Faculty Bibliography

The incidence of nonhealing wounds (diabetic foot, pressure, venous, and arterial ulcers) is reaching epidemic proportions, underscoring the need for new treatment modalities. Understanding hair follicle biology and its potential to accelerate wound healing may offer new treatment strategies. In this issue, Ansell et al. show that wounds on anagen skin heal faster than those on telogen skin, suggesting that hair cycle stages may influence healing outcome

An estimated 15% of patients with diabetes mellitus will develop a foot ulcer during their lifetime. Debridement is included in multiple guidelines and algorithms for the care of patients with diabetic neuropathic foot ulcers, and it has long been considered an essential step in the protocol for treating diabetic foot ulcers. In addition to altering the environment of the chronic wound, debridement is a technique aimed at removing nonviable and necrotic tissue, thought to be detrimental to healing. This is accomplished by removing abnormal wound bed and wound edge tissue, such as hyperkeratotic epidermis (callus) and necrotic dermal tissue, foreign debris, and bacteria elements known to have an inhibitory effect on wound healing. While the rationale for surgical debridement seems logical, the evidence for its role in enhancing healing is deficient. In this paper, we systematically review five published clinical trials, which met the criteria and investigated surgical debridement of diabetic foot ulcers to enhance healing. Most existing studies are not randomized clinical trials optimized to test the relationship between debridement of diabetic foot ulcers and wound healing. Therefore, a focused, well-designed study is needed to elucidate the effect of surgical debridement on the healing status of chronic wounds

TGFbeta is important in inflammation, angiogenesis, re-epithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from ten patients with non-healing 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 qPCR and immunohistochemistry, we found suppression of TGFbetaRI, RII and RIII and complete absence of phosphorylated Smad2 (pSmad2) in VUs epidermis. In contrast, pSmad2 was induced in the cells of 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 non-healing VUs. An inhibitor of Smad signaling, Smad7 was also down-regulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by down-regulation 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 beneficial treatment for VUs

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

MicroRNAs (miRNAs) are short (~22nt) noncoding RNAs that can suppress the expression of protein-coding genes and are found disregulated in various diseases. To determine the role of miRNAs in inhibition of wound healing we utilized biopsies obtained from non-healing venous ulcers (VUs) and molecular biology and bioinformatics approaches. miRNA fraction was isolated from full thickness skin biopsies obtained after surgical debridement of ten VUs and healthy skin. All VU biopsies were verified for established histological criteria for non-healing edges and nuclear presence of biomarker, beta-catenin. Quantification of specific miRNAs was performed using TaqManMicroRNA Assays, and miRNA expression was normalized between different samples based on the values of U48 RNA expression. PCR results revealed induction of miR-21 and miR-20a expression in VUs. To validate these findings we used ex-vivo human wound model in which we applied topically specific miRNAs and followed the rate of healing. Using dye-conjugated mimic we documented efficient penetration of miRNAs through epidermis and dermis within first 24hrs. Mimic-mir-21, but not mimic-control or a vehicle, significantly inhibited wound healing. Next, we used bioinformatics approach to identify genes that may be targeted by both, miR-21 and miR-20a. Interestingly, we found highest prediction for growth factor signaling molecules, including TGFbeta and EGF. To further determine if these targets are indeed affected in VUs we used qPCR and immunohistochemistry. Indeed, we found that all three TGFbeta and EGF receptors were down-regulated in non-healing edges of VUs. Interestingly, TGFbetaRI and III were also suppressed at mRNA level whereas TGFbetaRII mRNA levels remained unchanged. We conclude that induction of specific miRNAs in VUs contribute to loss of growth factor signaling leading to overall non-healing phenotype. Thus, targeting specific miRNA molecules may be intriguing novel approach to accelerate wound healing in patients

Because of its accessibility, skin has been among the first organs analyzed using DNA microarrays; psoriasis, melanomas, carcinomas, chronic wound biopsies, and epidermal keratinocytes in culture have been intensely investigated. Skin has everything: stem cells, differentiation, signaling, inflammation, diseases, cancer, etc. Here we provide step-by-step instructions for bioinformatics analysis of transcriptional profiling of skin. Specifically, we describe the use of GCOS and RMA programs for initial normalization and selection of differentially expressed genes, DAVID and LOLA programs for annotation of genes, and statistically relevant identification of over- and under-represented functional and biological categories in identified gene sets, L2L and Venn diagrams for comparing multiple lists of genes, and oPOSSUM for identification of statistically over-represented transcription factor binding sites in the promoter regions of gene sets. The work can be a primer for researchers embarking on skinomics, the comprehensive analysis of transcriptional changes in the skin

Glucocorticoids (GC) are potent antiinflammatory agents and known inhibitors of wound healing. We have shown that GCs inhibit keratinocyte migration by a unique molecular mechanism that involves beta-catenin as a co-regulator of the glucocorticoid receptor. Recently, we have verified that human epidermis expresses enzymes necessary for steroid synthesis and regulation (CYP11B1, HSD11B1, HSD11B2), suggesting endogenous extra-adrenal synthesis of corti-sol. We hypothesize that local glucocorticoid synthesis in skin may serve as a negative feedback loop that curbs initial pro-inflammatory response, preventing excessive inflammation. We used a partial-thickness porcine (in vivo) and human (ex vivo) wound models to test if wounding induces cortisol synthesis in epidermis. First, we confirmed that porcine skin expresses enzymes that participate in GC synthesis and its control. qPCR gene expression analysis, along with cloning and sequencing, confirmed that porcine epidermis expresses CYP11B1 and HSD11B1, genes encoding enzymes necessary for the synthesis and regulation of GCs. Next, we tested levels of CYP11B1, the enzyme that executes GC synthesis in both wound models. We found that CYP11B1 is significantly induced upon wounding in porcine and human wound models. The induction was evident at 24 hrs post-wounding and peaked at 48hrs, which correlates with the highest activity of pro-inflammatory cytokines, IL-1 and TNFa. To test if pro-inflammatory cytokines induce GC synthesis, we treated human skin with IL-1 and found induction of CYP11B1 and secretion of cortisol. We conclude that wounding induces anti-inflammatory response by inducing cortisol synthesis in epidermis. Modulation of cortisol production may be an important regulatory mechanism in wound healing that may participate in inhibition of wound closure

The intermediate filament cytoskeleton is essential for the development and maintenance of normal tissue function. A number of diverse recent observations implicate these filament systems in sensing stress and protecting cells against its worst consequences. Cells expressing severely disruptive keratin mutations, characteristic of Dowling-Meara EBS, were previously reported to show elevated responses to physiological stress, and partial disassembly of cell junctions was reported upon direct mechanical stress to the cells. Gene expression microarray analysis has therefore been used here to examine the broad spectrum of effects of mutant keratins. Many genes associated with keratins and other components of the cytoskeleton showed altered expression levels; in particular, many cell junction components are down-regulated in EBS cells. That this is due to the expression of the mutant keratins, and not to other genetic variables, is supported by observation of the same effects in isogenic cells generated from wild type keratinocytes transfected with the same keratin mutations in the helix boundary motifs of K14 or K5. Whilst the mechanism underlying this is unclear, these findings may help to explain other aspects of EBS-associated pathology, such as faster scratch wound migration, or acantholysis (cell-cell separation) in patients' skin. Constitutive stress combined with constitutively weakened cell junctions may also contribute to a recently reported increased risk of non-melanoma skin cancer in EBS patients

In this study, we hypothesize that local sustained release of vascular endothelial growth factor (VEGF), using adenovirus vector (ADV)-mediated gene transfer, accelerates experimental wound healing. This hypothesis was tested by determining the specific effects of VEGF(165) application on multiple aspects of the wound healing process, that is, time to complete wound closure and skin biomechanical properties. After showing accelerated wound healing in vivo, we studied the mechanism to explain the findings on multiple aspects of the wound healing cascade, including epithelialization, collagen deposition, and cell migration. Intradermal treatment of wounds in non-obese diabetic and db/db mice with ADV/VEGF(165) improves healing by enhancing tensile stiffness and/or increasing epithelialization and collagen deposition, as well as by decreasing time to wound closure. VEGF(165), in vitro, stimulates the migration of cultured human keratinocytes and fibroblasts, thus revealing a non-angiogenic effect of VEGF on wound closure. In conclusion, ADV/VEGF is effective in accelerating wound closure by stimulating angiogenesis, epithelialization, and collagen deposition. In the future, local administration and sustained, controlled release of VEGF(165) may decrease amputations in patients with diabetic foot ulcers and possibly accelerate closure of venous ulcers and pressure ulcers.Journal of Investigative Dermatology advance online publication, 12 March 2009; doi:10.1038/jid.2009.26