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Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

Systemic anthrax disease is characterized by vascular leakage pathologies. We previously reported that anthrax lethal toxin (LT) induces human endothelial barrier dysfunction in a cell death-independent manner with actin stress fiber formation and disruption of adherens junctions (AJs). In the present study, we further characterize the molecular changes in the AJ complex and investigate whether AJ structure and barrier function can be preserved by modulating key cytoskeletal signaling pathways. Here, we show that LT reduces total VE-cadherin protein and gene expression but the expression of the key linker protein beta-catenin remained unchanged. The changes in VE-cadherin expression correlated temporally with the appearance of actin stress fibers and a two-fold increase in phosphorylation of the stress fiber-associated protein myosin light chain (p-MLC) and cleavage of Rho-associated kinase-1 (ROCK-1). Co-treatment with ROCK inhibitors (H-1152 and Y27632), but not an inhibitor of MLC kinase (ML-7), blocked LT-induced p-MLC enhancement and stress fiber formation. This was accompanied by the restoration of VE-cadherin expression and membrane localization, and attenuation of the LT-induced increase in monolayer permeability to albumin. Together, these findings suggest the ROCK pathway may be a relevant target for countering LT-mediated endothelial barrier dysfunction.

Anthrax lethal toxin (LT) was previously shown to enhance transcriptional activity of NF-kappaB in tumor necrosis factor-alpha-activated primary human endothelial cells. Here we show that this LT-mediated increase in NF-kappaB activation is associated with the enhanced degradation of the inhibitory proteins IkappaBalpha and IkappaBbeta but not IkappaBepsilon. Moreover, this was accompanied by enhanced activation of the IkappaB kinase complex (IKK), which is responsible for targeting IkappaB proteins for degradation. Importantly, LT enhancement of IkappaBalpha degradation was completely blocked by a selective IKKbeta inhibitor, whereas IkappaBbeta degradation was attenuated, suggesting a mechanistic link. Consistent with the above data, LT-cotreated cells show elevated phosphorylation of two IKK substrates, IkappaBalpha and p65, both of which were blocked by incubation with the IKKbeta inhibitor. Consistent with NF-kappaB activation, LT increased transcription of the NF-kappaB regulated gene CD40. Conversely, LT inhibited transcription of another NF-kappaB-regulated gene, CCL2. This inhibition was linked to the LT-mediated suppression of another CCL2-regulating transcription factor, AP-1 (activator protein-1). These data suggest that LT-mediated enhancement of NF-kappaB is IKK-dependent, but importantly, the net effect of LT on the transcription of proinflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax.

Chemically modified or recombinant hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as oxygen therapeutics or 'blood substitutes' for use in a variety of clinical settings. Oxidative and nitrosative reactions of acellular Hb can limit the effectiveness and compromise the safety of HBOCs. The reactions between Hb and biologically relevant redox active molecules may also perturb redox sensitive signaling pathways. In recent years, systematic in vitro and in vivo structural and functional evaluation of several HBOCs has been carried out and, in some cases, delineated the 'structural' origin of their toxicity. This enables potential protective strategies against Hb-mediated side reactions to be rationally suggested. Here the authors provide an overview of their research experiences, novel insights into the molecular basis of toxicities of these products and some lessons learned.

Vascular endothelial dysfunction is thought to play a prominent role in systemic anthrax pathogenesis. We examined the effect of anthrax lethal toxin (LTx), a key virulence factor of Bacillus anthracis, on the expression of vascular cell adhesion molecule-1 (VCAM-1) on normal and cytokine-stimulated human lung microvascular endothelial cells. Confluent endothelial monolayers were treated with lethal factor (LF), protective antigen (PA), or both (LTx) in the presence or absence of tumor necrosis factor-alpha (TNFalpha). LTx enhanced cytokine-induced VCAM-1 expression and monocyte adhesion. LTx alone had no effect on VCAM-1 expression. LF, PA or the combination of a catalytically inactive mutant LF and PA failed to enhance cytokine-induced VCAM-1 expression. Treatment with inhibitors of mitogen-activated protein kinase kinases (MEKs) and mitogen-activated protein kinases did not reproduce the VCAM-1 enhancement effect of LTx, a known MEK metalloprotease, suggesting LTx-mediated MEK cleavage may not be a contributing factor.

Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection.