Abstract
ABSTRACTDysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.
Highlights
Endothelial tight junctions (TJ) regulate the transport of fluids and ions through the paracellular pathway and maintain vascular homeostasis (Claesson-Welsh et al, 2021)
Blood vessels were identified by claudin-5 positivity and high expression levels of von Willebrand factor (VWF), whereas lymphatic endothelial vessels were identified by positivity for claudin-5 and podoplanin
To investigate the expression of cingulin in capillaries and postcapillary venules, we stained for the expression of plasmalemma vesicle-associated protein (PLVAP), which is only detected in dermal capillaries (Sauter et al, 1998)
Summary
Endothelial tight junctions (TJ) regulate the transport of fluids and ions through the paracellular pathway and maintain vascular homeostasis (Claesson-Welsh et al, 2021). TJs consist of transmembrane proteins, such as claudins, occludin, tricellulin and junctional adhesion molecules (JAMs), and a dense network of cytoplasmic plaque proteins, including zonula occludens (ZO) proteins and cingulin, which links this junctional protein complex to the cytoskeleton and signalling pathways (Furuse, 2010; Van Itallie and Anderson, 2014). Handling Editor: Kathleen Green Received 15 February 2021; Accepted 20 July 2021 signalling molecules are important for junctional homeostasis. ZO-1 ( known as TJP1) and cingulin have been shown to form condensates that facilitate the repair of junctional complexes (Beutel et al, 2019). The local regulation of the permeability at TJs is not well understood
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