Evidence That Cingulin Regulates Endothelial Barrier Function In Vitro and In Vivo

Abstract

Objective-Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells. Approach and Results-We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites. The expression of cingulin in endothelial cells has been poorly characterized because early studies showed a low abundance of cingulin in endothelial cells when compared with epithelia. 18,19 However, the recently characterized role of GEF-H1 in regulating endothelial permeability 20,21 raises the possibility that cingulin is a key regulator of endothelial barrier function. Conclusions-WeHere, we studied the expression and molecular environment of cingulin in different types of endothelial cells. Furthermore, we characterized the effects of exogenous cingulin expression or deletion of the cingulin head domain on the expression of claudin-5 and endothelial barrier function and analyzed barrier function in cingulin-deficient mice. The results indicate that expression levels of cingulin contribute to regulating endothelial barrier function. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement. Results Cingulin Is Localized at Human Endothelial TJ In SituWe analyzed the expression of cingulin in different vascular beds by immunohistochemistry. Endothelial cells of large and small-sized veins and arteries in human skin, lung, and brain tissues stained positive with anticingulin antibodies ( Figure 1A). By immunofluorescence, cingulin labeling was detected in close proximity of VE-cadherin labeling with a Pearson correlation coefficient of 0.11, but was colocalizing with ZO-1 labeling resulting in a Pearson correlation coefficient of 0.31 ( Figure 1B), supporting the notion that cingulin is associated with TJ of endothelial cells. 22 Analysis of mRNA expression levels of cingulin relative to VE-cadherin in different tissues and vessels showed that cingulin expression was detectable in the brain, the vena cava, and the aorta, whereas no expression was detected in muscle an...

Role of Cingulin in Agonist-induced Vascular Endothelial Permeability

“…Cingulin overexpression in cultured ECs induced TJ formation and strengthened the endothelial barrier, although cingulin knock-out only minimally increased basal EC permeability to low molecular weight tracers, without affecting basal permeability to high molecular weight molecules (39). These results are consistent with our data showing no significant differences in basal macromolecular permeability between control and cingulin knockdown ECs.…”

Mind the gap: mechanisms regulating the endothelial barrier

“…Via their cytoplasmic tail, these proteins are anchored to the peripheral actin cytoskeleton by a platform of scaffolding proteins including ZO‐1, cingulin and paracingulin (Fig. ) and interact with regulatory proteins such as Rho GTPases as well as kinases and phosphatases …”

“…In this context, ZO‐1 was shown to form a complex with JACOP (a cingulin‐like protein 1), which then serves as a platform for junctional recruitment of the RhoA activator p114RHoGEF to modulate junctional actomyosin contractility and thereby to stimulate the coupling of the mechano‐transducer vinculin to VE‐cadherin for maintenance of junctional integrity . However, another study showed that overexpression of cingulin (CGN), a protein similar to JACOP, increased barrier properties and this effect was accompanied by decreased claudin‐5 expression in vitro whereas in vivo (in CGN −/− mice) the expression of TJ proteins was unchanged …”

Primary graft dysfunction: pathophysiology to guide new preventive therapies

“…Cingulin is a component of endothelial tight junctions and serves to prevent formation of stress-induced membrane alterations. Overexpression of cingulin in cultured endothelial cells induced tight junction formation and improves endothelial barrier function[56]. A study in a rat lung transplantation model showed that intravascular administration of a cingulin-derived peptide during donor lung procurement, followed by intraperitoneal administration of the same peptide at the time of reperfusion reduced pulmonary edema and improved oxygenation up to 1 month after surgery [57].…”