Tai-Lan Tuan

The molecular basis of keloid and hypertrophic scar formation

Excess scar formation secondary to traumatic or surgical injuries can have devastating consequences, ranging from body disfigurement to organ dysfunction. Hypertrophic scars and keloids are skin fibrotic conditions that can be caused by minor insults to skin, such as acne or ear piercing, or by severe injuries such as burns. Differences between keloids, hypertrophic scars and normal scars include distinct scar appearance, histologic morphology and cellular function in response to growth factors. Recent advances in our understanding of the wound healing process reveal possible causes for hypertrophic scars and keloids. This information might assist in the development of efficacious treatment for hypertrophic scar and keloid formation.

Modulation of Collagen Synthesis by Transforming Growth Factor-β in Keloid and Hypertrophic Scar Fibroblasts

Keloid and hypertrophic scars are fibrous growths characterized by overabundant collagen deposition. We examined the effect of transforming growth factor-β (TGF-β), a known stimulant for the production of connective tissue matrices, on the rate of collagen synthesis in keloid fibroblasts (KFs), hypertrophic scar fibroblasts (HSFs), and normal skin fibroblasts (NSFs). Fibroblasts were cultured in three-dimensional fibrin-gel matrices in the presence or absence of TGF-β (5 ng/ml) or anti–TGF-β neutralizing antibody (50 μ/ml). Secreted collagen levels, labeled with 3H-proline, were measured after 48 hours. KFs produced up to 12 times more collagen than NSFs, and up to 4 times more than HSFs. Although KFs increased their rate of collagen production by up to 2.7 times in response to TGF-β, HSFs and NSFs did not (p = 0.065). Anti–TGF-β antibody reduced the rate of collagen synthesis of KFs by 40% (p = 0.003), although it did not suppress collagen production in HSFs (p = 0.06) and NSFs (p = 0.75). We conclude that although KFs and HSFs are similar in that they both overproduce collagen, they are different in that only KFs display a marked sensitivity to TGF-β, which is abundant during the proliferative phase of wound healing.

PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts

A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explant-derived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfrβ, are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rho-associated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish.

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor‐1 and gelatinase‐B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor‐1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor‐ bound urokinase plasminogen activator–plasminogen activator inhibitor‐1 complex (≅ 80–116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor‐1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator–plasminogen activator inhibitor‐1 complex in blood‐ and plasma‐derived serum and mastectomy wound fluid. Plasminogen activator inhibitor‐1 was detected in blood‐derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase‐9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase‐9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood‐derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme‐ inhibitor complex, the level of expression of matrix metalloproteinase‐9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase‐9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase‐9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase‐9 appear to be useful biomarkers to determine clinical wound healing status.

Plasminogen activator/plasmin system: A major player in wound healing?

The role of the plasminogen activator/plasmin system in fibrinolysis has been well established. Indeed, clinicians worldwide have successfully utilized recombinant tissue‐type plasminogen activator as first‐line treatment of acute myocardial infarction for almost 2 decades. Outside the field of cardiology, there has been increasing excitement regarding the possible contribution of this system in many other important biological processes, including cell adhesion, cell migration, cell–cell signaling, tumor invasion and metastasis, ovulation, and wound healing. In this review, we present evidence in the current literature that the plasminogen activator/plasmin system does have a role in wound healing, looking at both normal and abnormal healing. Furthermore, the invaluable insights provided by numerous transgenic animal experiments are summarized. (WOUND REP REG 2003;11:239–247)

Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures

Proteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1. In contrast, keloid fibroblasts expressed a persistently high level of PAI-1 and a low level of uPA. The high PAI-1 activity of keloid fibroblasts correlated with their elevated collagen accumulation in fibrin gel cultures. Substituting collagen for fibrin in the gel matrix resulted in increased uPA activity and reduced collagen accumulation of keloid fibroblasts. Furthermore, decreasing PAI-1 activity of keloid fibroblasts in fibrin gel cultures with anti-PAI-1-neutralizing antibodies also resulted in a reduction in collagen accumulation by keloid fibroblasts. Cumulatively, these results suggest that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo, and PAI-1 may play a causative role in elevated collagen accumulation of keloid fibroblasts.

Regeneration of fat cells from myofibroblasts during wound healing

Hair follicles: Secret to prevent scars? Although some animals easily regenerate limbs and heal broken flesh, mammals are generally not so gifted. Wounding can leave scars, which are characterized by a lack of hair follicles and cutaneous fat. Plikus et al. now show that hair follicles in both mice and humans can convert myofibroblasts, the predominant dermal cell in a wound, into adipocytes (see the Perspective by Chan and Longaker). The hair follicles activated the bone morphogenetic protein (BMP) signaling pathway and adipocyte transcription factors in the myofibroblast. Thus, it may be possible to reduce scar formation after wounding by adding BMP. Science, this issue p. 748; see also p. 693 Hair follicles convert wound myofibroblasts to adipocytes through the bone morphogenetic protein signaling pathway, revealing a target to decrease scarring. Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.

Role of growth factors in scar contraction: an in vitro analysis.

Excessive scar contracture by wound fibroblasts can have devastating consequences, ranging from body disfigurement to joint immobility. The ability of fibroblasts isolated from lesions of hypertrophic scars, keloids, normal skin, or normal scars in contracting the provisional wound matrix (i.e., fibrin clot) was compared and analyzed. Hypertrophic scar fibroblasts showed a consistently higher basal level of fibrin matrix gel (FMG) contraction than other fibroblasts. This heightened basal level of contractility may be attributed partially to the autocrine effect of transforming growth factor-beta1 (TGF-β1). Normal and keloid fibroblasts exhibited similar basal rates of FMG contraction, and both responded to platelet-derived growth factor (PDGF) and TGF-β by increasing FMG contraction two- to threefold. However, 45% of the TGF-β-induced increase in FMG contraction by keloid fibroblasts, but not normal fibroblasts, was mediated by the autocrine production of PDGF. Therefore, fibroblasts isolated from different scars exhibit varied degrees of FMG contraction. In addition, the mechanism underlying growth factor-mediated contraction differed vastly among fibroblasts of different scar origin. The significance of these differences in growth factor-mediated FMG contraction is discussed.

Activation of NFκB signal pathways in keloid fibroblasts

Keloids are characterized as an “over-exuberant” healing response resulting in a disproportionate extracellular matrix (ECM) accumulation and tissue fibrosis. In view of the integral role of inflammation and cytokines in the healing response, it is logical to assume that they may play a part in orchestrating the pathology of this “abnormal” healing process. Tumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine involved in activation of signaling events and transcriptional programs, such as NFκB. This study attempts to determine the difference in NFκB and its related genes expression and DNA binding activity between keloid and normal skin fibroblasts. Three keloid and normal skin tissues (NSk) and their derived fibroblasts were used to determine NFκB signaling pathway expression using specific cDNA microarrays, Western blot analysis and immunohistochemistry. Electrophoretic mobility gel shift assay (EMSA) was used to assess NFκB-binding activity, all assays were performed in the presence and absence of TNF-α. TNF-α up-regulated 15% of NFκB signal pathway related genes in keloid fibroblast compared to normal skin. At the protein level, keloid fibroblasts and tissues showed higher basal levels of TNF- receptor–associated factors—TRAF1, TRAF2—TNF-α, inhibitor of apoptosis (c-IAP-1), and NFκB, compared with NSk. Keloid fibroblasts showed a constitutive increase in NFκB-binding activity in comparison to NSk both with and without TNF-α treatment. NFκB and its targeted genes, especially the antiapoptotic genes, could play a role in keloid pathogenesis; targeting NFκB could help in developing therapeutic interventions for the treatment of keloid scarring.

Contractility, Transforming Growth Factor-|[beta]|, and Plasmin in Fetal Skin Fibroblasts: Role in Scarless Wound Healing

The early fetus responds to cutaneous wounds in a fundamentally different way from the adult; fetal wounds heal without scars. Wound contraction is a vital component of wound healing. The cytokine transforming growth factor(TGF)-β promotes wound contraction and can be activated by the serine protease plasmin. Herein, we explored whether murine skin fibroblast contractile properties, TGF-β, and plasmin formation are developmentally regulated. Our results showed that early fetal mouse embryonic day 15 skin fibroblasts contracted a collagen gel less, secreted less active and total TGF-β, and generated less plasmin than either late fetal (embryonic day 17) or adult skin fibroblasts. Furthermore, there was a slight positive correlation between the formation of plasmin and the level of activation of TGF-β. We conclude that early fetal mouse skin fibroblasts contract a collagen gel and secrete and activate TGF-β to a lesser extent than do late fetal and adult skin fibroblasts. We speculate that the fetal skin fibroblast undergoes a developmental transition that causes wounds in mouse to contract at or after embryonic day 17. Further, this developmental transition is influenced by growth factor-fibroblast interactions and coincides with the emergence of the skin fibroblasts ability to generate plasmin and activate TGF-β.