Oral mucositis (OM) is a common complication of treatments for head and neck cancer, particularly radiotherapy with or without chemotherapy. OM is characterised by oral erythema, ulceration, and pain. The aim of this study was to evaluate the effect of azilsartan (AZT), an angiotensin II receptor antagonist, on 5-fluorouracil (5-FU)-induced oral mucositis (OM) in Syrian hamsters. OM was induced by the intraperitoneal administration of 5-FU on experimental days 1 (60 mg/Kg) and <em>2</em> (40 mg/Kg). Animals were pretreated with oral AZT (1, 5, or 10 mg/kg) or vehicle 30 min before 5-FU injection and daily until day 10. Experimental treatment protocols were approved by the Animal Ethics Committee Use/CEUA (Number <em>2</em>8/<em>2</em>01<em>2</em>) of the UFRN. Macroscopic analysis and cheek pouch samples were removed for histopathologic analysis. Myeloperoxidase (MPO), Malonyldialdehyde (MDA), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), and tumour necrosis <em>factor</em>-alpha (TNF-α) were analysed by Enzyme Linked Immuno Sorbent Assay (ELISA). Vascular endothelial <em>growth</em> <em>factor</em> (VEGF), fibroblast <em>growth</em> <em>factor</em> (FGF), <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), and transforming <em>growth</em> <em>factor</em> (TGF)-α were measured by immunohistochemistry. Analysis of variance followed by Bonferroni's test was used to calculate the means of intergroup differences (p ≤ 0.05). Treatment with 1 mg/kg AZT reduced levels MPO (p<0.01), MDA (p<0.5) and histological inflammatory cell infiltration, and increased the presence of granulation tissue. AZT treatment at 1 mg/kg reduced the TNF-α (p<0.05) and IL-1β (p<0.05) levels, increased the cheek pouch levels of IL-10 (p<0.01), and upregulated VEGF, FGF, KGF, and TGF-α. Administration of AZT at higher doses (5 and 10 mg/kg) did not significantly reverse the OM. AZT at a dose of 1 mg/kg prevented the mucosal damage and inflammation associated with 5-FU-induced OM, increasing granulation and tissue repair.

BACKGROUND

The exposure of skin keratinocytes to Ultraviolet (UV) irradiation leads to Akt phosphorylation at Ser-473, which is important for the carcinogenic effects of excessive sun exposure. The present study investigated the underlying mechanism of Akt Ser-473 phosphorylation by UVB radiation.

RESULTS

We found that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mammalian target of rapamycin (mTOR) complex 2 (mTORC2) were both required for UVB-induced Akt Ser-473 phosphorylation in keratinocytes. Inhibition of DNA-PKcs activity via its inhibitor NU7026, a dominant-negative kinase-dead mutation, RNA interference (RNAi) or gene depletion led to the attenuation of UVB-induced Akt Ser-473 phosphorylation. Meanwhile, siRNA silencing or gene depletion of SIN1, a key component of mTORC2, abolished Akt Ser-473 phosphorylation by UVB. Significantly, we discovered that DNA-PKcs was associated with SIN1 in cytosol upon UVB radiation, and this complexation appeared required for Akt Ser-473 phosphorylation. Meanwhile, this DNA-PKcs-SIN1 complexation by UVB was dependent on epidermal growth factor receptor (EGFR) activation, and was disrupted by an EGFR inhibitor (AG1478) or by EGFR depletion. UVB-induced complexation between DNA-PKcs and mTORC2 components was also abolished by NU7026 and DNA-PKcs mutation. Finally, we found that both DNA-PKcs and SIN1 were associated with apoptosis resistance of UVB radiation, and inhibition of them by NU7026 or genetic depletion significantly enhanced UVB-induced cell death and apoptosis.

CONCLUSIONS

Taken together, these results strongly suggest that DNA-PKcs-mTORC2 association is required for UVB-induced Akt Ser-473 phosphorylation and cell survival, and might be important for tumor cell transformation.

Thrombin receptor (ThrR) and protease-activated receptor-<em>2</em> (PAR-<em>2</em>) are members of a unique G protein-coupled receptor family, which are characterized by the unveiling of a tethered peptide ligand upon proteolysis of their NH<em>2</em> terminus. We have previously shown that cultured human basal <em>keratinocytes</em> express both receptors (R.J. Santulli et al., Proc. Natl. Acad. Sci. USA, 9<em>2</em>: 9151-9155, 1995); however, their functional role in epidermal physiology has yet to be described. In the present study, we determined the effects of receptor activation on <em>keratinocyte</em> cell <em>growth</em> and differentiation using thrombin (selective for ThrR), SLIGRL (selective for PAR-<em>2</em>), and SFLLRN (stimulates ThrR and PAR-<em>2</em>), as agonists. ThrR stimulation enhanced cell <em>growth</em> in a dose-dependent manner in the absence of <em>growth</em> <em>factors</em> (epidermal <em>growth</em> <em>factor</em> and bovine pituitary extract). In contrast, under the same conditions, activation of PAR-<em>2</em> led to the inhibition of cell <em>growth</em>. This inhibitory activity by PAR-<em>2</em> activation was also observed in the presence of <em>growth</em> <em>factors</em>. Activation of both receptors diminished protein expression of the differentiation marker transglutaminase type 1 induced by either calcium or IFN-gamma. Calcium-induced involucrin expression was also decreased. These results indicate that PAR-<em>2</em> and ThrR differentially modulate <em>keratinocyte</em> function and may provide an important regulatory function in the epidermis by altering the functional state of <em>keratinocytes</em>.

OBJECTIVE

To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes and transforming growth factor β (TGF-β)1 siRNA transfected fibroblasts seeded on bladder acellular matrix graft (BAMG) to reconstruct urethra.

METHODS

Autologous oral keratinocytes and TGF-β1 siRNA transfected fibroblasts were seeded onto BAMGs to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed using morphology and scanning electron microscopy. In 27 male rabbits, a ventral urethral mucosal defect was created. Urethroplasty was performed with autogenic oral keratinocyte and TGF-β1 siRNA transfected fibroblast-seeded BAMGs (9 rabbits, group 1), with autogenic oral keratinocyte-seeded BAMGs (9 rabbits, group 2) or with BAMGs with no cell seeding (9 rabbits, group 3). Retrograde urethrography and histological analyses were performed to evaluate the results of urethroplasty.

RESULTS

In vitro, oral keratinocytes and TGF-β1 siRNA transfected fibroblasts had good biocompatibility with BAMGs. In vivo, the urethra kept a wide caliber in groups 1 and 2. Strictures were observed in group 3. Histologically, the retrieved urethra in group 3 showed fibrosis and inflammation during 6 months. Stratified epithelial layer regenerated in group 2, whereas there was no evidence of formation of capillary in the epithelial lower layer during the study period. Stratified epithelial layer and formation of capillary in the epithelial lower layer were evident after 6 months in group 1.

CONCLUSIONS

Our study suggested that oral keratinocytes and TGF-β1 siRNA transfected fibroblasts could be used as a source of seed cells for urethral tissue engineering.

Desmosomes are the most important intercellular adhering junctions that adhere two adjacent <em>keratinocytes</em> directly with desmosomal cadherins, that is, desmogleins (Dsgs) and desmocollins, forming an epidermal sheet. Recently, two cell-cell adhesion states of desmosomes, that is, "stable hyper-adhesion" and "dynamic weak-adhesion" conditions have been recognized. They are mutually reversible through cell signaling events involving protein kinase C (PKC), Src and epidermal <em>growth</em> <em>factor</em> receptor (EGFR) during Ca(<em>2</em>+)-switching and wound healing. This remodeling is impaired in pemphigus vulgaris (PV, an autoimmune blistering disease), caused by anti-Dsg3 antibodies. The antibody binding to Dsg3 activates PKC, Src and EGFR, linked to generation of dynamic weak-adhesion desmosomes, followed by p38MAPK-mediated endocytosis of Dsg3, resulting in the specific depletion of Dsg3 from desmosomes and acantholysis. A variety of pemphigus outside-in signaling may explain different clinical (non-inflammatory, inflammatory, and necrolytic) types of pemphigus. Pemphigus could be referred to a "desmosome-remodeling disease involving pemphigus IgG-activated outside-in signaling events".

Ultraviolet light A (UVA) exposure is thought to cause skin aging mainly by singlet oxygen ((1)O(<em>2</em>))-dependent pathways. Using microarrays, we assessed whether pre-treatment with the (1)O(<em>2</em>) quencher beta-carotene (betaC; 1.5 microM) prevents UVA-induced gene regulation in HaCaT human <em>keratinocytes</em>. Downregulation of <em>growth</em> <em>factor</em> signaling, moderate induction of proinflammatory genes, upregulation of immediate early genes including apoptotic regulators and suppression of cell cycle genes were hallmarks of the UVA effect. Of the 568 UVA-regulated genes, betaC reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for <em>2</em>45 genes. The different interaction modes imply that betaC/UVA interaction involved multiple mechanisms. In unirradiated <em>keratinocytes</em>, gene regulations suggest that betaC reduced stress signals and extracellular matrix (ECM) degradation, and promoted <em>keratinocyte</em> differentiation. In irradiated cells, expression profiles indicate that betaC inhibited UVA-induced ECM degradation, and enhanced UVA induction of tanning-associated protease-activated receptor <em>2</em>. Combination of betaC-promoted <em>keratinocyte</em> differentiation with the cellular "UV response" caused synergistic induction of cell cycle arrest and apoptosis. In conclusion, betaC at physiological concentrations interacted with UVA effects in <em>keratinocytes</em> by mechanisms that included, but were not restricted to (1)O(<em>2</em>) quenching. The retinoid effect of betaC was minor, indicating that the betaC effects reported here were predominantly mediated through vitamin A-independent pathways.

Mesenchymal stromal cells (MSCs) play an important role in tissue regeneration mainly through the secretion of trophic <em>factors</em> that enhance the repair of damaged tissues. The main goal of this work was to study the paracrine mechanisms by which an umbilical cord tissue-derived MSC population (UCX(®)) promotes the migration capacity of human dermal fibroblasts and <em>keratinocytes</em>, which is highly relevant for skin regeneration. Furthermore, the differences between paracrine activities of MSCs from the umbilical cord tissue and the bone marrow (BM-MSCs) were also evaluated. In vitro scratch assays revealed that conditioned media (CM) obtained from both <em>growing</em> and stationary-phase UCX(®) cultures induced human dermal fibroblast (HDF) and <em>keratinocyte</em> (HaCaT) migration. These assays showed that the motogenic activity of UCX(®) CM to HaCaTs was significantly higher than to HDFs, in opposition to the effect seen with CM produced by BM-MSCs that preferentially induced HDF migration. Accordingly, a comparative quantification of key <em>factors</em> with vital importance in the consecutive stages of wound healing revealed very different secretome profiles between UCX(®) and BM-MSCs. The relatively higher UCX(®) expression of EGF, FGF-<em>2</em>, and KGF strongly supports early induction of <em>keratinocyte</em> migration and function, whereas the UCX(®)-specific expression of G-CSF suggested additional roles in mobilization of healing-related cells including CD34(-)/CD45(-) precursors (MSCs) known to be involved in tissue regeneration. Accordingly, in vitro chemotaxis assays and an in vivo transplantation model for chemoattraction confirmed that UCX(®) are chemotactic to CD34(-)/CD45(-) BM-MSCs via a cell-specific mobilization mechanism mediated by G-CSF. Overall, the results strongly suggest different paracrine activities between MSCs derived from different tissue sources, revealing the potential of UCX(®) to extend the regenerative capacity of the organism by complementing the role of endogenous BM-MSCs.

Terminally differentiated <em>keratinocytes</em> are dead enucleated squams. We showed previously that the mitochondria-dependent cell death pathway might be gradually activated as differentiation progresses. In this study, we demonstrated that protoporphyrin IX, staurosporine, and rotenone induced apoptotic-like changes in the mitochondria, and early differentiation of <em>keratinocytes</em> without inducing apoptosis. Kinetics studies established that differentiation-related changes, including <em>growth</em> arrest, flattened morphology, stratification, and keratin 10 (K10) expression, were downstream of mitochondrial depolarization and proliferation, reactive oxygen species (ROS) production, and release of cytochrome c and apoptosis-inducing <em>factor</em>. When these changes were prevented by overexpressing Bcl-<em>2</em> or pharmacologically decreasing the ROS level, K10 upregulation was inhibited, implying that the differentiated phenotype and K10 expression require apoptotic mitochondria, ROS being the most likely differentiation-mediating <em>factor</em>. Our data also suggest that the same mitochondria-affecting stimuli can induce either differentiation or apoptosis, depending on the keratinocyte's competency to undergo differentiation, a competency that may be controlled by Bcl-<em>2</em>.

Data from in-vitro, animal, and human lung injury models suggest that keratinocyte growth factor (KGF) might be beneficial in acute respiratory distress syndrome (ARDS). The objective of this trial was to investigate the effect of KGF in patients with ARDS.

We did a double-blind, allocation concealed, randomised, placebo-controlled phase 2 trial in two intensive care units in the UK, involving patients fulfilling the American-European Consensus Conference Definition of ARDS. Patients were randomly assigned (1:1) by computer-generated randomisation schedule with variable block size stratified by site and presence of severe sepsis requiring vasopressors to receive either recombinant human KGF (palifermin 60 μg/kg) or placebo (0·9% sodium chloride solution) daily for a maximum of 6 days. Both patients and investigators were masked to treatment. The primary endpoint was oxygenation index (OI) at day 7. Analyses were by intention to treat. The trial is registered with International Standard Randomised Controlled Trial Registry, number ISRCTN95690673.

Between Feb 23, 2011, and Feb 26, 2014, 368 patients were assessed for eligibility for inclusion in the trial. Of the 60 patients recruited, 29 patients were randomly assigned to receive KGF and 31 to placebo; all were included in the analysis of the primary outcome. There was no significant difference between the two groups in OI at day 7 (mean 62·3 [SD 57·8] in the KGF group, 43·1 [33·5] in the placebo group; mean difference 19·2, 95% CI -5·6 to 44·0, p=0·13). Of interest, although not defined as outcome measures a priori, the KGF group, compared with placebo, had fewer median ventilator-free days (1 day [IQR 0 to 17] in the KGF group vs 20 days [13-22] in the placebo group; difference -8 days, 95% CI -17 to -2; p=0·0002), a longer median duration of ventilation in survivors to day 90 (16 days [IQR 13-30] in the KGF group vs 11 days [8-16] in the placebo group; difference 6 days, 95% CI 2 to 14; p=0·002), and a higher mortality at 28 days (nine [31%] vs three [10%] deaths; risk ratio 3·2, 95% CI 1·0 to 10·7, p=0·054). Adverse events were more frequent in the KGF group than the placebo group (14 vs 5 events; odds ratio 4·9, 95% CI 1·3 to 20·3, p=0·008). The two adverse events assessed as related to KGF were due to pyrexia.

KGF did not improve physiological or clinical outcomes in ARDS and might be harmful to patient health.

The Northern Ireland Public Health Agency Research and Development Division.

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(<em>2</em>)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(<em>2</em>)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(<em>2</em>) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(<em>2</em>). Expression of splice variant <em>2</em> of the HA-binding proteoglycan versican was also increased by E(<em>2</em>). In search of candidate mediators of these effects, it was found that E(<em>2</em>) strongly induced the expression of epidermal <em>growth</em> <em>factor</em> (EGF) in UVB-irradiated epidermis in vivo and in <em>keratinocytes</em> in vitro. EGF in turn up-regulated the expression of HAS3 and versican V<em>2</em> in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V<em>2</em> induction by E(<em>2</em>) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase <em>2</em>, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(<em>2</em>) in the dermis. Collectively, these data suggest that E(<em>2</em>) treatment increases the amount of dermal HA and versican V<em>2</em> via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(<em>2</em>) during photoaging.

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as <em>keratinocytes</em>, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and <em>growth</em> of these cells in <em>2</em>-dimensional culture. Here we show that <em>keratinocytes</em> grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as <em>keratinocytes</em> propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, <em>growth</em> media developed for serum-free expansion of <em>keratinocytes</em> facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains <em>factors</em> that both aid and inhibit stratification.

Wound-healing represents a major health burden, such as diabetes-induced skin ulcers and burning. Many works are being tried to find ideal clinical wound-healing biomaterials. Especially, small molecules with low cost and function to promote production of endogenous wound healing agents (i.e. transforming <em>growth</em> <em>factor</em> beta, TGF-β) are excellent candidates. In this study, a small peptide (tiger17, c[WCKPKPKPRCH-NH<em>2</em>]) containing only 11 amino acid residues was designed and proved to be a potent wound healer. It showed strong wound healing-promoting activity in a murine model of full thickness dermal wound. Tiger17 exerted significant effects on three stages of wound healing progresses including (1) the induction of macrophages recruitment to wound site at inflammatory reaction stage; (<em>2</em>) the promotion of the migration and proliferation both <em>keratinocytes</em> and fibroblasts, leading to reepithelialization and granulation tissue formation; and (3) tissue remodeling phase, by promoting the release of transforming TGF-β1 and interleukin 6 (IL-6) in murine macrophages and activating mitogen-activated protein kinases (MAPK) signaling pathways. Considering its easy production, store and transfer and function to promote production of endogenous wound healing agents (TGF-β), tiger17 might be an exciting biomaterial or template for the development of novel wound-healing agents.

<em>2</em>-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid <em>2</em>-hydroxylase (FA<em>2</em>H), required for the synthesis of <em>2</em>-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human <em>keratinocytes</em>. Unexpectedly, however, mice deficient in FA<em>2</em>H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA<em>2</em>H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of <em>2</em>-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA<em>2</em>H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active <em>growth</em> phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal <em>growth</em> <em>factor</em> receptor ligand epigen in sebocytes. Loss of FA<em>2</em>H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA<em>2</em>H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA<em>2</em>H, in the hair follicle homeostasis.

BACKGROUND

Impaired wound healing is a frequent phenomenon in diabetes mellitus. However, little is known of the fundamental cause of this pathology. The present study examined the effect of human insulin-like growth factor (hIGF)-1 overexpression in combination with autologous cell transplantation to diabetic wounds in a preclinical large-animal model.

METHODS

Diabetes was induced in Yorkshire pigs with streptozotocin. Keratinocytes were cultured and transfected with hIGF-1 or LacZ transgene. Plasmids were lipoplexed with either Lipofectin or Lipofectamin 2000. Transgene expression was assessed by enzyme-linked immunosorbent assay or X-gal staining. For in vivo studies, full-thickness wounds were created and dressed with a sealed chamber. Transfected cells were transplanted into the wounds. Wound contraction was monitored and biopsies were obtained for measurement of re-epithelialization. Wound fluid was collected and analysed for IGF-1 concentrations.

RESULTS

Quantification showed up to 740 ng/ml IGF-1 in vitro and significantly higher concentrations over 14 days compared to controls for the Lipofectamin 2000 group. Lipofectin-mediated gene transfer showed peak expression on day 2 with 68.5 ng/ml. In vivo, transfected cells showed peak expression of 457 ng/ml at day 1, followed by subsequent decline to 5 ng/ml on day 12 with Lipofectamin 2000. For Lipofectin, no significant IGF-1 expression could be detected. Gene therapy caused significantly faster wound closure (83%) than both controls (native-cell therapy = 57%; control wounds = 32%).

CONCLUSIONS

The present study demonstrates that optimized nonviral gene transfer increased IGF-1 expression in diabetic wounds by up to 900-fold. This high IGF-1 concentration in combination with cell therapy improved diabetic wound healing significantly.

<em>Growth</em> <em>factors</em> of the transforming <em>growth</em> <em>factor</em>-beta superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming <em>growth</em> <em>factor</em>-beta related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic <em>keratinocytes</em> of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in <em>keratinocytes</em> at the wound edge and <em>keratinocytes</em> of the newly formed epithelium as well as in fibroblast shaped cells in the wound bed. BMP-6-specific RNA was induced within <em>2</em>4 h after injury, whereas significant upregulation of BMP-6 on the protein level was detected only <em>2</em>-3 d after injury. Protein was confined to outermost suprabasal epidermal layers, whereas BMP-6-specific RNA was distributed throughout all epidermal layers including basal <em>keratinocytes</em> and the leading edge of the migrating <em>keratinocytes</em>. We also detected high levels of BMP-6-specific RNA and protein in chronic human wounds of different etiology. In contrast to the overall distribution pattern of BMP-6-specific RNA, the protein was not detected in <em>keratinocytes</em> directly bordering the wound. In order to test the influence of BMP-6 abundance on the progress of wound healing, we analyzed the wound response of transgenic mice overexpressing BMP-6 in the epidermis. In these mice, reepitheliazation of skin wounds was significantly delayed, suggesting that strict spatial and temporal regulation of BMP-6 expression is necessary not only for formation but also for reestablishment of a fully differentiated epidermis.

Cultured skin substitutes (CSS) lack a vascular plexus, leading to slower vascularization after grafting than split-thickness skin autograft. CSS containing <em>keratinocytes</em> genetically modified to overexpress vascular endothelial <em>growth</em> <em>factor</em> (VEGF) were previously shown to exhibit enhanced vascularization up to <em>2</em> weeks after grafting to athymic mice. The present study examines whether enhanced vascularization compared with controls persists after stable engraftment is achieved and analyzes VEGF expression, wound contraction, and engraftment. Control and VEGF-modified (VEGF+) CSS were grafted onto full-thickness wounds in athymic mice. VEGF expression was detected in VEGF+ CSS 14 weeks after grafting. Graft contraction was significantly lower in VEGF+ CSS compared with controls, suggesting more stable engraftment and better tissue development. Positive HLA-ABC staining, indicating persistence of human cells, was seen in 86.7% (13/15) of grafted VEGF+ CSS, compared with 58.3% (7/1<em>2</em>) of controls. Differences in dermal vascularization between control and VEGF+ grafts were significant 1 week after surgery, but not at later times. However, the distribution of vessels was different, with more vessels in the upper dermis of VEGF+ grafts. These results suggest that VEGF overexpression in genetically modified CSS acts to accelerate early graft vascularization and can contribute to improved healing of full-thickness skin wounds.

Beta ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming <em>growth</em> <em>factor</em> (TGF)-beta1. Whereas beta ig-h3 is known to mediate <em>keratinocyte</em> adhesion and migration, its effects on <em>keratinocyte</em> differentiation remain unclear. In the present study, it was demonstrated that expression of both beta ig-h3 and TGF-beta1 was enhanced during <em>keratinocyte</em> differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in beta-h3 expression would affect <em>keratinocyte</em> differentiation. Indeed, down-regulation of beta ig-h3 by transfection with antisense beta ig-h3 cDNA constructs effectively inhibited <em>keratinocyte</em> differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was an approximately <em>2</em>-fold increase in mitotic capacity of the cells. Conversely, overexpression of beta ig-h3, either by transfection with beta ig-h3 expression plasmids or by exposure to recombinant beta ig-h3, enhanced <em>keratinocyte</em> differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant beta ig-h3 also promoted <em>keratinocyte</em> adhesion through interaction with integrin alpha3beta1. Changes in beta ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant beta ig-h3 but also blockage of Akt phosphorylation by LY<em>2</em>9400<em>2</em>, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced beta ig-h3, induced by enhanced TGF-beta during <em>keratinocyte</em> differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin alpha3beta1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that beta ig-h3-mediated <em>keratinocyte</em> differentiation was caused by promotion of cell adhesion and not by calcium regulation.

In summary, although TGF-alpha was initially found in tumors, a number of later studies, some of them from the author's laboratory, have shown that TGF-alpha should no longer be considered a tumor associated <em>growth</em> <em>factor</em>. Rather, TGF-alpha is a normal physiological ligand for the EGF receptor. Table <em>2</em> lists some of the normal cellular sources of TGF-alpha. Our list is incomplete, but we know that TGF-alpha is made in <em>keratinocytes</em> and a number of epithelial cells, including gut and breast epithelial cells. It seems very likely that TGF-alpha is a major <em>growth</em> <em>factor</em> secreted by cells of epithelial origin. Zena Werb's and Russell Ross's groups have shown that activated macrophages make TGF-alpha. We have shown that brain makes TGF-alpha and Jeff Kudlow has found TGF-alpha made in the pituitary. Data from several sources, including David Lee, the author's laboratory, and Zena Werb's laboratory has shown that TGF-alpha is made during embryonic development. Therefore, it is now important to look at TGF-alpha in its normal physiological context. Finally, it should be stressed that, as was mentioned above, TGF-alpha is not necessarily a secreted <em>growth</em> <em>factor</em> 50 amino acids long. There is quite a bit of processing of the larger precursor that may or may not take place. This processing, which determines the ultimate size and location of the molecule, is also likely to influence its physiological action.

The expression of mRNAs for <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) (also called FGF-7) and its receptor was evaluated in normal human endometrium and myometrium as well as in myoma and in endometrial adenocarcinoma cell lines using reverse transcriptase polymerase chain reaction. Both KGF and its receptor mRNA are expressed in the human endometrium throughout the menstrual cycle, whereas fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR-<em>2</em>) mRNA expression is low in this tissue. In endometrial stromal cell enriched preparations KGF mRNA dominates with little expression of KGF receptor (KGFR) and FGFR-<em>2</em>, whereas in the epithelial cell-enriched fraction the KGFR mRNA dominates. Human myometrium and myoma express mRNA for KGF, but not for KGFR. FGFR-<em>2</em> is expressed in both myometrial and myoma tissues. None of the five endometrial adenocarcinoma cell lines studied expressed KGF mRNA, whereas all cell lines expressed mRNA for either KGFR or FGFR-<em>2</em> or for both receptors. The results show a selective expression of KGFR and the closely related FGFR-<em>2</em> in the human uterus with the former being expressed in the endometrium and the latter predominantly in the adjacent myometrium. In the endometrial tissue, selective expression of KGF in stromal cells and KGFR in epithelial cells supports the paracrine function of KGF in epithelial tissue.

Skin complications and chronic non-healing wounds are common in obesity, metabolic disease, and type <em>2</em> diabetes. Epidermal γδ T cells normally produce <em>keratinocyte</em> <em>growth</em> <em>factors</em>, participate in wound repair, and are necessary for <em>keratinocyte</em> homeostasis. We have determined that in γδ T cell-deficient mice, there are reduced numbers of <em>keratinocytes</em> and the epidermis exhibits a flattened, thinner structure with fewer basal <em>keratinocytes</em>. This is important in obesity, where skin-resident γδ T cells are reduced and rendered dysfunctional. Similar to γδ T cell-deficient mice, <em>keratinocytes</em> are reduced and the epidermal structure is altered in two obese mouse models. Even in regions where γδ T cells are present, there are fewer <em>keratinocytes</em> in obese mice, indicating that dysfunctional γδ T cells are unable to regulate <em>keratinocyte</em> homeostasis. The impact of absent or impaired γδ T cells on epidermal structure is exacerbated in obesity as E-cadherin localization and expression are additionally altered. These studies reveal that γδ T cells are unable to regulate <em>keratinocyte</em> homeostasis in obesity and that the obese environment further impairs skin structure by altering cell-cell adhesion. Together, impaired <em>keratinocyte</em> homeostasis and epidermal barrier function through direct and indirect mechanisms result in susceptibility to skin complications, chronic wounds, and infection.

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in <em>keratinocytes</em>, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in <em>keratinocytes</em> in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal <em>keratinocytes</em> (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. <em>2</em>0-40mJ/cm(<em>2</em>) dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis <em>factor</em> alpha (TNFalpha) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in <em>keratinocytes</em> after UVB treatment.

BACKGROUND

Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles in neovascularization and development of tissues. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We have previously shown that keratinocytes from human normal skin express VEGFRs. This poses the question of whether these receptors are also expressed by epidermal appendages, as epidermal appendages are lined with epithelial cells.

OBJECTIVE

To investigate the expression of VEGFR-2 compare with VEGF in epidermal appendages, including hair follicles, eccrine sweat glands and sebaceous glands.

METHODS

Monoclonal antibodies to VEGF and VEGFR-2 were used for immunohistochemical examination of cryostat-cut sections of normal human skin specimens from 11 donors undergoing cosmetic surgery.

RESULTS

Immunoreactivities for VEGF and VEGFR-2 principally showed parallel intense expression in anagen hair follicle (including outer root sheat, inner root sheath, dermal papillae epidermal matrix), sebaceous glands (ductal and secretory portions) and eccrine sweat glands (ductal and secretory portions), respectively. In particular, abundant expression of VEGF was found in the follicular basement membrane zone surrounding the bulb matrix and in the ductal and secretory portions of eccrine sweat glands.

CONCLUSIONS

A potential VEGF/VEGFR-2 autocrine pathway may be defined by the coexpression of VEGF and VEGFR-2 in human skin epidermal appendages.

The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal <em>keratinocytes</em> from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 microm(<em>2</em>)) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 microm(<em>2</em>)), but the addition of <em>keratinocyte</em>-conditioned media or exogenous vascular endothelial <em>growth</em> <em>factor</em> induced their normalization toward a smaller size. Vascular endothelial <em>growth</em> <em>factor</em> neutralization inhibited the effect of <em>keratinocyte</em>-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) purified from platelets is a potent <em>growth</em> inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this <em>factor</em>. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal <em>keratinocyte</em> strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, <em>keratinocytes</em> were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their <em>growth</em> was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable <em>growth</em> inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within <em>2</em> days, permitting normal rates of <em>keratinocyte</em> <em>growth</em>. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this <em>factor</em> in vivo.