Peter Kwan

Toll-like receptors expressed by dermal fibroblasts contribute to hypertrophic scarring.

Hypertrophic scar (HTS), a fibroproliferative disorder (FPD), complicates burn wound healing. Although the pathogenesis is not understood, prolonged inflammation is a known contributing factor. Emerging evidence suggests that fibroblasts regulate immune/inflammatory responses through toll‐like receptor 4 (TLR4) activated by lipopolysaccharide (LPS) through adaptor molecules, leading to nuclear factor kappa‐light‐chain‐enhancer of activated B cells and mitogen‐activated protein kinases activation, cytokine gene transcription and co‐stimulatory molecule expression resulting in inflammation. This study explored the possible role of TLR4 in HTS formation. Paired normal and HTS tissue from burn patients was collected and dermal fibroblasts isolated and cultured. Immunohistochemical analysis of tissues demonstrated increased TLR4 staining in HTS tissue. Quantitative RT‐PCR of three pairs of fibroblasts demonstrated mRNA levels for TLR4 and its legend myeloid differentiation factor 88 (MyD88) in HTS fibroblasts were increased significantly compared with normal fibroblasts. Flow cytometry showed increased TLR4 expression in HTS fibroblasts compared with normal. ELISA demonstrated protein levels for prostaglandin E2, interleukin (IL)‐6, IL‐8 and monocyte chemotactic protein‐1 (MCP‐1) were significantly increased in HTS fibroblasts compared to normal. When paired normal and HTS fibroblasts were stimulated with LPS, significant increases in mRNA and protein levels for MyD88, IL‐6, IL‐8, and MCP‐1 were detected. However, when transfected with MyD88 small interfering RNA (siRNA), then stimulated with LPS, a significant decrease in mRNA and protein levels for these molecules compared to only LPS‐stimulated fibroblasts was detected. In comparison, a scramble siRNA transfection did not affect mRNA or protein levels for these molecules. Results demonstrate LPS stimulates proinflammatory cytokine expression in dermal fibroblasts and MyD88 siRNA eliminates the expression. Therefore, controlling inflammation and manipulating TLR signaling in skin cells may result in novel treatment strategies for HTS and other FPD. J. Cell. Physiol. 226: 1265–1273, 2011.

Scar and Contracture: Biological Principles

Dysregulated wound healing and pathologic fibrosis cause abnormal scarring, leading to poor functional and aesthetic results in hand burns. Understanding the underlying biologic mechanisms involved allows the hand surgeon to better address these issues, and suggests new avenues of research to improve patient outcomes. In this article, the authors review the biology of scar and contracture by focusing on potential causes of abnormal wound healing, including depth of injury, cytokines, cells, the immune system, and extracellular matrix, and explore therapeutic measures designed to target the various biologic causes of poor scar.

MicroRNA 181b regulates decorin production by dermal fibroblasts and may be a potential therapy for hypertrophic scar.

Hypertrophic scarring is a frequent fibroproliferative complication following deep dermal burns leading to impaired function and lifelong disfigurement. Decorin reduces fibrosis and induces regeneration in many tissues, and is significantly downregulated in hypertrophic scar and normal deep dermal fibroblasts. It was hypothesized that microRNAs in these fibroblasts downregulate decorin and blocking them would increase decorin and may prevent hypertrophic scarring. Lower decorin levels were found in hypertrophic scar as compared to normal skin, and in deep as compared to superficial dermis. A decorin 3’ un-translated region reporter assay demonstrated microRNA decreased decorin in deep dermal fibroblasts, and microRNA screening predicted miR- 24, 181b, 421, 526b, or 543 as candidates. After finding increased levels of mir-181b in deep dermal fibroblasts, it was demonstrated that TGF-β1 stimulation decreased miR-24 but increased miR-181b and that hypertrophic scar and deep dermis contained increased levels of miR-181b. By blocking miR-181b with an antagomiR, it was possible to increase decorin protein expression in dermal fibroblasts. This suggests miR-181b is involved in the differential expression of decorin in skin and wound healing. Furthermore, blocking miR-181b reversed TGF-β1 induced decorin downregulation and myofibroblast differentiation in hypertrophic scar fibroblasts, suggesting a potential therapy for hypertrophic scar.

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.

The therapeutic potential of a C‐X‐C chemokine receptor type 4 (CXCR‐4) antagonist on hypertrophic scarring in vivo

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell‐derived factor 1/CXCR4 signaling is up‐regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood‐borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell‐derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE‐9908 on dermal fibrosis was determined in vivo using a human HTS‐like nude mouse model, in which split‐thickness human skin is transplanted into full‐thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE‐9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down‐regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.

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.

Biological Principles of Scar and Contracture

Hypertrophic scar and contracture in burn patients is a complex process. Contributing factors include critical injury depth and activation of key cell subpopulations, including deep dermal fibroblasts, myofibroblasts, fibrocytes, and T-helper cells, which cause scarring rather than regeneration. These cells influence each other via cellular profibrotic and antifibrotic signals, which help to determine the outcome. These cells also both modify and interact with extracellular matrix of the wound, ultimately forming hypertrophic scar. Current treatments reduce hypertrophic scar formation or improve remodeling by targeting these pathways and signals.

Serum Decorin, Interleukin-1β, and Transforming Growth Factor-β Predict Hypertrophic Scarring Postburn

Hypertrophic scar after burn injury is a significant problem. Previous studies have examined the roles for decorin, interleukin-1&bgr;, and transforming growth factor-&bgr;1 in hypertrophic scar formation locally, but few have considered their systemic influence. The authors conducted a pilot study to examine whether serum levels of these molecules could predict hypertrophic scar formation. Serum levels were measured using enzyme-linked immunosorbent assay, and hypertrophic scar formation determined from chart reviews. Peripheral blood mononuclear cells and fibroblasts were stimulated with decorin, interleukin-1&bgr;, and transforming growth factor-&bgr;1, and expression of profibrotic molecules examined using flow cytometry, immunofluorescence microscopy, quantitative polymerase chain reaction, and mass spectrometry. Multiple linear regression analysis suggested early serum levels of decorin and interleukin-1&bgr;, and late serum levels of transforming growth factor-&bgr;1 were predictive of hypertrophic scar formation. Decorin up-regulated the expression of toll-like receptor 4 and C-X-C receptor 4 in peripheral blood mononuclear cells, and interleukin-1&bgr; up-regulated fibroblast production of C-X-C ligand 12. Transforming growth factor-&bgr;1 up-regulated, and interleukin-1&bgr; down-regulated, the production of profibrotic cytokines, collagen, and myofibroblast differentiation. The model predicting hypertrophic scar formation is supported by clinical results and limited in vitro experiments.

Synergistic effect of vitamin D and low concentration of transforming growth factor beta 1, a potential role in dermal wound healing

Dermal wound healing, in which transforming growth factor beta 1 (TGFβ1) plays an important role, is a complex process. Previous studies suggest that vitamin D has a potential regulatory role in TGFβ1 induced activation in bone formation, and there is cross-talk between their signaling pathways, but research on their effects in other types of wound healing is limited. The authors therefore wanted to explore the role of vitamin D and its interaction with low concentration of TGFβ1 in dermal fibroblast-mediated wound healing through an in vitro study. Human dermal fibroblasts were treated with vitamin D, TGFβ1, both, or vehicle, and then the wound healing functions of dermal fibroblasts were measured. To further explore possible mechanisms explaining the synergistic effect of vitamin D and TGFβ1, targeted gene silencing of the vitamin D receptor was performed. Compared to either factor alone, treatment of fibroblasts with both vitamin D and low concentration of TGFβ1 increased gene expression of TGFβ1, connective tissue growth factor, and fibronectin 1, and enhanced fibroblast migration, myofibroblast formation, and collagen production. Vitamin D receptor gene silencing blocked this synergistic effect of vitamin D and TGFβ1 on both collagen production and myofibroblast differentiation. Thus a synergistic effect of vitamin D and low TGFβ1 concentration was found in dermal fibroblast-mediated wound healing in vitro. This study suggests that supplementation of vitamin D may be an important step to improve wound healing and regeneration in patients with a vitamin D deficiency.

Subcutaneous white adipocytes express a light sensitive signaling pathway mediated via a melanopsin/TRPC channel axis

Subcutaneous white adipose tissue (scWAT) is the major fat depot in humans and is a central player in regulating whole body metabolism. Skin exposure to UV wavelengths from sunlight is required for Vitamin D synthesis and pigmentation, although it is plausible that longer visible wavelengths that penetrate the skin may regulate scWAT function. In this regard, we discovered a novel blue light-sensitive current in human scWAT that is mediated by melanopsin coupled to transient receptor potential canonical cation channels. This pathway is activated at physiological intensities of light that penetrate the skin on a sunny day. Daily exposure of differentiated adipocytes to blue light resulted in decreased lipid droplet size, increased basal lipolytic rate and alterations in adiponectin and leptin secretion. Our results suggest that scWAT function may be directly under the influence of ambient sunlight exposure and may have important implications for our current understanding of adipocyte biology. (150 words)