Jerkic Mirjana, Letarte Michelle
*Molecular Structure and Function Program, The Hospital for Sick Children, Toronto, Ontario, Canada; and Department of Immunology and Keenan Research Centre for Biomedical Science, Anesthesia Research, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada
*Molecular Structure and Function Program, The Hospital for Sick Children, Toronto, Ontario, Canada; and Department of Immunology and Keenan Research Centre for Biomedical Science, Anesthesia Research, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada.
FASEB J. 2015 Sep;29(9):3678-88. doi: 10.1096/fj.14-269258. Epub 2015 May 13.
Endoglin (ENG) is a TGF-β superfamily coreceptor essential for vascular endothelium integrity. ENG mutations lead to a vascular dysplasia associated with frequent hemorrhages in multiple organs, whereas ENG null mouse embryos die at midgestation with impaired heart development and leaky vasculature. ENG interacts with several proteins involved in cell adhesion, and we postulated that it regulates vascular permeability. The current study assessed the permeability of ENG homozygous null (Eng(-/-)), heterozygous (Eng(+/-)), and normal (Eng(+/+)) mouse embryonic endothelial cell (EC) lines. Permeability, measured by passage of fluorescent dextran through EC monolayers, was increased 2.9- and 1.7-fold for Eng(-/-) and Eng(+/-) ECs, respectively, compared to control ECs and was not increased by TGF-β1 or VEGF. Prolonged starvation increased Eng(-/-) EC permeability by 3.7-fold with no effect on control ECs; neutrophils transmigrated faster through Eng(-/-) than Eng(+/+) monolayers. Using a pull-down assay, we demonstrate that Ras homolog gene family (Rho) A is constitutively active in Eng(-/-) and Eng(+/-) ECs. We show that the endothelial barrier destabilizing factor thrombospondin-1 and its receptor-like protein tyrosine phosphatase are increased, whereas stabilizing factors VEGF receptor 2, vascular endothelial-cadherin, p21-activated kinase, and Ras-related C3 botulinum toxin substrate 2 are decreased in Eng(-/-) cells. Our findings indicate that ENG deficiency leads to EC hyperpermeability through constitutive activation of RhoA and destabilization of endothelial barrier function.
内皮糖蛋白（ENG）是转化生长因子-β（TGF-β）超家族的一种共受体，对血管内皮完整性至关重要。ENG突变会导致一种血管发育异常，伴有多个器官频繁出血，而ENG基因敲除的小鼠胚胎在妊娠中期死亡，心脏发育受损且血管渗漏。ENG与几种参与细胞黏附的蛋白质相互作用，我们推测它调节血管通透性。本研究评估了ENG纯合缺失（Eng(-/-)）、杂合（Eng(+/-)）和正常（Eng(+/+)）小鼠胚胎内皮细胞（EC）系的通透性。通过荧光葡聚糖透过EC单层的情况来测量通透性，与对照EC相比，Eng(-/-)和Eng(+/-) EC的通透性分别增加了2.9倍和1.7倍，且不受TGF-β1或血管内皮生长因子（VEGF）的影响。长时间饥饿使Eng(-/-) EC的通透性增加了3.7倍，而对对照EC没有影响；中性粒细胞通过Eng(-/-)单层的迁移速度比通过Eng(+/+)单层的更快。通过下拉实验，我们证明Ras同源基因家族（Rho）A在Eng(-/-)和Eng(+/-) EC中组成性激活。我们发现，在Eng(-/-)细胞中，内皮屏障破坏因子血小板反应蛋白-1及其受体样蛋白酪氨酸磷酸酶增加，而稳定因子VEGF受体2、血管内皮钙黏蛋白、p21激活激酶和Ras相关C3肉毒杆菌毒素底物2减少。我们的研究结果表明，ENG缺乏通过RhoA的组成性激活和内皮屏障功能的破坏导致EC高通透性。
