Abstract
Space microgravity condition has great physiological influence on astronauts’ health. The interaction of endothelial cells, which control vascular permeability and immune responses, is sensitive to mechanical stress. However, whether microgravity has significant effects on the physiological function of the endothelium has not been investigated. In order to address such a question, a clinostat-based culture model with a HUVEC monolayer being inside the culture vessel under the simulated microgravity (SMG) was established. The transmittance of FITC-tagged dextran was used to estimate the change of integrity of the adherens junction of the HUVEC monolayer. Firstly, we found that the permeability of the HUVEC monolayer was largely increased after SMG treatment. To elucidate the mechanism of the increased permeability of the HUVEC monolayer under SMG, the levels of total expression and activated protein levels of Rap1 and Rap2 in HUVEC cells, which regulate the adherens junction of endothelial cells, were detected by WB and GST pull-down after SMG. As the activation of both Rap1 and Rap2 was significantly decreased under SMG, the expression of Rap1GEF1 (C3G) and Rap1GAP in HUVECs, which regulate the activation of them, was further determined. The results indicate that both C3G and Rap1GAP showed a time-dependent increase with the expression of Rap1GAP being dominant at 48 h after SMG. The down-regulation of the expression of junctional proteins, VE-cadherin and β-catenin, in HUVEC cells was also confirmed by WB and immunofluorescence after SMG. To clarify whether up-regulation of Rap1GAP is necessary for the increased permeability of the HUVEC monolayer after SMG, the expression of Rap1GAP was knocked down by Rap1GAP-shRNA, and the change of permeability of the HUVEC monolayer was detected. The results indicate that knock-down of Rap1GAP reduced SMG-induced leaking of the HUVEC monolayer in a time-dependent manner. In total, our results indicate that the Rap1GAP-Rap signal axis was necessary for the increased permeability of the HUVEC monolayer along with the down-regulation of junctional molecules including VE-cadherin and β-catenin.
Highlights
The endothelium formed by an endothelial cell monolayer functions as a selective barrier between the bloodstream and tissues, preventing the leaking of small molecules, macromolecules, and cells from blood to the underlying tissue [1,2]
The expression and localization of VE-cadherin stand the focal point of the permeability regulation, as it dictates the level of expression and the localization of other junctional molecules, including claudin-5, N-cadherin, and so on [10]
The results indicate that, similar to VE-cadherin, the expression of β-catenin showed a pattern of an upward curve with the valley of VE-cadherin at 24 h simulated microgravity (SMG) treatment
Summary
The endothelium formed by an endothelial cell monolayer functions as a selective barrier between the bloodstream and tissues, preventing the leaking of small molecules, macromolecules, and cells from blood to the underlying tissue [1,2]. Adherens junctions are highly dynamic, and the exchange of molecules or extravasation and intravasation of white blood cells across the endothelial barrier could be achieved by regulation of the permeability of the endothelium [4]. The trans-interaction of VE-cadherin between endothelial cells could recruit β-catenin and p120-catenin to the cytosolic domain of VE-cadherin to stabilize the endothelial adherens junctions. The interaction of α-catenin, vinculin, and EPLIN with the cytosolic domain of VE-cadherin could mediate the connection of adherens junctions to the actin cytoskeleton to form zonula adherens and maintain the functional integrity of the vascular barrier of the endothelium by facilitating the formation of adherens junctions between endothelial cells [6,7,8,9,10,11]. The expression and localization of VE-cadherin stand the focal point of the permeability regulation, as it dictates the level of expression and the localization of other junctional molecules, including claudin-5, N-cadherin, and so on [10]
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