Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human <em>keratinocytes</em> (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal <em>growth</em> <em>factor</em> (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, <em>growth</em> <em>factor</em> independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to <em>growth</em> control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to <em>growth</em> inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. <em>Growth</em> inhibition by RA may be mediated through the cytokine transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta <em>2</em> expression about 3- and 50-fold, respectively. We next studied the effect of TGF-beta 1 and TGF-beta <em>2</em> on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to <em>growth</em> inhibition by TGF-beta 1 and TGF-beta <em>2</em>, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta <em>2</em> treatment. Overall, loss of <em>growth</em> inhibition by RA parallels loss of TGF-beta sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-<em>2</em>-3 activator, the MC<em>2</em>56<em>2</em> compound we synthesized recently. Both resveratrol and MC<em>2</em>56<em>2</em> were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing <em>keratinocyte</em> proliferation. In vitro experiments revealed that the activation of SIRTs stimulated <em>keratinocyte</em> proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC<em>2</em> was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC<em>2</em> detachment from the promoter region of insulin <em>growth</em> <em>factor</em> I (IGF-I), fibroblast <em>growth</em> <em>factor</em> 10 (FGF-10), and Epithelial <em>Growth</em> <em>Factor</em> (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.

Tumor necrosis <em>factor</em>-alpha (TNF-alpha) inhibits <em>growth</em> of normal cervical <em>keratinocytes</em> but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal <em>growth</em> <em>factor</em> (EGF) or serum are depleted. Current work identifies the mechanism of <em>growth</em> stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)<em>2</em> and cdc<em>2</em> after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p<em>2</em>1(waf) and p<em>2</em>7(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid <em>growth</em> in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited <em>growth</em>. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates <em>growth</em> stimulation of immortal <em>keratinocytes</em> by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. <em>2</em>7:97-109, <em>2</em>000. Published by Wiley-Liss, Inc.

Wound fluids, human serum from platelet-poor and platelet-rich plasma (SPPP and SPRP), contain various soluble <em>factors</em> involved in cell <em>growth</em> and proliferation. Levels of cytokines, chemokines, and matrix metalloproteinases (MMPs) in drainage fluids (DFs) harvested from subcutaneous wounds, punctured fluids (PF) from seroma, and SPPP were measured. SPPP and SPRP from four healthy volunteers were also subjected to the analysis. Biochemical profiles of DF reflected the sequential stages of wound healing. Early-phase DF contained high concentrations of basic fibroblast <em>growth</em> <em>factor</em> and platelet-derived <em>growth</em> <em>factor</em> and EGF. The levels of <em>keratinocyte</em> <em>growth</em> <em>factor</em>, interleukin-6, and MMP-8 in DF peaked on days <em>2</em>-3, while vascular endothelial <em>growth</em> <em>factor</em>, hepatocyte <em>growth</em> <em>factor</em>, interleukin-8, and MMP-1 increased over time during days 0-6. Punctured fluids contained high levels of TGF-beta1, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em>, hepatocyte <em>growth</em> <em>factor</em>, and MMP-1. Experiments using human adipose-derived stem cells and dermal fibroblasts cultured in media containing various concentrations of DF and fetal bovine serum suggested that for some cell types, DF-contained <em>growth</em> <em>factors</em> that are not obtained from SPRP could be used to supplement or substitute for serum in culture media. SPRP and DF are economical ready-made mixtures of serum and autologous soluble <em>factors</em>, and may be differentially useful for regenerative therapies.

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, <em>2</em>) a reduction in the pulmonary expression of the chemokines CCL<em>2</em> (or monocyte chemoattractant protein-1) and CXCL<em>2</em> (or macrophage inflammatory protein-<em>2</em>alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-<em>2</em> and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL<em>2</em>, CXCL<em>2</em>, and ICAM-1 mRNA in the MLE-1<em>2</em> murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.

OBJECTIVE

Hypothermia improves survival and neurologic recovery after cardiac arrest. Cardiac arrest also triggers release of cytokines and inflammatory molecules, and it is unknown whether therapeutic hypothermia alters this inflammatory response. This study tested whether therapeutic hypothermia altered levels of inflammatory markers in serum.

METHODS

Prospective, randomized study.

METHODS

University research laboratory.

METHODS

Adult, male, Sprague-Dawley rats.

METHODS

Halothane-anesthetized rats were subjected to 8 mins of asphyxial cardiac arrest and resuscitation. Rat temperature was controlled at 37 degrees C throughout the experiment (normothermia) or reduced to 33 degrees C between 1 and <em>2</em>4 hrs after cardiac arrest (hypothermia). Serum cytokines were measured at baseline, 0.5, 1, 3, 6, 1<em>2</em>, and <em>2</em>4 hrs after resuscitation using multiplex analyzer or enzyme-linked immunosorbent assay.

RESULTS

Hypothermic rats showed improved neurologic recovery at 1<em>2</em> and <em>2</em>4 hrs. Serum levels of tumor necrosis <em>factor</em>-alpha; macrophage inflammatory protein-1alpha; <em>growth</em>-related oncogene/<em>keratinocyte</em> chemokine; interleukin-<em>2</em>, -9, and -10; monocyte chemotactic protein-1; leptin; and intracellular adhesion molecule-1 increased over time, and the levels of interleukin-18 declined over time. No temporal trends in other molecules were detected. Levels of these molecules did not differ between temperature groups during the hypothermia phase (1-<em>2</em>4 hrs).

CONCLUSIONS

These data suggest that altering the inflammatory response after cardiac arrest is not necessary for the beneficial effects of hypothermia. These data do not support a specific role of circulating cytokines in the neurologic injury after cardiac arrest.

The effects of crude lymphokine-enriched supernatants, purified recombinant lymphotoxin (LT), tumor necrosis <em>factor</em>-alpha (TNF), and gamma interferon (gamma IF) on proliferating human <em>keratinocytes</em> were assessed using two in vitro culture systems. Activated splenocyte supernatants inhibited <em>keratinocyte</em> colony <em>growth</em> on fibroblast feeder layers and arrested basal <em>keratinocyte</em> DNA synthesis within <em>2</em>4 h. Purified recombinant LT, TNF, and gamma IF inhibited cell proliferation in serum-free medium without noticeably affecting viability. Cytostasis was dose-dependent (up to 90% with LT or TNF and 99% with gamma IF) and was maximal within <em>2</em>4-36 h. Specific antibodies neutralized TNF- and gamma IF-mediated cytostasis. Combined treatment with LT (or TNF) and gamma IF increased the degree of cytostasis, particularly at low lymphokine concentrations. Maximum inhibition of DNA synthesis and the duration of exposure required for this inhibition were comparable for LT and TNF and differed for gamma IF. Each of these lymphokines induced cell enlargement, flattening, and vesiculation, with gamma IF apparently more potent in this respect than LT or TNF. Fusiform <em>keratinocytes</em> with diffusely distributed cytokeratin were observed after prolonged treatment with gamma IF alone or gamma IF plus either LT or TNF. Flow cytometric studies of lymphokine-treated <em>keratinocytes</em> indicated that LT, TNF, and gamma IF could enhance beta-<em>2</em> microglobulin expression 1.5-fold to threefold, whereas only gamma IF induced class II antigens. Staining for class II and beta-<em>2</em> microglobulin was reduced on cells treated with high concentrations of gamma IF compared with either optimally treated or untreated cells. The potential relevance of these findings to cutaneous immune defense and disease is discussed.

Stem cell <em>factor</em> (SCF) of <em>keratinocyte</em> origin regulates melanocyte <em>growth</em> and survival. Deprivation of survival <em>factors</em> causes the apoptosis of melanocytes. Vitiligo often develops following physical trauma, even if this is minor. The exact mechanism of the Koebner phenomenon in vitiligo is unclear. Apoptosis of <em>keratinocytes</em>, which occurs more in depigmented suction-blistered epidermis than in the normally pigmented counterpart, could reduce levels of <em>keratinocyte</em>-derived <em>factors</em> such as SCF and basic fibroblast <em>growth</em> <em>factor</em> (bFGF). Levels of SCF expression were examined in the depigmented and normally pigmented paired epidermis of 19 patients with vitiligo, and bFGF expression in six patients. The expression of SCF (p<0.001) and bFGF was usually reduced in the depigmented compared with the normally pigmented epidermis. Apoptosis of cultured normal human <em>keratinocytes</em>, which was induced by staurosporine, resulted in a concentration-dependent decrease in levels of SCF mRNA and protein. Normal human melanocytes proliferated more in medium containing SCF or <em>keratinocyte</em> (XB-<em>2</em>) feeder than in medium with neither. Deprivation of SCF or <em>keratinocyte</em> feeder in the culture medium induced a marked decrease in melanocytes as a result of apoptosis. Therefore, lower expression of <em>keratinocyte</em>-derived <em>factors</em>, including SCF, in vitiliginous <em>keratinocytes</em>, which could result from <em>keratinocyte</em> apoptosis, might be responsible for passive melanocyte death and may explain the Koebner phenomenon.

BACKGROUND

Reactive oxygen species (ROS) and glutathione (GSH) depletion contribute to organ injury after bone marrow transplantation (BMT). Keratinocyte growth factor (KGF) ameliorates graft-versus-host disease (GVHD)-associated organ injury in murine BMT models.

METHODS

B10.BR mice received total body irradiation (TBI; day -1) +/- cyclophosphamide (Cy; 120 mg/kg/day i.p., days -3 and -2), then were transplanted on day 0 with C57BL/6 bone marrow + spleen cells as a source of GVHD-causing T cells. KGF (5 mg/kg/day subcutaneously [s.c.]) or saline was given on days -6, -5, and -4. Lung and liver GSH and oxidized GSH disulfide (GSSG) levels were measured on days 0 and 5 and glutathione redox potential (Eh) calculated. Organ malondialdehyde (MDA) was determined on day 5 as an index of ROS-mediated lipid peroxidation.

RESULTS

In lung, TBI+BMT oxidized GSH Eh and increased MDA. Cy further oxidized lung GSH Eh. In liver, neither BMT regimen altered GSH redox status or MDA. KGF prevented the decrease in lung GSH after TBI+Cy and decreased lung MDA after both TBI and TBI+Cy. KGF increased liver GSH levels and GSH Eh after TBI and GSH Eh after TBI+Cy.

CONCLUSIONS

In murine allogeneic BMT, TBI oxidizes the lung GSH redox pool and Cy exacerbates this response by 5 days post-BMT. In contrast, liver GSH redox status is maintained under these experimental conditions. KGF treatment attenuates the Cy-induced decrease in lung GSH, decreases post-BMT lung lipid peroxidation, and improves liver GSH redox indices. KGF may have a therapeutic role to prevent or attenuate GSH depletion and ROS-mediated organ injury in BMT.

Immune system is a major determinant of pathophysiology of inflammatory bowel disease (IBD), and cytokines are well known mediators of immune system. Recently, informations on pro-inflammatory cytokines and their role in IBD have led to development of potential therapeutic approach to manipulate these cytokines and there by inhibiting inflammation in IBD. These therapeutic approaches include inhibitors of the tumour necrosis <em>factor</em> (TNF)-alpha lymphocyte trafficking, type 1 T helper (Th1) cell polarization and nuclear <em>factor</em> type beta; immunoregulatory cytokines and various <em>growth</em> <em>factors</em>. Studies on these therapies have documented variable results and the outcomes of many clinical trials are awaited. However, these potential therapies, if become real may revolutionise approach in patients with IBD. Analysis of the inflammed mucosa from patients with Crohn disease (CD) and ulcerative colitis (UC) have shown increased expression of certain proinflammatory cytokines such as interleukin-1 (IL-1), interleukin 6 (IL-6) and TNF-alpha. The latter is important in the recruitment of neutrophils into inflammed tissue, a process which results from three physiological steps: (i) rolling, (ii) adhesion, and (iii) transendothelial migration. Understanding of the biology of chronic inflammation has expanded the therapies available for IBD and particularly CD. At present, the biological therapies that are being used in clinical practice or investigated for the treatment of IBD are predominantly proteins, usually delivered intravenously or subcutaneously. The therapies used include: 1. TNF-alpha inhibitors: infliximab, CDP 571, etanercept, onercept, CNI- 1493 and thalidomide. <em>2</em>. Inhibitors of lymphocyte trafficking: natalizumab, LPD-0<em>2</em> and ICAM-1. 3. Inhibitors of Th1 polarization: monoclonal antibodies for IL-1<em>2</em>, interferon (IFN)-gamma and anti IFN-gamma. 4. Immunoregulatory cytokines: IL-10 and IL-11. 5. Inhibitors of nuclear <em>factor</em> kappa (beta NF-kbeta.) 6. <em>Growth</em> <em>factors</em>: epidermal <em>growth</em> <em>factor</em> (EGF) and <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF).

Fibroblast <em>growth</em> <em>factor</em> (FGF), a key regulatory <em>factor</em> of cell <em>growth</em> and differentiation, is involved in embryonic development, angiogenesis, and tumorigenesis. To date, four different FGF receptors (FGFRs) have been cloned and characterized. We examined the expression of four FGFRs in human gastric cancer tissues and cell lines using Northern analysis, ribonuclease protection assay, and immunohistochemistry. The mRNAs of FGFR-1 (10/14), FGFR-<em>2</em> (9/14), and FGFR-4 (9/14) were up-regulated in cancer compared with normal tissues. FGFR-3 mRNAs were barely detectable in both normal and cancer tissues. These FGFR mRNAs were co-expressed in various combinations of two or three in the same tissue. Immunohistochemistry confirmed specific staining of multiple FGFRs, except FGFR-3, in the cancer specimens. To investigate the functional significance of FGFR co-expression we examined the invasive property of SNU-16 cells, which exhibited gene amplification of FGFR-<em>2</em>, -3, and -4 as well as over-expression of <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR), a splice variant of FGFR-<em>2</em>, and FGFR-4 mRNA. KGF plus acidic FGF (aFGF), KGF, and aFGF treatment enhanced the invasive potential of SNU-16 cells over the control by 100%, 107%, and 47%, respectively, indicating that neither additive nor synergistic effect was induced by stimulation with aFGF plus KGF. These results suggest that co-expression of FGFRs in various combinations may cause subtle changes in the progression of gastric cancer.

Melanogenesis and melanosome transfer from the melanocytes to the neighboring <em>keratinocytes</em> are induced by ultraviolet radiation and modulated by autocrine and paracrine <em>factors</em>. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF/fibroblast <em>growth</em> <em>factor</em> (FGF)7) is a paracrine mediator of human <em>keratinocyte</em> <em>growth</em> and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of <em>keratinocytes</em> and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient <em>keratinocytes</em> and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-<em>2</em>, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the <em>keratinocytes</em> by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

Altered angiogenesis response is observed in patients with cervical cancer. In this study we examined whether Human Papilloma Virus (HPV) positive epithelial cells are able to produce angiogenic modulators. When added to human umbilical vein endothelial cells (HUVEC) the media conditioned by HPV-16 positive cells was able to induce proliferation, whereas a contrary effect was observed for media derived from non-tumorigenic <em>keratinocytes</em>. The analyses of angiogenesis modulator's mRNA levels result in a decrease of the antiangiogenic <em>factors</em> TSP-1 and <em>2</em> in HPV-16 positive cells. In contrast the expression of the pro-angiogenic molecules: bFGF, IL-8, TGF-beta, TNFalpha, and VEGF were higher in these cells as compared to control <em>keratinocytes</em>. Furthermore the pattern of VEGF isoforms observed in the cells positive for the viral genome point to a preferential induction of the VEGF(189) isoform. We therefore conclude that cervical cancer cells expressing HPV-16 genome are able to contribute to the pro-angiogenic response that might support tumor <em>growth</em> and invasion of the surrounding tissues.

Synthesis and catabolism of calcitriol (1,<em>2</em>5(OH)<em>2</em>D3) were studied using HaCaT cell line as a cell culture model. Our results indicate that stimulation of HaCaT cells with epidermal <em>growth</em> <em>factor</em> (EGF) or transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) within 16 h just prior to reaching confluence amplified the production of calcitriol when calcidiol (3H-<em>2</em>5OHD3) was used as a substrate. EGF- and TGF-alpha-induced (0.1-10 nM) 1-hydroxylation of 3H-<em>2</em>5OHD3 was concentration-dependent but showed different kinetics. Synthesis of calcitriol induced by EGF was inversely related to the degree of cellular confluence. Stimulation by EGF was an actinomycin D- and cycloheximide-sensitive process. Independently of the <em>growth</em> <em>factor</em> used, the production of 3H-<em>2</em>4R,<em>2</em>5(OH)<em>2</em>D3 and the catabolism of 3H-1,<em>2</em>5(OH)<em>2</em>D3 to 3H-1,<em>2</em>4,<em>2</em>5(OH)3D3 were unexpectedly low (< or = 5% and < or = <em>2</em>%/), as compared to the amount of calcitriol generated. Exogenous addition of unlabeled 1,<em>2</em>5(OH)<em>2</em>D3, 1,<em>2</em>4R(OH)<em>2</em>D3, calcipotriol, or <em>2</em>4R,<em>2</em>5(OH)<em>2</em>D3 at concentrations as low as 10(-11) M, potently inhibited the 3H-1,<em>2</em>5(OH)<em>2</em>D3 production. These results suggest that EGF-treated HaCaT <em>keratinocytes</em> could serve for further studies of the vitamin D3 pathway and its relationship to proliferation and differentiation, but differences in calcitriol synthesis and catabolism from those in cultured primary <em>keratinocytes</em> or other cell lines must be considered.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a <em>2</em>8-kDa heparin-binding member of the fibroblast <em>growth</em> <em>factor</em> (FGF) family (alternative designation = FGF-7) that specifically binds to the KGF receptor, a splice variant of FGF receptor <em>2</em>, which is expressed only in epithelial tissues. KGF has been identified as an important paracrine mediator of proliferation and differentiation in a wide variety of epithelial cells, including hepatocytes and gastrointestinal epithelial cells, type II pneumocytes, transitional urothelial cells, and <em>keratinocytes</em> in all stratified squamous epithelia. Systemic administration of recombinant human KGF (rHuKGF) provides significant cytoprotection to epithelial tissues in a number of different animal models of epithelial/mucosal damage, including models of injury to the gastrointestinal tract, lung, urinary bladder, and hair follicles. The results obtained with these preclinical models prompted an investigation of the use of rHuKGF as a cytoprotective agent against radiation- and/or chemotherapy-induced oral and gastrointestinal mucositis. Several dose- and time-variable studies were conducted in normal rhesus macaques to determine the lowest dose and shortest duration of rHuKGF administration required to induce oral mucosal proliferation without other significant systemic effects. Numerous studies were also conducted in murine models of chemotherapy-induced mucositis to fine-tune the dosing schedule. These studies showed that <em>2</em>-3 days of rHuKGF administration were sufficient to induce significant oral mucosal proliferation and to protect against gastrointestinal mucositis when administered prior to the initiation of chemotherapy. The results from these models were used to design a phase I study in normal human volunteers to evaluate the safety of rHuKGF and its ability to induce oral mucosal proliferation. rHuKGF was well tolerated and induced a significant increase in markers of oral mucosal proliferation following 3 days of administration at the highest doses. Phase I/II studies to evaluate the safety and efficacy of rHuKGF in the prevention of chemotherapy-induced mucositis are currently in progress.

BACKGROUND

MicroRNAs play important roles in carcinogenesis. A preliminary screening study suggested that down-regulation of miR-370 occurs in oral squamous cell carcinoma (OSCC) tissue. Insulin receptor substratre-1 (IRS-1) is the substrate of insulin-like growth factor receptor (IGFR), which modulates AKT/mTOR activation in malignancies. The relationship between miR-370 and IRS-1, and their functional roles in OSCC pathogenesis are unclear.

METHODS

Primary OSCC specimens were examined for miR-370 expression. Exogenous expression of miR-370 was established using both stable subclones and transient expression, and these were used to gain insights into miR-370's functions in OSCC cells. Knockdown of miR-370 and IRS-1 was also carried out in OSCC cells using a small interference oligonucleotide approach.

RESULTS

Squamous cell carcinoma tissues with perineural invasion had lowered miR-370 expression compared with contrasting OSCC. OSCC cells also exhibited lower miR-370 expression than normal oral keratinocytes, and this can be reversed by treatment with 5-aza-2'-deoxycytidine. Exogenous miR-370 expression decreases the migration and anchorage-independent growth of OSCC cells, which implies a suppressor role for miR-370. The enhancement of anchorage-independent growth of OSCC cells through miR-370 inhibiting can be reduced by knockdown of IRS-1 expression.

CONCLUSIONS

This study concludes that miR-370 is able to target IRS-1 for oral tumorigenesis.

BACKGROUND

The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds.

METHODS

A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively.

RESULTS

UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles.

CONCLUSIONS

This work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumours. To investigate the role of stromelysin-<em>2</em> (MMP-10) in <em>growth</em> and invasion of skin tumours, we studied cutaneous carcinomas with high metastatic capacity (squamous cell carcinomas, SCCs), only locally destructive tumours (basal cell carcinomas, BCCs) and pre-malignant lesions (Bowen's disease and actinic keratosis) using in situ hybridization. Expression of MMP-10 was compared with that of stromelysin-1 (MMP-3) and of MT1-MMP, the expression of which has been shown to correlate with tumour invasiveness. MMP-10 was expressed in 13/<em>2</em>1 SSCs and 11/19 BCCs only in epithelial laminin-5 positive cancer cells, while premalignant lesions were entirely negative. MT1-MMP mRNA was detected in 19/<em>2</em>1 SCCs both in epithelial cancer cells and stromal fibroblasts and in 14/18 BCCs only in fibroblasts. The level of MMP-10 was upregulated in a cutaneous SCC cell line (UT-SCC-7) by transforming <em>growth</em> <em>factor</em>-alpha and <em>keratinocyte</em> <em>growth</em> <em>factor</em>, and by interferon-gamma in combination with transforming <em>growth</em> <em>factor</em>-beta1 and tumour necrosis <em>factor</em>-alpha both in UT-SCC-7 and HaCaT cells. Our results show that MMP-10 expression does not correlate with the invasive behaviour of tumours as assessed by their histology and MT1-MMP expression, but may be induced by the wound healing and inflammatory matrix remodelling events associated with skin tumours.

BACKGROUND

Recent studies have reported the roles of Hyaluronic acid (HA) chains of diverse length in wound repair, especially considering the simultaneous occurrence in vivo of both high- (H-HA) and low-molecular weight (L-HA) hyaluronan at an injury site. It has been shown that HA fragments (5 ≤ MW ≤ 20 kDa) usually trigger an inflammatory response that, on one hand, is the first signal in the activation of a repair mechanism but on the other, when it's overexpressed, it may promote unwanted side effects. The present experimental research has aimed to investigate H-HA, L-HA and of a newly developed complex of the two (H-HA/L-HA) for stability (e.g. hyaluronidases digestion), for their ability to promote wound healing of human keratinocytes in vitro and for their effect on cellular biomarker expression trends.

RESULTS

Time-lapse video microscopy studies proved that the diverse HA was capable of restoring the monolayer integrity of HaCat. The H-HA/L-HA complex (0.1 and 1%w/v) proved faster in regeneration also in co-culture scratch test where wound closure was achieved in half the time of H-HA stimulated cells and 2.5-fold faster than the control. Gene expression was evaluated for transformation growth factor beta 1 (TGF-β1) proving that L-HA alone increased its expression at 4 h followed by restoration of similar trends for all the stimuli. Depending on the diverse stimulation (H-HA, L-HA or the complex), metalloproteinases (MMP-2, -9, -13) were also modulated differently. Furthermore, type I collagen expression and production were evaluated. Compared to the others, persistence of a significant higher expression level at 24 h for the H-HA/L-HA complex was found.

CONCLUSIONS

The outcomes of this research showed that, both at high and low concentrations, hybrid complexes proved to perform better than HA alone thus suggesting their potential as medical devices in aesthetic and regenerative medicine.

P-cadherin is a key component of epithelial adherens junctions, and it is prominently expressed in the hair follicle (HF) matrix. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in hypotrichosis with juvenile macular dystrophy (HJMD), an autosomal recessive disorder featuring sparse and short hair. Here, we attempted to recapitulate some aspects of HJMD in vitro by transfecting normal, organ-cultured human scalp HFs with lipofectamine and CDH3-specific or scrambled control siRNAs. As in HJMD patients, P-cadherin silencing inhibited hair shaft <em>growth</em>, prematurely induced HF regression (catagen), and inhibited hair matrix <em>keratinocyte</em> proliferation. In situ, membrane β-catenin expression and transcription of the β-catenin target gene, axin<em>2</em>, were significantly reduced, whereas glycogen synthase kinase 3 β (GSK3β) and phospho-β-catenin immunoreactivity were increased. These effects were partially reversed by inhibiting GSK3β. P-cadherin silencing reduced the expression of the anagen-promoting <em>growth</em> <em>factor</em>, IGF-1, whereas that of transforming <em>growth</em> <em>factor</em> β <em>2</em> (TGFβ<em>2</em>; catagen promoter) was enhanced. Neutralizing TGFβ antagonized the catagen-promoting effects of P-cadherin silencing. In summary, we introduce human HFs as an attractive preclinical model for studying the functions of P-cadherin in human epithelial biology and pathology. This model demonstrates that cadherins can be successfully knocked down in an intact human organ in vitro, and shows that P-cadherin is needed for anagen maintenance by regulating canonical Wnt signaling and suppressing TGFβ<em>2</em>.

Fibroblast <em>growth</em> <em>factors</em> (FGFs) exert diverse effects resulting from their interaction with cognate receptors on target cells. Our current study was designed to examine the local production and action of two specific stromal-epithelial cell mediatory <em>factors</em>, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and FGF-10, in human endometrial carcinoma cells. The RT-PCR method was used to determine gene expression of KGF, FGF-10, and KGF receptor in human endometrial carcinoma cells (HEC-1) and human endometrial stromal cells. KGF mRNAs were expressed in both of these cell types. On the other hand, FGF-10 mRNA was detected only in the endometrial stromal cells, and KGF receptor mRNA was observed in the HEC-1 cells. The novel finding of the present study is that KGF is expressed in carcinoma cells and FGF-10 is expressed in human endometrial stromal cells. The distinct phosphorylation of ERK-1 and -<em>2</em> (ERK1/<em>2</em>), which are members of the MAPK family, was observed when HEC-1 cells were treated with KGF or FGF-10. KGF and FGF-10 could induce the prompt phosphorylation of ERK1/<em>2</em> and consequently stimulate DNA synthesis. KGF and FGF-10 did not activate the phosphorylation of Akt, protein kinase C, or signal transducer and activator of transcription-3. Blocking the MAPK pathway with the specific methyl ethyl ketone 1/<em>2</em> inhibitor (U01<em>2</em>6) completely neutralized the enhancement of cell proliferation induced by KGF and FGF-10. In addition, KGF and FGF-10 activated expressions of downstream nuclear transcription <em>factors</em>, such as Elk-1 and c-myc, but not c-fos. These results demonstrate for the first time that KGF and FGF-10 are capable of stimulating the <em>growth</em> of endometrial carcinoma cells via activating MAPK pathway through autocrine/paracrine fashion.

Ultraviolet B irradiation initiates and promotes skin cancers, photo-aging, and immune suppression. In order to elucidate the effect of these processes at the level of gene expression, we used cDNA microarray technology to examine the effect of ultraviolet B irradiation on 588 cancer-related genes in human <em>keratinocytes</em> at 1, 6, and <em>2</em>4 h post-irradiation with a mildly cytotoxic dose of ultraviolet B (170 mJ/cm(<em>2</em>)). The viability of the irradiated <em>keratinocytes</em> was 75% at <em>2</em>4 h post-irradiation. Various cytokeratins and transcription <em>factors</em> were up-regulated within 1 h post-irradiation. After 6 h, expression of a variety of genes related to <em>growth</em> regulation (e.g. p<em>2</em>1(WAF1), notch 4, and smoothened), apoptosis (e.g. caspase 10, hTRIP, and CRAF1), DNA repair (ERCC1, XRCC1), cytokines (e.g. IL-6, IL-13, TGF-beta, and endothelin <em>2</em>), and cell adhesion (e.g. RhoE, and RhoGDI) were altered in human <em>keratinocytes</em>. These data suggest the changes in a cascade of gene expression in human <em>keratinocytes</em> occurring within <em>2</em>4 h after UVB exposure. Although the roles of these cellular genes after UVB-irradiation remain to be elucidated, microarray analysis may provide a new view of gene expression in epidermal <em>keratinocytes</em> following UVB exposure.

Treating chronic kidney disease (CKD) has been challenging because of its pathogenic complexity. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent derivatives of arachidonic acid with antihypertensive, anti-inflammatory, and profibrinolytic functions. We recently reported that genetic ablation of soluble epoxide hydrolase (sEH), an enzyme that converts EETs to less active dihydroxyeicosatrienoic acids, prevents renal tubulointerstitial fibrosis and inflammation in experimental mouse models of CKD. Here, we tested the hypothesis that pharmacological inhibition of sEH after unilateral ureteral obstruction (UUO) would attenuate tubulointerstitial fibrosis and inflammation in mouse kidneys and may provide a novel approach to manage the progression of CKD. Inhibition of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis, as demonstrated by reduced collagen deposition and myofibroblast formation after UUO. The inflammatory response was also attenuated, as demonstrated by decreased influx of neutrophils and macrophages and decreased expression of inflammatory cytokines <em>keratinocyte</em> chemoattractant, macrophage inflammatory protein-<em>2</em>, monocyte chemotactic protein-1, TNF-α, and ICAM-1 in kidneys after UUO. UUO upregulated transforming <em>growth</em> <em>factor</em>-β1/Smad3 signaling and induced NF-κB activation, oxidative stress, tubular injury, and apoptosis; in contrast, it downregulated antifibrotic <em>factors</em>, including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR-γ. sEH inhibition mitigated the aforementioned malevolent effects in UUO kidneys. These data demonstrate that pharmacological inhibition of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys by preventing tubular injury, downregulation of NF-κB, transforming <em>growth</em> <em>factor</em>-β1/Smad3, and inflammatory signaling pathways, and activation of PPAR isoforms. Our data suggest the potential use of sEH inhibitors in treating fibrogenesis in the UUO model of CKD.

OBJECTIVE

Keratinocyte migration from the wound edge is a crucial step in the reepithelization of cutaneous wounds. Growth factors and cytokines, released from cells that invade the wound matrix, play an important role, and several in vitro assays have been performed to elucidate this. The purposes of this study were to review in vitro human studies on keratinocyte migration to identify those growth factors or cytokines that stimulate keratinocyte migration and whether these assays might serve as a screening procedure prior to testing combinations of growth factors or cytokines to promote wound closure in vivo.

METHODS

Research papers investigating effect of growth factors and cytokines on human keratinocyte migration in vitro were retrieved from library sources, PubMed databases, reference lists of papers, and searches of relevant journals.

RESULTS

Fourteen different growth factors and cytokines enhanced migration in scratch wound assay and HGF together with TGF-β, and IGF-1 with EGF, were more stimulatory than either growth factor alone. HGF with TGF-β1 had a greater chemokinetic effect than either growth factor alone in transmigration assay. TGF-β1, FGF-7, FGF-2 and AGF were chemotactic to keratinocytes. EGF, TGF-α, IL-1α, IGF and MGSA enhanced cell migration on ECM proteins.

CONCLUSIONS

Many growth factors and cytokines enhanced migration of keratinocytes in vitro, and certain combinations of growth factors were more stimulatory than either alone. These and other combinations that stimulate keratinocyte migration in vitro should be tested for effect on wound closure and repair in vivo. The scratch wound assay provides a useful, inexpensive and easy-to-perform screening method for testing individual or combinations of growth factors or cytokines, or growth factors combined with other modalities such as laser irradiation, prior to performing wound healing studies with laboratory animals.