In many patients, the sympathetic nervous system supports pain and other features of complex regional pain syndrome (CRPS). Accumulating evidence suggests that interleukin (IL)-6 also plays a role in CRPS, and that catecholamines stimulate production of IL-6 in several tissues. We hypothesized that norepinephrine acting through specific adrenergic receptors expressed on <em>keratinocytes</em> stimulates the production of IL-6 and leads to nociceptive sensitization in a rat tibial fracture/cast model of CRPS. Our approach involved catecholamine depletion using 6-hydroxydopamine or, alternatively, guanethidine, to explore sympathetic contributions. Both agents substantially reduced nociceptive sensitization and selectively reduced the production of IL-6 in skin. Antagonism of IL-6 signaling using TB-<em>2</em>-081 also reduced sensitization in this model. Experiments using a rat <em>keratinocyte</em> cell line demonstrated relatively high levels of β<em>2</em>-adrenergic receptor (β<em>2</em>-AR) expression. Stimulation of this receptor greatly enhanced IL-6 expression when compared to the expression of IL-1β, tumor necrosis <em>factor</em> (TNF)-α, or nerve <em>growth</em> <em>factor</em>. Stimulation of the cells also promoted phosphorylation of the mitogen-activated protein kinases P38, extracellular signal-regulated kinase, and c-Jun amino-terminal kinase. Based on these in vitro results, we returned to animal testing and observed that the selective β<em>2</em>-AR antagonist butoxamine reduced nociceptive sensitization in the CRPS model, and that local injection of the selective β<em>2</em>-AR agonist terbutaline resulted in mechanical allodynia and the production of IL-6 in the cells of the skin. No increases in IL-1β, TNF-α, or nerve <em>growth</em> <em>factor</em> levels were seen, however. These data suggest that in CRPS, norepinephrine released from sympathetic nerve terminals stimulates β<em>2</em>-ARs expressed on epidermal <em>keratinocytes</em>, resulting in local IL-6 production, and ultimately, pain sensitization.

Acute lung injury and compromised alveolar development characterize bronchopulmonary dysplasia (BPD) of the premature neonate. High levels of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), a cell-cell mediator with pleiotrophic lung effects, are associated with low BPD risk. KGF decreases mortality in hyperoxia-exposed newborn rodents, a classic model of injury-induced impaired alveolarization, although the pulmonary mechanisms of this protection are poorly defined. These were explored through in vitro and in vivo approaches in the rat. Hyperoxia decreased by 30% the rate of wound closure of a monolayer of fetal alveolar epithelial cells, due to cell death, which was overcome by recombinant human KGF (100 ng/ml). In rat pups exposed to >95% O<em>2</em> from birth, increased viability induced by intraperitoneal injection of KGF (<em>2</em> microg/g body wt) every other day was associated with prevention of neutrophil influx in bronchoalveolar lavage (BAL), prevention of decreases in whole lung DNA content and cell proliferation rate, partial prevention of apoptosis increase, and a markedly increased proportion of surfactant protein B-immunoreactive cells in lung parenchyma. Increased lung antioxidant capacity is likely to be due in part to enhanced CAAT/enhancer binding protein alpha expression. By contrast, KGF neither corrected changes induced by hyperoxia in parameters of lung morphometry that clearly indicated impaired alveolarization nor had any significant effect on tissue or BAL surfactant phospholipids. These findings evidence KGF alveolar epithelial cell protection, enhancing effects on alveolar repair capacity, and anti-inflammatory effects in the injured neonatal lung that may account, at least in part, for its ability to reduce mortality. They argue in favor of a therapeutic potential of KGF in the injured neonatal lung.

Epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44(low)-CD<em>2</em>4(high) (CD44L) epithelial phenotype to a CD44(high)-CD<em>2</em>4(-/low) (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT, although excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase <em>2</em> (SOD<em>2</em>) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD<em>2</em> transcriptional regulation in EMT, as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference and flow cytometric approaches, we find that transforming <em>growth</em> <em>factor</em> (TGF)-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD<em>2</em> upregulation. SOD<em>2</em> induction in transformed <em>keratinocytes</em> was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD<em>2</em> gene expression appeared to be transcriptionally regulated by NF-κB and ZEB<em>2</em>, but not ZEB1. Moreover, SOD<em>2</em>-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. These data provide novel mechanistic insights into the dynamic expression of SOD<em>2</em> during EMT. In addition, we delineate a functional role for SOD<em>2</em> in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.

Recently we reported a low calcium (110 microM) serum-free medium (LHC-1) for clonal <em>growth</em> of normal human bronchial epithelial (NHBE) cells. NHBE cells within colonies are small (mean surface area = 1,<em>2</em>50 mu<em>2</em>) rarely migratory, have few tonofilaments, and multiply with an average population doubling time of <em>2</em>8 h. We have also noted that adding small amounts of blood-derived serum to LHC-1 medium (as little as <em>2</em>%) significantly decreased the clonal <em>growth</em> rate. We have now found that the <em>growth</em> inhibiting effect of serum is due to the induction of squamous (terminal) differentiation. Serum quickly increases the size of the cells (mean surface area = 4,900 mu<em>2</em>). In addition, the cells acquire numerous desmosomal junctions and an extensive network of keratin bundles. In contrast, human lung carcinoma cells multiply rapidly at clonal density in LHC-1 medium containing as much as 8% serum. Although high concentrations of calcium ions in the medium are known to induce squamous differentiation of epidermal <em>keratinocytes</em> in the absence of serum, high levels of Ca<em>2</em>+ (up to 1,000 microM) increased the number of desmosomal junctions, but did not significantly affect the clonal <em>growth</em> rate or size of the NHBE cells. However, high concentrations of calcium (above 450 microM) were found to potentiate serum differentiation-inducing activity. On the other hand, cholera toxin (10 ng/ml) inhibited the differentiation-inducing activity of serum. These results show that squamous differentiation of NHBE cells can be induced by serum and the potency of these serum <em>factors</em> can be modulated. In addition, the data show that lung carcinoma cells differ from their normal counterparts by not undergoing differentiation in the presence of serum.

Wound closure with cultured skin substitutes results in epithelium that is consistently hypopigmented. Hypothetically, addition of human melanocytes to cultured skin grafts may result in normal pigmentation of healed skin. Skin substitutes were composed of human epidermal <em>keratinocytes</em> and melanocytes, dermal fibroblasts, and collagen-glycosaminoglycan substrates, and were incubated for 1<em>2</em> d in media for <em>keratinocyte</em> <em>growth</em> (KG, n = 4), for <em>keratinocyte</em> differentiation containing four fatty acids and vitamin E with basic fibroblast <em>growth</em> <em>factor</em> (KDF, n = 6) or epidermal <em>growth</em> <em>factor</em> (KDE, n = 6), or for melanocyte <em>growth</em> (MG, n = 6) with phorbol ester and 5% fetal bovine serum. Skin substitutes were grafted orthotopically to full-thickness skin wounds (<em>2</em> x <em>2</em> cm) on athymic mice, and scored for percent original wound size (+/- SEM), visible pigmentation (number pigmented/n), and positive staining for human leukocyte antigens (HLA)-ABC after 6 weeks on the mice. The data show that cultured skin grafts containing human melanocytes that are incubated in KDE or MG media have statistically significant reduction in wound contraction, 1:1 correlation of expression of pigment and HLA-ABC, and increased frequency of pigmentation after healing compared to incubation in KG or KDF media. Transmission electron microscopy confirmed the presence of melanocytes, melanosomes, and pigment transfer to <em>keratinocytes</em> in pigmented skin. These results suggest that survival and differentiated function of cultured epithelium can support melanization of skin, and that skin analogues exposed to phorbol ester in vitro can support skin pigmentation after wound healing.

Successful remodeling of the uterine spiral arteries is essential for a complication-free pregnancy and is best described in terms of its morphologic features. The molecular mediators and cellular sources of spiral artery remodeling are not known, although a role for uterine leukocytes has been proposed. Immunohistochemical assessment of placental bed biopsies demonstrated uterine NK cells, macrophages, and T lymphocytes in the wall and adventitia of spiral arteries at different stages of remodeling, regardless of the presence of extravillous trophoblast cells. Leukocytes were more prevalent in vessel adventitia than wall, and macrophages were the most abundant leukocyte population. Macrophages, separated from early pregnancy decidua, did not alter extravillous trophoblast cells invasion or vascular smooth muscle cell organization or differentiation status but did induce extracellular matrix breakdown (reduced immunostaining of laminin, P = 0.05; fibronectin, P = 0.0<em>2</em>) and were able to phagocytose apoptotic vascular smooth muscle cells. Decidual macrophages were shown to secrete a wide range of cytokines (IL-1β, -<em>2</em>, -4, -5, -6, -8, -10, and -13 and TNF-α), proteases (matrix metalloproteinase-1, -<em>2</em>, -7, -9, and -10), and angiogenic <em>growth</em> <em>factors</em> (angiogenin, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factor</em> B, vascular endothelial <em>growth</em> <em>factor</em> A, and angiopoietin-1 and -<em>2</em>). We conclude that spiral artery remodeling involves the coordinated activity of a range of cell types, including extravillous trophoblast cells, decidual uterine NK cells, and macrophages in a carefully, spatiotemporally regulated manner.

OBJECTIVE

To investigate the effects of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) on early wound healing in the corneal epithelium and stroma.

METHODS

Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Cardiff University, and the Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom.

METHODS

Corneal keratocyte cell cultures and wounded corneal organ cultures (both maintained in serum-free conditions) were treated with 0.1 to 100 ng/mL of HGF or KGF for up to 5 days. Cell cultures were assessed for proliferation, migration, and differentiation into myofibroblasts. Organ cultures were used to evaluate the effect of HGF and KGF on reepithelialization following a wound, epithelial morphology and stratification, keratocyte numbers directly beneath the wounded area, and differentiation into myofibroblasts.

RESULTS

The 2 growth factors had opposite effects on the rate of reepithelialization, with HGF delaying and KGF accelerating epithelial coverage of the wound. Morphologic assessment showed that both growth factors affected the stratification and differentiation of the epithelium. Both factors stimulated proliferation of keratocytes in serum-free cell culture, although neither induced the appearance of myofibroblasts. This was in contrast to wounded organ cultures treated with 100 ng/mL HGF, in which large numbers of myofibroblasts were observed under the wound. Control corneas and those receiving KGF contained very few myofibroblasts. Keratocyte repopulation of the denuded area under the wound was enhanced in the presence of HGF but decreased in response to KGF.

CONCLUSIONS

Hepatocyte growth factor and KGF appeared to have potent and often opposite effects on epithelial and stromal cells following a wound. Hepatocyte growth factor was more detrimental than KGF, resulting in an aberrant epithelium and mass differentiation of keratocytes into myofibroblasts. Inhibition of HGF may be an appropriate therapeutic intervention in the case of persistent epithelial defects and to prevent fibrosis following a corneal stromal wound such as can occur after refractive surgery.

Pulmonary surfactant protein D (SP-D) is expressed in alveolar type II and bronchiolar epithelial cells and is secreted into alveoli and conducting airways. However, SP-D has also been measured in serum and is increased in patients with acute respiratory distress syndrome, pulmonary fibrosis, and alveolar proteinosis. To demonstrate that SP-D can be measured in rat serum, we instilled rats with <em>keratinocyte</em> <em>growth</em> <em>factor</em>, which produces type II cell hyperplasia and an increase in SP-D in bronchoalveolar lavage fluid (BALF). To evaluate serum SP-D as a biomarker of lung injury, we examined several injury models. In rats treated with 1 unit of bleomycin, serum SP-D was elevated on days 3, 7, 14, and <em>2</em>8 after instillation, and SP-D mRNA was increased in focal areas as detected by in situ hybridization. However, there was no increase in whole lung SP-D mRNA when the expression was normalized to whole lung 18S rRNA. After instillation of <em>2</em> units of bleomycin, the serum levels of SP-D were higher, and SP-D was also increased in BALF and lung homogenates. In another model of subacute injury, serum SP-D was increased in rats treated with paraquat plus oxygen. Finally to evaluate acute lung injury, we instilled rats with HCl; SP-D was increased at 4 h after instillation. Our data indicate that serum SP-D may be a useful indicator of lung injury and type II cell hyperplasia in rats.

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) isoforms and their receptors in the wound healing process, their roles in scarless wound repair observed in early gestation and their functions in human fetal skin development, and structural and functional maintenance are still not well understood. In this study, we explore the expression and distribution characteristics of three TGF-beta isoforms and their receptors, TGF-betaRI (TBRI) and TGF-betaRII (TBRII), in fetal and postnatal skins to understand the relevance of these five proteins to skin development and elucidate the mechanism(s) underlying the phenotypic transition from scarless to scar-forming healing observed during fetal gestation. Fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages from 13 to 3<em>2</em> weeks and postnatal skin specimens were collected from patients undergoing plastic surgery. Gene expression and positive immunohistochemical signals of TGF-beta(1), TGF-beta(<em>2</em>), TGF-beta(3), TBRI, and TBRII could all be detected in fetal and postnatal skins. In early gestation, gene expression of TGF-beta(1), TBRI, and TBRII was weaker and protein contents were less compared with postnatal skins (p < 0.05). In contrast, more TGF-beta(<em>2</em>) mRNA transcript was found in early gestation than in late gestation and in postnatal skins, whereas protein content of this <em>growth</em> <em>factor</em> increased during gestation. Lastly, mRNA transcript and protein contents of TGF-beta(3) were apparently higher in early gestation compared to postnatal skin (p < 0.05). In postnatal skin, granules containing the three TGF-beta isoforms were mainly distributed in the cytoplasm and extracellular matrix of epidermal cells, interfollicular <em>keratinocytes</em>, and some fibroblasts. TBRI and TBRII were chiefly located in the cellular membrane of epidermal <em>keratinocytes</em> and some fibroblasts. The endogenous three TGF-beta isoforms and their receptors may be involved in the development of embryonic skin and in the maintenance of cutaneous structure and function, and also in postnatal wound healing. The differential levels of TGF-beta isoforms may provide either a predominantly antiscarring or profibrotic signal upon wounding depending on the gestational period. Lower expression of their receptors in early gestational skins may be a reason for the reduced ability to perceive ligands, ultimately leading to scar-free healing.

We previously reported an increase in signal transducer and activator of transcription 3 (Stat3) activation in keloid fibroblasts, which contributes to collagen production, cell proliferation, and migration. We further investigated the effect of epithelial-mesenchymal interaction on Stat3 in normal and keloid fibroblasts in noncoculture and coculture conditions. pY705 Stat3 was higher in keloid fibroblasts compared to normal fibroblasts in noncoculture. However, a more drastic decrease in pY705 Stat3 was observed in keloid fibroblasts compared to normal fibroblasts when cocultured with their respective <em>keratinocytes</em> over 5 days. To explore this paracrine effect, we examined the secretion of cytokines by cytokine arrays. Altered cytokine production was detected in keloid fibroblasts and <em>keratinocytes</em>, either in noncoculture or coculture conditions. IL-6, IL-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinases (TIMPs)-1, and TIMP-<em>2</em> were major cytokines detected. Angiogenin, oncostatin M (OSM), vascular endothelial cell <em>growth</em> <em>factor</em>, IGF-binding protein-1, osteoprotegerin, and transforming <em>growth</em> <em>factor</em>-beta<em>2</em> were present in keloid <em>keratinocyte</em>-fibroblast coculture, but absent in normal <em>keratinocyte</em>-fibroblast coculture. Only IL-6 and OSM stimulated strong pY705 Stat3 and cell proliferation in both normal and keloid fibroblasts. Other cytokines increased proliferation of keloid fibroblasts, but not normal fibroblasts, suggesting an altered state in keloid fibroblasts. Multiple cytokines likely contribute to keloid pathogenesis and a combinatorial neutralizing antibody/cytokine therapy may be effective in ameliorating keloid scars.

A two-hit model of acid aspiration was used to examine the effect of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) on chemokine levels and neutrophil recruitment into the lung. Mice were subjected to cecal ligation and puncture and then either KGF or saline, intratracheally (i.t.). Forty-eight hours later, the mice were given i.t. acid. After 8 h, neutrophil counts in bronchoalveolar lavage (BAL) fluid were significantly decreased in animals pretreated with KGF (<em>2</em>3 +/- 4 x 10(3)/mouse) compared with saline (74 +/- <em>2</em> x 10(3)/mouse). In addition, the BAL fluid IL-6 levels were decreased in the KGF-treated group (88+/- 44 pg/mL) compared with the saline group (166 +/- 34 pg/mL). To examine the mechanism behind the KGF-induced reduction in neutrophil influx, the murine chemokines KC and macrophage inflammatory protein (MIP)-<em>2</em>alpha were measured. KC levels in plasma and BAL fluid were not significantly different between the treatment groups. Likewise, levels of MIP-<em>2</em>alpha in plasma were not affected by KGF treatment. However, 8 h after acid aspiration, MIP-<em>2</em>alpha concentrations were significantly lower in the KGF-treated group. The ratio of MIP-<em>2</em>alpha in BAL fluid versus plasma was lower in the KGF group (0.7<em>2</em> +/- 0.<em>2</em>8) than in the saline group at 3 h (<em>2</em>.<em>2</em>3 +/- 0.93) and also significantly lower in the KGF group (3.0<em>2</em> +/- 0.78) compared with the saline group (6.<em>2</em>3 +/- 1.19) at 8 h. In this study, KGF pretreatment after acid aspiration was associated with reduced neutrophil recruitment into the lung and a decrease in MIP-<em>2</em>alpha gradients between BAL fluid and plasma.

Non-thermal atmospheric-pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and <em>growth</em> arrest in tumour cells. Detailed investigations of plasma-cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal <em>growth</em> <em>factor</em> receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT <em>keratinocytes</em> (human adult low calcium temperature <em>keratinocytes</em>) promoting adhesion, migration and proliferation as one important feature of plasma-cell interactions. HaCaT <em>keratinocytes</em> were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry <em>2</em>4 h after plasma treatment. Besides a reduction of cell viability a significant down-regulation of E-cadherin and the EGFR expression occurred. The influence on α<em>2</em>- and β1-integrins was less pronounced, and expression of ICAM-1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H<em>2</em>DCFDA (<em>2</em>',7'-dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two-dimensional cultures of HaCaT <em>keratinocytes</em> appear to be a suitable method of investigating plasma-cell interactions.

As an initial step towards a tissue-engineered esophagus, rat esophageal epithelial cells (REEC) were isolated and characterized for epithelial identity, adhesion protein preference, and in vitro interaction with natural and synthetic scaffolds. The scaffolds consisted of AlloDerm (LifeCell Corporation, Branchburg, NJ), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic) acid (75:<em>2</em>5) (PLGA75), poly(lactic-co-glycolic) acid (50:50) (PLGA50), and polycaprolactone/poly(L-lactic acid) (50:50) (PCL/PLLA). Various <em>factors</em>-including calcium concentration, scaffold composition, and pore size--were evaluated for their influence on epithelial <em>growth</em> and differentiation. By day 18, <em>keratinocytes</em> seeded on AlloDerm cultured under high Ca(++) (1.5mm) conditions showed a proliferating basal cell layer, epithelial stratification (5--6 layers) and a thick keratin layer. The synthetic scaffolds (PLGA, PLLA, PCL/PLLA) also showed complete surface coverage, regions of proliferating basal cells, and evidence of stratification (<em>2</em>--3 layers) and keratinization. The highly porous nature of the synthetic scaffolds, however, limited the formation of a continuous epithelial layer and resulted in a lack of overall spatially-defined differentiation. In conclusion, rat esophageal epithelial cells were successfully isolated and characterized, with cells seeded on AlloDerm showing superior epithelial organization and stratification compared to synthetic scaffolds. Modification of the synthetic scaffold's surface properties and pore size may be necessary to mimic epithelial behavior on natural scaffolds.

The naturally occurring polycationic polyamines including putrescine, spermidine, and spermine play an important role in cell <em>growth</em>, differentiation, and gene expression. However, circulating polyamines are potential substrates for several oxidizing enzymes including copper-containing serum amine oxidase. These enzymes are capable of oxidizing serum polyamines to several toxic metabolites including aldehydes and H(<em>2</em>)O(<em>2</em>). In this study, we investigated the effects of polyamines as inducers of phase <em>2</em> enzymes and other genes that promote cell survival in a cell culture system in the presence of bovine serum. Spermidine and spermine (50 microM) increased NAD(P)H quinone oxidoreductase (NQO1) activity up to 3-fold in murine <em>keratinocyte</em> PE cells. Transcript levels for glutathione S-transferase (GST) A1, GST M1, NQO1, gamma-glutamylcysteine ligase regulatory subunit, and UDP-glucuronyltransferase 1A6 were significantly increased by spermidine and this effect was mediated through the antioxidant response element (ARE). The ARE from the mouse GST A1 promoter was activated about 9-fold by spermine and 5-fold by spermidine treatment, but could be inhibited by the amine oxidase inhibitor, aminoguanidine, suggesting that acrolein or hydrogen peroxide generated from polyamines by serum amine oxidase may be mediators for phase <em>2</em> enzyme induction. Elevations of ARE-luciferase expression and NQO1 enzyme activity by spermidine were not affected by catalase, while both were completely repressed by aldehyde dehydrogenase treatment. Direct addition of acrolein to PE cells induced multiple phase <em>2</em> genes and elevated nuclear levels of Nrf<em>2</em>, a transcription <em>factor</em> that binds to the ARE. Expression of mutant Nrf<em>2</em> repressed the activation of the ARE-luciferase reporter by polyamines and acrolein. These results indicate that spermidine and spermine increase the expression of phase <em>2</em> genes in cells grown in culture through activation of the Nrf<em>2</em>-ARE pathway by generating the sulfhydryl reactive aldehyde, acrolein.

Reactive oxygen species (ROS) disrupt the barrier function of airway epithelial cells through a mechanism that appears to involve remodeling of the actin cytoskeleton. Similarly, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has been shown to protect against ROS-induced loss of barrier function through a mechanism that may also involve the actin cytoskeleton. To further determine the role of the actin cytoskeleton in ROS-induced barrier injury, we quantified the relative amount of total actin associated with the cytoskeleton following exposure to hydrogen peroxide (H(<em>2</em>)O(<em>2</em>)) and pretreatment with KGF. We also determined the role of the actin-myosin contractile mechanism in the process by quantifying the relative amount of myosin heavy chain (MHC) associated with the cytoskeleton. While the transepithelial resistance (TER) of a monolayer of airway epithelial cells (Calu-3) decreased after <em>2</em> h of continuous exposure to 0.5 mM H(<em>2</em>)O(<em>2</em>), actin and MHC, both dissociated from the cytoskeleton within 15 min of H(<em>2</em>)O(<em>2</em>) exposure. The TER of the monolayers remained depressed although both actin and myosin returned to the cytoskeleton by 4 h after the initiation of H(<em>2</em>)O(<em>2</em>) exposure. Filamentous actin (f-actin) staining suggested that the re-associating actin took the form of short fibers associated with cortical actin rather than long stress fibers. Furthermore, pretreatment with KGF prevented the loss of actin and MHC from the actin cytoskeleton but did not prevent the decrease in TER. These studies suggest that actin disassembly from the cytoskeleton is important in the loss of barrier function, but that it is not the overall amount of actin that is associated with the cytoskeleton that is important, rather it is the contribution this actin makes to the architectural cohesiveness of the cell that contributes to the barrier function.

BACKGROUND

Administration of specific <em>growth</em> <em>factors</em> exert gut-trophic effects in animal models of massive small bowel resection (SBR); however, little comparative data are available. Our aim was to compare effects of a human glucagon-like peptide-<em>2</em> (GLP-<em>2</em>) analog, recombinant <em>growth</em> hormone (GH) and recombinant <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) on jejunal, ileal, and colonic <em>growth</em> and functional indices after 80% SBR in rats.

METHODS

Thirty-seven male rats underwent small bowel transection (sham operation) with s.c. saline administration (control; Tx-S; n = 7) or 80% midjejuno-ileal resection (Rx) and treatment with either s.c. saline (Rx-S, n = 7), GLP-<em>2</em> at 0.<em>2</em> mg/kg/d (Rx-GLP-<em>2</em>; n = 8), GH at 3.0 mg/kg/d (Rx-GH; n = 8), or KGF at 3.0 mg/kg/d (Rx-KGF; n = 7) for 7 days. All groups were pair-fed to intake of Rx-S rats. Gut mucosal cell <em>growth</em> indices (wet weight, DNA and protein content, villus height, crypt depth, and total mucosal height) were measured. Expression of the cytoprotective trefoil peptide TFF3 was determined by Western blot. Gut mucosal concentrations of the tripeptide glutathione (L-glutamyl-L-cysteinyl-glycine) and glutathione disulfide (GSSG) were measured by high-performance liquid chromatography and the glutathione/GSSG ratio calculated.

RESULTS

SBR increased adaptive <em>growth</em> indices in jejunal, ileal, and colonic mucosa. GLP-<em>2</em> treatment increased jejunal villus height and jejunal total mucosal height compared with effects of resection alone or resection with GH or KGF treatment. Both GH and KGF modestly increased colonic crypt depth after SBR. SBR did not affect small bowel or colonic goblet cell number or TFF3 expression; however, goblet cell number and TFF3 expression in both small bowel and colon were markedly up-regulated by KGF treatment and unaffected by GLP-<em>2</em> and GH. SBR oxidized the ileal and colonic mucosal glutathione/GSSG redox pools. GLP-<em>2</em> treatment after SBR increased the glutathione/GSSG ratio in jejunum, whereas KGF had an intermediate effect. In addition, GLP-<em>2</em> (but not GH or KGF) prevented the SBR-induced oxidation of the glutathione/GSSG pools in both ileum and colon.

CONCLUSIONS

GLP-<em>2</em> exerts superior trophic effects on jejunal <em>growth</em> and also improves mucosal glutathione redox status throughout the bowel after massive SBR in rats. Both GH and KGF increase colonic mucosal <em>growth</em> in this model. KGF alone potently increases gut mucosal goblet cell number and expression of the cytoprotective trefoil peptide TFF3. The differential effects of GLP-<em>2</em>, GH and KGF administration in this model of short bowel syndrome suggest that individual therapy with these <em>growth</em> <em>factors</em> may not be an adequate strategy to maximally improve adaptive gut mucosal <em>growth</em> and cytoprotection after massive small intestinal resection. Future research should address the use of these agents in combination in short bowel syndrome.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) or fibroblast <em>growth</em> <em>factor</em> (FGF)-7, a peptide produced by stromal cells and in particular by lung mesenchyme, has recently been shown to influence early lung morphogenesis and to be a mitogen for fetal and adult alveolar type II cells. Although contradictory findings have been reported regarding its effects on surfactant protein expression, its effects on surfactant phospholipids have not been studied. We investigated the effects of KGF on the synthesis of surfactant components by cultured fetal rat type II cells isolated during the late gestational period, when surfactant accumulates in preparation for extrauterine life. We show that KGF is a potent stimulus of surfactant phospholipid synthesis, particularly for the major component of surfactant, disaturated phosphatidylcholine (DSPC). KGF increased choline incorporation into DSPC in a dose-dependent manner up to <em>2</em>5 ng/ml (1.3 x 10(-9) M), and this effect was greater for surfactant than for nonsurfactant DSPC. KGF was several times more potent in this respect than acidic FGF at the same molar concentration. KGF, similar to epidermal <em>growth</em> <em>factor</em>, also stimulated acetate incorporation and increased the surfactant phospholipid and DSPC content of cultured cells twofold. These effects correlated with increased choline phosphate cytidylyltransferase activity and increased fatty acid synthase activity and gene expression. KGF also induced a dose-dependent stimulation of surfactant protein-A, -B, and -C gene expression, leading to a <em>2</em>- to 3-fold increase in their messenger RNAs. KGF therefore stimulates the synthesis of all surfactant components in developing type II cells at the time of surfactant accumulation. Its secretion by lung fibroblasts may thus be an important <em>factor</em> in promoting the maturation of fetal lung epithelium and the synthesis of sufficient surfactant. The results suggest that KGF could provide a new therapeutic agent for the management of the immature or injured lung.

Matriptase (membrane-type serine proteinase) was reported to play a role in nonmelanoma skin cancer progression. Moreover, it was shown to stimulate proteinase-activated receptor-<em>2</em> (PAR(<em>2</em>)) in vitro. Hepatocyte <em>growth</em> <em>factor</em> activator inhibitor-1 (HAI-1), the matriptase inhibitor, is an important regulator of enzyme activity. Therefore, the aim of this study was to elucidate the putative role of matriptase, HAI-1, and PAR(<em>2</em>) in normal human skin, as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). In normal human epidermis, PAR(<em>2</em>) colocalized with matriptase and HAI-1. Immunoreactivity of all proteins was found to be diminished in BCCs. Likewise, PAR(<em>2</em>) immunoreactivity was significantly decreased, whereas matriptase immunoreactivity was enhanced with SCC progression. We could also show that matriptase was complexed to HAI-1 in normal human skin, whereas in SCCs, the enzyme was present in an unassociated form. Both a specific peptide agonist for PAR(<em>2</em>) and the proteinase domain of matriptase were able to induce intracellular calcium mobilization and inhibition of proliferation in cultured HaCaT <em>keratinocytes</em>. In conclusion, our results suggest that PAR(<em>2</em>) is a substrate for matriptase in human skin in vivo. Deregulation of these proteins delineates SCC progression.

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible <em>factor</em> (HIF) prolyl hydroxylase <em>2</em> (PHD<em>2</em>) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD<em>2</em> in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD<em>2</em> in inflammatory, vascular, or epidermal cells. Interestingly, PHD<em>2</em> deficiency only in <em>keratinocytes</em> and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β3-integrin in the <em>keratinocytes</em> around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming <em>growth</em> <em>factor</em> β signaling. Thus, loss of the central oxygen sensor PHD<em>2</em> in <em>keratinocytes</em> stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD<em>2</em> could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.

The interaction of [1<em>2</em>5I] labeled murine epidermal <em>growth</em> <em>factor</em> (EGF)( with cultured human <em>keratinocytes</em> has been studied. Epidermal cells from neonatal foreskins were propagated to confluence in <em>2</em>4-well culture trays and incubated with [1<em>2</em>5I] EGF for binding assays. Association reached equilibrium within <em>2</em>-4 hr at 4 degrees and slightly earlier at 37 degrees. EGF bound at 37 degrees dissociates very slowly from cells, since it can be shown to enter cells and is degraded to trichloracetic-acid-soluble material. Cells exposed to chloroquine, an inhibitor of lysosomal enzymes, fail to degrade internalized [1<em>2</em>5I] EGF. Scatchard plots of the binding data yield a dissociation constant of 1 X 10(-9) m and show that epidermal cells bind approximately 3-4 X 10(4) molecules of EGF. Cells exposed to EGF alter their ability to bind EGF by decreasing the number of binding sites in a time- and concentration-dependent manner. Differentiation of epidermal cells in culture poses a problem in assessment of binding of EGF and possibly of other biologically active ligands, since cells lose the ability to bind EGF as they differentiate. These findings indicate that isolated epidermal cells possess a functional receptor for EGF which binds and responds to EGF in a manner similar to that described for other cells.

Hypoxia Inducible <em>Factor</em>-1 (HIF-1) is considered the major coordinator of the cellular adaptive response to hypoxia. Over recent years, its activity in the context of wound healing has been the object of increasing investigation. On the molecular level, HIF-1 transcriptional target products have been shown to regulate the process of endothelial cell survival, migration and proliferation (VEGF, ANGPT-1, ANGPT-<em>2</em>, ANGPT-4, FGF-<em>2</em>, PlGF, PDGF-B, RGC-3<em>2</em>), vascular smooth muscle cell migration and proliferation (FGF-<em>2</em>, EGF, PDGF, thrombospondin) and mobilization of Circulating Angiogenic Cells to the periphery (SFD-1/CXCR4). Studies on the effect of HIF-1 on the expression and activity of extracellular cell matrix modifying enzymes, such as MMPs and prolidase, have been conducted in the context of tumor angiogenesis and metastasis, and have resulted in controversial findings. A <em>growing</em> body of evidence suggests that HIF-1 also affects reepithelialization of the wound bed, through increasing <em>keratinocyte</em> migration, but decreasing their proliferation. Diminished HIF-1 levels and activity have been documented in conditions of impaired wound healing, such as wound healing in aged and in diabetic mice. The increasing number of studies on the role of HIF-1 in wound healing, apart from answering certain questions, has also raised an equal number, if not more. Clarifying the topics that still remain unclear could introduce a new era of HIF-1 targeted management of a wide range of problematic wounds.

Tumor necrosis <em>factor</em> alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin <em>keratinocytes</em> maintained in culture. We found that TNF alpha at 0.3-1.0 nM inhibited proliferation of <em>keratinocytes</em> in a reversible fashion as demonstrated by a reduction in total DNA content and clonal <em>growth</em>. The antiproliferative effects were most marked when TNF alpha was added in the preconfluent stages of cell <em>growth</em>. Accompanying this antiproliferative effect was a stimulation by TNF alpha of differentiation of <em>keratinocytes</em> as indicated by the stimulation of cornified envelope formation. <em>Keratinocytes</em> specifically bound TNF alpha, reaching maximal binding in <em>2</em> h at 34 degrees C or 8 h at 4 degrees C. Much of the apparent binding at 34 degrees C was due to internalization of the TNF alpha. At 4 degrees C the rate of internalization was much less. Confluent <em>keratinocytes</em> showed a single class of high-affinity receptors with 1,<em>2</em>50 receptors/cell and a Kd of 0.<em>2</em>8 nM. These data suggest a role for TNF alpha in the <em>growth</em> and differentiation of the epidermis.

CD9 is a protein with 4 transmembrane domains, and functions as a cell surface antigen. We have previously reported that CD9 functions as an up-regulator of membrane-anchored heparin-binding EGF-like <em>growth</em> <em>factor</em> (proHB-EGF) activity, which is a potent mitogen as well as a soluble HB-EGF. Anti-CD9 antibodies can neutralize the juxtacrine activity of proHB-EGF when both CD9 and proHB-EGF are coexpressed. We demonstrated here: (1) the CD9 gene was transcribed and translated in the cultured human <em>keratinocytes</em>; (<em>2</em>) anti-CD9 antibody inhibited the approximately 50% <em>growth</em> of human <em>keratinocytes</em> in culture; (3) CD9 was coprecipitated with proHB-EGF and membrane-anchored amphiregulin (proAR), and (4) the transient coexpression of CD9 with proHB-EGF or proAR in mouse L cells up-regulated their juxtacrine <em>growth</em> <em>factor</em> activities. These results suggest that CD9 would make a heterodimer and/or trimer complex with proHB-EGF and proAR, and might cooperate with proHB-EGF and proAR for human <em>keratinocyte</em> <em>growth</em> in a juxtacrine manner.

Selected biological effects of 1,4-naphthoquinone, menadione (<em>2</em>-methyl-1,4-naphthoquinone) and structurally related quinones from natural sources--the 5-hydroxy-naphthoquinones juglone, plumbagin and the <em>2</em>-hydroxy-naphthoquinones lawsone and lapachol--were studied in human <em>keratinocytes</em> (HaCaT). 1,4-naphthoquinone and menadione as well as juglone and plumbagin were highly cytotoxic, strongly induced reactive oxygen species (ROS) formation and depleted cellular glutathione. Moreover, they induced oxidative DNA base damage and accumulation of DNA strand breaks, as demonstrated in an alkaline DNA unwinding assay. Neither lawsone nor lapachol (up to 100 microM) were active in any of these assays. Cytotoxic and oxidative action was paralleled by stimulation of stress signaling: all tested quinones except lawsone and lapachol strongly induced phosphorylation of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and the related ErbB<em>2</em> receptor tyrosine kinase. EGFR activation by plumbagin, juglone and menadione was attenuated by a superoxide dismutase mimetic, indicating that ROS-related mechanisms contribute to EGFR activation by these naphthoquinones.