Tumor Necrosis Factor Disrupts Claudin-5 Endothelial Tight Junction Barriers in Two Distinct NF-κB-Dependent Phases

Abstract

Capillary leak in severe sepsis involves disruption of endothelial cell tight junctions. We modeled this process by TNF treatment of cultured human dermal microvascular endothelial cell (HDMEC) monolayers, which unlike human umbilical vein endothelial cells form claudin-5-dependent tight junctions and a high-resistance permeability barrier. Continuous monitoring with electrical cell-substrate impedance sensing revealed that TNF disrupts tight junction-dependent HDMEC barriers in discrete steps: an ~5% increase in transendothelial electrical resistance over 40 minutes; a decrease to ~10% below basal levels over 2 hours (phase 1 leak); an interphase plateau of 1 hour; and a major fall in transendothelial electrical resistance to < 70% of basal levels by 8–10 hours (phase 2 leak), with EC50 values of TNF for phase 1 and 2 leak of ~30 and ~150 pg/ml, respectively. TNF leak is reversible and independent of cell death. Leak correlates with disruption of continuous claudin-5 immunofluorescence staining, myosin light chain phosphorylation and loss of claudin-5 co-localization with cortical actin. All these responses require NF-κB signaling, shown by inhibition with Bay 11 or overexpression of IκB super-repressor, and are blocked by H-1152 or Y-27632, selective inhibitors of Rho-associated kinase that do not block other NF-κB-dependent responses. siRNA combined knockdown of Rho-associated kinase-1 and -2 also prevents myosin light chain phosphorylation, loss of claudin-5/actin co-localization, claudin-5 reorganization and reduces phase 1 leak. However, unlike H-1152 and Y-27632, combined Rho-associated kinase-1/2 siRNA knockdown does not reduce the magnitude of phase 2 leak, suggesting that H-1152 and Y-27632 have targets beyond Rho-associated kinases that regulate endothelial barrier function. We conclude that TNF disrupts TJs in HDMECs in two distinct NF-κB-dependent steps, the first involving Rho-associated kinase and the second likely to involve an as yet unidentified but structurally related protein kinase(s).

Prognostic Value of Plasma Tight-Junction Proteins for Sepsis in Emergency Department

“…The pathogenesis of MODS is a result of a complex network of events involving immune cell activation, inflammation, coagulation abnormalities, as well as endothelial dysfunction (21,22). Recently, experimental and clinical studies illustrated that inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-8, and IL-6, destabilized endothelial cell-cell interactions and crippled vascular barrier function in sepsis (10,12,23). Widespread damage of vascular endothelium contributes not only to vascular leak and edema, but also to the shock, microvascular thrombosis, and organ failure of the disease (24).…”

“…Studies have illustrated that plasma levels of VE-cadherin, a major component of AJs, were related to the severity and prognosis of severe sepsis (27). Although the defect of TJs has been proved to contribute to the endothelial barrier dysfunction in sepsis (10,12,22), the prognostic value of circulation TJ proteins in the disease was not explored.…”

“…Claudin (CLDN) is a family of transmembrane proteins with four transmembrane domains (9). Previously studies indicated that endothelial TJs specifically and highly express CLDN-5, with a few exceptions (7,10). CLDNs and occludin (OCLN), another plasma membrane component, bind to the cytosolic protein zonula occludens (ZO)-1, which links to the cytoskeletion and thereby provides junctional stability (6,7,11).…”

“…It has been demonstrated that disruption of TJ-associated proteins contributed to the breakdown of TJs and an increase in vascular permeability in response to stimulation of inflammatory cytokines (10,12,13). Additionally, the breakdown of TJs and release of TJ-associated proteins into the circulation were identified to occur during celiac disease, type 1 diabetes, as well as hemorrhagic transformation after ischemic stroke (14)(15)(16).…”

IL-17 Promotes Neutrophil-Mediated Immunity by Activating Microvascular Pericytes and Not Endothelium

“…Human umbilical vein endothelial cells (HUVECs), human placental pericytes (PCs), human dermal microvascular endothelial cells (HDMECs), and human neutrophils were isolated and cultured using methods previously described (2, 13-15). The neutrophil population derived from this protocol is >99.9% CD15 + and CD68 − , indicating the purity of isolated cells.…”