Sulfur mustard corneal injury is associated with alterations in the epithelial basement membrane and stromal extracellular matrix.

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8-17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45+ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14-28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.

SM1997 downregulates mustard-induced enzymes that disrupt corneal epithelial attachment.

Amino-Plex (SM1997) is a spray or liquid cosmeceutical that has been used for skin dryness, aging, or sun exposure. Its formulation includes electrolytes, trace minerals, amino acids, peptides, nucleosides and nucleotides, all substances that are <10 kDa. It is designed to increase oxygen levels in cells, improve glucose transport, stimulate ATP synthesis, and stimulate collagen formation, actions that can help facilitate repair of damaged cells. It also supports collagen synthesis and formation of healthy granulation tissue, accelerating reepithelization of damaged skin. Here, SM1997 has been tested as an agent to improve the healing of mustard injury to the cornea. The results indicate that SM1997 facilitates the retention of corneal epithelial attachment when applied to corneal organ cultures after nitrogen mustard exposure. In addition, it reduces the activation of enzymes that lead to epithelial-stromal separation, namely, ADAM17 and MMP-9. Therefore, SM1997 should be further investigated as a potential therapy sulfur mustard and nitrogen mustard exposure.

A type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), suppresses skin injury induced by sulfur mustard.

Sulfur mustard (SM) is a highly toxic blistering agent thought to mediate its action, in part, by activating matrix metalloproteinases (MMPs) in the skin and disrupting components of the basement membrane zone (BMZ). Type IV collagenases (MMP-9) degrade type IV collagen in the skin, a major component of the BMZ at the dermal-epidermal junction. In the present studies, a type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), was tested for its ability to protect the skin against injury induced by SM in the mouse ear vesicant model. SM induced inflammation, epidermal hyperplasia and microblistering at the dermal/epidermal junction of mouse ears 24-168 h post-exposure. This was associated with upregulation of MMP-9 mRNA and protein in the skin. Dual immunofluorescence labeling showed increases in MMP-9 in the epidermis and in the adjacent dermal matrix of the SM injured skin, as well as breakdown of type IV collagen in the basement membrane. Pretreatment of the skin with BiPS reduced signs of SM-induced cutaneous toxicity; expression of MMP-9 mRNA and protein was also downregulated in the skin by BiPS. Following BiPS pretreatment, type IV collagen appeared intact and was similar to control skin. These results demonstrate that inhibiting type IV collagenases in the skin improves basement membrane integrity after exposure to SM. BiPS may hold promise as a potential protective agent to mitigate SM induced skin injury.

Expression of Laminin γ2 Proteolytic Fragments in Murine Skin Following Exposure to Sulfur Mustard.

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.

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

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 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days 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 α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-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.

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.

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.

Doxycycline hydrogels as a potential therapy for ocular vesicant injury.

PURPOSE: The goals of this study were (1) to compare the injury at the basement membrane zone (BMZ) of rabbit corneal organ cultures exposed to half mustard (2 chloroethyl ethyl sulfide, CEES) and nitrogen mustard with that of in vivo rabbit eyes exposed to sulfur mustard (SM); (2) to test the efficacy of 4 tetracycline derivatives in attenuating vesicant-induced BMZ disruption in the 24-h period postexposure; and (3) to use the most effective tetracycline derivative to compare the improvement of injury when the drug is delivered as drops or hydrogels to eyes exposed in vivo to SM. METHODS: Histological analysis of hematoxylin and eosin­stained sections was performed; the ultrastructure of the corneal BMZ was evaluated by transmission electron microscopy; matrix metalloproteinase-9 was assessed by immunofluorescence; doxycycline as drops or a hydrogel was applied daily for 28 days to eyes exposed in vivo to SM. Corneal edema was assessed by pachymetry and the extent of neovascularization was graded by length of longest vessel in each quadrant. RESULTS: Injury to the BMZ was highly similar with all vesicants, but varied in degree of severity. The effectiveness of the 4 drugs in retaining BMZ integrity did not correlate with their ability to attenuate matrix metalloproteinase-9 expression at the epithelial­stromal border. Doxycycline was most effective on organ cultures; therefore, it was applied as drops or a hydrogel to rabbit corneas exposed in vivo to SM. Eyes were examined at 1, 3, 7, and 28 days after exposure. At 7 and 28 days after SM exposure, eyes treated with doxycycline were greatly improved over those that received no therapy. Corneal thickness decreased somewhat faster using doxycycline drops, whereas the hydrogel formulation decreased the incidence of neovascularization. CONCLUSIONS: Corneal cultures exposed to 2-chloroethyl ethyl sulfide and nitrogen mustard were effective models to simulate in vivo SM exposures. Doxycycline as drops and hydrogels ameliorated vesicant injury. With in vivo exposed animals, the drops reduced edema faster than the hydrogels, but use of the hydrogels significantly reduced neovascularization. The data provide proof of principle that a hydrogel formulation of doxycycline as a daily therapy for ocular vesicant injury should be further investigated.

Doxycycline loaded poly(ethylene glycol) hydrogels for healing vesicant-induced ocular wounds.

Half mustard (CEES) and nitrogen mustard (NM) are commonly used surrogates and vesicant analogs of the chemical warfare agent sulfur mustard. In the current study, in situ forming poly(ethylene glycol) (PEG)-based doxycycline hydrogels are developed and evaluated for their wound healing efficacy in CEES and NM-exposed rabbit corneas in organ culture. The hydrogels, characterized by UV-Vis spectrophotometry, rheometry, and swelling kinetics, showed that the hydrogels are optically transparent, have good mechanical strength and a relatively low degree of swelling (<7%). In vitro doxycycline release from the hydrogel disks (0.25% w/v) was found to be biphasic with release half times of approximately 12 and 72h, respectively, with 80-100% released over a 7-day period. Permeation of doxycycline through vesicant wounded corneas was found to be 2.5 to 3.4 fold higher than non-wounded corneas. Histology and immunofluorescence studies showed a significant reduction of matrix metalloproteinase-9 (MMP-9) and improved healing of vesicant-exposed corneas by doxycycline hydrogels compared to a similar dose of doxycycline delivered in phosphate buffered saline (PBS, pH 7.4). In conclusion, the current studies demonstrate that the doxycycline-PEG hydrogels accelerate corneal wound healing after vesicant injury offering a therapeutic option for ocular mustard injuries.

Collagens.

The collagens represent a family of trimeric extracellular matrix molecules used by cells for structural integrity and other functions. The three alpha chains that form the triple helical part of the molecule are composed of repeating peptide triplets of glycine-X-Y. X and Y can be any amino acid but are often proline and hydroxyproline, respectively. Flanking the triple helical regions (i.e., Col domains) are non-glycine-X-Y regions, termed non-collagenous domains. These frequently contain recognizable peptide modules found in other matrix molecules. Proper tissue function depends on correctly assembled molecular aggregates being incorporated into the matrix. This review highlights some of the structural characteristics of collagen types I-XXVIII.

Expression of type XXIII collagen mRNA and protein.

Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.

Extracellular matrix changes in the umbilical arteries of growth-restricted fetuses.

OBJECTIVE: This study was undertaken to determine whether the expression of extracellular matrix components (ECM) is altered in the umbilical arteries from preterm fetal growth-restricted (FGR) pregnancies. STUDY DESIGN: Preterm pregnancies with FGR were compared with appropriately grown preterm pregnancies. Umbilical artery messenger RNA (mRNA) levels for fibrillar collagens I and III, nonfibrillar collagen XIV, and decorin were determined by using relative reverse transcriptase polymerase chain reaction (RT-PCR). The mRNA expression was compared between cases and controls by using the Student t test. P < or = .05 was considered significant. RESULTS: Eight FGR cases and 5 control pregnancies were analyzed. Mean counts per minute (cpm) +/- SEM for collagen I and collagen III were increased in FGR pregnancies. There were no differences in mRNA expression of collagen XIV or decorin. CONCLUSION: Increased mRNA expression of collagen I and III, but not collagen XIV or decorin, is found in the umbilical arteries of preterm FGR pregnancies.

Collagen XXIV, a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone.

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.

Collagen messenger RNA expression in the human amniochorion in premature rupture of membranes.

OBJECTIVE: It has been suggested that there is a decrease in the collagen content of the fetal membranes when there is premature rupture of the membranes before the onset of labor. This study was designed to determine whether decreased amniochorion collagen production (as measured by reduced amounts of messenger RNA) or alterations in relative production of different fibrillar and nonfibrillar collagens are associated with premature rupture of the membranes. STUDY DESIGN: Fetal membranes were collected after preterm (24-36 weeks of gestation) and term (> or =37 weeks of gestation) deliveries both with and without premature rupture of the membranes. Specimens with evidence of histologic chorioamnionitis were excluded. The messenger RNA levels for fibrillar collagen types I, III, and V and fibril-associated collagens with interrupted triple-helices types XII and XIV were measured with relative quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The messenger RNA levels for fibrillar collagens decreased with advancing gestational age. Preterm premature rupture of membranes was associated with increased messenger RNA levels for fibrillar collagens and fibril-associated collagens with interrupted triple-helices collagen XII, but not type XIV. The greatest change in relative amounts of collagen messsenger RNA was demonstrated by an increased type I/XIV ratio, which was due to the up-regulation of type I levels, but not type XIV levels. CONCLUSION: A rise in fibrillar collagen production (messenger RNA) for types I, III, and V and fibril-associated collagens with interrupted triple-helices collagen type XII is observed with preterm premature rupture of the membranes. There is no evidence for a similar up-regulation of messenger RNA for fibril-associated collagens with interrupted triple-helices collagen type XIV. The rise in the collagen I/XIV messenger RNA ratio in preterm premature rupture of the membranes may result in collagen fibrils without enough stabilizing fibril-associated collagens with interrupted triple-helices type XIV on the fibril surface to maintain structural integrity.