Dariush Honardoust

Small leucine-rich proteoglycans, decorin and fibromodulin, are reduced in postburn hypertrophic scar.

Small leucine‐rich proteoglycans (SLRPs) are extracellular matrix molecules that regulate collagen fibrillogenesis and inhibit transforming growth factor‐β activity; thus, they may play a critical role in wound healing and scar formation. Hypertrophic scarring is a dermal form of fibroproliferative disorders, which occurs in over 70% of burn patients and leads to disfigurement and limitations in function. By understanding the cellular and molecular mechanisms that lead to scarring after injury, new clinical therapeutic approaches can by developed to minimize abnormal scar formation in hypertrophic scarring and other fibroproliferative disorders. To study the expression and localization of SLRPs with connective tissue cells in tissue immunohistochemistry, immunofluorescence staining, immunoblotting, and reverse‐transcription polymerase chain reaction were used in normal skin and hypertrophic scar (HTS). In normal skin, there was more decorin and fibromodulin accumulation in the superficial layers than in the deeper dermal layers. The levels of decorin and fibromodulin were significantly lower in HTS, whereas biglycan was increased when compared with normal skin. There was an increased expression of biglycan, fibromodulin, and lumican in the basement membrane and around basal epithelial cells. In contrast, these proteoglycans were absent or weakly expressed in HTS. The findings suggest that down‐regulation of SLRPs after wound healing in deep injuries to the skin plays an important role in the development of fibrosis and HTS.

Human hypertrophic scar-like nude mouse model: characterization of the molecular and cellular biology of the scar process.

Hypertrophic scar (HTS) following thermal injury and other forms of trauma is a dermal fibroproliferative disorder that leads to considerable morbidity. Because of the lack of an ideal animal model, research is difficult. We have established an HTS model that involves transplanting human split‐thickness skin graft (STSG) or full‐thickness skin graft (FTSG) onto the backs of nude mice. The animals developed raised, firm, and reddish scars 2 months following transplantation. Histology and micromeasurement indicate raised, thickened engrafted skin with STSG and FTSG. In contrast, thickening was not observed with full‐thickness rat skin grafts used as controls. Massons trichrome staining demonstrates increased accumulations of collagen fibrils in the dermis in both scars grafted with STSG and FTSG. Staining cells with toludine blue and an antibody for F4/80 showed an increase in the infiltration of mast cells and macrophages. Quantification of fibrocytes reveals increased fibrocytes. Moreover, STSG grafted skin had significantly more macrophages, mast cells, and fibrocytes than FTSG. Real‐time polymerase chain reaction analysis showed significantly elevated mRNA levels for type I collagen, transforming growth factor‐β, connective tissue growth factor and heat shock protein 47 in both types of engrafted skin. These data demonstrate that human skin grafted onto nude mice develops red raised and thickened scars having intrinsic properties that closely resemble HTS formation as seen in humans. Interestingly, STSG developed more scar than FTSG. Furthermore, inflammatory cells and bone marrow‐derived fibrocytes may play a critical role in HTS development in this animal model.

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in the formation of postburn hypertrophic scar (HTS).

Recent data support the involvement of stromal cell‐derived factor 1 (SDF‐1) in the homing of bone marrow‐derived stem cells to wound sites during skeletal, myocardial, vascular, lung, and skin wound repair as well as some fibrotic disorders via its receptor CXCR4. In this study, the role of SDF‐1/CXCR4 signaling in the formation of hypertrophic scar (HTS) following burn injury and after treatment with systemic interferon α2b (IFNα2b) is investigated. Studies show SDF‐1/CXCR4 signaling was up‐regulated in burn patients, including SDF‐1 level in HTS tissue and serum as well as CD14+CXCR4+ cells in the peripheral blood mononuclear cells. In vitro, dermal fibroblasts constitutively expressed SDF‐1 and deep dermal fibroblasts expressed more SDF‐1 than superficial fibroblasts. Lipopolysaccharide increased SDF‐1 gene expression in fibroblasts. Also, recombinant SDF‐1 and lipopolysaccharide stimulated fibroblast‐conditioned medium up‐regulated peripheral blood mononuclear cell mobility. In the burn patients with HTS who received subcutaneous IFNα2b treatment, increased SDF‐1/CXCR4 signaling was found prior to treatment which was down‐regulated after IFNα2b administration, coincident with enhanced remodeling of their HTS. Our results suggest that SDF‐1/CXCR4 signaling is involved in the development of HTS by promoting migration of activated CD14+CXCR4+ cells from the bloodstream to wound sites, where they may differentiate into fibrocyte and myofibroblasts and contribute to the development of HTS.

Reduced Decorin, Fibromodulin, and Transforming Growth Factor-β3 in Deep Dermis Leads to Hypertrophic Scarring

Hypertrophic scar (HTS) occurs after injuries involving the deep dermis, while superficial wounds (SWs) to the skin heal with minimal or no scarring. The levels of transforming growth factor (TGF)-&bgr;1 and small leucine-rich proteoglycans (SLRPs) with fibroblast subtype and function may influence the development of HTS. The aim of this study was to characterize the expression and localization of factors that regulate wound healing including SLRPs, TGF-&bgr;1, and TGF-&bgr;3 in an experimental human SW and deep wound (DW) scar model including fibroblasts from superficial and deep layers of normal dermis. A 6-cm horizontal dermal scratch experimental wound was created, which consisted of progressively deeper wounds that were superficial at one end (0–0.75 mm deep) and deep (0.75–3 mm deep) at the other end, located on the anterior thigh of an adult male. Immunofluorescence staining, immunoblotting, reverse transcription polymerase chain reaction, and flow cytometry were performed to analyze the cellular and molecular differences between the SW scar and DW scar as well as fibroblasts isolated from superficial layer (L1) and deep layer (L5) of normal dermis. Comparing SWs and L1 fibroblasts, the expression of decorin, fibromodulin, and TGF-&bgr;3 was considerably lower than in DWs and L5 fibroblasts; however, TGF-&bgr;1 was higher in the deeper dermal wounds. When compared with L1 fibroblasts, L5 fibroblasts had lower Thy-1 immunoreactivity and significantly higher expression of TGF-&bgr; receptor type II. Decreased antifibrotic molecules in matrix of deep dermis of the skin and the unique features of the associated fibroblasts including an increased sensitivity to TGF-&bgr;1 stimulation contribute to the development of HTS after injuries involving the deep dermis.

Deep dermal fibroblasts refractory to migration and decorin-induced apoptosis contribute to hypertrophic scarring.

Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders. Fibroblasts from the deep dermis are implicated in the development of HTS after injuries that involve deeper areas of the skin. However, fibroblasts that reside in the superficial layer of the skin show antifibrotic properties, and injuries limited to this area heal with little or no scarring. Previously, cellular and molecular characteristics of superficial fibroblasts and deep dermal fibroblasts that may influence HTS formation were analyzed. In this study, differences in cellular behavior between superficial fibroblasts and deep dermal fibroblasts that may also affect the development of HTS or tissue fibrosis were further characterized. Immunostaining and migration, adhesion, apoptosis, and cell viability assays were performed in fibroblasts from the superficial and deep dermis. Reverse-transcription polymerase chain reaction was used to examine the gene expression of molecules involved in cell death after treatment of fibroblasts with decorin. When compared with superficial fibroblasts, deep dermal fibroblasts showed lower migration rates. Although all the fibroblasts tested showed no difference in adhesion to fibronectin, superficial fibroblasts demonstrated increased apoptotic and dead cells when treated with decorin. Decorin resulted in a significant increase in the expression of apoptosis markers, histone-1, caspase-1, caspase-8, and p53 in superficial fibroblasts when compared with deep dermal fibroblasts. Taken together, the findings suggest that reduced migration, lack of decorin, and resistance of deep dermal fibroblasts to decorin-induced apoptosis may result in hypercellularity in injuries involving the deep dermis, leading to deposition of excess extracellular matrix and HTS formation.

A nude mouse model of hypertrophic scar shows morphologic and histologic characteristics of human hypertrophic scar

Hypertrophic scar (HSc) is a fibroproliferative disorder that occurs following deep dermal injury. Lack of a relevant animal model is one barrier toward better understanding its pathophysiology. Our objective is to demonstrate that grafting split‐thickness human skin onto nude mice results in survival of engrafted human skin and murine scars that are morphologically, histologically, and immunohistochemically consistent with human HSc. Twenty nude mice were xenografted with split‐thickness human skin. Animals were euthanized at 30, 60, 120, and 180 days postoperatively. Eighteen controls were autografted with full‐thickness nude mouse skin and euthanized at 30 and 60 days postoperatively. Scar biopsies were harvested at each time point. Blinded scar assessment was performed using a modified Manchester Scar Scale. Histologic analysis included hematoxylin and eosin, Massons trichrome, toluidine blue, and picrosirius red staining. Immunohistochemistry included anti‐human human leukocyte antigen‐ABC, α‐smooth muscle actin, decorin, and biglycan staining. Xenografted mice developed red, shiny, elevated scars similar to human HSc and supported by blinded scar assessment. Autograft controls appeared morphologically and histologically similar to normal skin. Xenografts survived up to 180 days and showed increased thickness, loss of hair follicles, adnexal structures and rete pegs, hypercellularity, whorled collagen fibers parallel to the surface, myofibroblasts, decreased decorin and increased biglycan expression, and increased mast cell density. Grafting split‐thickness human skin onto nude mice results in persistent scars that show morphologic, histologic, and immunohistochemical consistency with human HSc. Therefore, this model provides a promising technique to study HSc formation and to test novel treatment options.

Novel Methods for the Investigation of Human Hypertrophic Scarring and Other Dermal Fibrosis

Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders that occur after injury involving the deep dermis while superficial wounds to the skin heal with minimal or no scarring. HTS is characterized by progressive deposition of collagen that occurs with high frequency in adult dermal wounds following traumatic or thermal injury. Increased levels of transforming growth factor-β1 (TGF-β1), decreased expression of small leucine-rich proteoglycans (SLRPs), and/or fibroblast subtypes may influence the development of HTS. The development of HTS is strongly influenced by the cellular and molecular properties of fibroblast subtypes, where cytokines such as fibrotic TGF-β1 and CTGF as well as the expression of SLRPs, particularly decorin and fibromodulin, regulate collagen fibrillogenesis and the activity of TGF-β1. Reduced anti-fibrotic molecules in the ECM of the deep dermis and the distinctive behavior of the fibroblasts in this region of the dermis which display increased sensitivity to TGF-β1s biological activity contribute to the development of HTS following injury to the deep dermis. By comparing the cellular and molecular differences involved in deep and superficial wound healing in an experimental wound scratch model in humans that has both superficial and deep injuries within the same excisional model, our aim is to increase our understanding of how tissue repair following injury to the deep dermis can be changed to promote healing with a similar pattern to healing that occurs following superficial injury that results in no or minimal scarring. Studying the characteristics of superficial dermal injuries that heal with minimal scarring will help us identify therapeutic approaches for tissue engineering and wound healing. In addition, our ability to develop novel therapies for HTS is hampered by limitations in the available animal models used to study this disorder in vivo. We also describe a nude mouse model of transplanted human skin that develops a hypertrophic proliferative scar consistent morphologically and histologically with human HTS, which can be used to test novel treatment options for these dermal fibrotic conditions.

Differential expression of class I small leucine-rich proteoglycans in an animal model of partial bladder outlet obstruction.

PURPOSE Partial bladder outlet obstruction has been shown in a rat model to progress from inflammation to hypertrophy to fibrosis. Small leucine-rich proteoglycans are extracellular matrix components associated with collagen fibrillogenesis and resultant scar formation. Two such critical small leucine-rich proteoglycans are decorin and biglycan. We hypothesized that in keeping with other scar models, decorin would be down-regulated and biglycan would be up-regulated with the onset of fibrosis compared to sham. MATERIALS AND METHODS We challenged our hypothesis with female Fisher rats that underwent ligation of the bladder neck or sham surgery. Animals were sacrificed at 4, 8 and 12 weeks, and bladders were harvested. Frozen sections were stained for immunofluorescence for decorin and biglycan. mRNA expression for decorin and biglycan was analyzed using quantitative reverse transcriptase polymerase chain reaction. RESULTS All rats survived to specified experimental end points in good health. Immunofluorescent stains showed progressive down-regulation of decorin and up-regulation of biglycan during the 12-week course by 0.36 and 1.82-fold, respectively (p = 0.02 and p = 0.02), compared to shams. Quantitative real-time reverse transcriptase polymerase chain reaction confirmed these findings in 12-week specimens, showing a down-regulation of decorin by a factor of 0.45 (p = 0.02) and up-regulation of biglycan by a factor of 2.04-fold (p = 0.08). CONCLUSIONS We present the first identification to our knowledge of small leucine-rich proteoglycans in normal and abnormal bladder tissue, and their differential expression in the process of bladder fibrosis, consistent with experimental findings in other anatomical sites. Further investigation into small leucine-rich proteoglycan expression and regulation may allow for the development of new antifibrotic therapeutics.

Morphologic and Histologic Comparison of Hypertrophic Scar in Nude Mice, T-Cell Receptor, and Recombination Activating Gene Knockout Mice.

Background: Proliferative scars in nude mice have demonstrated morphologic and histologic similarities to human hypertrophic scar. Gene knockout technology provides the opportunity to study the effect of deleting immune cells in various disease processes. The authors’ objective was to test whether grafting human skin onto T-cell receptor (TCR) &agr;&bgr;-/-&ggr;&dgr;-/-, recombination activating gene (RAG)-1-/-, and RAG-2-/-&ggr;c-/- mice results in proliferative scars consistent with human hypertrophic scar and to characterize the morphologic, histologic, and cellular changes that occur after removing immune cells. Methods: Nude TCR&agr;&bgr;-/-&ggr;&dgr;-/-, RAG-1-/-, and RAG-2-/-&ggr;c-/- mice (n = 20 per strain) were grafted with human skin and euthanized at 30, 60, 120, and 180 days. Controls (n = 5 per strain) were autografted with mouse skin. Scars and normal skin were harvested at each time point. Sections were stained with hematoxylin and eosin, Masson’s trichrome, and immunohistochemistry for anti-human leukocyte antigen-ABC, &agr;-smooth muscle actin, decorin, and biglycan. Results: TCR&agr;&bgr;-/-&ggr;&dgr;-/-, RAG-1-/-, and RAG-2-/-&ggr;c-/- mice grafted with human skin developed firm, elevated scars with histologic and immunohistochemical similarities to human hypertrophic scar. Autografted controls showed no evidence of pathologic scarring. Knockout animals demonstrated a capacity for scar remodeling not observed in nude mice where reductions in &agr;-smooth muscle actin staining pattern and scar thickness occurred over time. Conclusions: Human skin transplanted onto TCR&agr;&bgr;-/-&ggr;&dgr;-/-, RAG-1-/-, and RAG-2-/-&ggr;c-/- mice results in proliferative scars with morphologic and histologic features of human hypertrophic scar. Remodeling of proliferative scars generated in knockout animals is analogous to changes in human hypertrophic scar. These animal models may better represent the natural history of human hypertrophic scar.