Date of Award

January 2016

Document Type


Degree Name

Medical Doctor (MD)



First Advisor

Martin S. Kluger

Second Advisor

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).


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