Collagen degradation in cultured keloid and hypertrophic scar tissue

SUMMARY In order to study collagen catabolism of normal human skin, keloid and hypertrophic scars, explants of these tissues have been cultured for periods of up to 10 days. All three tissues released a similar amount of neutral collagenase activity into the culture medium with maximal yield between days 3 and 7, and the collagenase from scar tissues was found to be identical to the normal skin enzyme with regard to its inhibition by EDTA, cysteine and human serum. The major site of collagenase production in keloid specimens appeared to be, as in normal skin, the upper dermal or epidermal layer, with minimal production occurring in the lower fibrous or nodular areas. Prior to culture the collagen content of each tissue was found to be similar, with approximately 1%, of the total being acid-soluble. During the culture period considerable amounts of insoluble tissue collagen were degraded in all three tissues, as judged by the release of hydroxyproline into the culture medium. These results suggest that the persistence of keloids and hypertrophic scars is attributable neither to an inability of the tissues to produce an active collagenase molecule nor to any resistance of the tissue collagen to degradation.     

Reduced collagenase gene expression in fibroblasts from hypertrophic scar tissue

Summary The major histopathological feature of hypertrophic scar lesions is fibrosis. characterized by excessive accumulation of collagen. The purpose of this study was to determine if there is not only increased expression of collagen but also decreased expression of collagenase in hypertrophic scar fibroblasts. We compared the expression of mRNA for of α₁ (I) and α₁ (III) collagen, and collagenase in cultured fibroblasis from different portions of hypertrophic scars and normal dermis. the hypertrophic scar fibroblasts. increased levels of α₁ (I) and α₁ (III) collagen mRNAs were observed in fibroblasts from the edge and outside of scar tissue, while normal levels were noted in fibroblasts from the centre of this tissue. In contrast. decreased levels of collagenase mRNA were found in the hypertrophic scar fibroblasts. The reductions were centre (25% of the control) greater than the edge (43% of the control) greater than the outside (84% of the control). The changes in the collagenase mRNA levels of the hypertrophic scar fibroblasts correlated well with decreased collagenolytic activity as determined by the degradation rate of fluorescent isothiocyanate-labelled type I collagen in fibroblast culture supernatant. These results suggest that decreased expression of collagenase in hypertrophic scar fibroblasts may be one possible cause for the excessive accumulation of collagen in the skin lesions of hypertrophic scars.     

Suppressed inflammatory gene expression during human hypertrophic scar compared to normotrophic scar formation

Hypertrophic scar formation is a result of adverse cutaneous wound healing. The pathogenesis of hypertrophic scar formation is still poorly understood. A problem next to the lack of suitable animal models is that often normal skin is compared to hypertrophic scar (HTscar) and not to normotrophic scar (NTscar) tissue. Another drawback is that often only one time period after wounding is studied, while scar formation is a dynamic process over a period of several months. In this study, we compared the expression of genes involved in inflammation, angiogenesis and extracellular matrix (ECM) formation and also macrophage infiltration in biopsies obtained before and up to 52 weeks after standard surgery in five patients who developed HTscar and six patients who developed NTscar. It was found that HTscar formation coincided with a prolonged decreased expression of inflammatory genes (TNFα, IL‐1α, IL‐1RN, CCL2, CCL3, CXCL2, CXCR2, C3 and IL‐10) and an extended increased expression of ECM‐related genes (PLAU, Col3A1, TGFβ3). This coincided with a delayed but prolonged infiltration of macrophages (type 2) in HTscar tissue compared to NTscar tissue. These findings were supported by immunohistochemical localization of proteins coding for select genes named above. Our study emphasizes that human cutaneous wound healing is a dynamic process that is needed to be studied over a period of time rather than a single point of time. Taken together, our results suggest innate immune stimulatory therapies may be a better option for improving scar quality than the currently used anti‐inflammatory scar therapies.     

Immunolocalization of collagenase and tissue inhibitor of metalloproteinases (TIMP) in hypertrophic scar tissue

Normal, mature and hypertrophic dermal scars were examined by indirect immunofluorescence for the presence of collagenase, tissue inhibitor of metalloproteinases (TIMP) and cathepsin D. Significant extracellular immunoprecipitation of both collagenase and TIMP were found in areas of all scars judged to be actively remodelling, whereas inactive areas were predominantly negative. TIMP was also present in endothelial cells of patent blood vessels, but found not to be present in the enlarged endothelial cells of occluded vessels. Negligible amounts of extracellular cathepsin D were found.     

The proteoglycans in hypertrophic scar

Sulfated proteoglycans of hypertrophic scar (6 months old after a burn) were extracted with 6 M hot urea, 0.02 M phosphate buffer, pH 7.0, containing protease inhibitors. Two types of proteoglycans, dermatan sulfate proteoglycans and chondroitinsulfate rich proteoglycans, were recovered. The dermatan sulfate proteoglycans, which are capable of binding to concanavalin A, were composed of Mr=53000 core protein and iduronate rich dermatan sulfate. The chondroitin sulfate rich proteoglycans did not bind to concanavalin A but had the same size core proteins as the dermatan sulfate proteoglycans, based on SDS-polyacrylamide gel electrophoresis. The tryptic peptide mappings showed that the core proteins from dermatan sulfate have the same structural sequences as those from chondroitin sulfate rich proteoglycans.     

Steroid-induced 'granulomas' in hypertrophic scar

Intralesional steroids injected into hypertrophic scars and keloids can result in histiocytic and foreign body granulomatous reaction which may be confused with focal mucinosis or necrobiotic process such as rheumatoid nodule. An awareness of this possibility might avoid unnecessary investigations in the patient.     

Studies on human scar tissue proteoglycans

The proteoglycans (PGs) in pooled normal scars and pooled hypertrophic scars were extracted with 4 M guanidinium chloride and isolated by DEAE-cellulose chromatography. The PG samples were then fractionated further by dissociative CsCl density gradient sedimentation. Following cleavage of the density gradient PG fractions with alkaline NaB3H4, the glycosaminoglycan (GAG) constituents were isolated and analyzed by quantitative cellulose acetate electrophoresis. In addition, single samples of normal skin and a keloid scar were also analyzed. The results showed that the hypertrophic scars had a higher average content of extractable and also residual PGs than did the normal scars but a wide range of values was obtained for each type of scar. Some differences were noted in the amounts and distribution of the GAGs in CsCl gradient fractions, in the different types of scar tissue. The PGs in tissues were distributed in low-, medium-, and high density fractions and are, therefore, heterogeneous. Dermatan sulfate (DS) was the major GAG in each tissue and smaller quantities of chondroitin sulfate (CS), heparan sulfate (HS), and heparin (HP) were also present. In addition, 2 other GAG constituents were also detected. Based on the susceptibility of these GAGs to enzymes and nitrous acid treatments, one was a HS or HP while the second was a DS. The major differences in the PG composition of the scar tissues were the higher proportions of low-density CS-PGs in the keloid scar and of low density DS-PGs in hypertrophic and keloid scars.     

Where we stand with human hypertrophic and keloid scar models

We have yet to create a human scar model that demonstrates the complex nature of hypertrophic scar and keloid formation as well as ways to prevent them despite emerging advances in our understanding of the immune system, the inflammatory response, and proteomic and genomic changes after injury. Despite more complex in vitro models, we fail to explain the fundamental principles to scar formation, and the timeline of their development. The solution to developing the ideal in vitro scar model is one that mimics the heterogeneous cellular and molecular interactions, as well as the evolving structure and function of human skin.     

Fibronectin. Localization in normal human skin, granulation tissue, hypertrophic scar, mature scar, progressive systemic sclerotic skin, and other fibrosing dermatoses

The histologic localization of fibronectin (FN) in normal human skin, granulation tissue, hypertrophic scar, mature scar, progressive systemic sclerotic skin, and tissue of other fibrotic disorders was investigated by an indirect immunofluorescence technique using specific antiserum prepared in rabbits against purified human plasma FN. In granulation tissue that developed just after traumatic wounding, FN seemed to increase remarkably in the wound as a fibrillar network. In the hypertrophic scar, one to five years after wounding, FN was detected in a linear or curling arrangement throughout the dermis. On the contrary, FN gradually decreased in the wound of the mature scar five to 20 years after wounding. There were some interesting observations among other diseases. In the skin of patients with progressive systemic sclerosis and morphea, FN was found to be localized faintly on the dermoepidermal junction and papillary dermis. In the involved skin of dermatofibroma, FN was observed in a curling arrangement throughout the dermis.     

Mechanical analysis of hypertrophic scar tissue: structural basis for apparent increased rigidity

The mechanical behavior of normal human skin and hypertrophic scar tissue (HST) is compared using constant-strain-rate and successive stress-relaxation uniaxial loading programs in vitro. HST is less extensible, requires more energy to be stretched in the physiologic range, and stores strain energy less efficiently than normal skin. The explanations for the differences observed between the mechanical behavior of normal skin and HST are based on the differences in their composition and structure. We suggest that the collagen fiber network is partially "prealigned" in a crimped tendon-like organization in HST, which reduces its extensibility and raises the strain energy required to stretch it. It is further hypothesized that an incomplete elastic fiber network, an abnormal glycosaminoglycan content, and/or abnormal collagen fiber slippage are responsible for the reduced capacity to return strain energy in the hypertrophic scar tissue. The results of these studies indicate that although HST has been described as stiffer than normal skin, the maximum stiffness of skin and HST are similar. The "apparent" increased rigidity of HST is a result of reduced extensibility rather than a change in its stiffness. This inexensibility may manifest itself by limiting joint mobility in the patient with HST.     

Crosslink of collagen in hypertrophic scar.

It is conceivable that intramolecular and intermolecular crosslinks of collagen may be involved in the formation of hypertrophic scar, but little is known about the relationship between hypertrophic scar and crosslinks of collagen. We have isolated a new crosslinking amino acid from collagen and have named it pyridinoline. In this investigation, we examined the content of pyridinoline in human normal skin, mature scar and hypertrophic scar. An appreciable amount of pyridinoline was found in collagen of hypertrophic scar, but pyridinoline is virtually absent in collagen of normal skin.     

Imbalance between activin A and follistatin drives postburn hypertrophic scar formation in human skin

Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF-beta, play an important role in this process. Activin A, a member of the TGF-beta family, shares the same intra-cellular Smad signalling pathway with TGF-beta, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of alpha-SMA(+) myofibroblasts and DC-SIGN(+) dendritic cells coexpressing activin A. Ex-vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF-betas stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF-betas. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced alpha-SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin-induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.     

增生性瘢痕的预防与治疗进展

瘢痕是人体创伤修复过程的自然产物,但过度修复将导致病理性瘢痕的形成。增生性瘢痕是常见的病理性瘢痕,引起瘙痒、疼痛、烧灼感和不适,严重影响患者生活。目前可从手术设计、术后应用硅胶膜及注射肉毒素方面预防瘢痕发生,新的治疗方法如局部注射药物、激光治疗及中药治疗等都展现了很好的疗效,有望成为治疗瘢痕的主要选择。本文将结合国内外最新文献对增生性瘢痕防治的进展作一综述。     

Multinucleate cell angiohistiocytoma with hypertrophic nerves

Multinucleate cell angiohistiocytoma (MCAH) represents a rare benign skin lesion characterized by multiple papules that are usually found on the distal extremities or face of middle‐aged women. We report on a 60‐year‐old male with a history of monoclonal gammopathy and severe rheumatoid arthritis who had several asymptomatic red‐to‐livid papules grouped on the right side of his trunk. The lesions had been present for a few years and were gradually enlarging. Biopsies from three lesions showed a spectrum of changes consisting of a proliferation of small venules together with thickened collagen bundles and increased numbers of interstitial cells including bizarre, multinucleated giant cells. Immunohistochemically, the multinucleated cells were positive for vimentin and lysozyme while the interstitial cells expressed CD68, factor XIIIa and lysozyme. Interestingly, strikingly enlarged dermal nerves were seen in all the three biopsies and two of the biopsies showed an accompanying infiltrate of lymphocytes and plasma cells.     

Elastic fibers in scar tissue

The capacity of the skin to be stretched and to return to its resting position is correlated to the quantity and to the quality of the elastic fiber network. Although elastic fibers have been demonstrated in scars, the time course of their appearance in scars and their role in scar elasticity has not been elucidated. A study was therefore undertaken to evaluate the elastic fiber network in scars.
The scars studied were from re-excision specimens following a biopsy performed for a benign or a malignant process. A total of 182 scars were evaluated in patients of different age groups. Miller's elastic tissue stain, considered to be superior to Verhoeff's van Giesen stain, was used. No elastic fibers were detected in any of 116 scars which were of less than 3 months' duration. In 66 scars present for over 3 months, a progressive increase in elastic fibers was present, first as focal and thin fibers, then as diffuse and thicker fibers. For scars of the same duration, a regional difference was noted in that scars from the back contained more and thicker elastic fibers than those from the cheek. When patients were stratified according to age, no appreciable difference was noted in the density of elastic fibers in both new and old scars between the different age groups. These results show that the synthesis of elastic tissue fibers in scars is a function of duration and site of the scar.     

Shedding of proangiogenic microvesicles from hypertrophic scar myofibroblasts

Hypertrophic scars are a common complication of burn injuries and represent a major challenge in terms of prevention and treatment. These scars are characterized by a supraphysiological vascular density and by the presence of pathological myofibroblasts (Hmyos) displaying a low apoptosis propensity. However, the nature of the association between these two hallmarks of hypertrophic scarring remains largely unexplored. Here, we show that Hmyos produce signalling entities known as microvesicles that significantly increase the three cellular processes underlying blood vessel formation: endothelial cell proliferation, migration and assembly into capillary‐like structures. The release of microvesicles from Hmyos was dose‐dependently induced by the serum protein α‐2‐macroglobulin. Using flow cytometry, we revealed the presence of the α‐2‐macroglobulin receptor—low‐density lipoprotein receptor‐related protein 1—on the surface of Hmyos. The inhibition of the binding of α‐2‐macroglobulin to its receptor abolished the shedding of proangiogenic microvesicles from Hmyos. These findings suggest that the production of microvesicles by Hmyos contributes to the excessive vascularization of hypertrophic scars. α‐2‐Macroglobulin modulates the release of these microvesicles through interaction with low‐density lipoprotein receptor‐related protein 1.     

Histopathological differential diagnosis of keloid and hypertrophic scar

Distinguishing hypertrophic scar (HS) from keloid histopathologically is sometimes difficult because thickened hyalinized collagen (keloidal collagen), the hallmark of keloid, is not always detectable and alpha-smooth muscle actin (alpha-SMA), a differentiating marker of HS, is variably expressed in both forms of scar. The aim of this study was to investigate additional distinguishing features to facilitate differentiation between keloid and HS. We compared various histologic features and the expression of alpha-SMA in 40 specimens of keloid and 10 specimens of HS. The features more commonly seen in keloids were: (a) no flattening of the overlying epidermis, (b) no scarring of the papillary dermis, (c) presence of keloidal collagen, (d) absence of prominent vertically oriented blood vessels, (e) presence of prominent disarray of fibrous fascicles/nodules, (f) presence of a tongue-like advancing edge underneath normal-appearing epidermis and papillary dermis, (g) horizontal cellular fibrous band in the upper reticular dermis, and (h) prominent fascia-like fibrous band. The last three features were found in keloid specimens only, including the ones lacking detectable keloidal collagen. Our study confirmed the diagnostic value of keloidal collagen, but it was only found in 55% of keloid specimens. Alpha-SMA expression was found in both HS (70%) and keloid (45%), thus it would not be a differentiating marker. In scars with no detectable keloidal collagen, the presence of the following feature(s) favors the diagnosis of keloid: non-flattened epidermis, non-fibrotic papillary dermis, a tongue-like advancing edge, horizontal cellular fibrous band in the upper reticular dermis, and prominent fascia-like band.