Treatment-related morbidity, and in some cases, mortality, associated with autologous and allogeneic bone marrow transplantation has decreased in the past decade largely due to the use of blood stem cells combined with hematopoietic <em>growth</em> <em>factors</em>. However, these procedures remain morbid, with several series documenting regimen-related injury to the oral mucous membranes, the worst form of toxicity from a patient perspective. The pathophysiology of transplant-related mucositis is related to two major events: direct mucosal basal cell injury leading to atrophy and ulcerations, and local infections that can become systemic, the latter of which are exacerbated by the severe neutropenia accompanying high-dose chemotherapy. Recent investigational agents designed to interfere with these two aspects of mucositis have been developed and are showing promise in early clinical trials. In particular, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and interleukin-11 appear active. They increase basal cell proliferation, prevent apoptosis due to the preparative regimen, and appear to ameliorate the mucositis seen with high-dose chemotherapy regimens. Oral, nonabsorbable anti-infective agents are also being tested in an attempt to prevent both local and systemic infections. Devoid of significant side-effects, KGF is now in large phase <em>2</em> trials that, if positive, will be a significant advance in promoting less morbid transplants by reducing pain and the risk of secondary infections and thus may reduce supportive care costs.

Stromelysin-<em>2</em> is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-<em>2</em> (matrix metalloproteinase-8), as well as 9<em>2</em> kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and <em>keratinocyte</em> cultures, which <em>factors</em> contribute to stromelysin-<em>2</em> expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-<em>2</em> mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-<em>2</em> is invariably expressed by <em>keratinocytes</em> migrating over dermal matrix. The number of <em>keratinocytes</em> expressing stromelysin-<em>2</em> was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In <em>keratinocyte</em> cultures, tumor necrosis <em>factor</em>-alpha, epidermal <em>growth</em> <em>factor</em>, and transforming <em>growth</em> <em>factor</em>-beta1 induced stromelysin-<em>2</em> expression as measured by quantitative reverse transcriptase-polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming <em>growth</em> <em>factor</em>-beta1 in contact with the stromelysin-<em>2</em>-positive <em>keratinocytes</em>. Our results suggest that stromelysin-<em>2</em> expression is important for the normal repair process and is upregulated by cytokines rather than cell-matrix interactions. Stromelysin-<em>2</em> is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.

BACKGROUND

Laminin 5 is known to induce the adhesion, spreading and migration of human keratinocytes. In skin wound healing, laminin 5 deposition beneath migrating keratinocytes occurs early and is followed by the formation of hemidesmosomes and then basement membrane.

OBJECTIVE

To identify factors that regulate the synthesis and secretion of laminin 5 by human keratinocytes during acute wound healing.

METHODS

Laminin 5 synthesis by human keratinocytes was determined by a specific sandwich enzyme-linked immunosorbent assay. To determine the total amount of laminin 5 synthesized, laminin 5 deposited on culture dishes and inside cells was solubilized by detergent solution and determined separately from conditioned medium, and the total laminin 5 synthesis was calculated. A quantitative polymerase chain reaction method was used to measure the expression levels of laminin 5 genes, LAMA3, LAMB3 and LAMC2, which correspond to the alpha3, beta3 and gamma2 chains of laminin 5. We also examined the effects of lysophospholipids, proinflammatory cytokines and growth factors, which are components in acute wound fluids, on laminin 5 synthesis in keratinocytes.

RESULTS

Human acute wound fluid at days 1, 2 and 3 stimulated laminin 5 synthesis in cultured human keratinocytes in a concentration-dependent manner, although findings are restricted to one case. Human serum also increased laminin 5 production by human keratinocytes as strongly as the wound fluid did, suggesting that the major active components in acute wound fluid may be derived from those in human serum. Lysophospholipids such as lysophosphatidic acid (LPA), lysophosphatidylcholines (LPCs) and sphingosine-1-phosphate (S1P) increased laminin 5 synthesis in a concentration-dependent manner. Among growth factors, epidermal growth factor, insulin-like growth factor-1, interferon-gamma and keratinocyte growth factor increased laminin 5 production in keratinocytes, while platelet-derived growth factor, hepatocyte growth factor and basic fibroblast growth factor were ineffective. Although interleukin-1alpha had no effect, transforming growth factor (TGF)-alpha, tumour necrosis factor (TNF)-alpha and TGF-beta1 also stimulated laminin 5 synthesis, and TGF-alpha and TGF-beta1 showed a synergistic effect. Neutralizing antibodies to TGF-alpha and TGF-beta1 markedly inhibited the enhanced laminin 5 synthesis by human serum, suggesting that TGF-alpha and TGF-beta1 are important components to increase laminin 5 in human serum. In line with the increase of laminin 5 synthesis, the expression levels of all three laminin 5 genes were also augmented by TGF-alpha and TGF-beta1.

CONCLUSIONS

Laminin 5 synthesis in human keratinocytes was augmented by inflammatory cytokines and growth factors such as TGF-alpha, TGF-beta1 and TNF-alpha, and lysophospholipids such as S1P, LPA and LPCs, which are supposed to be present in acute wound fluid. The increased laminin 5 protein in the wound area presumably enhances wound repair by stimulating adhesion and migration of keratinocytes on the wound bed and by facilitating basement membrane formation at the dermal-epidermal junction.

The complement system plays an important part in host defense and inflammation. Locally synthesized complement may perform these functions at tissue and organ level. In skin the <em>keratinocyte</em> is the major cell type, it is known to produce two soluble complement components, C3 and <em>factor</em> B. In this study we investigated the regulation of synthesis of these components in foreskin <em>keratinocytes</em> by cytokines. Human <em>keratinocytes</em> were cultured in the presence of supernatant of activated peripheral blood mononuclear cells, interleukin-1alpha, interleukin-<em>2</em>, interleukin-6, transforming <em>growth</em> <em>factor</em>-beta1, tumor necrosis <em>factor</em>-alpha, or interferon-gamma. C3 and <em>factor</em> B proteins were measured in culture supernatant by enzyme-linked immunosorbent assay and C3 and <em>factor</em> B transcripts in harvested cells by reverse transcriptase-polymerase chain reaction. Cultured <em>keratinocytes</em> constitutively produced C3 and <em>factor</em> B. Supernatant of activated mononuclear cells upregulated C3 and <em>factor</em> B production by <em>2</em>7- and 15-fold, respectively. interleukin-1alpha, interferon-gamma, and tumor necrosis <em>factor</em>-alpha upregulated C3 synthesis by 7-, 8-, and <em>2</em><em>2</em>-fold, and interleukin-1alpha, interleukin-6, and interferon-gamma upregulated <em>factor</em> B synthesis by 3-, 3-, and 34-fold, respectively. Tumor necrosis <em>factor</em>-alpha induced production of C3 and interferon-gamma induced production of <em>factor</em> B were inhibited by cycloheximide. Cytokine induced upregulation of C3 and <em>factor</em> B proteins was always associated with the upregulation of levels of C3 and <em>factor</em> B mRNA. This indicated that, as expected, cytokine-induced enhancement in C3 and <em>factor</em> B levels was due to an increase in synthesis rather than their possible release from intracellular stores. In conclusion, synthesis of C3 and <em>factor</em> B in <em>keratinocytes</em> is regulated by some cytokines, known to be produced by inflammatory cells and <em>keratinocytes</em>.

An inducible bovine KIV* keratin gene promoter was used to target expression of latent or activated transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1) to <em>keratinocytes</em> in transgenic mice. This short (<em>2</em>.<em>2</em>-kb) keratin 6 (K6) promoter element was generally silent in untreated animals but was induced in <em>keratinocytes</em> when placed in culture or, in vivo, in response to hyperplasia that follows topical application of the tumor promoter, 1<em>2</em>-O-tetradecanoylphorbol-13-acetate. All of the K6-TGF beta 1 transgenic lines studied showed attenuation of the basal <em>keratinocyte</em> proliferative response to 1<em>2</em>-O-tetradecanoylphorbol-13-acetate as a consequence of inducible TGF beta 1 gene expression. One of the six lines studied showed constitutive transgene expression at low levels in the skin, and this line had a <em>2</em>- to 3-fold increase in epidermal DNA labeling index over control mice. Although in vitro TGF beta 1 is known to be a potent negative regulator of epithelial cell proliferation, in vivo TGF beta 1 has complex biological activities and can act as either a positive or negative regulator of <em>keratinocyte</em> proliferation.

Although sphingosine-1-phosphate (S1P) is a well-known mitogen, it has only recently been demonstrated that S1P is able to inhibit cell proliferation in human epidermal <em>keratinocytes</em> and hepatic myofibroblasts. In the present study, we investigated the possible signalling pathways involved in the <em>growth</em> inhibition of human <em>keratinocytes</em>. Our results show that S1P potently inhibits <em>keratinocyte</em> proliferation, and that this leads to the inhibition of DNA synthesis. Interestingly, the prolonged activation of extracellular signal-regulated protein kinase (ERK) and the transient inactivation of Akt/protein kinase B (PKB) were also observed in concert with the inhibition of <em>keratinocyte</em> proliferation by S1P. To verify further the antiproliferative action of S1P, we examined changes in cell cycle-related proteins. S1P inhibited cyclin D(<em>2</em>) synthesis but stimulated p<em>2</em>1(WAF1/CIP1) (p<em>2</em>1) and p<em>2</em>7(KIP1) (p<em>2</em>7) synthesis; all are inhibitors of cyclin-dependent kinase. Furthermore, we found that the <em>growth</em> inhibition by S1P was in part abolished by pertussis toxin (PTX) treatment, but that ERK activation and Akt/PKB inhibition were not abrogated, suggesting that S1P functions both intracellularly, as a second messenger, and extracellularly, as a ligand for cell surface receptors. Insulin-like <em>growth</em> <em>factor</em> I (IGF-I) is a well-established human <em>keratinocyte</em> mitogen and is known to stimulate Akt/PKB in various cell types. In the present study, S1P was found to inhibit the <em>keratinocyte</em> proliferation and Akt/PKB activation induced by IGF-I. Our results suggest that S1P may play an important role in the negative regulation of <em>keratinocyte</em> proliferation by inhibiting the Akt/PKB pathway.

Estrogen affects proliferation and migration of different skin components, thus influencing wound healing processes. The human <em>keratinocyte</em> cell line NCTC <em>2</em>544 has been used to examine the effects of estrogen, dissect its mechanism of action and characterize receptor subtypes involved. Western blot and immunocytochemical analyses confirmed the expression of estrogen receptors (ERs) alpha and beta, with prevalence in the nuclear and extranuclear compartment, for ER alpha and ER beta respectively. Treatment with 10 nM 17beta-estradiol (17beta-E(<em>2</em>)) and the ER alpha and ER beta selective agonists, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; 100 nM), and diarylpropionitrile (DPN; 1 nM) produced a slight but significant increase in cell proliferation, as by 3-(4,5-dimethylthiazol-<em>2</em>-yl)-<em>2</em>,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays, only after a long-term treatment (96 h). Analysis of cell migration by a scratch wound assay showed that 17beta-E(<em>2</em>) (10 nM) accelerated migration between 5 and <em>2</em>4 h after scratching, an effect confirmed by the transwell migration assay. PPT and DPN elicited similar effects. Pre-treatment with the mitogen-activated protein kinase inhibitor, U01<em>2</em>6 (1 microM), abolished the ability of 17beta-E(<em>2</em>) and DPN, but not of PPT, to accelerate wound closure. TGF-beta1 (10 ng/ml) produced a similar positive effect on wound closure and the TGF-beta1 receptor antagonist, SB43154<em>2</em> (10 microM), reduced the ability of 17beta-E(<em>2</em>) and PPT to accelerate cell migration, but did not modify DPN effect. It is suggested that estrogen positively affects in vitro wound healing by stimulating cell proliferation after long-term exposure but mainly by accelerating cell migration within a few hours from treatment. Selective activation of ER beta may result in favorable stimulation of wound healing without any increase of transforming <em>growth</em> <em>factor</em>-beta1 production.

We identified the IIIb C<em>2</em> epithelial cell-specific splice variant of fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em> IIIb C<em>2</em>) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR<em>2</em> IIIb C<em>2</em> expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR<em>2</em> IIIc splice variant in invasive breast carcinomas. FGFR<em>2</em> IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR<em>2</em> IIIb is expressed in epithelial cells, previous studies on FGFR<em>2</em> IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR<em>2</em> IIIb C<em>2</em> in RIE-1 intestinal cells and several mammary epithelial cells. FGFR<em>2</em> IIIb C<em>2</em> caused <em>growth</em> transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR<em>2</em> IIIb C<em>2</em>-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express <em>keratinocyte</em> <em>growth</em> <em>factor</em>, a ligand for FGFR<em>2</em> IIIb C<em>2</em>. Ectopic treatment with <em>keratinocyte</em> <em>growth</em> <em>factor</em> caused FGFR<em>2</em> IIIb C<em>2</em>-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR<em>2</em> IIIb C<em>2</em> transformation.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has recently been shown to protect rats from hyperoxia-induced lung injury. However, the mechanism of the protective effect of KGF remains unclear. To elucidate the mechanism of action of KGF, we determined the effect of KGF on the barrier function of epithelial monolayers exposed to H(<em>2</em>)O(<em>2</em>). Calu-3 (human airway epithelial cells) were grown on Transwell membranes, and the permeability to fluorescein isothiocyanate-albumin was measured. Exposure to 0.5 mM H(<em>2</em>)O(<em>2</em>) significantly increased permeability from 1.50 +/- 0.09 to <em>2</em>4.8 +/- 1.5 (mean +/- SE x 10(-6) cm/s; P < 0.001). Incubation of monolayers with 50 ng/ml KGF for <em>2</em>4 h significantly reduced basal albumin flux (0.85 +/- 0.09; P < 0.001), and pretreatment with KGF completely abolished the H(<em>2</em>)O(<em>2</em>)-induced permeability increase (1.08 +/- 0.09). The protective effect of KGF was dose dependent and was observed at concentrations as low as 1 ng/ml. Partial amelioration of the H(<em>2</em>)O(<em>2</em>)-induced permeability increase occurred after 1 h of exposure to KGF. Treatment of cells with calphostin C, an inhibitor of protein kinase C (PKC), had no effect on the permeability of control or H(<em>2</em>)O(<em>2</em>)-treated cells. Calphostin C abolished both the KGF-mediated decrease in basal albumin flux and the protective effect of KGF against H(<em>2</em>)O(<em>2</em>)-induced increases in permeability. KGF pretreatment also prevented H(<em>2</em>)O(<em>2</em>)-induced disruption of F-actin staining patterns, suggesting stabilization of the cytoskeleton. These studies demonstrate that KGF decreases albumin flux across airway epithelial monolayers and prevents H(<em>2</em>)O(<em>2</em>)-induced increases in permeability by a PKC-dependent process that may involve stabilization of the cytoskeleton.

<em>Keratinocyte</em> derived T-cell <em>growth</em> <em>factor</em> was initially described as a product of cultured neonatal <em>keratinocytes</em> and <em>keratinocyte</em> cell lines that induced the proliferation of HT-<em>2</em> cells, a murine T-cell line that responds to IL-<em>2</em> and IL-4 by incorporating 3H-Thymidine. Subsequently, KTGF has been purified to high specific activity and found to be distinct from IL-<em>2</em> and IL-4 by a variety of biochemical, immunologic, and immunochemical criteria. Because it was found that certain HT-<em>2</em> cell lines also proliferated in response to GM-CSF, the present study asked whether KTGF was related to GM-CSF. In this study, we demonstrate that antibodies to recombinant murine GM-CSF completely neutralize the capacity of KTGF to induce HT-<em>2</em> proliferation without interfering with IL-<em>2</em> or IL-4 induced HT-<em>2</em> proliferation. Furthermore, poly-A+ RNA homologous to murine GM-CSF cDNA as judged by S1 nuclease analysis was detected in Pam <em>2</em>1<em>2</em> cells, and protein serologically homologous to GM-CSF was found in Pam <em>2</em>1<em>2</em> conditioned medium. We conclude that KTGF is identical to GM-CSF. The T-cell activating properties of GM-CSF require further exploration.

Signaling through the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) has been primarily implicated in the <em>growth</em> of epithelial cells including <em>keratinocytes</em>. However, the mechanism by which EGFR stimulation promotes <em>keratinocyte</em> cell <em>growth</em> is poorly understood. Here we report that human <em>keratinocytes</em> undergo apoptosis when incubated with the blocking EGFR monoclonal antibody <em>2</em><em>2</em>5 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-<em>2</em> family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming <em>growth</em> <em>factor</em> (TGF)-alpha and epidermal <em>growth</em> <em>factor</em> (EGF), increased the expression of Bcl-XL in quiescent <em>keratinocytes</em> and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT <em>keratinocytes</em> transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of <em>keratinocyte</em> survival in response to EGFR signaling.

OBJECTIVE

To investigate the effect of desiccation on secretion of inflammatory cytokines in corneal epithelial cells and in the rat desiccation model.

METHODS

A human corneal epithelial cell line (CEPI) was grown in <em>keratinocyte</em> <em>growth</em> medium <em>2</em> (KGM<em>2</em>) to approximately 80% confluence. The medium was aspirated and dishes were left for 0 to 30 min with the cover left open to dry the cells (short-term desiccation). After desiccation, KGM<em>2</em> was added to the dishes and collected from the dishes 15 min later to measure the concentrations of cytokines in the medium by sandwich enzyme immunoassay (ELISA). Viability of the cells was estimated with alamer blue. To study the effect of long-term desiccation, cultivated cells on transwells were used. After dessiccation for up to 8 h, the viability of the cells and levels of cytokines in the culture medium were examined. The expression of cytokines in the cornea of the dry eye model rat was measured by real-time PCR.

RESULTS

Short-term dessication of CEPI cells significantly increased the interleukin (IL)-6 level and slightly increased the tumor necrosis factor (TNF)-α level. Anti-IL-6 antibody partially suppressed cell death caused by desiccation. Upon long-term desiccation, IL-6 and IL-8 levels were increased. In the dry eye model rats, the IL-6 mRNA level in the cornea significantly increased, whereas TNF-α mRNA level slightly increased.

CONCLUSIONS

Desiccation induced IL-6 expression in corneal epithelial cells, suggesting that IL-6 participates in desiccation-induced cell death.

BACKGROUND

Rodent mesenchymal stem/stromal cells (MSCs) enhance repair after ventilator-induced lung injury (VILI). We wished to determine the therapeutic potential of human MSCs (hMSCs) in repairing the rodent lung.

METHODS

In series 1, anesthetized rats underwent VILI (series 1A, n = 8 to 9 per group) or protective ventilation (series 1B, n = 4 per group). After VILI, they were randomized to intravenous administration of (1) vehicle (phosphate-buffered saline); (<em>2</em>) fibroblasts (1 × 10 cells/kg); or (3) human MSCs (1 × 10 cells/kg) and the effect on restoration of lung function and structure assessed. In series <em>2</em>, the efficacy of hMSC doses of 1, <em>2</em>, 5, and 10 million/kg was examined (n = 8 per group). Series 3 compared the efficacy of both intratracheal and intraperitoneal hMSC administration to intravascular delivery (n = 5-10 per group). Series 4 examined the efficacy of delayed hMSC administration (n = 8 per group).

RESULTS

Human MSC's enhanced lung repair, restoring oxygenation (131 ± 19 vs. 103 ± 11 vs. 95 ± 11 mmHg, P = 0.004) compared to vehicle or fibroblast therapy, respectively. hMSCs improved lung compliance, reducing alveolar edema, and restoring lung architecture. hMSCs attenuated lung inflammation, decreasing alveolar cellular infiltration, and decreasing cytokine-induced neutrophil chemoattractant-1 and interleukin-6 while increasing keratinocyte growth factor concentrations. The lowest effective hMSC dose was <em>2</em> × 10 hMSC/kg. Intraperitoneal hMSC delivery was less effective than intratracheal or intravenous hMSC. hMSCs enhanced lung repair when administered at later time points after VILI.

CONCLUSIONS

hMSC therapy demonstrates therapeutic potential in enhancing recovery after VILI.

OBJECTIVE

Mesenchymal stem cells-based therapies have shown promising effects in experimental acute respiratory distress syndrome. Different mesenchymal stem cells sources may result in diverse effects in respiratory diseases; however, there is no information regarding the best source of mesenchymal stem cells to treat pulmonary acute respiratory distress syndrome. We tested the hypothesis that mesenchymal stem cells derived from bone marrow, adipose tissue, and lung tissue would lead to different beneficial effects on lung and distal organ damage in experimental pulmonary acute respiratory distress syndrome.

METHODS

Animal study and primary cell culture.

METHODS

Laboratory investigation.

METHODS

Seventy-five Wistar rats.

METHODS

Wistar rats received saline (control) or Escherichia coli lipopolysaccharide (acute respiratory distress syndrome) intratracheally. On day <em>2</em>, acute respiratory distress syndrome animals were further randomized to receive saline or bone marrow, adipose tissue, or lung tissue mesenchymal stem cells (1 × 10 cells) IV. Lung mechanics, histology, and protein levels of inflammatory mediators and <em>growth</em> <em>factors</em> were analyzed 5 days after mesenchymal stem cells administration. RAW <em>2</em>64.7 cells (a macrophage cell line) were incubated with lipopolysaccharide followed by coculture or not with bone marrow, adipose tissue, and lung tissue mesenchymal stem cells (10 cells/mL medium).

RESULTS

Regardless of mesenchymal stem cells source, cells administration improved lung function and reduced alveolar collapse, tissue cellularity, collagen, and elastic fiber content in lung tissue, as well as decreased apoptotic cell counts in liver. Bone marrow and adipose tissue mesenchymal stem cells administration also reduced levels of tumor necrosis factor-α, interleukin-1β, keratinocyte-derived chemokine, transforming growth factor-β, and vascular endothelial growth factor, as well as apoptotic cell counts in lung and kidney, while increasing expression of keratinocyte growth factor in lung tissue. Additionally, mesenchymal stem cells differently modulated the secretion of biomarkers by macrophages depending on their source.

CONCLUSIONS

Mesenchymal stem cells from different sources led to variable responses in lungs and distal organs. Bone marrow and adipose tissue mesenchymal stem cells yielded greater beneficial effects than lung tissue mesenchymal stem cells. These findings may be regarded as promising in clinical trials.

The <em>growth</em> <em>factor</em>- and phorbol ester-inducible prostaglandin H synthase (PGHS)-<em>2</em> has been found to be constitutively overexpressed in epidermal tumors generated by the initiation-promotion protocol in murine skin, whereas the expression of PGHS-1 does not change under these conditions. In this paper we report the intra-tumor distribution of the aberrantly expressed PGHS-<em>2</em> and the cancer chemopreventive activity of a specific PGHS-<em>2</em> inhibitor. By immunohistochemical methods using isoenzyme-specific antibodies, we found that the PGHS-1 protein was expressed in <em>keratinocytes</em> and Langerhans cells dispersed throughout the epithelial part of papillomas and squamous cell carcinomas and in inflammatory infiltrates occasionally seen in these tumors. A uniform pattern of PGHS-<em>2</em> expression was observed in the basal <em>keratinocytes</em> of papillomas and in the follicular <em>keratinocytes</em> of carcinomas. In addition, Langerhans cells as well as tumor-associated inflammatory infiltrates exhibited PGHS-<em>2</em>-specific immunoreactivity. PGHS-<em>2</em>-catalyzed prostaglandin synthesis stimulated by the phorbol ester 1<em>2</em>-O-tetradecanoylphorbol-13 acetate (TPA) in mouse epidermis in vivo was dose-dependently suppressed by topical administration of SC-581<em>2</em>5, a specific PGHS-<em>2</em> inhibitor. TPA-induced edema formation, epidermal DNA synthesis, and mitotic activity were not impaired by SC-581<em>2</em>5 applied at a dose that inhibited TPA-induced prostaglandin E<em>2</em> synthesis. However, the repetitive epicutaneous administration of SC-581<em>2</em>5 substantially and significantly suppressed papilloma development. Malignant progression of papillomas was slightly retarded by the drug. These results indicate that aberrant expression of PGHS-<em>2</em> in epidermal tumors may be a relevant target for prevention of epidermal cancer development in experimental animals and that the PGHS-<em>2</em>-specific inhibitor SC-581<em>2</em>5, which is a potent inhibitor of tumor promotion in mouse skin, may be important for cancer chemoprevention in humans as well.

Malignant gliomas are highly angiogenic and aggressive tumors. IFN-beta has been used for the treatment of patients with malignant glioma; however, its antitumor mechanism in vivo remains unclear. To understand the in vivo antitumor effect and mechanism of recombinant human IFN-beta (rhIFN-beta) depending on the stages of tumor development or progression, we used orthotopic xenograft brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days (group 1) and <em>2</em>1 days (group <em>2</em>) postimplant were treated with <em>2</em> x 10(5) IU/day of rhIFN-beta or saline i.p. for 15 days, respectively. Tumor <em>growth</em> was suppressed by 69.6% in group 1 and 10.8% in group <em>2</em> compared with tumors of each control group treated with saline. rhIFN-beta-treated group 1 animals showed 38% reduction in vascularization along with a <em>2</em>.5-fold increase of the apoptotic index and no change in the proliferative index as compared with untreated tumors. The expression level of vascular endothelial cell <em>growth</em> <em>factor</em> and basic fibroblast <em>growth</em> <em>factor</em> was not affected by rhIFN-beta treatment. rhIFN-beta showed inhibitory activity on proliferation of U-87 cells, human umbilical vein endothelial cells, and PAM <em>2</em>1<em>2</em> murine <em>keratinocytes</em> in vitro. Our results indicate that the in vivo antitumor effect of rhIFN-beta on malignant gliomas may be mediated, at least in part, via angiogenesis inhibition rather than antiproliferative activity and that rhIFN-beta may be more effective for the treatment of malignant glioma patients at an early stage with minimal or microscopic tumor burdens rather than at an advanced stage of tumor development.

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(<em>2</em>+) signaling, <em>growth</em>, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many <em>factors</em> have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(<em>2</em>+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-<em>2</em>-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-1<em>2</em> cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(<em>2</em>+) concentration ([Ca(<em>2</em>+)](i)) oscillations in HaCaT cells and Ca(<em>2</em>+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(<em>2</em>+)](i). The K6PC-5-induced [Ca(<em>2</em>+)](i) oscillations were dependent on thapsigargin-sensitive Ca(<em>2</em>+) stores and Ca(<em>2</em>+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on <em>keratinocytes</em> in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of <em>keratinocytes</em> via [Ca(<em>2</em>+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.

Monoclonal antibodies were used to localize immunohistochemically epidermal <em>growth</em> <em>factor</em> receptor and HER-<em>2</em>/neu in normal and neoplastic frozen tissue samples from the lower genital tract of women. In squamous epithelia of the cervix, vulva, and vagina, epidermal <em>growth</em> <em>factor</em> receptor and HER-<em>2</em>/neu both were expressed most strongly by basal <em>keratinocytes</em>. Expression of both of these cell surface molecules decreased as cells underwent differentiation toward the mucosal surface. In contrast, both epidermal <em>growth</em> <em>factor</em> receptor and HER-<em>2</em>/neu were expressed throughout the entire thickness of the epithelium by undifferentiated squamous cells in squamous metaplasia, raised condyloma, and carcinoma in situ. In 34 squamous cancers of the cervix, vulva, and vagina, all malignant cells were found to have moderate to heavy staining for epidermal <em>growth</em> <em>factor</em> receptor. Staining of 33 of these cancers for HER-<em>2</em>/neu was light, although one patient who presented with distant metastases had heavy staining for HER-<em>2</em>/neu. These data suggest that although overexpression of HER-<em>2</em>/neu in squamous cancers of the lower genital tract is a rare event, it may be associated with aggressive biologic behavior.

The c-MET oncogene encodes the receptor for hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF), which is known to stimulate the invasive <em>growth</em> of epithelial cells cultured in vitro. The Met/HGF receptor is a heterodimeric transmembrane tyrosine kinase, which is a prototype for a new family of <em>growth</em> <em>factor</em> receptors. The c-MET oncogene is expressed in several types of epithelial tissue including <em>keratinocytes</em> and is over-expressed in a number of human carcinomas. Studies on various carcinoma cell lines have shown that over-expression and structural alteration of the receptor result in its activation and confer tumorigenesis. We have studied Met/HGF receptor expression in tissue specimens from 34 patients with head and neck squamous cell carcinomas (HNSCC) and in 17 regional lymph node metastases. Western blot analysis was employed, using monoclonal antibodies directed against either the intracellular or extracellular domain of the receptor. Each sample was compared to its normal counterpart. The receptor did not show any major structural alterations in HNSCC tissues, but its expression was increased from <em>2</em>- to 50-fold in about 70% of tumors. Immunohistochemistry then showed that the same antibodies stained only a few cells in the basal layer of normal squamous epithelium but intensely marked tumor cells. In the lymph node metastases of Met-positive tumors, receptor expression was maintained and sometimes increased with respect to primary tumors. Immunohistochemical analysis of the metastatic lymph nodes showed that cells were negative in the normal lymphatic tissue and strongly stained in tumor cells. Over-expression of the Met/HGF receptor was found at all tumor stages but was more significant in those associated with enlarged or multiple (N<em>2</em>-N3) lymph node metastases. These data show that expression of the Met/HGF receptor may be involved in the progression of HNSCC towards a metastatic phenotype and may be a useful marker of head and neck tumor cell spread to regional lymph nodes.

BACKGROUND

Exposure to ultraviolet (UV) radiation causes a complex cellular response, mostly mediated by the production of reactive oxygen species (ROS), which can be counteracted by exogenous treatments and endogenous mechanisms with anti-oxidant and scavenger properties. Keratinocyte growth factor (KGF/FGF7), a member of the fibroblast growth factor family, promotes epithelial growth and differentiation and is involved in cell survival after oxidant injuries.

OBJECTIVE

We analyzed the role of KGF in the control of intracellular ROS production and oxidative stress after UVB exposure on KGF receptor (KGFR) transfected cells and human immortalized and primary keratinocytes.

METHODS

We assessed the intracellular ROS production measuring the intensity of the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) by confocal microscopy, as well as the catalase activity by spectrophotometric assay. Moreover, morphological and biochemical analysis of actin cytoskeleton reorganization was evaluated as a further marker of oxidative damage.

RESULTS

Our data show that KGF significantly reduces intracellular ROS generation in response to UVB, preserves the decrease of catalase activity and prevents actin cytoskeleton rearrangement.

CONCLUSIONS

Our results provide a further evidence that KGF may be crucial for an efficient skin photoprotection, demonstrating a direct role for KGF in the reduction of intracellular ROS content following UVB exposure.

Oxygen is an essential micronutrient. Unlike many internal tissues, human epidermis obtains much of its oxygen supply from the atmosphere (<em>2</em>1% oxygen), and it ordinarily experiences higher oxygen levels than internal tissues (estimated approximately 5%). To test whether epidermal cell <em>growth</em> and differentiation depend upon this higher oxygen level, <em>keratinocyte</em> cultures were studied at <em>2</em>1, 5, and <em>2</em>% oxygen concentrations. Compared to <em>2</em>1% oxygen, culture in 5% had little effect on <em>growth</em> but led to profound suppression of the differentiation program as assessed by expression of differentiation markers and formation of squames in the superficial layers. Culture in <em>2</em>% oxygen reduced the <em>growth</em> rate as well as stratification and differentiation. In low-oxygen conditions, the cells exhibited increased colony-forming ability, consistent with a lower proportion of differentiated cells, and increased expression of vascular endothelial <em>growth</em> <em>factor</em> and cyclooxygenase-<em>2</em>. <em>Growth</em> in <em>2</em>1% oxygen led to higher levels of glutathione and expression of oxidant-responsive genes. Electrophoretic mobility supershift assay using an involucrin activator protein 1 (AP1) response element sequence revealed altered binding by proteins of the Jun and Fos families in nuclear extracts. The present data thus demonstrate oxygen-dependent differentiation in human <em>keratinocytes</em>, to which altered utilization of AP1 transcriptional response elements may contribute.

Transforming <em>growth</em> <em>factor</em> (TGF)-β is an important cytokine that negatively regulates <em>keratinocyte</em> proliferation. Deregulation of TGF-β signalling has been reported in psoriasis, where despite increased expression of TGF-β, psoriatic <em>keratinocytes</em> continue to hyperproliferate. Recently, we have identified CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein, as a novel co-receptor and negative regulator of TGF-β signalling. In the current work, we demonstrate that release of CD109 from the cell surface or the addition of CD109 protein results in downregulation of TGF-β signalling and TGF-β receptor expression in human <em>keratinocytes</em>. Moreover, these effects are associated with an increase in phospho-STAT3 levels, enhanced total STAT3 and Bcl-<em>2</em> expression and an increase in cell <em>growth</em> and survival, suggesting that released/soluble CD109 is able to induce molecular changes that are known to occur in psoriasis. Analysis of CD109 expression in psoriasis patients reveals that CD109 protein expression is markedly decreased in psoriatic epidermis as compared to adjacent uninvolved skin. In contrast, CD109 mRNA expression is unchanged in psoriatic plaques in comparison with normal skin. This raises a possibility that CD109 protein release is enhanced in psoriatic <em>keratinocytes</em>. Furthermore, psoriatic epidermis displays decreased expression of TGF-β receptors, consistent with the results obtained in vitro in <em>keratinocytes</em> with CD109 release or addition of CD109 recombinant protein. Together our findings suggest that aberrant CD109 release from the cell surface in human <em>keratinocytes</em> may induce molecular changes that are usually observed in psoriasis and may explain TGF-β receptor downregulation and decrease in TGF-β signalling in psoriasis.

Matriptase, a type <em>2</em> transmembrane serine protease, and its inhibitor hepatocyte <em>growth</em> <em>factor</em> activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of <em>2</em>3 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in <em>keratinocytes</em> at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(<em>2</em>)O(<em>2</em>) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.

Advances in understanding the complex process of wound healing and development of novel <em>growth</em> <em>factor</em> and gene therapies would benefit from models that mimic closely the physiology of human wounds. To this end, we developed a hybrid wound-healing model based on human tissue-engineered skin transplanted onto athymic mice. Grafted tissues were infiltrated with mouse mesenchymal cells as native and foreign dermal regions fused together. Immunohistochemical staining for human involucrin revealed that the transplanted epithelium maintained its human origin, whereas the dermis was infiltrated by numerous mouse fibroblasts and blood vessels. Grafted tissues were wounded with a 4-mm punch to create full-thickness excisional wounds. At 1 and <em>2</em> weeks, the tissues were excised and assessed for reepithelialization, differentiation, and neovascularization. Interestingly, the average rate of <em>keratinocyte</em> migration (1<em>2</em>0 microm/day) was similar to migration rates observed in human subjects and significantly lower than migration in mouse epidermis. Immunohistochemical staining for keratin 10, laminin, and involucrin revealed a normal pattern of differentiation in the neoepidermis. Neovascularization was significantly elevated in the granulation tissue at 1 week and subsided to the level of unwounded tissue at <em>2</em> weeks postwounding. Our data suggest that skin equivalents grafted to a mouse model may serve as a realistic model of human wound regeneration. Because skin equivalents can be prepared with patient cells and genetically modified to stimulate or suppress gene expression, this model may be ideal for addressing mechanistic questions and evaluating the efficacy of biomaterials and gene therapeutics for promoting wound healing.