Yoke-Chen Chang

Differential gene expression profiling of mouse skin after sulfur mustard exposure: Extended time response and inhibitor effect

Sulfur mustard (HD, SM), is a chemical warfare agent that within hours causes extensive blistering at the dermal-epidermal junction of skin. To better understand the progression of SM-induced blistering, gene expression profiling for mouse skin was performed after a single high dose of SM exposure. Punch biopsies of mouse ears were collected at both early and late time periods following SM exposure (previous studies only considered early time periods). The biopsies were examined for pathological disturbances and the samples further assayed for gene expression profiling using the Affymetrix microarray analysis system. Principal component analysis and hierarchical cluster analysis of the differently expressed genes, performed with ArrayTrack showed clear separation of the various groups. Pathway analysis employing the KEGG library and Ingenuity Pathway Analysis (IPA) indicated that cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), and hematopoietic cell lineage are common pathways affected at different time points. Gene ontology analysis identified the most significantly altered biological processes as the immune response, inflammatory response, and chemotaxis; these findings are consistent with other reported results for shorter time periods. Selected genes were chosen for RT-PCR verification and showed correlations in the general trends for the microarrays. Interleukin 1 beta was checked for biological analysis to confirm the presence of protein correlated to the corresponding microarray data. The impact of a matrix metalloproteinase inhibitor, MMP-2/MMP-9 inhibitor I, against SM exposure was assessed. These results can help in understanding the molecular mechanism of SM-induced blistering, as well as to test the efficacy of different inhibitors.

Upregulation of gamma-2 laminin-332 in the mouse ear vesicant wound model†

Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin‐332 (formerly LN‐5), a heterodimer formed from α, β, and γ polypeptide chains. Under static conditions, laminin‐332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin γ2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin‐332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real‐time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin‐γ2 in comparison to the other two chains. Protein production of laminin‐γ2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin‐332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin‐γ2.

Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model

The endoplasmic reticulum (ER) stress response is a cell survival pathway upregulated when cells are under severe stress. Severely damaged mouse ear skin exposed to the vesicant, sulfur mustard (bis-2-chloroethyl sulfide, SM), resulted in increased expression of ER chaperone proteins that accompany misfolded and incorrectly made proteins targeted for degradation. Time course studies with SM using the mouse ear vesicant model (MEVM) showed progressive histopathologic changes including edema, separation of the epidermis from the dermis, persistent inflammation, upregulation of laminin γ2 (one of the chains of laminin-332, a heterotrimeric skin glycoprotein required for wound repair), and delayed wound healing from 24h to 168h post exposure. This was associated with time related increased expression of the cell survival ER stress marker, GRP78/BiP, and the ER stress apoptosis marker, GADD153/CHOP, suggesting simultaneous activation of both cell survival and non-mitochondrial apoptosis pathways. Dual immunofluorescence labeling of a keratinocyte migration promoting protein, laminin γ2 and GRP78/BIP, showed colocalization of the two molecules 72h post exposure indicating that the laminin γ2 was misfolded after SM exposure and trapped within the ER. Taken together, these data show that ER stress is induced in mouse skin within 24h of vesicant exposure in a defensive response to promote cell survival; however, it appears that this response is rapidly overwhelmed by the apoptotic pathway as a consequence of severe SM-induced injury.

Therapeutic potential of a non-steroidal bifunctional anti-inflammatory and anti-cholinergic agent against skin injury induced by sulfur mustard.

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 h post-SM exposure. After 96 h, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermal-epidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure.

Tryptophan fluorescence quenching in rabbit skeletal myosin rod

The solvent accessibility of the four tryptophans of rabbit skeletal muscle myosin rod was investigated using steady-state and time-resolved fluorescence quenching by iodide, acrylamide, and cesium. The quenching by iodide and acrylamide was biphasic; the discrete, long lifetime component was quenched with bimolecular collision constants (kq) of 1 x 10(9) M-1 s-1 and 1.6 x 10(9) M-1 s-1, respectively, while the Gaussian distributed, short lifetime component was quenched with a kq value of 0.3 x 10(9) M-1 s-1 and 0.04 x 10(9) M-1 s-1, respectively. Comparison with kq values for N-acetyl-tryptophanamide indicated that the fractional solvent accessibility was about 25% for the long and less than 10% for the short lifetime component. Cesium quenching was monophasic and provided evidence of an excess of positive charge around these tryptophans. Our findings cast doubt on the general application of the simple coiled-coil model to describe coiled-coil interactions in this protein in solution.

Tryptophan photophysics in rabbit skeletal myosin rod

The fibrous region of myosin (myosin rod) is an alpha-helical, two-stranded coiled-coil made up of identical hydrophobic d sites in the heptad repeat that forms the basis for hydrophobic dimerization. The fluorescence excitation and emission spectra of rod in high salt buffer (where the rod exists as a coiled-coil monomer) at 20 degrees C are red- and blue-shifted, respectively, from the comparable spectra of N-acetyl-tryptophanamide or L-tryptophan. These spectral shifts, as well as red-shifts in the emission spectra induced by excitation on the red edge of the absorption or by increases in temperature, indicate that (on average) the tryptophans are partially exposed to aqueous solvent yet in contact with the protein matrix. The tryptophan intensity decays show an unusual bimodal distribution; the major species has a discrete lifetime of about 5.2 ns while the minor species exhibits a complex decay with a broad (3.4 ns full width at half maximum) Gaussian distribution of lifetimes centered around 1.3 ns. The long lifetime species has a blue-shifted excitation and red-shifted emission characteristic of the indole chromophore in a polar (probably aqueous) environment while the short lifetime species has the spectral parameters characteristic of indole in a non-polar environment. Although assignment of these lifetime species to particular tryptophans in the rod is problematic, this study indicates that the coiled-coil interface presents a complex heterogeneous environment that may undergo rapid conformational mobility.

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.

The molecules in the corneal basement membrane zone affected by mustard exposure suggest potential therapies

Mustard exposures result in epithelial–stromal separations in the cornea and epidermal–dermal separations in the skin. Large blisters often manifest in skin, while the cornea develops microblisters, and, when enough form, the epithelium sloughs. If the exposure is severe, healing can be imperfect and can result in long‐term adverse consequences. For the cornea, this could manifest as recurrent corneal erosions. Since the corneal epithelial–stromal separations are in the region identified by electron microscopy as the lamina lucida, the same region affected by the blistering disease junctional epidermolysis bullosa (JEB), we postulated that the molecules that are defective in JEB would be the same ones cleaved by mustard compounds. These molecules are α6β4 integrin and collagen XVII, which can be cleaved by matrix metalloproteinase‐9 (MMP‐9) and ADAM17, respectively. Therefore, our laboratory has tested MMP‐9 and ADAM17 inhibitors as potential therapies to attenuate corneal mustard injury. Our results demonstrated that inhibiting MMP‐9 and ADAM17 resulted in less epithelial–stromal separation in the corneas at 24 h postexposure, as compared with using only medium as a therapy.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard

ABSTRACT Sulfur mustard (2,2′‐dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM‐induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7days. This was associated with a 2–15 fold increase in expression of the proinflammatory cytokines interleukin‐1&bgr;, interleukin‐6, and tumor necrosis factor &agr; at time points up to 7days post‐SM exposure. Marked increases (20–1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth‐regulated oncogene &agr; (GRO&agr;/CXCL1), monocyte chemoattractant protein 1 (MCP‐1/CCL2), granulocyte‐colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1&agr; (MIP1&agr;/CCL3), and IFN‐&ggr;‐inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell‐derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM‐induced cutaneous injury. HIGHLIGHTSWe found elevated mRNA and protein expression of cytokines/chemokines in SM injured skin.We identified an array of significant inflammatory mediators in skin post‐exposure.The infiltration of inflammatory cells was maximum in skin at 168h post‐SM.The alteration of mediator expression was coordinate with the Inflammatory cells.Inflammatory cell‐derived mediators contribute to the SM induced skin damage.

Temperature dependence of tryptophan photophysics in rabbit skeletal myosin rod

The fluorescence intensity decays of the two tryptophans in rabbit skeletal myosin rod were fit to double exponential functions at six temperatures ranging from 5 to 62 degree(s)C. The radiative decay rate (kf) was estimated from the ratio of the average lifetime and quantum yield (0.11) at 23 degree(s)C. This value (0.024 ns-1 was used to calculate the non-radiative decay rates (equals knr) for each lifetime species at each temperature. Plots of log (knr) versus 1/T for each species showed distinctive breaks at 45 degree(s)C. The Arrhenius activation energies (Ea) for non-radiative quenching of the two lifetime species were significantly different below 45 degree(s)C, with values of 85 J/mole for the long and 31 J/mole for the short lifetime species, while the values were similar, 210 J/mole for the long and 230 J/mole for the short species, above 45 degree(s)C. The thermal unfolding of rod is known to be complex with transitions at 43 degree(s), 47 degree(s), and 53 degree(s)C; only the 43 degree(s) and 53 degree(s)C transitions involve the tryptophan-containing light meromyosin region (LMM) (King & Lehrer (1989) Biochemistry 28, 3498). Our results indicate that the two lifetime components are quenched by different mechanisms below, and similar mechanisms above, 45 degree(s)C. Although the environment of the two pairs of tryptophans differ in the folded protein, they have similar environments in the unfolded protein.