Use of an in vitro model of tissue-engineered skin to investigate the mechanism of skin graft contraction

Skin graft contraction leading to loss of joint mobility and cosmetic deformity remains a major clinical problem. In this study we used a tissue-engineered model of human skin, based on sterilized human adult dermis seeded with keratinocytes and fibroblasts, which contracts by up to 60% over 28 days in vitro, as a model to investigate the mechanism of skin contraction. Pharmacologic agents modifying collagen synthesis, degradation, and cross-linking were examined for their effect on contraction. Collagen synthesis and degradation were determined using immunoassay techniques. The results show that skin contraction was not dependent on inhibition of collagen synthesis or stimulation of collagen degradation, but was related to collagen remodelling. Thus, reducing dermal pliability with glutaraldehyde inhibited the ability of cells to contract the dermis. So did inhibition of matrix metalloproteinases and inhibition of lysyl oxidase-mediated collagen cross-linking, but not transglutaminase-mediated cross-linking. In summary, this in vitro model of human skin has allowed us to identify specific cross-linking pathways as possible pharmacologic targets for prevention of graft contracture in vivo.     

The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system

Acute and chronic pulmonary diseases are characterized by impaired fibrinolytic activity within the lung. To determine the role of the fibrinolytic system in regulating the pathologies associated with lung injury, we examined the effect of bleomycin, an agent that induces the development of pulmonary fibrosis, in mice deficient for plasminogen (Pg(-)(/-)), urokinase (u-PA(-)(/-)), urokinase receptor (u-PAR(-)(/-)), or tissue plasminogen activator (t-PA(-)(/-)), and in control wild-type (WT) mice. Pg(-)(/-) and t-PA(-)(/-) mice demonstrated an enhanced increase in lung collagen content relative to that observed in WT mice. Levels in u-PA(-)(/-) and u-PAR(-)(/-) mice were similar to those in WT mice. Histological analysis 14 days after lung injury confirmed enhanced interstitial fibrosis in Pg(-)(/-), u-PA(-)(/-), and t-PA(-)(/-) mice relative to WT and u-PAR(-)(/-) mice. Areas of pulmonary hemorrhage were observed in bleomycin-treated WT mice and not in Pg(-)(/-), u-PA(-)(/-), and u-PAR(-)(/-) mice or saline controls. Instead, extensive areas of fibrosis were present throughout the lungs of bleomycin-treated Pg(-)(/-) and u-PA(-)(/-) mice. A mixed phenotype (hemorrhage and fibrosis) was observed in t-PA(-)(/-) and Pg(+/-) mice. Hemosiderin-laden macrophages were abundant in the lungs of mice exhibiting hemorrhage and these mice were prone to an early death. Enhanced macrophage levels in the lungs and activation of matrix metalloelastase (MMP-12) were found in mice with a hemorrhage phenotype. The results of these studies indicate a role for the fibrinolytic system in acute lung injury and suggests that intra-alveolar hemorrhage is the result of basement membrane degradation through cell-mediated u-PA activation of Pg with possible involvement of matrix metalloproteinases. Absence of these two components of the fibrinolytic system, either urokinase or plasminogen, results in accelerated fibrosis.     

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing

Excessive proteolytic activity is a feature of chronic wounds such as venous ulcers, in which resolution of the inflammatory response fails and restorative matrix accumulation is delayed as a consequence. The inflammatory actions of native androgens during the healing of acute skin wounds have lately been characterized. We have now investigated the hypothesis that such activities may impact upon the balance between anabolic and catabolic processes during wound healing. We report that wound deposition of both type I collagen and fibronectin is increased in castrated rats compared with control animals. This response is accompanied by early increases and later decreases in overall wound levels of the key collagenolytic enzymes, matrix metalloproteinase (MMP)-1 and MMP-13. Moreover, the activities of MMP-2 and MMP-9, two further enzymes that contribute to collagen digestion during venous ulceration, were significantly decreased in the wounds of castrated rats. Additional analyses provide evidence that androgens directly stimulate dermal fibroblast collagen production, which supports the suggestion that increased wound collagen deposition in androgen-deprived rats results from reduced matrix degradation (as opposed to enhanced matrix protein biosynthesis). Androgen-mediated dysregulation of the parallel processes of collagen deposition and turnover may underscore the delayed healing of cutaneous wounds in elderly male patients and further contribute to the increased incidence of non-healing wounds in this population.     

Atrophie blanche. A disorder associated with defective release of tissue plasminogen activator

Atrophie blanche is a skin disorder characterized by recurrent ulceration and fibrosis. It is one of a group of cutaneous vasculitides associated with decreased blood fibrinolytic activity. We examined pre- and post-venous occlusion plasma from eight patients with atrophie blanche for release of vascular (tissue-type) plasminogen activator (t-PA). The average plasma level of releasable t-PA was only 0.03 IU/mL, compared with 0.70 IU/mL for 118 healthy controls. Therefore, the fibrinolytic disorder in atrophie blanche is associated with defective release of t-PA from blood vessel walls.     

Reaction of glycation and human skin: The effects on the skin and its components,reconstructed skin as a model

Skin is affected by the aging process and numerous modifications are observed. In human, with time the skin becomes drier, thinner, spots appear, elasticity decreases and stiffening increases, together with the appearance of wrinkles. These observations result from the overlapping of an intrinsic chronological aging (individual, genetic) and of an extrinsic aging (dependent on external factors like UV, pollution and lifestyle). One of the causes of aging is the appearance of the Advanced Glycosylation End Products (AGEs) during life. The glycation reaction results from a non-enzymatic reaction between a sugar and a free amine group of Lys, Arg amino acids in proteins. This reaction does not occur only in the skin, indeed, AGEs are also found in the kidney, lens, vessels, etc. These products are also responsible, because of their localization, of some pathologies related to diabetes. AGEs provoke biological modifications implying an activation of molecules synthesis (extracellular matrix, cytokines) and enzyme activation of matrix degradation (metalloproteinases). The UV effect on AGEs (like pentosidine) generates reactive oxygen species (ROS) in the extracellular matrix which could lead to additional deleterious effects. Molecules are described in the literature as inhibitors to this irreversible reaction i.e. aminoguanidine. To understand the consequences of the glycation in the skin, a system of reconstructed skin was developed with a collagen modified by glycation for the dermal component. In this system we observed that dermis and epidermis are both modified due to glycation (macromolecules synthesis, cytokines, metalloproteinases) and it is possible to test inhibitors of this reaction. In conclusion, in skin, glycation is involved in a very complex aging process and simultaneously affect, directly and indirectly, certain cells, their synthesis and the organization of the matrix.     

The fibrinolytic system and thrombotic tendency

The deposition of the insoluble protein matrix, fibrin is temporary. The mainly known mechanism of proteolytic removal is orchestrated by a cascade type of proteolytic process involving ultimately the formation from plasminogen of the active degradation enzyme plasmin. The occurrence of plasminogen deficiency without a massive deposition of fibrin and thrombotic events indicates the occurrence of alternate routes of fibrin degradation. In the literature, data have been reported about the direct fibrinolytic activity of various other enzymes including leucocytal elastase and cathepsin G and three metalloproteinases (MMP-3,MMP-7, MT1-MMP). The importance of each of these pathways and the possible differences in importance in various diseases, in acute situations and at different locations in the circulation, in tissues and organs is not known in detail. It is suggested that multiple combined knock-outs be created to evaluate the situation for various well-defined phenotypes. It is concluded that fibrin removal is an important biological process with various buffering mechanisms and only combinations of abnormalities in the various mechanisms and special situations will lead to fibrin accumulation and thrombotic events.     

Plasminogen activation in healing human wounds.

Tissue injury is followed by formation of a provisional, fibrin-containing matrix. It is later on replaced by granulation tissue. Replacement involves extracellular proteolysis by fibrinolytic enzymes. Plasmin is a fibrinolytic proteinase and is generated from ubiquitous plasminogen by cell-derived urokinase-type (uPA) or tissue-type (tPA) plasminogen activator. To explore the cells and components involved in plasminogen activation, we have performed a combined immunohistological and zymographic study on human skin wounds produced iatrogenically by debridement. The fibrin(ogen)-specific staining indicated the progressive removal of a fibrin-containing provisional matrix. Plasmin(ogen) was present over the entire observation period. It was diffusely distributed and also displayed a conspicuous association with cells of the granulation tissue, in particular with monocytes/macrophages and fibroblasts. Also, uPA was associated with monocytes/macrophages and fibroblasts, whereas the uPA-receptor (uPA-R) was stained in monocytes/macrophages only. The uPA was potentially active as indicated by zymography. No tPA-specific staining was found. The findings point at the importance of monocytes/macrophages and fibroblasts in uPA-mediated plasminogen activation in healing human skin wounds.     

Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease

Multiphoton microscopy was used to study the extracellular matrix of keloid at the molecular level without tissue fixation and staining. Direct imaging of collagen and elastin was achieved by second harmonic generation and two-photon excited fluorescence, respectively. The morphology and quantity of collagen and elastin in keloid were characterized and quantitatively analyzed in comparison to normal skin. The study demonstrated that in keloid, collagen content increased in both the upper dermis and the deep dermis, while elastin mostly showed up in the deep dermis and its quantity is higher compared to normal skin. This suggests the possibility that abnormal fibroblasts synthesized an excessive amount of collagen and elastin at the beginning of keloid formation, corresponding to the observed deep dermis, while after a certain time point, the abnormal fibroblast produced mostly collagen, corresponding to the observed upper dermis. The morphology of collagen and elastin in keloid was disrupted and presented different variations. In the deep dermis, elastic fibers showed node structure, while collagen showed obviously regular gaps between adjacent bundles. In the upper dermis, collagen bundles aligned in a preferred direction, while elastin showed as sparse irregular granules. This new molecular information provided fresh insight about the development process of keloid.     

Increased TIMP/MMP ratio in varicose veins: a possible explanation for extracellular matrix accumulation

Primary varicose veins are functionally characterized by venous back-flow and blood stagnation in the upright position. Dilatation and tortuosity provide evidence for progressive venous wall remodelling, with disturbance of smooth muscle cell/extracellular matrix organization. Affected areas are not uniformly distributed, some areas being hypertrophic, whereas others are atrophic or unaffected. In 12 varicose veins and ten control veins, the proteolytic enzyme/inhibitor balance which may participate in the remodelling of the venous wall was investigated. For this purpose, the presence and enzymatic activity of matrix metalloproteinases (MMP-2, MMP-9), tissue inhibitors of MMPs (TIMP-1, TIMP-2), urokinase-type (uPA) and tissue-type (tPA) plasminogen activators (PAs), and plasminogen activator inhibitor-1 (PAI-1) were quantified by western blot and gelatin or plasminogen-casein zymography. In addition, MMP-2, TIMP-1, TIMP-2, and PAI-1 levels were measured by ELISA. A high TIMP-1 level and a low MMP-2 level/activity were found in varicose veins (p<0.005), resulting in a three-fold increase in the TIMP-1/MMP-2 ratio in varicose versus control veins. Levels of PAs (uPA and tPA) as well as PAI-1 were both lower in varicose veins (p<0.005), with minimal change in the PAI/PA ratio. These results demonstrate that varicose veins are characterized by a higher than normal TIMP/MMP ratio, which may facilitate extracellular matrix accumulation in the diseased venous wall.     

Expression of nitric oxide synthase isoforms and arginase in normal human skin and chronic venous leg ulcers

Chronic venous ulcers, an example of abnormal wound healing, show chronic inflammation with defective matrix deposition which together with the underlying vascular pathology, result in delayed healing. L-arginine is known to be metabolized by one of two pathways: nitric oxide synthase (NOS), producing nitric oxide (NO), or arginase, producing ornithine. NO is involved in many pathological conditions including vascular and inflammatory disorders. This study therefore investigated the distribution, level and activity of NOS and arginase in chronic venous ulcers in comparison with normal skin, using immunocytochemistry, western blotting, and enzyme assays. The results demonstrated an increased distribution of both NOS and arginase in chronic venous ulcer tissue compared with normal skin, with inflammatory cells and vascular endothelial cells as the main sources. These data were confirmed by western blot analysis, which showed increased levels of both enzymes in chronic venous ulcers. Moreover, there was significantly increased activity of both total NOS (p<0.04) and inducible NOS (p<0.05) in chronic venous ulcer tissue compared with normal skin, and significantly increased activity of arginase (p<0.01) in chronic venous ulcer tissue in comparison with normal skin. NO is known to combine with hydroxyl free radicals forming peroxynitrite, a potent free radical which causes tissue destruction. NO overexpression in chronic venous ulcers may be involved directly or indirectly (through production of peroxynitrite) in the pathogenesis and delayed healing of chronic venous ulcers, through its effects on vasculature, inflammation, and collagen deposition. Arginase is known to enhance matrix deposition. Thus, increased levels of arginase in chronic venous ulcers could contribute to the pathogenesis of lipodermatosclerosis associated with chronic venous insufficiency, predisposing to the formation of chronic venous ulcers and also to matrix cuff formation around blood vessels.     

Matrix metalloproteinases in skin pathology (Review)

Human tissue is composed in part of cells and in part of amorphous matrix components. Equilibrium exists between the synthesis and degradation of connective tissue under physiological conditions, which serves both the formation and the maintenance of tissue architecture. Synthesis progresses via mesenchymal cells, degradation is controlled by the proteolytic effect of a group of enzymes, which belong to the protein family of matrix metalloproteinases. In biological development, matrix metalloproteinases play an important role in all essential configurative processes in embryo- and histogenesis. As a result of the importance of matrix metalloproteinases in creation of connective tissue, dysregulation with excessive proteolytic activity may result in tissue damage. Histological structural changes are set in relation to the molecular expression pattern of matrix metalloproteinases using matrix relevant diseases of human skin. Discussion includes fibrosing processes, skin inflammations, wound healing, blistering diseases, premature sun-induced skin aging and primary cutaneous malignomas and their metastases.     

Remodeling of elastic fiber components in scleroderma skin

The skin of patients with scleroderma is characterized by an excess accumulation of collagen in the extracellular matrix of the fibrotic reticular dermis. Elastic fibers are also disrupted in this disease, however, in contrast to collagen, relatively few studies have provided information concerning the changes that occur to elastic fiber components in scleroderma. In the present study, the extracellular matrix in scleroderma skin was examined with a specific focus on the integrity of elastic fibers. Electron microscopic observations confirmed an excess of 10 nm microfibrils present in small bundles independent of amorphous elastin in the fibrotic reticular dermis. In the same area, a population of stellate-shaped fibroblasts was identified in close association with the dermal elastic fibers. In contrast to the uniform black appearance of the elastic fibers seen in the papillary dermis and in areas of the reticular dermis not infiltrated by these cells, the elastic fibers apposed to the cells were mottled in density and often almost electron-lucent. These observations suggest that the elastic fibers in the reticular dermis were being actively degraded. Results from this study provide evidence for disintegration of elastic fibers in the skin of scleroderma patients and suggest the possibility that degradation products from the elastic matrix in the diseased tissues may act as a feedback signal for increased matrix production.     

Heparin fragments modulate the collagen phenotype of fibroblasts from radiation-induced subcutaneous fibrosis

Acute local gamma irradiation of porcine skin induces, as in human skin, an extensive and mutilating sclerosis characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of the macromolecular components of the extracellular matrix. Collagen synthesis, content, and types were studied in the presence of heparin fragments (100 micrograms/10(6) cells) in the culture medium, by measuring the incorporation of the radiolabeled precursor [3H]proline into confluent primary cultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. Enhancement in collagen biosynthesis and deposition and preferential increase in collagen type III synthesis were observed in fibrotic fibroblast cultures when compared to those in normal dermis fibroblasts. The total collagen synthesis and the rate of collagen hydroxylation appear unmodified by heparin fragments both in normal and in fibrotic fibroblast cultures. But heparin fragments induce a 10- and 2-fold decrease, respectively, in collagen type III and type V syntheses by fibrosis fibroblasts. As only minor effects upon collagen type III and V are observed in cultures of normal dermis fibroblasts, these results highly suggest that heparin fragments are capable of specifically modulating the collagen phenotype of fibroblasts derived from radiation-induced dermis fibrosis and thus are able to regulate the fibrotic process.     

Plasmin induces apoptosis of aortic valvular myofibroblasts

Previous studies have described remodelling of the extracellular substratum by matrix metalloproteinases (MMPs) in aortic valves. However, involvement of the fibrinolytic system has not yet been analysed. We hypothesized that plasminogen and plasminogen activator(s) are present in aortic valves and that plasminogen activation could induce the degradation of adhesive proteins and apoptosis of the valvular myofibroblasts. We employed ELISA, western blotting, fibrin‐agar zymography, and immunochemistry to detect components of the plasminogen activation system, in samples of aortic valves and valvular myofibroblasts in primary culture. Using myofibroblast cultures, real‐time measurement of plasminogen activation was performed in the absence and presence of inhibitors (amiloride, ε‐aminocaproic acid, and an MMP inhibitor); the degradation of fibronectin was visualized on western blots; and the apoptotic process was assessed by detection of phosphatidylserine exposure (binding of FITC‐annexin V) and DNA fragmentation (TUNEL and ELISA). We demonstrate that a time‐ and plasminogen concentration‐dependent generation of plasmin occurs on the surface of cultured valvular myofibroblasts expressing both u‐PA and t‐PA. Only u‐PA appears to activate plasminogen as t‐PA is essentially found in complex with PAI‐1. Plasmin‐dependent degradation of pericellular proteins, such as fibronectin, leads to cell detachment and apoptosis. In conclusion, various proteins of the fibrinolytic system are synthesized in vitro by cultured myofibroblasts from aortic valves, leading to plasmin‐dependent cell detachment‐induced apoptosis, a biological process named anoikis. The presence of plasminogen in aortic valves suggests that this process may be operating in vivo and may participate in valvular tissue remodelling, as also suggested by the finding of apoptotic cells in valvular tissue. This is the first demonstration of the presence and potential role of enzymes of the fibrinolytic system in aortic valves. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Plasmin-Coated Borrelia burgdorferi Degrades Soluble and Insoluble Components of the Mammalian Extracellular Matrix

Borrelia burgdorferi, the spirochetal agent of Lyme disease, binds plasminogen in vitro. Exogenously provided urokinase-type plasminogen (PLG) activator (uPA) converts surface-bound PLG to enzymatically active plasmin. In this study, we investigated the capacity of a B. burgdorferi human isolate, once complexed with plasmin, to degrade purified extracellular matrix (ECM) components and an interstitial ECM. In a modified enzyme-linked immunosorbent assay using immobilized, soluble ECM components, plasmin-coated B. burgdorferi degraded fibronectin, laminin, and vitronectin but not collagen. Incubation of plasmin-coated organisms with biosynthetically radiolabeled native ECM resulted in breakdown of insoluble glycoprotein, other noncollagenous proteins, and collagen, as measured by release of solubilized radioactivity. Radioactive release did not occur with untreated spirochetes or spirochetes treated with uPA or PLG alone. Kinetic and inhibition studies suggested that the breakdown of collagen was indirect and due to prior disruption of supportive ECM proteins. B. burgdorferi is an invasive bacterial pathogen that may benefit by use of the host's plasminogen activation system. The results of this study have identified mechanisms in which the spirochete can use this borrowed proteolytic activity to enhance invasiveness.     

胶原基人工真皮替代物的材料与结构设计

介绍了胶原基人工真皮替代物的结构模型、材料选择和微观结构控制。仿照人体天然的皮肤结构与功能，人工皮肤也应具有双层结构：上层以透气不透水的聚合物弹性体构成，发挥临时表皮的作用，下层由可降解的基于胶原的海绵体构成，作为自体可吸收的人工真皮层。除胶原外，多糖类化合物如氨基葡聚糖也是构成人工真皮替代物的重要物质之一，使人工真皮的成分更接近于天然皮肤，并调控胶原的降解速率与组织的再生同步。通过控制PH值、浓度和冻干温度等成膜条件，可调控所得胶原多孔膜的大小和形态结构。如此制备的基于胶原的人工真皮替代物可望成为皮肤移植的一种替代方法，用以治疗全厚皮损伤及皮肤溃疡。     

The matrix metalloproteinases and their inhibitors.

A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.     

EXPRESSION OF PLASMINOGEN ACTIVATORS IN BASAL CELL CARCINOMA

The plasminogen activators, tissue type and urokinase type (tPA and uPA, respectively), have been identified in various malignancies and have been implicated in both local growth and metastatic spread. To characterize plasminogen activator expression more fully in human basal cell carcinoma, the localization of uPA and tPA mRNAs was evaluated by in situ hybridization. Nodular basal cell carcinomas demonstrated uPA expression in most cases, whereas the non-nodular subtypes were negative. Message for uPA was identified within tumour islands (11/12 cases), scattered fibroblast-like stromal cells (6/12 cases), and the basal layer of the overlying epidermis (10/12 cases). In addition, signal for uPA was elevated and pronounced in areas where the epidermis merged into invasive basal cell carcinoma in the superficial papillary dermis in some cases. Message for uPA was often associated with ulceration or erosion of the overlying epithelium. Expression of tPA was noted in the epidermis (3/12 cases) and in tumour cells (4/12 cases), but tended to be focal and sparse. These results suggest that complex interactions involving uPA expression occur between the tumour, the stroma, and the overlying epidermis. Both the stroma and the epidermis may contribute to local spread of the tumour through production of uPA and consequent plasmin-mediated activation of collagenases and metalloproteinases.     

Cleavage of surfactant-incorporating fibrin by different fibrinolytic agents. Kinetics of lysis and rescue of surface activity

Incorporation of surfactant into polymerizing fibrin causes loss of surface activity and marked retardation of clot lysis by plasmin (Günther and colleagues, Am. J. Physiol. 1994;267:L618-L624). We compared the efficacy of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), activated anisoylated streptokinase-plasminogen activator complex (APSAC), and plasmin to dissolve surfactant-incorporating fibrin. Alveofact was employed as a natural surfactant source, and plasminogen was coincorporated into the fibrin matrix at a physiologic ratio to fibrin. Fibrinolysis was quantified by the release of tracer from (125)I-labeled fibrin, and the pattern of split products was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, we investigated the fibrinolysis-related restoration of surface activity by measurement in the pulsating bubble surfactometer. Concentrations of all fibrinolytic agents were chosen to effect approximately 40% lysis of clot material in the absence of surfactant (control). When incorporated into the fibrin matrix, but not when admixed after clot formation, surfactant inhibited the cleavage of fibrin by all fibrinolytic agents in a dose-dependent manner. Interestingly, t-PA and u-PA were significantly less inhibited than was plasmin or APSAC. The pattern of arising fibrin scission products was identical for all fibrinolytic approaches and was independent of surfactant incorporation. Adsorption and minimum surface tension-lowering properties of Alveofact were almost completely lost upon incorporation into fibrin, but surface activity was fully restored upon sustained clot lysis with all fibrinolytic agents. We conclude that the fibrinolytic capacity of all agents investigated is markedly inhibited by surfactant incorporation in fibrin, but this inhibition is significantly less pronounced in the agents employing preincorporated plasminogen (t-PA and u-PA), as compared with plasmin and APSAC. The plasminogen activators may thus proffer to "rescue" pulmonary surfactant function by induction of fibrinolysis in the alveolar compartment.     

Granuloma annulare: an elastic tissue disease? Case report and literature review

Granuloma annulare (GA) is a condition characterized by the presence of palisading granulomas usually in the dermis. Traditionally, the histopathological changes are described as consisting of focal degeneration of dermal collagen fibers. However, no convincing evidence for such alteration is found in the literature. A histopathological study was done to ascertain the nature of the dermal abnormality. Ten skin biopsies showing lesions of GA were studied by light and electron microscopy. On light microscopy, all lesions showed focal dermal degeneration with near total loss of elastic fibers. Ultrastructural examination in nine cases demonstrated degenerated elastic fibers with loss of peripheral microfibrils and abnormal elastic matrix. The degenerated elastic fibers were surrounded by intact collagen fibers. These findings show that the main alteration in GA is elastic fiber degeneration and strongly suggest that the primary target leading to the development of this disorder is injury to the elastic tissue.