Allison E. Parent

Mast Cells Contribute to Scar Formation during Fetal Wound Healing

Scar formation is a potentially detrimental process of tissue restoration in adults, affecting organ form and function. During fetal development, cutaneous wounds heal without inflammation or scarring at early stages of development, but begin to heal with significant inflammation and scarring as the skin becomes more mature. One possible cell type that could regulate the change from scarless to fibrotic healing is the mast cell. We show here that dermal mast cells in scarless wounds generated at embryonic day 15 (E15) are fewer in number, less mature and do not degranulate in response to wounding as effectively as mast cells of fibrotic wounds made at embryonic day 18 (E18). Differences were also observed between cultured mast cells from E15 and E18 skin with regard to degranulation and preformed cytokine levels. Injection of mast cell lysates into E15 wounds disrupted scarless healing, suggesting that mast cells interfere with scarless repair. Finally, wounds produced at E18, which normally heal with a scar, healed with significantly smaller scars in mast cell-deficient KitW/W-v mice compared to Kit+/+ littermates. Together, these data suggest that mast cells enhance scar formation, and that these cells may mediate the transition from scarless to fibrotic healing during fetal development.

Cutaneous wound reepithelialization is compromised in mice lacking functional Slug (Snai2)

BACKGROUND Keratinocytes at wound margins undergo partial epithelial to mesenchymal transition (EMT). Based on previous in vitro and ex vivo findings, Slug (Snai2), a transcriptional regulator of EMT in development, may play an important role in this process. OBJECTIVES This study was designed to validate an in vivo role for Slug in wound healing. METHODS Excisional wounds in Slug null and wild type mice were examined histologically at 6, 24, 48, and 72h after wounding; reepithelialization was measured and immunohistochemistry for keratins 8, 10, 14, and 6 and E-cadherin was performed. In 20 Slug null and 20 wild type mice exposed three times weekly to two minimal erythemal doses of UVR, the development of non-healing cutaneous ulcers was documented. Ulcers were examined histologically and by immunohistochemistry. RESULTS The reepithelialization component of excisional wound healing was reduced 1.7-fold and expression of the Slug target genes keratin 8 and E-cadherin was increased at wound margins in Slug null compared to wild type mice. In contrast, no differences in expression of keratins 10 or 14 or in markers of proliferation K6 and Ki-67 were observed. Forty per cent of Slug null mice but no wild type mice developed non-healing cutaneous ulcers in response to chronic UVR. Keratinocytes at ulcer margins expressed high levels of keratin 8 and retained E-cadherin expression, thus resembling excisional wounds. CONCLUSION Slug is an important modulator of successful wound repair in adult tissue and may be critical for maintaining epidermal integrity in response to chronic injury.

The Developmental Transcription Factor Slug Is Widely Expressed in Tissues of Adult Mice

The Slug transcription factor plays an important role in epithelial-mesenchymal transformation during embryogenesis and is expressed in adult tissues during carcinogenesis. By detecting expression of a Slug–β-galactosidase fusion protein, we have now demonstrated that Slug is also re-expressed in a variety of normal tissues in the adult mouse. Slug is expressed at relatively high levels in patchy stretches of basal cells in stratified and pseudostratified epithelium, including skin, oral mucosa, esophagus, stomach, rectum, cervix, and trachea. Slug is also found at variable levels in fibroblasts and stromal smooth muscle cells in many tissues. Sites of more intense Slug expression in mesenchymal tissues include cartilage, kidney glomeruli, lung, ovary, and uterus. Therefore, Slug expression is not restricted to the period of embryonic development or to pathological processes. The pattern of localization to basal cells in various epithelia suggests that Slug may play a role in the cell migration that occurs during continual renewal of these tissues. (J Histochem Cytochem 52:959–965, 2004)

Ultraviolet Radiation-Induced Corneal Degeneration in 129 Mice

Ultraviolet radiation (UVR) is a risk factor for the development of ocular disease in humans, including acute photokeratitis, chronic corneal spheroidal degeneration, and cataract formation. This report describes the ocular lesions seen in 21 mice chronically exposed to UVR as part of a skin carcinogenicity study. All globes were affected to varying degrees. The primary lesion, not previously reported in UVR-exposed mice, was marked loss of keratocytes relative to age-matched controls. Secondary lesions included corneal stromal thinning, keratoconus, corneal vascularization and fibrosis, keratitis, globe rupture, and phthisis bulbi. In addition, more than 90% of UVR-exposed and unexposed lenses had evidence of cataract formation; this is the first report of the occurrence of spontaneous cataracts in 129 mice. In a subsequent study, apoptotic cells were identified histologically and by cleaved caspase 3 immunoreactivity in the corneal epithelium and, less commonly, in the corneal stroma after acute UVR exposure. Based on this finding, we propose that the loss of keratocytes observed in the chronic study was due to UVR-induced apoptosis.

The acute cutaneous inflammatory response is attenuated in Slug-knockout mice

We previously reported ultraviolet radiation (UVR) induction of Slug, a Snail family zinc-finger transcription factor, in the epidermis of mice; we now report that Slug-knockout mice are, unexpectedly, more resistant to sunburn than wild-type mice. There was a marked difference between the cutaneous inflammatory response in the skin of Slug-knockout and wild-type mice from 12 h to 1 week following a single exposure to 3 minimal erythemal doses of UVR. Slug-knockout mice showed a much reduced immediate increase in skin thickness and neutrophil infiltration compared to wild-type mice. However, there were as many or more intraepidermal T cells, dermal mast cells, and dermal blood vessels in the UVR-exposed skin of Slug-knockout mice as in the skin of wild-type mice. Differences in cytokine and chemokine expression following UVR appeared to account for at least some differences between the genotypes in cutaneous inflammatory response. Despite the reported antiapoptotic and antiproliferative role for Slug in some cell types, we observed little difference between the genotypes in UVR-induced keratinocyte apoptosis or proliferation. Our findings indicate an unexpected but important role for Slug in the acute cutaneous inflammatory response to UVR.

Novel differences in the expression of inflammation-associated genes between mid- and late-gestational dermal fibroblasts.

While cutaneous wounds of late‐gestational fetuses and on through adulthood result in scar formation, wounds incurred early in gestation have been shown to heal scarlessly. Unique properties of fetal fibroblasts are believed to mediate this scarless healing process. In this study, microarray analysis was used to identify differences in the gene expression profiles of cultured fibroblasts from embryonic day 15 (E15; midgestation) and embryonic day 18 (E18; late‐gestation) skin. Sixty‐two genes were differentially expressed and 12 of those genes are associated with inflammation, a process that correlates with scar formation in fetal wounds. One of the differentially expressed inflammatory genes was cyclooxygenase‐1 (COX‐1). COX‐1 was more highly expressed in E18 fibroblasts than in E15 fibroblasts, and these differences were confirmed at the gene and protein level. Differences in COX‐1 protein expression were also observed in fetal skin by immunohistochemical and immunofluorescence staining. The baseline differences in gene expression found in mid‐ and late‐gestational fetal fibroblasts suggest that developmental alterations in fibroblasts could be involved in the transition from scarless to fibrotic fetal wound healing. Furthermore, baseline differences in the expression of inflammatory genes by fibroblasts in E15 and E18 skin may contribute to inflammation and scar formation late in gestation.

Slug (Snai2) Expression during Skin and Hair Follicle Development

TO THE EDITOR Our previous investigations showed that Slug (Snai2), a member of the Snail family of developmental transcription factors, is expressed in unperturbed adult murine epidermis, where it regulates a wide variety of gene targets (Newkirk et al., 2008b). Slug expression is induced by a number of growth factors and environmental stimuli (Hudson et al., 2007; Kusewitt et al., 2009). Slug enhances cutaneous wound reepithelialization, skin tumor progression, and the sunburn response (Savagner et al., 2005; Newkirk et al., 2007, 2008a; Hudson et al., 2009). The present studies document Slug expression in embryonic and neonatal epidermis and hair follicles, and significant alterations in hair growth kinetics in Slug knockout mice during the first postnatal hair cycle, indicating a further contribution of Slug to the maintenance of skin homeostasis. Microwave antigen retrieval was performed in citrate buffer on deparaffinized E10–E17 CD1 mouse embryo sections (Zyagen, San Diego, CA). Slides were incubated overnight at 4 1C in monoclonal rabbit anti-Slug antibody (#9585, Cell Signaling, Danvers, MA) diluted 1:50, treated with Biocare Rabbit on Rodent HRP-Polymer (Biocare Medical, Concord, CA) and 3,30-diaminobenzidine chromagen, then counterstained with hematoxylin. Of the many anti-Slug antibodies we have tested, only this antibody consistently gives single bands of appropriate size on protein isolated from HaCaT human epidermal cells (data not shown). Keratin 14 immunostaining was performed similarly, applying first the primary antibody (Covance, Princeton, NJ) at a dilution of 1:500 for 30 minutes at room temperature, and then Envision Plus labeled polymer anti-rabbit-HRP (DAKO, Carpinteria, CA) for 30 minutes. In embryonic tissues, Slug staining was exclusively nuclear. On E10 and E11, Slug was expressed in scattered cells of the single-layered epidermis and in most of the underlying primitive mesenchymal cells, a time when keratin 14 immunoreactivity was seen in only a few cells (Figure 1). At the beginning of periderm formation at E12, when strong keratin 14 immunoreactivity was first observed, Slug was expressed in essentially all skin epithelial cells and in the underlying mesenchyme. As the epithelium underwent stratification, Slug and keratin 14 expression was progressively confined to basal keratinocytes, with Slug localization occurring earlier. Slug staining was observed in progressively fewer dermal cells as primitive mesenchymal cells matured. However, a substantial number of dermal cells continued to express Slug. In the placode, hair germ, and peg stages of hair follicle development, Slug expression was prominent in the thickened and invaginating epithelium, but was absent from the underlying mesenchymal cells that ultimately form the dermal papillae (Figure 1). In pigmented Slug-lacZ 129 mice, the Slug locus was inactivated by an inframe insertion of the b-galactosidase gene into the zinc finger coding region of the Slug gene (Jiang et al., 1998). Mice homozygous for the Slug-lacZ allele are functional Slug knockout animals, but heterozygous Slug knockout mice are phenotypically normal. Daily examination of newborn mice (Figure 2a) revealed that darkening of the skin occurred in all wild-type and heterozygous Slug knockout mice by postnatal days 2–3; however, darkening of the skin of Slug knockout neonates was not seen until postnatal days 4–7. In wild-type and heterozygous knockout mice, hair emergence occurred on postnatal days 5–7, but in knockout mice the emergence was delayed until days 8–10. Differences between knockout and wild-type/heterozygous mice were highly significant for both skin darkening and hair emergence (Po10 7 using the log-rank statistic). Skin darkening in pigmented neonatal mice occurs during early hair follicle growth and hair shafts emerge from follicles during mid to late follicle maturation (Muller-Rover et al., 2001). Thus, our findings suggest delayed hair follicle development in neonatal Slug knockout mice. Immunohistochemical analysis for Slug in 129 wild-type mice showed that Slug was expressed in many, but not all, basal keratinocytes at birth. As previously reported for adult epidermis (Parent et al., 2004), Slug-expressing keratinocytes were clustered around hair follicles. In developing follicles, Slug was stably expressed in the developing external root sheath, hair matrix cells, and some mesenchymal cells of the dermal papilla (Figure 2b). Most interfollicular epidermal cells were Slug-positive at 3 days after birth, but staining progressively declined. At 18 days after birth, follicles in catagen expressed little or no Slug, and Slug expression was also absent from the interfollicular epidermis. Early telogen follicles and interfollicular epidermis showed no Slug immunoreactivity. Localization of Slug was confirmed by immunohistochemical analysis for

Hyperoxemic reperfusion after prolonged cardiac arrest in a rat cardiopulmonary bypass resuscitation model

BACKGROUND The effect of hyperoxygenation at reperfusion, particularly in the setting of cardiac arrest, remains unclear. This issue was studied in a prolonged cardiac arrest model consisting of 25 min cardiac arrest in a rat resuscitated with cardiopulmonary bypass (CPB). The objective of this study was to determine the effect of hyperoxygenation following prolonged cardiac arrest resuscitation on mitochondrial and cardiac function. METHODS Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine and xylazine and instrumented for closed chest cardiopulmonary bypass (CPB). Following a 25-min KCl-induced cardiac arrest, the animals were resuscitated by CPB with 100% oxygen. Three minutes after successful return of spontaneous circulation (ROSC), the animals received either normoxemic reperfusion (CPB with 40-50% oxygen) or hyperoxemic reperfusion (CPB with 100% oxygen) for 1 h. Post-resuscitation hemodynamics, cardiac function, mitochondrial function and immunostaining of 3-nitrotyrosine were compared between the two different treatment groups. RESULTS At 1 h after ROSC, the hyperoxemic reperfusion group had a significant higher mean arterial pressure, less metabolic acidosis and better diastolic function than the normoxemic reperfusion group. Cardiac mitochondria from the hyperoxemic reperfusion group had a higher respiratory control ratio (RCR) and cardiac tissue showed less nitroxidative stress compared to the normoxemic reperfusion group. CONCLUSIONS One hour of hyperoxemic reperfusion after 25 min of cardiac arrest in an in vivo CPB model resulted in significant short-term improvement in myocardial and mitochondrial function compared with 1h of normoxemic reperfusion. This myocardial response may differ from previously reported post-arrest hyperoxia mediated effects following shorter arrest times.

Abstract 2536: VEGF as a potential survival factor for keratinocytes following UV exposure in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Non-melanoma skin cancer (NMSC) is the most prevalent type of cancer. These cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are primarily caused by chronic exposure to ultraviolet light (UV) from the sun. Vascular endothelial growth factor (VEGF) is produced by the skin in response to UV and is known to promote NMSC indirectly through the induction of angiogenesis. Evidence is emerging that VEGF can also promote skin carcinogenesis by directly affecting epidermal keratinocytes and tumor cells. In addition, epidermal keratinocytes express VEGFR-1 and proliferate and migrate directly in response to VEGF. In endothelial cells, VEGF functions as a potent survival factor by inhibiting apoptosis; however, it is not known if VEGF promotes survival in keratinocytes. Keratinocytes undergo apoptosis in response to UV exposure, a process that can be disrupted during carcinogenesis. Here, the role of VEGF and VEGFR-1 in keratinocyte and SCC-13 tumor cell survival after UV exposure was explored. Neutralizing VEGF or VEGFR-1 activity or adding VEGF did not affect UV-induced apoptosis, suggesting that VEGF-VEGFR-1 signaling is not important for keratinocyte or SCC-13 survival in vitro. The role of VEGFR-1 in keratinocyte survival was also examined in vivo. Mice with VEGFR-1-deficient keratinocytes (K14-Cre/VEGFR-1fl/fl) were generated and exposed to a single acute dose of UV light. Keratinocytes in the epidermis of K14-Cre/VEGFR-1fl/fl mice showed a significant increase in apoptosis 24 hours following UV exposure compared to K14-Cre/VEGFR-1+/+ controls. Future studies will investigate skin carcinogenesis in K14-Cre/VEGFR-1fl/fl mice chronically exposed to UV light. Overall, these results suggest that VEGFR-1 mediates the survival of keratinocytes exposed to acute UV in vivo and points to a mechanism by which VEGF could directly promote skin carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2536. doi:1538-7445.AM2012-2536