ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure.

PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.

DEP induction of ROS in capillary-like endothelial tubes leads to VEGF-A expression.

Inhalation of diesel exhaust particles (DEPs) is associated with pulmonary and cardiovascular disease. One contributor to pathogenesis is inhaled particles reaching and injuring the lung capillary endothelial cells, and possibly gaining access to the blood stream. Using in vitro capillary tubes as a simplified vascular model system for this process, it was previously shown that DEPs induce the redistribution of vascular endothelial cell-cadherin (VE-Cad) away from the plasma membrane to intracellular locations. This allowed DEPs into the cell cytoplasm and tube lumen, suggesting the tubes may have become permeable (Chao et al., 2011). Here some of the mechanisms responsible for endothelial tube changes after DEP exposure were examined. The results demonstrate that endothelial tube cells mounted an oxidative stress response to DEP exposure. Hydrogen peroxide and oxidized proteins were detected after 24h of exposure to DEPs. Particles induced relocalization of Nrf2 from the cytoplasm to the nucleus, upregulating the expression of the enzyme heme oxygenase-1 (HO-1). Surprisingly, vascular endothelial cell growth factor-A (VEGF-A), initially termed "vascular permeability factor" (VPF), was found to be up-regulated in response to the HO-1 expression induced by DEPs. Similar to DEPs, applied VEGF-A induced relocalization of VE-Cadherin from the cell membrane surface to an intracellular location, and relocalization of VE-cadherin was associated with permeability. These data suggest that the DEPs may induce or contribute to the permeability of capillary-like endothelial tube cells via induction of HO-1 and VEGF-A.

Diesel exhaust particle exposure causes redistribution of endothelial tube VE-cadherin.

Whether diesel exhaust particles (DEPs) potentially have a direct effect on capillary endothelia was examined by following the adherens junction component, vascular endothelial cell cadherin (VE-cadherin). This molecule is incorporated into endothelial adherens junctions at the cell surface, where it forms homodimeric associations with adjacent cells and contributes to the barrier function of the vasculature (Dejana et al., 2008; Venkiteswaran et al., 2002; Villasante et al., 2007). Human umbilical vein endothelial cells (HUVECs) that were pre-formed into capillary-like tube networks in vitro were exposed to DEPs for 24h. After exposure, the integrity of VE-cadherin in adherens junctions was assessed by immunofluorescence analysis, and demonstrated that increasing concentrations of DEPs caused increasing redistribution of VE-cadherin away from the cell-cell junctions toward intracellular locations. Since HUVEC tube networks are three-dimensional structures, whether particles entered the endothelial cells or tubular lumens was also examined. The data indicate that translocation of the particles does occur. The results, obtained in a setting that removes the confounding effects of inflammatory cells or blood components, suggest that if DEPs encounter alveolar capillaries in vivo, they may be able to directly affect the endothelial cell-cell junctions.