High temperature requirement factor A1 (HTRA1) regulates the activation of latent TGF-ß1 in keloid fibroblasts.

Scar treatments are considered a major issue in the plastic surgery field. Activation of the transforming growth factor-ß (TGF-ß)-mediated signaling pathway plays a key role in the scar pathogeneses, and high temperature requirement factor A1 (HTRA1) inhibits TGF-ß1 activation in tumor cells. Our study aims to investigate the role of HTRA1 in the pathogenesis of scars. The mRNA levels of HTRA1 was evaluated by real time PCR, HTRA1 protein expression was determined using western blot and immunohistochemistry, and a luciferase assay was applied to measure dynamic changes of TGF-ß1 activity. We found that the expression of HTRA1 was significantly elevated in keloid tissues, compared to normal skin, and TGF-ß1 mRNA levels slightly increase in the keloid tissue. Furthermore, active TGF-ß1 protein levels and Smad2 phosphorylation significantly increased in the keloid tissue. Treatment with the latent TGF-ß1 or recombinant human HTRA1 (rhHTRA1), alone or in combination, increased Smad2 phosphorylation levels in keloid fibroblasts and active TGF-ß1 contents of associated supernatants. Our results suggest that HTRA1 is involved in the pathogenesis of scars through regulating activation of latent TGF-ß1 in keloid fibroblasts, and our study reveals that HTRA1 is a novel target that regulates scar formation.

A Role for Neuregulin-1 in Promoting Keloid Fibroblast Migration via ErbB2-mediated Signaling.

Keloid disease is a fibroproliferative tumour characterised by aggressive local invasion, evident from a clinically and histologically active migrating margin. During combined laser capture microdissection and microarray analysis-based in situ gene expression profiling, we identified upregulation of the polypeptide growth factor neuregulin-1 (NRG1) and ErbB2 oncogene in keloid margin dermis, leading to the hypothesis that NRG1 contributed to keloid margin migration through ErbB2-mediated signalling. The aim of this study was to probe this hypothesis through functional in vitro studies. Exogenous NRG1 addition to keloid and normal skin fibroblasts altered cytokine expression profiles, significantly increased in vitro migration and keloid fibroblast Src and protein tyrosine kinase 2 (PTK2/FAK) gene expression. ErbB2 siRNA knockdown attenuated both keloid fibroblast migration and Src/PTK2 expression, which were not recovered following NRG1 administration, suggesting the NRG1/ErbB2/Src/PTK2 signaling pathway may be a novel regulator of keloid fibroblast migration, and representing a potential new therapeutic target.

Growth Differentiation Factor-9 Promotes Fibroblast Proliferation and Migration in Keloids through the Smad2/3 Pathway.

BACKGROUND: Keloids are fibroproliferative scars that develop as a result of a dysregulated wound healing process; however, the molecular mechanisms of keloid pathogenesis remain unclear. Keloids are characterized by the ability to spread beyond the original boundary of the wound, and they represent a significant clinical challenge. Previous work from our group suggested that growth differentiation factor (GDF)-9 plays a role in the invasive behavior of keloids. Here, we examined the involvement of GDF-9 in keloid formation and spread and elucidated a potential underlying mechanism. METHODS: The expression of GDF-9, cyclooxygenase (COX)-2, vascular epidermal growth factor (VEGF)-C, matrix metalloprotease (MMP)-2, MMP-9, transforming growth factor (TGF)-ß1, and the related signaling pathway components in human keloid tissues or keloid fibroblasts (kFBs) was monitored by qRT-PCR and western blot. A series of overexpression and silencing experiments in normal and keloid fibroblasts were used to modify the expression of GDF-9. The effects of GDF-9 on kFB proliferation and migration were assessed using the CCK-8, cell cycle and scratch wound healing assays. RESULTS: GDF-9 promotes fibroblast proliferation and migration. GDF-9 silencing in kFBs decreased cell proliferation, blocked cell cycle progression, downregulated the angiogenic markers COX-2 and VEGF-C, and downregulated MMP-2 and MMP-9 expression, whereas it had no effect on the levels of TGF-ß1. GDF-9 silencing significantly inhibited Smad2 and Smad3 phosphorylation in kFBs. CONCLUSIONS: GDF-9 promotes the proliferation and migration of kFBs via a mechanism involving the Smad2/3 pathway.

NADPH oxidase-2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis.

The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor-beta1 (TGF-ß1) exerts pro-oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF-ß1-induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real-time Q-PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock-down analysis. Hydrogen peroxide production and 2',7'-dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non-phagocytic NADPH oxidase mRNA. TGF-ß1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF-ß1 phosphorylated MAPKs and increased activator protein-1 (AP-1) in a redox-sensitive manner, and NOX2 suppression inhibited baseline and TGF-ß1-mediated stimulation of Smad2 phosphorylation. Moreover, TGF-ß1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI-1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.

Keloid disease can be inhibited by antagonizing excessive mTOR signaling with a novel dual TORC1/2 inhibitor.

Keloid disease (KD) is a fibroproliferative lesion of unknown etiopathogenesis that possibly targets the PI3K/Akt/mTOR pathway. We investigated whether PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which targets both mammalian target of rapamycin complex 1 (mTORC-1) and mTORC-2 signaling, could exert anti-KD effects in a novel KD organ culture assay and in keloid fibroblasts (KF). Treatment of KF with P529 significantly (P < 0.05) inhibited cell spreading, attachment, proliferation, migration, and invasive properties at a low concentration (5 ng/mL) and induced substantial KF apoptosis when compared with normal dermal fibroblasts. P529 also inhibited hypoxia-inducible factor-1α expression and completely suppressed Akt, GSK3ß, mTOR, eukaryotic initiation factor 4E-binding protein 1, and S6 phosphorylation. P529 significantly (P < 0.05) inhibited proliferating cell nuclear antigen and cyclin D and caused considerable apoptosis. Compared with rapamycin and wortmannin, P529 also significantly (P < 0.05) reduced keloid-associated phenotypic markers in KF. P529 caused tissue shrinkage, growth arrest, and apoptosis in keloid organ cultures and substantially inhibited angiogenesis. pS6, pAkt-Ser473, and mTOR phosphorylation were also suppressed in situ. P529 reduced cellularity and expression of collagen, fibronectin, and α-smooth muscle actin (substantially more than rapamycin). These pre-clinical in vitro and ex vivo observations are evidence that the mTOR pathway is a promising target for future KD therapy and that the dual PI3K/Akt/mTOR inhibitor P529 deserves systematic exploration as a candidate agent for the future treatment of KD.

Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production.

Keloid represents overgrowth of granulation tissue, which is characterized by collection of atypical fibroblasts with excessive deposition of extracellular matrix components, after skin injury, but its etiology is still largely unknown. We recently performed genome-wide association study (GWAS) of keloid and identified NEDD4 to be one of candidate molecules associated with keloid susceptibility. Here we demonstrate a possible mechanism of NEDD4 involvement in keloid formation through enhancement of the proliferation and invasiveness of fibroblasts as well as upregulation of type 1 collagen expression. Activation of NEDD4 affected subcellular localization and protein stability of p27 which was implied its critical role in contact inhibition. It also induced accumulation of ß-catenin in the cytoplasm and activated the TCF/ß-catenin transcriptional activity. Furthermore, NEDD4 upregulated expressions of fibronectin and type 1 collagen and contributed to the excessive accumulation of extracellular matrix. Our findings provide new insights into mechanism developing keloid and can be applied for development of a novel treatment for keloid.

Hepatoma-derived growth factor and its role in keloid pathogenesis.

Hepatoma-derived growth factor (HDGF) is a novel mitogenic growth factor that has been implicated in many different carcinomas. Its role in keloid biology has not yet been investigated. The present study is aimed at examining the role of HDGF in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine in vivo localization and expression of HDGF in keloid and normal skin tissue. This was followed by the detection of HDGF expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were co-cultured with the fibroblasts. HDGF expression levels in all cell extracts and conditioned media were assayed through Western blot analysis. In another set of experiments, the effect of exogenous recombinant HDGF on keloid fibroblasts (KF) and normal fibroblasts (NF) was examined. Cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by quantifying proliferating cell nuclear antigen (PCNA) expression. Downstream targets of HDGF were identified by detecting their expression through Western blot analysis. Our results indicate that there was an increase in HDGF expression in the dermis of keloid compared with normal skin tissue. The application of serum and epithelial-mesenchymal interactions did not seem to have any effect on intracellular HDGF expression levels. However, co-culturing keloid keratinocytes with KFs resulted in increased HDGF secretion when compared with monoculture or normal controls. Furthermore, treatment with exogenous recombinant HDGF was found to increase the proliferation of KFs, activate the extracellular signal-regulated kinase (ERK) pathway and up-regulate the secretion of vascular endothelial growth factor (VEGF).

Telomere shortening may be associated with human keloids.

BACKGROUND: Keloids are benign skin tumors that are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are inherited with an autosomal dominant mode with incomplete clinical penetrance and variable expression. Keloids are characterized by formation of excess scar tissue beyond the boundaries of the wound. The exact etiology is still unknown and there is currently no appropriate treatment for keloid disease. METHODS: We analyzed sample tissues were obtained from 20 patients with keloid skin lesions and normal skin was obtained from 20 healthy donors. The telomeres were measured by Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay. Quantitative Real-Time RT-PCR analysis of hTERT gene expression was performed and intracellular ROS generation was measured. RESULTS: In this study, we determined whether telomeric shortening and the expression of human telomerase reverse transcriptase (hTERT) occurs in keloid patients. Using Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay, we detected a significant telomere shortening of 30% in keloid specimens compared to normal skin. Using quantitative Real-Time RT-PCR, telomerase activity was found absent in the keloid tissues. Moreover, an increase in ROS generation was detected in fibroblasts cell cultures from keloid specimens as more time elapsed compared to fibroblasts from normal skin. CONCLUSION: Telomere shortening has been reported in several metabolic and cardiovascular diseases. We found that telomere shortening can also be associated with human keloids. Chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases. Here we found increased ROS generation in fibroblasts from keloid fibroblasts cell cultures when compared to normal skin fibroblasts. Hence we conclude that oxidative stress might be an important modulator of telomere loss in keloid because of the absence of active telomerase that counteracts telomere shortening.

Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression.

AIMS: Keloid is characterized by excessive deposition of collagen, resulting from aberrant extracellular matrix (ECM) production and degradation. The aim was to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in pathological wound healing in keloids. METHODS AND RESULTS: Semiquantitative analysis of 60 keloid tissue samples and 25 mature scar tissue samples demonstrated significantly increased expression of MMP-2, TIMP-2 and TIMP-3 in keloids compared with mature scars. Within keloid regions, MMP-2 expression was significantly higher in collagen bundle regions than in non-collagen bundle regions. Double immunofluorescence revealed that keloid fibroblasts between collagen bundles exhibited MMP-2, TIMP-2 and membrane-type 1 MMP (MT1-MMP) co-expression, whereas only MMP-2 expression was evident on the edge of collagen bundles. Western blot analysis and gelatin zymography of 13 keloid-derived fibroblasts (KFbs) and six normal skin dermal-derived fibroblasts (NFbs) demonstrated that unstimulated KFbs exhibited significantly increased MMP-2 activity and expression compared with NFbs under the same conditions. CONCLUSIONS: These results together indicate that MMP-2 activity can be promoted in keloid fibroblasts between collagen bundles in cooperation with TIMP-2 and MT1-MMP. This could contribute to remodelling of collagen bundle regions and invasion of fibroblasts into peripheral normal regions through promoted degradation of ECM.

Matrix metalloproteinase-2 and -9 activities in human keloids, hypertrophic and atrophic scars: a pilot study.

Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP-9 activity was undetectable in gelatin zymography analysis. Pro-MMP-2 levels (median) were highest in normal skin group 53.58 (36.40-75.11) OD microg(-1) protein, while active MMP-2 levels were highest in keloid group 52.53 (42.47-61.51) OD microg(-1) protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two-phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex.

TGF-beta1 antisense therapy modulates expression of matrix metalloproteinases in keloid-derived fibroblasts.

Transforming growth factor-beta1 (TGF-beta1) has been identified as an important regulator of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta1. The purpose of this study was to analyze the effect of TGF-beta1 targeting on the expression of matrix metalloproteinases (MMPs) in fibroblasts cultured from earlobe keloids. The expression of MMP-2 and -9 in tissue samples from keloids was investigated by immunohistochemistry. The effect of TGF-beta1 targeting using antisense oligonucleotides on the expression of MMPs in keloid-derived fibroblasts was analysed by ELISA and multiplex RT-PCR. Immunohistochemical studies demonstrated an increased expression of MMP protein in tissue samples from keloids compared to normal human skin. Antisense TGF-beta1 oligonucleotide treatment significantly downregulated MMP-9 secretion in vitro. In conclusion, TGF-beta1 antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in keloids.

[Activity detection and immunohistochemistry study on xanthine oxidase in pathological scars].

OBJECTIVE: To study the change of xanthine oxidase in pathological scars. METHODS: The tissues of hypertrophic scar (10 cases), keloid (10 cases) and normal skin (8 cases) were collected for this project, in which the content of malonaldehyde, the activity of xanthine oxidase and total of antioxidant capacity were detected by spectrophotometric method. The expression of xanthine oxidase was evaluated by immunohistochemistry technique. RESULTS: Compared with normal skin tissue, the content of malonaldehyde and xanthine oxidase activity were significantly higher in pathological scars (P < 0.05), but no significant difference was observed in the decrease of total antioxidant capacity. Differences in above-mentioned indexes all were not remarkable between hypertrophic scars and keloids. Immunohistochemical study indicated that the expressions of xanthine oxidase in the epidermal keratinocytes and dermal fibroblasts of pathological scars increased markedly in contrast with normal skin (P < 0.05). CONCLUSION: The xanthine oxidase activity shows to be increased in pathological scars, and the xanthine oxidase expressions are enlarged in epidermal keratinocytes and dermal fibroblasts of pathological scars, which may be a reason resulting in the increase of free excessive growth of scar tissues. radical level, and may play a role in pathogenesis of excessive growth of scar tissues.

Lipo-PGE1 suppresses collagen production in human dermal fibroblasts via the ERK/Ets-1 signaling pathway.

Dysregulation of collagen production contributes to various pathological processes, including tissue fibrosis as well as impaired wound healing. Lipo-prostaglandin E1 (Lipo-PGE1), a lipid microsphere-incorporated prostaglandin E1, is used as a vasodilator for the treatment of peripheral vascular diseases. Lipo-PGE1 was recently shown to enhance human dermal fibroblast (HDF) migration and in vivo wound healing. No published study has characterized the role of Lipo-PGE1 in collagen regulation in HDFs. Here, we investigated the cellular signaling mechanism by which Lipo-PGE1 regulates collagen in HDFs. Collagen production was evaluated by the Sircol collagen assay, Western blot analysis of type I collagen and real time PCR. Unexpectedly, Lipo-PGE1 decreased mRNA expression of collagen 1A1, 1A2, and 3A1. Lipo-PGE1 markedly inhibited type I collagen and total soluble collagen production. In addition, Lipo-PGE1 inhibited transforming growth factor-ß-induced collagen expression via Smad2 phosphorylation. To further investigate whether extracellular signal-regulated kinase (ERK)/Ets-1 signaling, a crucial pathway in collagen regulation, is involved in Lipo-PGE1-inhibited collagen production, cells were pretreated with an ERK-specific inhibitor, PD98059, prior to the addition of Lipo-PGE1. Lipo-PGE1-inhibited collagen mRNA expression and total soluble collagen production were recovered by pretreatment with PD98059. Moreover, Lipo-PGE1 directly induced the phosphorylation of ERK. Furthermore, silencing of Ets-1 recovered Lipo-PGE1-inhibited collagen production and PD98059 blocked Lipo-PGE1-enhanced Ets-1 expression. The present study reveals an important role for Lipo-PGE1 as a negative regulator of collagen gene expression and production via ERK/Ets-1 signaling. These results suggest that Lipo-PGE1 could potentially be a therapeutic target in diseases with deregulated collagen turnover.

Emerging new drugs for scar reduction.

Hypertropic and keloid scars cause both functional and cosmetic problems for those afflicted. Although people of all ages suffer from these conditions, the patients are often young and otherwise healthy, and become burdened with an activity limiting lesion or psychosocial stresses associated with a perceived aesthetic defect. Currently available treatment modalities are often inconvenient, occasionally painful, and have unwanted side effects. Despite the highest standard of care, treatment protocols are prone to failure with high rates of scar recurrence. Hypertropic and keloid scars are the result of an abnormal healing response and may result from an extended inflammatory phase in the wound healing process. Regardless of the causes, which remain elusive, excessive collagen deposition occurs relative to normal wounds. This extracellular matrix collagen accumulation makes a logical target for pharmacological interventions, and researchers are attempting to modify collagen-synthetic and -degradative pathways. In addition, growth factors and cytokines have been implicated in scar formation, and these factors are targeted for potential therapeutic use in scar management. Cytotoxic agents are also being evaluated for their potential utility in the reduction of tissue bulk associated with these excessive scar states. Given the wide range of potential therapeutic agents, the future market for scar therapy remains highly promising.

Hepatocyte growth factor-expressing adenovirus upregulates matrix metalloproteinase-1 expression in keloid fibroblasts.

BACKGROUND: Keloids are marked by an overabundance of extracellular matrix. The antifibrotic effect of hepatocyte growth factor (HGF) is achieved by increasing the expression of matrix metalloproteinases (MMPs) that drive extracellular matrix catabolism. As such, we cultivated an RGD-modified HGF-expressing adenovirus (dE1-RGD/lacZ/HGF) for introduction into keloid fibroblasts (KFs), looking at the subsequent impact on MMP-1 expression. METHODS: KFs infected with either test virus as experimental group (dE1-RGD/lacZ/HGF) or its counterpart (dE1-RGD/lacZ) as control group were examined for HGF protein expression using an enzyme-linked immunosorbent assay (ELISA). Collagen (types I and III) and MMP-1 mRNA levels were also determined by reverse transcriptase-polymerase chain reaction, and ELISA was used to monitor MMP-1 protein expression. RESULTS: In KFs harboring the test virus, high levels of HGF were induced at a multiplicity of infection ratio of 50 (3260.6 ± 162.7 pg/ml) after 72 hours of incubation. Furthermore, reverse transcriptase-polymerase chain reaction and ELISA confirmed that MMP-1 mRNA and protein expression rose significantly in KFs after transduction by the test virus (P < 0.05). However, mRNA levels of collagen were unaffected by the experimental group. CONCLUSION: These results suggest that an HGF-expressing adenovirus may be therapeutic for keloids by increasing MMP-1 expression.

Meprin α and meprin ß: Procollagen proteinases in health and disease.

Metalloproteases meprin α and meprin ß were recently discovered as procollagen proteinases, capable of cleaving off the globular C- and N-terminal prodomains of fibrillar collagen type I and type III. This proteolytic process is indeed sufficient to induce collagen fibril assembly as visualized by transmission electron microscopy. The biological relevance was demonstrated with the help of meprin α and meprin ß knock-out mice, which exhibit decreased collagen deposition in skin resulting in impaired tensile strength. On the other hand, overexpression of meprin metalloproteases was found under fibrotic conditions in the skin (keloids) and the lung (pulmonary hypertension). Thus, regulation of meprin activity by specific inhibition to reduce collagen maturation might be a suitable approach for the treatment of certain pathological conditions.

Role of chymase in the local renin-angiotensin system in keloids: inhibition of chymase may be an effective therapeutic approach to treat keloids.

BACKGROUND: Histologically, keloids contain excess fibroblasts and an overabundance of dermal collagen. Recently, it was reported that chymase induced a profibrotic response via transforming growth factor-ß1 (TGF-ß1)/Smad activation in keloid fibroblasts (KFs). However, the role of chymase in the local renin-angiotensin system (RAS) in keloids has not been elucidated. This study aims to determine whether chymase plays an important role in the local RAS in keloids. METHODS: We compared the expression and activity of chymase in keloids and normal skin tissues using Western blotting and radioimmunoassay, and studied the expression of TGF-ß1, interleukin-1ß, collagen I, hydroxyproline, and angiotensin II in KFs after chymase and inhibitors' treatment. RESULTS: The results revealed an increased activity of chymase in keloid tissues, and that chymase enhanced the expression of angiotensin II, collagen I, TGF-ß1, and interleukin-1ß in KFs. Blockade of the chymase pathway involved in the local RAS lowered the expression of these signaling factors. CONCLUSION: This research suggests that inhibition of chymase might be an effective therapeutic approach to improve the clinical treatment of keloids.

PKC412 induces apoptosis through a caspase-dependent mechanism in human keloid-derived fibroblasts.

There is no established pharmacological therapy for skin keloids, a wound healing disorder. In this study, we investigated the effect of N-benzoyl staurosporine (PKC412), a protein kinase C inhibitor, on human keloid-derived fibroblasts to examine whether this agent is applicable for the treatment of keloid formation. Although PKC412 induced apoptosis in keloid fibroblasts in a time- and dose-dependent manner, the effective concentration of this agent was much higher than that of staurosporine. Western blotting showed that both PKC412 (10 microM) and staurosporine (100 nM) cleaved pro-caspase-3 to active forms. An in vitro caspase assay also showed that PKC412 and staurosporine elevated caspase-3 activities. Carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), a caspase inhibitor with a broad spectrum, inhibited caspase-3 activities stimulated by PKC412 and staurosporine; however, only PKC412-induced apoptosis, but not staurosporine-induced apoptosis, was prevented by Z-VAD-FMK. These results suggested that PKC412-induced apoptosis, but not staurosporine-induced apoptosis, is mainly mediated by the caspase-dependent mechanism.

Expression of urokinase-type plasminogen activator and its receptor in keloids.

BACKGROUND: The urokinase-mediated plasminogen activation (uPA) system plays a central role in a number of cellular processes including tissue remodeling, cell migration, and angiogenesis. Elevated uPA activity has also been seen with tumor invasion and metastasis in a variety of malignancies. Keloids represent an aberrant form of wound healing characterized by uncontrolled growth with invasion beyond the margins of the original wound. The regulation of this cellular process remains poorly understood. We hypothesize that keloids will have increased staining percentage for uPA and its receptor (uPAR) compared with normal scars. To our knowledge, no previous studies have examined the relationship of uPAR in keloid formation. DESIGN: Analysis of uPAR expression by immunohistochemistry in paraffin sections from 20 keloids and 18 normal scars. Expression was graded by a dermatopathologist according to percentage of cells staining for uPAR. SETTING: University Medical Center (Division of Otolaryngology-Head and Neck Surgery) and the Department of Dermatology at the University of Rochester Medical and Dental School, Rochester, NY. RESULTS: Of the 20 keloids, 8 (40%) strongly expressed uPAR (>50% of cells), while only 4 (22%) of 18 normal scars had similar staining. More than half of the normal scars stained minimally for uPAR (<5% staining). There was a strong expression of uPAR in the extracellular matrix in 14 (70%) of the 20 keloids, while no scar showed uPAR in the extracellular matrix. CONCLUSION: Our observation suggests that the uPA system is involved in the expansion of keloids beyond the wound margins in part through the degradation of the extracellular matrix, a finding that is supported by the strong expression of uPAR in the extracellular matrix and collagenous cords in most keloids studied.