ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor‐induced leak

Abstract

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)‐dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ‐forming human dermal microvascular ECs (HDMECs) and adherens junction only forming‐human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction‐disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF‐induced decrease in trans‐endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA‐GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP‐bound Rap1A after four hours but increased GTP‐bound RhoA after 12 h. In cell‐free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A‐C, RhoB‐C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ‐forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP‐mediated Epac1 activation.

Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

“…Junctional protein complexes recruit RhoGEFs (Juettner et al, 2019; Timmerman et al, 2015; Ngok et al, 2012; Acharya et al, 2018; Ngok et al, 2013) and RhoGAPs (Zebda et al, 2013), but they could also prevent the accessibility of these regulators, as it happens for the Rho activity suppressor p190B RhoGAP in columnar epithelial cells (Priya et al, 2015). These accessibility is regulated, for example, by mechanotransduction (Acharya et al, 2018) and may be a relevant target for endothelial hyperpermeability caused by pathological inflammation (Khan et al, 2021; Pierce et al, 2017; Khan et al, 2022)…”

MicroRNA-1 protects the endothelium in acute lung injury

“…Total RNA was extracted using TRIzol (16096040, Thermo Fisher Scientific) and the miRNeasy Kit (Qiagen). mRNA levels were analyzed by RNA sequencing ( 115 ). Genes with differential expression above 0.5 log(fold change) and P value less than 0.05 were considered for further analysis.…”

“…RNA-sequencing data were deposited in the NCBI Gene Expression Omnibus (GEO) database under accession numbers GSE190181, GSE161021 ( 115 ), GSE236263, and GSE239928. Values for all data points found in graphs can be found in the Supporting Data Values file and are available from the corresponding author upon request.…”

cAMP: A master regulator of cadherin‐mediated binding in endothelium, epithelium and myocardium

“…Inhibition of Epac also increased the basal permeability in bovine retinal endothelium and the Epac‐Rap1 pathway preserved TJ organization of ZO1, claudin‐5 and occludin 119 . Independent of cAMP/Epac1 activation, Rap1A can be also triggered by ArhGEF12 to limit disruption of TJ‐dependent integrity induced by inflammation 120 . Furthermore, it was shown that FLRT2/LPHN2/cAMP/Rap1 signaling disassemble integrin‐based focal adhesions and promotes ZO1‐containing TJ.…”

“…119 Independent of cAMP/Epac1 activation, Rap1A can be also triggered by ArhGEF12 to limit disruption of TJ-dependent integrity induced by inflammation. 120 Furthermore, it was shown that FLRT2/LPHN2/cAMP/ Rap1 signaling disassemble integrin-based focal adhesions and promotes ZO1-containing TJ. These effects were associated with restriction of YAP/TAZ signaling cascade known to regulate an angiogenic blood vessel formation and function.…”