Simulated Microgravity Increases the Permeability of HUVEC Monolayer through Up-Regulation of Rap1GAP and Decreased Rap2 Activation

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.

Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research

“…While a plethora of experimental evidence conducted on numerous cell types has demonstrated that under mechanical unloading cells present cytoskeletal reorganization 12 , 48 , 54 , 58 – 61 followed by transcription and translation of cytoskeletal proteins 62 , very few studies focused on the mechanical unloading impact on VE-Cad molecule in endothelial cells and its role in barrier functionality 63 , 64 .…”

“…A significant contribution was provided by Shi et al 64 , who found that fluorescence dye passage through a HUVEC monolayer cultured in trans well chamber was largely increased after 24 h of 2D clinostat-simulated μG condition. The study also proposed the possible molecular pathways responsible for the enhanced HUVECs permeability being the up-regulation of Ras-related protein 1 (RAP1) and the decrease activation of Ras-related protein 2 (RAP2), which are molecules that antagonistically regulate the adherents junction in endothelial cells 65 .…”

The Blood-Brain Barrier in Space: Implications for Space Travelers and for Human Health on Earth

“…Other changes involved the cytoskeleton, extracellular matrix, mitochondrial distribution, angiogenic response, apoptosis, cell growth, and cell cycle regulation (da Silveira et al, 2020;Dittrich et al, 2018;Maier et al, 2015;Morbidelli et al, 2005;Barravecchia et al, 2021;Crawford-Young, 2006;Grenon et al, 2013;Janmaleki et al, 2016;Kapitonova et al, 2013;Kapitonova et al, 2012;Wehland et al, 2013). In human umbilical vein endothelial cells (HUVEC), microgravity increased the permeability to fluorescein isothiocyanate (FITC)-tagged dextran (Shi et al, 2022) and promoted activation of inflammatory reactions with a shift towards senescence (Versari et al, 2013).…”

Loss of RAP2A Aggravates Cartilage Degradation in TMJOA via YAP Signaling

“…HEK293A cells lose the capacity to respond to ECM or mechanical stress stimulation after RAP2 knockout, and YAP signaling results in continuous activation (Meng et al 2018). The molecular mechanisms of RAP2A have been studied in rare diseases, and only a few studies have investigated RAP2A in tumor migration and vascular remodeling (He et al 2018; Shi et al 2022). As a recently identified critical regulator of YAP signaling, RAP2A may contribute greatly to TMJOA, but research investigating the molecular role of RAP2A in chondrocytes during TMJOA is lacking.…”