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ABSTRACT: Diabetic foot ulcers (DFUs) represent an important clinical problem resulting in significant morbidity and mortality. Ongoing translational research studies strive to better understand molecular/cellular basis of DFU pathology that may lead to identification of novel treatment protocols. Tissue at the non-healing wound edge has been identified as one of major contributors to the DFU pathophysiology that provides important tool for translational and clinical investigations. To evaluate quality of tissue specimens and their potential use, we obtained 81 DFU specimens from 25 patients and performed histological analyses, immunohistochemistry and RNA quality assessments. We found that depth of the collected specimen is important determinant of research utility, and only specimens containing a full-thickness epidermis could be utilized for immunohistochemistry and RNA isolation. We showed that only two-thirds of collected specimens could be utilized in translational studies. This attrition rate is important for designs of future studies involving tissue specimen collection from DFU.
Experimental Dermatology 03/2013; 22(3):216-218. · 3.54 Impact Factor
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ABSTRACT: Glucocorticoids are important regulators of epidermal tissue homeostasis. As such, their clinical applications are widespread, ranging from inflammatory skin disorders to keloids and cancer. Glucocorticoids exert their effect by binding to glucocorticoid receptor (GR) which translocates to the nucleus and regulates gene expression (genomic effect). In addition, GR has rapid non- genomic effects that are mediated by cell signaling proteins and do not involve gene transcription. Although genomic effects of GR in the epidermis are well documented, the non-genomic effects are not completely understood. Therefore, we utilized immunostaining and immunoprecipitations to determine specific localization of the GR in human keratinocytes that may contribute to non-genomic effects of glucocorticoid action. Here we describe a novel finding of GR localization to the plasma membrane of keratinocytes. Immunocytochemistry showed co-localization of GR with α-catenin. Immunoprecipitation of the membranous fraction revealed an association of GR with α-catenin, confirming its localization to adherens junctions. We conclude that GR localization to adherens junctions of keratinocytes provides a new mechanism of non-genomic signaling by glucocorticoids which may have significant biological and clinical impact.
PLoS ONE 01/2013; 8(4):e63453. · 4.09 Impact Factor
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ABSTRACT: Understanding the pathology resulting from and polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. showed an inhibitory effect on USA300 growth while both species co-existed in cutaneous wounds . Polymicrobial wound infection in the presence of resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.
PLoS ONE 01/2013; 8(2):e56846. · 4.09 Impact Factor
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ABSTRACT: Chronic wounds represent a significant burden to patients, health care professionals, and the health care system. Micro-RNAs (miRNAs) have recently emerged as a novel class of gene expression modulators involved in regulation of multiple biological processes, including development, differentiation, organogenesis, inflammation, cell proliferation, growth control, and apoptosis. Importantly, aberrant expression or activity of miRNAs can lead to a disease state. However, the role of miRNAs in chronic wounds remains to be elucidated. This article reviews available literature on the role of miRNAs in a range of processes important for successful wound healing including epidermal differentiation and proliferation, inflammation and angiogenesis. The potential role of miRNAs in normal wound healing and their contribution to chronic wound pathology has been anticipated. The prospective use of miRNAs as markers for surgical debridement, and as novel diagnostic and therapeutic targets for chronic wounds is also discussed.
Surgical technology international 12/2012; XXI:51-60.
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ABSTRACT: Chronic nonhealing wounds, such as venous ulcers (VUs), are a widespread and serious medical problem with high morbidity and mortality. The molecular pathology of VUs remains poorly understood, impeding the development of effective treatment strategies. Using mRNA expression profiling of VUs biopsies and computational analysis, we identified a candidate set of microRNAs with lowered target gene expression. Among these candidates, miR-16, -20a, -21, -106a -130a, and -203 were confirmed to be aberrantly overexpressed in a cohort study of 10 VU patients by quantitative PCR and in situ hybridizations. These microRNAs were predicted to target multiple genes important for wound healing, including early growth response factor 3, vinculin, and leptin receptor (LepR). Overexpression of the top up-regulated miRNAs, miR-21 and miR-130a, in primary human keratinocytes down-regulated expression of the endogenous LepR and early growth response factor 3. The luciferase reporter assay verified LepR as a direct target for miR-21 and miR-130a. Both miR-21 and miR-130a delayed epithelialization in an acute human skin wound model. Furthermore, in vivo overexpression of miR-21 inhibited epithelialization and granulation tissue formation in a rat wound model. Our results identify a novel mechanism in which overexpression of specific set of microRNAs inhibits wound healing, resulting in new potential molecular markers and targets for therapeutic intervention.
Journal of Biological Chemistry 07/2012; 287(35):29324-35. · 4.77 Impact Factor
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ABSTRACT: Utilizing a three-dimensional in vitro glycated collagen model, we evaluated the therapeutic effects of a peroxisome proliferator-activated receptor-γ ligand, rosiglitazone, and its potential as a topical treatment of diabetic chronic wounds. Rosiglitazone induced fibroblast migration, α-smooth muscle actin production, and transformation into myofibroblasts in the presence of advanced glycation end products. Both transforming growth factor β and peroxisome proliferator-activated receptor-γ expression were induced, while the receptor for advanced glycation end products was suppressed. Lastly, the reduced activities of matrix metalloproteinase-2 and matrix metalloproteinases-9 in the carboxymethyllysine-modified collagen matrices by rosiglitazone increases extracellular matrix deposition. Our findings identify rosiglitazone as a candidate for localized topical treatment of diabetic chronic wounds.
Wound Repair and Regeneration 05/2012; 20(3):435-43. · 2.91 Impact Factor
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ABSTRACT: Diabetic foot ulcers (DFUs) are a significant and rapidly growing complication of diabetes and its effects on wound healing. Over half of diabetic patients who develop a single ulcer will subsequently develop another ulcer of which the majority will become chronic non-healing ulcers. One-third will progress to lower extremity amputation. Over the past decade, the outcomes for patients with DFUs ulcers have not improved, despite advances in wound care. Successful treatment of diabetic foot ulcers is hindered by the lack of targeted therapy that hones in on the healing processes dysregulated by diabetes. Stem cells are a promising treatment for DFUs as they are capable of targeting, as well as bypassing, the underlying abnormal healing mechanisms and deranged cell signaling in diabetic wounds and promote healing. This review will focus on existing stem cell technologies and their application in the treatment of DFUs.
Diabetes research and clinical practice 12/2011; 96(1):1-9. · 2.16 Impact Factor
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ABSTRACT: Hydroxyurea (HU) has been shown to induce a variety of cutaneous adverse reactions, including severe leg ulcers. This report shows a successful treatment of a HU-induced chronic wound associated with squamous cell carcinomas (SCC). A 62-year-old patient affected with polycythemia vera and treated with HU for 10 years, presented with a non healing ulcer on a left heel. The patient gave a history of suffering from the wound for over 2 years. Biopsy showed evidence of invasive SCC. The patient underwent Mohs surgery and a greater saphenous vein ablation for polycythemia vera-associated vascular complications. The wound consistently decreased in size following successive debridements and coverage with human skin equivalent. The wound healed completely after a 6-month period. A multidisciplinary team approach to the treatment proved to be effective resulting in healing of this multifactorial chronic ulcer.
International Wound Journal 11/2011; 9(3):324-9. · 1.46 Impact Factor
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ABSTRACT: The objective of the study was to inform wound care practitioners of mesenchymal stem cell application for nonhealing wounds. Recent advances in delivery systems are also discussed in order to highlight potential improvements toward clinical application of stem cell therapy for chronic wounds.
MEDLINE and PubMed Central were searched for scientific studies regarding the use of mesenchymal stem cells and delivery systems in wound healing.
Preclinical studies using stem cells as therapeutic modality for chronic wounds were selected for this review.
Information on study design, sample size and characteristics, stem cell source, type of delivery systems, and rate and time of wound closure was abstracted.
Application of mesenchymal stem cells improved wound healing in experimental and clinical settings. Advances in stem cell therapy and delivery vehicles offer promising alternatives to current limited therapeutic modalities for chronic wounds.
Stem cell therapy has recently emerged as a promising therapeutic strategy for nonhealing wounds. Further research is needed to evaluate the relationship between the various delivery systems and stem cells in order to maximize their therapeutic effects. Development of novel delivery vehicles for stem cells can open new opportunities for more effective cell therapy of chronic wounds.
Advances in skin & wound care 11/2011; 24(11):524-32; quiz 533-4.
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ABSTRACT: Glucocorticoids (GCs) are known inhibitors of wound healing. In this study we report the novel finding that both keratinocytes
in vitro and epidermis in vivo synthesize cortisol and how this synthesis regulates wound healing. We show that epidermis expresses enzymes essential for
cortisol synthesis, including steroid 11 β-hydroxylase (CYP11B1), and an enzyme that controls negative feedback mechanism,
11β-hydroxysteroid dehydrogenase 2 (11βHSD2). We also found that cortisol synthesis in keratinocytes and skin can be stimulated
by ACTH and inhibited by metyrapone (CYP11B1 enzyme inhibitor). Interestingly, IL-1β, the first epidermal signal of tissue
injury, induces the expression of CYP11B1 and increases cortisol production by keratinocytes. Additionally, we found induction
of CYP11B1 increased production of cortisol and activation of GR pathway during wound healing ex vivo and in vivo using human and porcine wound models, respectively. Conversely, inhibition of cortisol synthesis during wound healing increases
IL-1β production, suggesting that cortisol synthesis in epidermis may serve as a local negative feedback to proinflammatory
cytokines. Local GCs synthesis, therefore, may provide control of the initial proinflammatory response, preventing excessive
inflammation upon tissue injury. Inhibition of GC synthesis accelerated wound closure in vivo, providing the evidence that modulation of cortisol synthesis in epidermis may be an important regulatory mechanism during
wound healing.
Journal of Biological Chemistry 03/2011; 286(12):10265-10275. · 4.77 Impact Factor
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ABSTRACT: Glucocorticoids (GCs) are known inhibitors of wound healing. In this study we report the novel finding that both keratinocytes in vitro and epidermis in vivo synthesize cortisol and how this synthesis regulates wound healing. We show that epidermis expresses enzymes essential for cortisol synthesis, including steroid 11 β-hydroxylase (CYP11B1), and an enzyme that controls negative feedback mechanism, 11β-hydroxysteroid dehydrogenase 2 (11βHSD2). We also found that cortisol synthesis in keratinocytes and skin can be stimulated by ACTH and inhibited by metyrapone (CYP11B1 enzyme inhibitor). Interestingly, IL-1β, the first epidermal signal of tissue injury, induces the expression of CYP11B1 and increases cortisol production by keratinocytes. Additionally, we found induction of CYP11B1 increased production of cortisol and activation of GR pathway during wound healing ex vivo and in vivo using human and porcine wound models, respectively. Conversely, inhibition of cortisol synthesis during wound healing increases IL-1β production, suggesting that cortisol synthesis in epidermis may serve as a local negative feedback to proinflammatory cytokines. Local GCs synthesis, therefore, may provide control of the initial proinflammatory response, preventing excessive inflammation upon tissue injury. Inhibition of GC synthesis accelerated wound closure in vivo, providing the evidence that modulation of cortisol synthesis in epidermis may be an important regulatory mechanism during wound healing.
Journal of Biological Chemistry 01/2011; 286(12):10265-75. · 4.77 Impact Factor
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ABSTRACT: Statins, HMG-CoA reductase inhibitors, are common cholesterol-lowering drugs. Recent studies suggest that statins may have potential as novel treatments for diverse conditions, ranging from sepsis and inflammatory diseases to chronic wounds and bone fractures. The diverse pleiotropic actions of statins are probably related to reduced isoprenylation of downstream targets of the mevalonate pathway and their binding to several nuclear hormone receptors. Statins exert their anti-inflammatory effect by inhibiting the release of C-reactive peptide, chemokines, cytokines and adhesion molecules, which may make them a powerful addition to the dermatologic anti-inflammatory medication arsenal. Along with reducing inflammation, statins have the potential to heal chronic wounds by decreasing farnesyl pyrophosphate, facilitating vascular relaxation, promoting neovascularization and reducing bacterial load. A review of the literature elucidates that route of administration, dose and type of statin appear to impact the outcome. A better understanding of their effects at the cellular and molecular level in skin is necessary for their future use.
Expert Review of Dermatology 11/2010; 5(6):689-698.
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ABSTRACT: Transforming growth factor beta (TGFbeta) is important in inflammation, angiogenesis, reepithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from 10 patients with nonhealing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFbeta-treated keratinocytes, we found deregulation of TGFbeta target genes in VUs. Using quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, we found suppression of TGFbeta RI, TGFbeta RII and TGFbeta RIII, and complete absence of phosphorylated Smad2 (pSmad2) in VU epidermis. In contrast, pSmad2 was induced in the cells of the migrating epithelial tongue of acute wounds. TGFbeta-inducible transcription factors (GADD45beta , ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFbeta (FABP5, CSTA and S100A8) were induced in nonhealing VUs. An inhibitor of Smad signaling, Smad7 was also downregulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by downregulation of TGFbeta receptors and attenuation of Smad signaling resulting in deregulation of TGFbeta target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFbeta may not be a beneficial treatment for VUs.
Molecular Medicine 03/2010; 16(3-4):92-101. · 3.76 Impact Factor
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ABSTRACT: Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist
for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist
for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA)
or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing
inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte
migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for
wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin
6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation,
not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition
of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP
is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins.
Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms
by which they may accelerate wound healing.
Journal of Biological Chemistry 01/2010; 285(3):1980-1988. · 4.77 Impact Factor
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ABSTRACT: Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.
Journal of Biological Chemistry 11/2009; 285(3):1980-8. · 4.77 Impact Factor
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ABSTRACT: Epidermal morphology of chronic wounds differs from that of normal epidermis. Biopsies of non-healing edges obtained from patients with venous ulcers show thick and hyperproliferative epidermis with mitosis present in suprabasal layers. This epidermis is also hyper-keratotic and parakeratotic. This suggests incomplete activation and differentiation of keratinocytes. To identify molecular changes that lead to pathogenic alterations in keratinocyte activation and differentiation pathways we isolated mRNA from non-healing edges deriving from venous ulcers patients and determined transcriptional profiles using Affymetrix chips. Obtained transcriptional profiles were compared to those from healthy, unwounded skin. As previously indicated by histology, we found deregulation of differentiation and activation markers. We also found differential regulation of signalling molecules that regulate these two processes. Early differentiation markers, keratins K1/K10 and a subset of small proline-rich proteins, along with the late differentiation marker filaggrin were suppressed, whereas late differentiation markers involucrin, transgultaminase 1 and another subset of small proline-rich proteins were induced in ulcers when compared to healthy skin. Surprisingly, desomosomal and tight junction components were also deregulated. Keratinocyte activation markers keratins K6/K16/K17 were induced. We conclude that keratinocytes at the non-healing edges of venous ulcers do not execute either activation or differentiation pathway, resulting in thick callus-like formation at the edge of a venous ulcers.
Journal of Cellular and Molecular Medicine 04/2008; 12(6B):2675-90. · 4.13 Impact Factor
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ABSTRACT: The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.
Molecular and Biochemical Parasitology 02/2008; 157(1):73-82. · 2.55 Impact Factor
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ABSTRACT: Keratinocytes are the major cellular component of epidermis, and they have several critical roles in the wound healing process. They are involved in the intricate mechanisms of initiation, maintenance, and completion of wound healing. The properties of keratinocytes vary depending upon their location and circumstances within chronic wounds. Keratinocytes at the non-healing edges of chronic wounds differ from normal, healthy keratinocytes. The cross-talk between healthy keratinocytes and other cell types participating in wound healing is critical for successful wound closure. This discovery provides the biological foundation for debridement: Removing "bad" cells from a quiescent wound edge and exposing or even replacing them with "healthy" cells with a high regenerative potential can enhance epithelialization and healing of chronic wounds. This paper will review the biological and pathological properties of keratinocytes as they relate to wound healing, and the ways in which they may provide highly efficacious therapy for patients with chronic wounds.
Surgical technology international 02/2008; 17:105-12.
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ABSTRACT: Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination in bacteria and yeast contain cytidine deaminase-conserved motifs, suggesting an evolutionary link between the two reactions. In trypanosomatids, tRNAs undergo both cytidine-to-uridine and adenosine-to-inosine editing, but the relationship between the two reactions is unclear. Here we show that down-regulation of the Trypanosoma brucei tRNA-editing enzyme by RNAi leads to a reduction in both C-to-U and A-to-I editing of tRNA in vivo. Surprisingly, in vitro, this enzyme can mediate A-to-I editing of tRNA and C-to-U deamination of ssDNA but not both in either substrate. The ability to use both DNA and RNA provides a model for a multispecificity editing enzyme. Notably, the ability of a single enzyme to perform two different deamination reactions also suggests that this enzyme still maintains specificities that would have been found in the ancestor deaminase, providing a first line of evidence for the evolution of editing deaminases.
Proceedings of the National Academy of Sciences 06/2007; 104(19):7821-6. · 9.68 Impact Factor
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ABSTRACT: Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNgamma-regulated genes, including IFNgamma receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced keratinocyte apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of keratinocytes. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic factor, vascular endothelial growth factor. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGFbeta1 and -2 and MMP1, -2, -9, and -10 and inducing TIMP-2. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies.
Journal of Biological Chemistry 03/2007; 282(6):4021-34. · 4.77 Impact Factor
