Jennifer M. Hahn

Keloid-derived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology

Keloids are disfiguring scars that extend beyond the original wound borders and resist treatment. Keloids exhibit excessive extracellular matrix deposition, although the underlying mechanisms remain unclear. To better understand the molecular basis of keloid scarring, here we define the genomic profiles of keloid fibroblasts and keratinocytes. In both cell types, keloid‐derived cells exhibit differential expression of genes encompassing a diverse set of functional categories. Strikingly, keloid keratinocytes exhibited decreased expression of a set of transcription factor, cell adhesion, and intermediate filament genes essential for normal epidermal morphology. Conversely, they exhibit elevated expression of genes associated with wound healing, cellular motility, and vascular development. A substantial number of genes involved in epithelial–mesenchymal transition were also up‐regulated in keloid keratinocytes, implicating this process in keloid pathology. Furthermore, keloid keratinocytes displayed significantly higher migration rates than normal keratinocytes in vitro and reduced expression of desmosomal proteins in vivo. Previous studies suggested that keratinocytes contribute to keloid scarring by regulating extracellular matrix production in fibroblasts. Our current results show fundamental abnormalities in keloid keratinocytes, suggesting they have a profoundly more direct role in keloid scarring than previously appreciated. Therefore, development of novel therapies should target both fibroblast and keratinocyte populations for increased efficacy.

Inhibition of hyaluronan synthase 2 reduces the abnormal migration rate of keloid keratinocytes.

Keloids are fibroproliferative scars that spread beyond the original wound boundary and are very resistant to treatment. Development of highly effective therapies requires a comprehensive understanding of the mechanisms regulating keloid formation. Previous studies indicated that keloid keratinocytes have abnormal expression of genes involved in differentiation and adhesion, and increased migration rates. The objective of the current study was to better understand the role of hyaluronan synthase 2 (HAS2) in keloid keratinocyte migration and gene expression. Keratinocytes were isolated from keloid scars and normal skin. Migration rates of keloid keratinocytes were quantified using an in vitro scratch assay. Expression levels of HAS2, related HAS1, and HAS3 genes, and genes aberrantly expressed in keloid keratinocytes, were quantified using real-time polymerase chain reaction. Treatment with 4-methylumbelliferone (4MU) was used to inhibit hyaluronic acid synthesis. The expression of HAS2 was significantly increased in keloid vs normal keratinocytes. Treatment with 4MU caused a dose-dependent reduction in keloid keratinocyte migration and HAS2 expression; HAS3 expression was moderately inhibited by 4MU and HAS1 was not expressed. Keloid keratinocytes displayed a motile phenotype in vitro, including loose colonies and widely separated refractile cells; this phenotype was normalized by 4MU. Further, 4MU altered gene expression in keloid keratinocytes. The results suggest that HAS2 overexpression contributes to increased migration and altered gene expression in keloid keratinocytes. Abnormal keratinocyte migration may contribute to the overhealing of keloid scars beyond the original wound boundaries. Therefore, inhibition of HAS2 expression using 4MU may represent a novel strategy for treatment of keloid scarring.

Culture medium and cell density impact gene expression in normal skin and abnormal scar-derived fibroblasts.

Fibroblasts, the main cell type of the dermis, are responsible for production and remodeling of extracellular matrix during wound healing. Disruption of either production or degradation of extracellular matrix can lead to abnormal scarring, resulting in hypertrophic scar or keloid scar. Aberrations in proliferation and gene expression have been observed in fibroblasts isolated from abnormal scars, but differences observed may be related to biologic responses to growth conditions and media formulations. This study examined gene expression in primary human fibroblasts from normal skin or abnormal scar in two culture media formulations and three relative cell densities. In general, higher expression of collagen type 1 alpha-1 (COL1A1) and alpha-2 (COL1A2) and matrix metalloproteinase 3 (MMP3) and lower levels of MMP1 were observed in all cell strains cultured in standard medium containing 10% fetal bovine serum compared with cells cultured in medium optimized for proliferation. Normal and scar-derived fibroblasts exhibited differences in gene expression in specific response to media formulations and cell density. COL1A1 and COL1A2 were increased, and MMP1 and MMP3 were decreased, in keloid cells compared with normal fibroblasts under most conditions analyzed. However, expression of plasminogen activator inhibitor 1 in keloid fibroblasts, which was significantly different than in normal fibroblasts, was either increased or decreased in response to the medium formulation and relative cell density. A related gene, plasminogen activator inhibitor 2, was shown for the first time to be significantly increased in keloid fibroblasts compared with normal fibroblasts, in both media formulations and at all three cell densities. The results emphasize the critical role of culture conditions in interpretation of cell behavior and expression data and for comparison of cells representing normal and fibrotic phenotypes.

Partial epithelial-mesenchymal transition in keloid scars: regulation of keloid keratinocyte gene expression by transforming growth factor-β1.

BackgroundKeloids are an extreme form of abnormal scarring that result from a pathological fibroproliferative wound healing process. The molecular mechanisms driving keloid pathology remain incompletely understood, hindering development of targeted, effective therapies. Recent studies in our laboratory demonstrated that keloid keratinocytes exhibit adhesion abnormalities and display a transcriptional signature reminiscent of cells undergoing epithelial-mesenchymal transition (EMT), suggesting a role for EMT in keloid pathology. In the current study, we further define the EMT-like phenotype of keloid scars and investigate regulation of EMT-related genes in keloid.MethodsPrimary keratinocytes from keloid scar and normal skin were cultured in the presence or absence of transforming growth factor beta 1 (TGF-β1) +/− inhibitors of TGF-β1 and downstream signaling pathways. Gene expression was measured using quantitative polymerase chain reaction. Migration was analyzed using an in vitro wound healing assay. Proteins in keloid scar and normal skin sections were localized by immunohistochemistry. Statistical analyses utilized SigmaPlot (SyStat Software, San Jose, CA) or SAS® (SAS Institute, Cary, NC).ResultsIn keloid and normal keratinocytes, TGF-β1 regulated expression of EMT-related genes, including hyaluronan synthase 2, vimentin, cadherin-11, wingless-type MMTV integration site family, member 5A, frizzled 7, ADAM metallopeptidase domain 19, and interleukin-6. Inhibition of canonical TGF-β1 signaling in keloid keratinocytes significantly inhibited expression of these genes, and TGF-β1 stimulation of normal keratinocytes increased their expression. The inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway or the p38 mitogen-activated protein kinase pathway attenuated TGF-β1-induced expression of subsets of these genes. Migration of keloid keratinocytes, previously shown to be increased compared with normal keratinocytes, was significantly reduced by inhibition of TGF-β1 or ERK1/2 signaling. Biomarkers of EMT, including reduced E-cadherin and increased active β-catenin, were observed in keloid epidermis in vivo. However, evidence of basement membrane breakdown in keloid scar was not observed.ConclusionsThe results suggest that keloid keratinocytes exist in an EMT-like metastable state, similar to activated keratinocytes in healing wounds. The EMT-like gene expression pattern of keloid keratinocytes is regulated by canonical and non-canonical TGF-β1 signaling pathways. Therefore, interventions targeting TGF-β1-regulated EMT-like gene expression in keloid keratinocytes may serve to suppress keloid scarring.

Soluble Epoxide Hydrolase Inhibition and Epoxyeicosatrienoic Acid Treatment Improve Vascularization of Engineered Skin Substitutes

Background: Autologous engineered skin substitutes comprised of keratinocytes, fibroblasts, and biopolymers can serve as an adjunctive treatment for excised burns. However, engineered skin lacks a vascular plexus at the time of grafting, leading to slower vascularization and reduced rates of engraftment compared with autograft. Hypothetically, vascularization of engineered skin grafts can be improved by treatment with proangiogenic agents at the time of grafting. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase (sEH). EETs have multiple biological activities and have been shown to promote angiogenesis. Inhibitors of sEH (sEHIs) represent attractive therapeutic agents because they increase endogenous EET levels. We investigated sEHI administration, alone or combined with EET treatment, for improved vascularization of engineered skin after grafting to mice. Methods: Engineered skin substitutes, prepared using primary human fibroblasts and keratinocytes, were grafted to full-thickness surgical wounds in immunodeficient mice. Mice were treated with the sEHI 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered in drinking water throughout the study period, with or without topical EET treatment, and were compared with vehicle-treated controls. Vascularization was quantified by image analysis of CD31-positive areas in tissue sections. Results: At 2 weeks after grafting, significantly increased vascularization was observed in the TPPU and TPPU + EET groups compared with controls, with no evidence of toxicity. Conclusions: The results suggest that sEH inhibition can increase vascularization of engineered skin grafts after transplantation, which may contribute to enhanced engraftment and improved treatment of full-thickness wounds.

Inflammatory response and biomechanical properties of coaxial scaffolds for engineered skin in vitro and post-grafting

Engineered skin (ES) offers many advantages over split-thickness skin autografts for the treatment of burn wounds. However, ES, both in vitro and after grafting, is often significantly weaker, less elastic and more compliant than normal human skin. Biomechanical properties of ES can be tuned in vitro using electrospun co-axial (CoA) scaffolds. To explore the potential for coaxial scaffold-based ES use in vivo, two CoA scaffolds were fabricated with bioactive gelatin shells and biodegradable synthetic cores of polylactic acid (PLA) and polycaprolactone (PCL), and compared with gelatin monofilament scaffolds. Fibroblast and macrophage production of inflammatory cytokines interleukin 6 (IL-6) and transforming growth factor β-1 was significantly higher when cultured on PLA and PCL monofilament scaffolds compared to gelatin monofilament scaffolds. The core-shell fiber configuration significantly reduced production of pro-inflammatory cytokines to levels similar to those of gelatin monofilament scaffolds. In vitro, ES mechanical properties were significantly enhanced using CoA scaffolds; however, after grafting CoA- and gelatin-based ES to full-thickness excisional wounds on athymic mice, the in vitro mechanical advantage of CoA grafts was lost. A substantially increased inflammatory response to CoA-based ES was observed, with upregulation of IL-6 expression and a significant M2 macrophage presence. Additionally, expression of matrix metalloproteinase I was upregulated and collagen type I alpha 1 was downregulated in CoA ES two weeks after grafting. These results suggest that while coaxial scaffolds provide the ability to regulate biomechanics in vitro, further investigation of the inflammatory response to core materials is required to optimize this strategy for clinical use. STATEMENT OF SIGNIFICANCE: Engineered skin has been used to treat very large burn injuries. Despite its ability to heal these wounds, engineered skin exhibits reduced biomechanical properties making it challenging to manufacture and surgically apply. Coaxial fiber scaffolds have been utilized to tune the mechanical properties of engineered skin while maintaining optimal biological properties but it is not known how these perform on a patient especially with regards to their inflammatory response. The current study examines the biomechanical and inflammatory properties of coaxial scaffolds and uniaxial scaffolds in vitro and in vivo. The results show that the biological response to the scaffold materials is a critical determinant of tissue properties after grafting with reduced inflammation and rapid scaffold remodeling leading to stronger skin.

Abnormal expression of the vitamin D receptor in keloid scars

Keloids are abnormal fibroproliferative scars that pose a significant challenge to patients and clinicians. The molecular basis for keloid formation remains incompletely understood, and currently no universally effective treatments exist. It is well recognized that keloids are more prevalent in populations with darkly pigmented skin, such as African Americans, but the basis for the link between skin color and keloid risk is not known. Pigmentation reduces vitamin D production in the skin. Because most of the bodys vitamin D is produced in the skin, rates of vitamin D deficiency are higher in populations with darker skin pigmentation. In addition to regulation of calcium homeostasis, vitamin D plays important roles in cell proliferation, differentiation, cancer progression, inflammation, and fibrosis. The activities of vitamin D are dependent on the vitamin D receptor (VDR), a member of the steroid nuclear receptor superfamily. The ligand-bound VDR acts as a transcription factor; thus, nuclear localization is required for ligand-dependent regulation of target gene expression. The current study investigated expression and nuclear localization of VDR in keloid scars (N=24) and biopsies of normal skin (N=24). Immunohistochemistry with two different antibodies demonstrated reduced VDR protein levels in a majority of keloid scars. Further, the percentage of epidermal cells displaying nuclear VDR localization was significantly lower in keloid scars compared with normal skin samples. Interestingly, analysis of VDR-positive nuclei among different normal skin samples showed a significant reduction in nuclear localization in epidermis of black donors compared with white donors. The results suggest that VDR may play a role in keloid pathology, and hint at a possible role for VDR in the increased susceptibility to keloid scarring in individuals with darkly pigmented skin.