data found that the inhibition of CD151 protein expression or CD151 gene knockout could cause the damage of vascular stability in vivo and in vitro, and the mutation of the CD151 YXXφ sequence damaged the cell-cell adhesion and tube formation on the Matrigel. Further, we found that the decreased expression of CD151 could also lead to abnormal VE-cadherin distribution. Therefore, it is speculated that CD151 may influence the endothelial cell barrier integrity and vascular stability through the regulation of the VE-cadherin trafficking by CD151 YXXφ sequence. In this study, we will construct the endothelial cell specific CD151 YXXφ sequence mutant transgenic mice and the endothelial cell specific CD151 knockout mice. The purpose of the present study is to investigate the mechanism of CD151 in maintaining the vascular stability through the regulation of the vesicle trafficking. We will provide new strategies for clinical treatment and prevention of cardiovascular and tumor diseases.

维持新生血管的稳定性是血管生成的关键步骤，而调控VE-cadherin的囊泡运输被认为是维持血管稳定性和内皮细胞屏障的重要机制之一。本课题组及国内外多项研究证实CD151高表达可促进体内外血管生成，但其机制并不清楚。研究报道CD151C末端包含囊泡运输靶基序YXXφ，该序列突变可导致囊泡运输功能受损。我们前期研究发现CD151基因敲除或表达下降可导致体内外血管稳定性显著被破坏，CD151YXXφ序列突变则破坏细胞黏附和Matrigel上类血管结构形成，而CD151表达下降还可致VE-cadherin分布异常。故推测CD151可能通过其YXXφ序列调控VE-cadherin的囊泡运输影响内皮细胞屏障和血管稳定性。本研究分别构建内皮细胞特异性CD151YXXφ序列突变转基因小鼠和内皮细胞特异性CD151基因敲除小鼠，探讨CD151通过囊泡运输在维持血管稳定性中的机制。为临床疾病防治提供新的策略

维持新生血管的稳定性是血管生成的关键步骤， CD151高表达可促进体内外血管生成，但其机制并不清楚。本课题组主要探讨CD151通过调控VE-cadherin的囊泡运输影响血管生成稳定性的机制。.1. CD151 及其囊泡运输在维持血管稳定性方面发挥重要作用：1）CD151基因敲除或表达下降可导致体内外血管稳定性显著被破坏，CD151囊泡运输突变则破坏细胞黏附和Matrigel上类血管结构形成。内皮细胞特异性CD151基因敲除小鼠在VEGF-A诱导下，背部和耳朵皮肤渗透性较CD151+/+鼠均显著升高。2）体外研究发现，CD151下调显著增加VEGF-A诱导的内皮细胞单层通透性，导致内皮屏障功能障碍。3）CD151囊泡运输突变增加VEGF-A诱导的内皮细胞单层高通透性。.2. CD151囊泡运输基序参与调控 VE-cadherin 的囊泡运输：研究结果发现CD151囊泡运输突变改变了CD151蛋白的分布，阻碍了CD151由细胞膜向细胞核内的转运过程，导致CD151在细胞浆及细胞核周的表达明显减少。CD151缺失改变了粘附连接血管内皮钙粘附蛋白(VE-cadherin)的分布，显著破坏了VE-cadherin在细胞-细胞接触处的表达，促进细胞-细胞单层渗漏增加。CD151囊泡运输突变导致VE-cadherin在细胞膜表面的分布破坏并且VE-cadherin的内化和肌动蛋白细胞骨架重组受到阻碍，该突变破坏VE-cadherin 囊泡运输的分布。.3. CD151囊泡运输调控内皮细胞屏障和血管稳定性的机制： 1）clathrin通路抑制剂使CD151组的VE-cadherin在细胞-细胞接触处的表达明显破坏，证明囊泡运输调控VE-cadherin依赖于clathrin途径。2 CD151YALA突变体增加RhoA活性，下调 Rac1的活性。3）CD151 YALA突变体影响F-肌动蛋白重塑，通过肌动蛋白细胞骨架介导的内皮-内皮粘附连接的屏障作用和血管稳定性。4）CD151显著增加了FAK、Src的磷酸化水平，这说明CD151通过激活FAK/Src信号来维持内皮细胞屏障和血管稳定性，CD151YALA破坏该信号通路的磷酸化激活。.本研究将通过对CD151囊泡运输突变的机制研究为血管生成的病理生理提供新的思路。