Date of Award
Medical Doctor (MD)
Martin S. Kluger
Jordan S. Pober
Capillary leak in severe sepsis involves disruption of endothelial cell tight junctions in an NF-kB dependent manner. We modeled this process by interleukin-1β (IL-1β) and tumor necrosis factor (TNF) treatment of cultured human dermal microvascular endothelial cell (HDMEC) monolayers which, unlike human umbilical vein endothelial cells (HUVEC), form claudin-5-dependent tight junctions. Continuous monitoring with electrical cell-substrate impedance sensing revealed that the two cytokines disrupt tight junction-dependent HDMEC barriers in two phases, including a decrease to ~10% below basal transendothelial electrical resistance levels over 2 hours (phase 1) and a major fall to < 70% of basal levels by 7-9 hours (phase 2), separated by an interphase plateau of 1 hour. The EC50 values of IL-1β and TNF for phase 1 and 2 leak vary. IL-1β and TNF leak are reversible and independent of cell death. ARNO inhibition with SecinH3 decreased Arf6 activation, but did not significantly abrogate IL-1β-induced leak. TNF-induced-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-kB signaling, shown by inhibition with Bay11 or overexpression of IkB super-repressor, and are blocked by H-1152 or Y-27632, selective inhibitors of Rho-associated kinase that do not block other NF-kB-dependent responses. siRNA knockdown of Rho-associated kinase -1 and -2 also prevents claudin-5 reorganization, myosin light chain phosphorylation, loss of claudin-5/actin co-localization, and reduces phase 1 leak. However, it does not reduce the magnitude of phase 2 leak. IL-1β and TNF disrupts tight junctions in HDMEC in two distinct NF-kB-dependent steps, the first involving Rho-associated kinase and the second likely to involve unidentified but structurally related protein kinase(s).
Kim, Richard, "Interleukin-1β And Tumor Necrosis Factor-Induced Leak Of Human Microvascular Endothelial Tight Junction Barriers" (2016). Yale Medicine Thesis Digital Library. 2060.