The Anti-Scar Effects of Basic Fibroblast Growth Factor on the Wound Repair In Vitro and In Vivo

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.     

Fetal wound healing: a biochemical study of scarless healing

Human fetal surgery is being successfully performed today in a small number of highly selected patients for conditions that may lead to irreversible damage to the fetus and threaten the viability of the newborn. Following surgical repair, fetal wounds heal without scarring. This study was initiated to characterize fetal wounds both histologically and biochemically. Gore-Tex tubing was implanted into the subcutaneous tissue of the back of fetal, newborn, and adult New Zealand white rabbits. Light microscopic examination of healed wounds revealed no evidence of scar formation. Electron microscopy demonstrated a striated fibrillar structure suggestive of collagen within the lumen of the Gore-Tex tubing implants. Amino acid analysis (sensitivity 40 pmol) confirmed the presence of hydroxylysine and hydroxyproline within the Gore-Tex wound chambers indicating the presence of collagen in fetal wounds. The small amount of collagen precluded the typing of the collagen using cyanogen bromide peptide analysis. The absence of scarring and the small amounts of detectable collagen suggest a high degree of reorganization of the connective tissues involved in repair. The fetal wound matrix is rich in hyaluronic acid. Topical hyaluronic acid has been associated experimentally with a reduced amount of scarring in postnatal wound healing. Hyaluronic acid extracted from human skin and scar tissue is associated with collagen and other proteins. We propose that a hyaluronic acid-collagen-protein complex may play a role in fetal wound healing.     

In vitro wounding: effects of hypoxia and transforming growth factor β<Subscript>1</Subscript> on proliferation,migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model

The adequate reconstitution of human soft tissue wounds requires the coordinated interaction of endothelial cells and fibroblasts during the proliferation phase of healing. Endothelial cells assure neoangiogenesis, fibroblasts fill the defect and provide extracellular matrix proteins, and myofibroblasts are believed to support the reconstitution of microvessels. In the present study, we combined in vitro-wound size measurement and multicolour immunocytochemical staining of co-cultured human dermal microvascular endothelial cells and normal human dermal fibroblasts, recently introduced as co-culture scratch-wound migration assay. Applying antibodies for α-smooth-muscle actin, von Willebrand factor, extra domain A fibronectin and endothelin-1, we were able to monitor proliferation, migration and the differentiation process from fibroblasts to myofibroblasts as a response to hypoxia. Furthermore, we verified, whether transforming growth factor β₁ (TGFβ₁) and endothelin-1 are able to mediate this response. We show, that proliferation and migration of endothelial cells and fibroblasts decreased under hypoxia. The additional administration of TGFβ₁ did not significantly attenuate this decrease. Solely the myofibroblast population in co-culture adapted well to hypoxia, when cultures were supplemented with TGFβ₁. Considerating the data concerning TGFβ₁ and endothelin-1, we propose a model explaining the cellular interaction during early and late proliferation phase of human wound healing.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

Mutual inter-regulation between iNOS and TGF-β1: Possible molecular and cellular mechanisms of iNOS in wound healing

Abnormal wound healing with excessive scarring is a major health problem with socioeconomic and psychological impacts. In human, chronic wounds and scarring are associated with upregulation of the inducible nitric oxide synthase (iNOS). Recently, we have shown physiological regulation of iNOS in wound healing. Here, we sought to investigate the possible mechanistic role of iNOS in wound healing using biochemical and immunohistochemical assays. We found: (a) iNOS is the main source of wound nitric oxide (NO), (b) NOS inhibition in the wound, downregulated iNOS protein, mRNA and enzymatic activity, and reduced wound NO, and (c) iNOS inhibition resulted in delayed healing at early time points, and excessive scarring at late time points. Furthermore, molecular and cellular analysis of the wound showed that iNOS inhibition significantly (P < 0.05) increased TGF-β1 mRNA and protein levels, fibroblasts and collagen deposition. These latter findings suggest that iNOS might be exerting its action in the wound by signaling through TGF-β1 that activates wound fibroblasts to produce excessive collagen. Our current findings provide further support that iNOS is crucial for physiological wound healing, and suggest that dysregulation of iNOS during the inflammatory phase impairs healing, and results in disfiguring post-healing scarring. Thus, the mutual feedback regulation between iNOS and TGF-β1 at the gene, protein and functional levels might be the mechanism through which iNOS regulates the healing. Monitoring and maintenance of wound NO levels might be important for healing and avoiding long-term complications in susceptible people including patients with diabetic wounds, venous ulcers or keloid prone.     

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.     

Immunohistochemical localization of growth factors in fetal wound healing

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.     

Collagen-binding peptidoglycans inhibit MMP mediated collagen degradation and reduce dermal scarring

Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing.     

Growth modulation of fibroblasts by chitosan-polyvinyl pyrrolidone hydrogel: Implications for wound management?

Wounds in adults and fetuses differ in their healing ability with respect to scar formation. In adults, wounds lacking the epidermis exhibit excess collagen production and scar formation. Fibroblasts synthesize and deposit a collagen rich extracellular matrix. The early migration and proliferation of fibroblasts in the wound area is implicated in wound scarring. We have synthesized a hydrogel from chitosan-polyvinyl pyrrolidone (PVP) and examined its effect on fibroblast growth modulation in vitro. The hydrogel was found to be hydrophilic as seen from its octane contact angle (141.2+/-0.37 degrees). The hydrogel was non-toxic and biocompatible with fibroblasts and epithelial cells as confirmed by the 3(4,5-dimethylthiazolyl-2)-2, 5-diphenyl tetrazolium bromide (MTT) as-say. It showed dual properties by supporting growth of epithelial cells (SiHa) and selectively inhibiting fibro-blast (NIH3T3) growth. Growth inhibition of fibroblasts resulted from their inability to attach on to the hydrogel. These findings are supported by image analysis, which revealed a significant difference (P<0.05) between the number of fibroblasts attached to the hydrogel in tissue culture as compared to tissue culture treated polystyrene (TCPS) controls. However, no significant difference was observed (P>0.05) in the number of epithelial (SiHa) cells attached on to the hydrogel as compared to the TCPS control. Although in vivo experiments are awaited, these findings point to the possible use of chitosan-PVP hydrogels in wound-management.     

The effect of myofibroblast on contracture of hypertrophic scar

Wound contraction in humans has both positive and negative effects. It is beneficial to wound healing by narrowing the wound margins, but the formation of undesirable scar contracture brings cosmetic and even functional problems. The entire mechanism of wound healing and scar contracture is not clear yet, but it is at least considered that both the fibroblasts and the myofibroblasts are responsible for contraction in healing wounds. The myofibroblast is a cell that possesses all the morphologic and biochemical characteristics of both a fibroblast and a smooth muscle cell. Normally, the myofibroblasts appear in the initial wound healing processes and generate contractile forces to pull both edges of an open wound until it disappears by apoptosis. But as an altered regulation of myofibroblast disappearance, they remain in the dermis and continuously contract the scar, eventually causing scar contracture. In this research, to compare and directly evaluate the influence on scar contracture of the myofibroblast versus the fibroblast, dermal tissues were taken from 10 patients who had highly contracted hypertrophic scars. The myofibroblasts were isolated and concentrated from the fibroblasts using the magnetic activating cell-sorting column to obtain the myofibroblast group, which contained about 28 to 41 percent of the myofibroblasts, and the fibroblast group, which contained less than 0.9 percent of the myofibroblasts. Each group was cultured in the fibroblast-populated collagen lattice for 13 days, and the contraction of the collagen gel was measured every other day. In addition, they were selectively treated with tranilast [N-(3',4'-dimethoxycinnamoyl) anthranilic acid] to evaluate the influence on the contraction of the collagen gel lattice. During the culture, the myofibroblast group, compared with the fibroblast group, showed statistically significant contraction of the collagen gel lattice day by day, except on the first day, and only the myofibroblast group was affected by tranilast treatment, showing significant inhibition of gel contraction. By utilizing an in vitro model, the authors have demonstrated that myofibroblasts play a more important role in the contracture of the hypertrophic scar.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Mechanical Boundary Conditions Bias Fibroblast Invasion in a Collagen-Fibrin Wound Model

Because fibroblasts deposit the collagen matrix that determines the mechanical integrity of scar tissue, altering fibroblast invasion could alter wound healing outcomes. Anisotropic mechanical boundary conditions (restraint, stretch, or tension) could affect the rate of fibroblast invasion, but their importance relative to the prototypical drivers of fibroblast infiltration during wound healing—cell and chemokine concentration gradients—is unknown. We tested whether anisotropic mechanical boundary conditions affected the directionality and speed of fibroblasts migrating into a three-dimensional model wound, which could simultaneously expose fibroblasts to mechanical, structural, steric, and chemical guidance cues. We created fibrin-filled slits in fibroblast-populated collagen gels and applied uniaxial mechanical restraint along the short or long axis of the fibrin wounds. Anisotropic mechanical conditions increased the efficiency of fibroblast invasion by guiding fibroblasts without increasing their migration speed. The migration behavior could be modeled as a biased random walk, where the bias due to multiple guidance cues was accounted for in the shape of a displacement orientation probability distribution. Taken together, modeling and experiments suggested an effect of strain anisotropy, rather than strain-induced fiber alignment, on fibroblast invasion.     

Mechanical Boundary Conditions Bias Fibroblast Invasion in a Collagen-Fibrin Wound Model

Because fibroblasts deposit the collagen matrix that determines the mechanical integrity of scar tissue, altering fibroblast invasion could alter wound healing outcomes. Anisotropic mechanical boundary conditions (restraint, stretch, or tension) could affect the rate of fibroblast invasion, but their importance relative to the prototypical drivers of fibroblast infiltration during wound healing—cell and chemokine concentration gradients—is unknown. We tested whether anisotropic mechanical boundary conditions affected the directionality and speed of fibroblasts migrating into a three-dimensional model wound, which could simultaneously expose fibroblasts to mechanical, structural, steric, and chemical guidance cues. We created fibrin-filled slits in fibroblast-populated collagen gels and applied uniaxial mechanical restraint along the short or long axis of the fibrin wounds. Anisotropic mechanical conditions increased the efficiency of fibroblast invasion by guiding fibroblasts without increasing their migration speed. The migration behavior could be modeled as a biased random walk, where the bias due to multiple guidance cues was accounted for in the shape of a displacement orientation probability distribution. Taken together, modeling and experiments suggested an effect of strain anisotropy, rather than strain-induced fiber alignment, on fibroblast invasion.     

Induction of tenascin in healing wounds

The distribution of the extracellular matrix glycoprotein, tenascin, in normal skin and healing skin wounds in rats, has been investigated by immunohistochemistry. In normal skin, tenascin was sparsely distributed, predominantly in association with basement membranes. In wounds, there was a marked increase in the expression of tenascin at the wound edge in all levels of the skin. There was also particularly strong tenascin staining at the dermal-epidermal junction beneath migrating, proliferating epidermis. Tenascin was present throughout the matrix of the granulation tissue, which filled full-thickness wounds, but was not detectable in the scar after wound contraction was complete. The distribution of tenascin was spatially and temporally different from that of fibronectin, and tenascin appeared before laminin beneath migrating epidermis. Tenascin was not entirely codistributed with myofibroblasts, the contractile wound fibroblasts. In EM studies of wounds, tenascin was localized in the basal lamina at the dermal-epidermal junction, as well as in the extracellular matrix of the adjacent dermal stroma, where it was either distributed homogeneously or bound to the surface of collagen fibers. In cultured skin explants, in which epidermis migrated over the cut edge of the dermis, tenascin, but not fibronectin, appeared in the dermis underlying the migrating epithelium. This demonstrates that migrating, proliferating epidermis induces the production of tenascin. The results presented here suggest that tenascin is important in wound healing and is subject to quite different regulatory mechanisms than is fibronectin.     

pGlcNAc Nanofiber Treatment of Cutaneous Wounds Stimulate Increased Tensile Strength and Reduced Scarring via Activation of Akt1

Treatment of cutaneous wounds with poly-N-acetyl-glucosamine containing nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increased wound closure, antibacterial activities and innate immune responses. We have shown that Akt1 plays a central role in the regulation of these activities. Here, we show that pGlcNAc treatment of cutaneous wounds results in a smaller scar that has increased tensile strength and elasticity. pGlcNAc treated wounds exhibit decreased collagen content, increased collagen organization and decreased myofibroblast content. A fibrin gel assay was used to assess the regulation of fibroblast alignment in vitro. In this assay, fibrin lattice is formed with two pins that provide focal points upon which the gel can exert force as the cells align from pole to pole. pGlcNAc stimulation of embedded fibroblasts results in cellular alignment as compared to untreated controls, by a process that is Akt1 dependent. We show that Akt1 is required in vivo for the pGlcNAc-induced increased tensile strength and elasticity. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in the proper alignment of fibroblasts, decreased scarring, and increased tensile strength during cutaneous wound healing.     

Mathematical modelling of nitric oxide activity in wound healing can explain keloid and hypertrophic scarring

Keloid and hypertrophic lesions are both types of scarring pathologies which arise as a consequence of excess collagen deposition during the wound healing process. The exact mechanism by which this occurs is not understood and currently no effective treatment exists. In this paper, we study the possible role of nitric oxide in excess scar formation. In recent years, the physiological role of this free radical in mammalian tissue has been extensively studied; in particular numerous groups have studied its role in wound healing. We describe a mathematical model which offers a possible explanation for keloid scarring in terms of the presence of higher than normal nitric oxide concentrations related to the fact that nitric oxide stimulates synthesis of collagen by fibroblasts. As a consequence of this, we put forward a suggestion for a treatment strategy involving the surgical excision of the keloid lesion combined with the application of a low-dose nitric oxide inhibitor. In addition, we show that a quasi-steady-state analysis of our model reveals a possible approach to distinguishing between hypertrophic and keloid lesions, a task which has to date proven to be clinically difficult. We also present an extended model which confirms these results in the context of a more complicated and biologically more realistic system. The fuller model demonstrates additional features of keloid and hypertrophic scarring which we were not able to consider in the basic model, and as a consequence further supports our hypothesis that nitric oxide activity could play a key role in keloid scarring.     

Elevated CCN2 expression in scleroderma: a putative role for the TGFβ accessory receptors TGFβRIII and endoglin

The ability of TGFβ1 to act as a potent pro-fibrotic mediator is well established, potently inducing the expression of fibrogenic genes including type I collagen (COL1A2) and CCN2. Previously we have shown elevated expression of the TGFβ accessory receptor, endoglin on Systemic Sclerosis (SSc) dermal fibroblasts. Here we sought to assess the cell surface expression of the TGFβ receptor complex on SSc dermal fibroblasts (SDF), and investigate their role in maintaining the elevated expression of CCN2. SDF exhibited elevated expression of the TGFβ accessory receptors betaglycan/TGFβRIII and endoglin, but not type I or type II receptors. To determine the effect of altered receptor repertoire on TGFβ responses, we investigated the effect of exogenous TGFβ on expression of two pro-fibrotic genes. SDF exhibited higher basal expression of COL1A2 and CCN2 compared to healthy controls. TGFβ induced a marked increase in the expression of these genes in normal dermal fibroblasts, whereas SDF exhibited only a modest increase. We next sought to determine if higher basal expression in SDF was a result of autocrine expression of TGFβ. Surprisingly basal expression was not affected by a pan-neutralizing TGFβ antibody. To explore if altered accessory receptor expression alone could account for these changes, we determined their effects on CCN2 promoter activity. Endoglin inhibited CCN2 promoter activity in response to TGFβ. TGFβRIII alone or in combination with endoglin was sufficient to enhance basal CCN2 promoter activity. Thus TGFβ accessory receptors may play a significant role in the altered expression of fibrogenic genes in SDF.