Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA (P.Y., F.C., P.B., H.W., V.M.D., V.M., D.S.M., T.N.M.).
Department of Medicine, Massachusetts General Hospital, Boston (V.Y., K.K., A.P.).
Arterioscler Thromb Vasc Biol. 2022 Jul;42(7):886-902. doi: 10.1161/ATVBAHA.122.317565. Epub 2022 Apr 28.
The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)-an unconventional Rho family GTPase GEF in vascular function.
We generated mice deficient in DOCK4' used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells' used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation.
Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)-induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation.
Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1-mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.
血管内皮通过控制血液和组织间隙之间大分子和液体的通过来维持组织液平衡。连环蛋白和肌动蛋白细胞骨架与血管内皮钙黏蛋白（VE-钙黏蛋白）的相互作用是稳定 AJs（黏附连接）的主要机制，从而防止肺血管屏障破坏。Rho（Ras 同源）家族的 GTPases 成员和这些 GTPases 的常规 GEF（鸟嘌呤交换因子）已被证明在调节内皮通透性方面发挥重要作用。在这里，我们评估了 DOCK4（胞质分裂的 dedicator 4）-一种非典型 Rho 家族 GTPase GEF 在血管功能中的作用。
我们生成了 DOCK4 缺陷的小鼠，并用 DOCK4 沉默和重组方法在人肺动脉内皮细胞中进行了实验，以评估蛋白定位、内皮细胞通透性和小 GTPase 激活。
我们的数据表明 DOCK4 缺陷的小鼠是存活的。然而，这些小鼠的肺出血是选择性的，肺血管中的平滑肌细胞覆盖不完全，基础微血管通透性增加，以及对 S1P（鞘氨醇-1-磷酸）诱导的血栓素诱导通透性逆转的反应受损。与此一致，DOCK4 在 S1P 处理后迅速向细胞外周易位并与去垢剂不溶性部分结合，其缺失可防止 S1P 诱导的 Rac-1 激活和增强屏障功能。此外，DOCK4 沉默的肺动脉内皮细胞在体外表现出增强的基础通透性，这与增强的 Rho GTPase 激活有关。
我们的发现表明，DOCK4 通过建立 RhoA（Ras 同源家族成员 A）和 Rac-1 介导的肌动蛋白细胞骨架重塑之间的正常平衡来维持肺血管屏障功能所必需的 AJs，这是一种以前未被认识到的非典型 GEF 家族分子的功能。我们的研究还确定 S1P 是 DOCK4 活性的潜在上游调节剂。
