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.

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.

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.

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.

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.

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.

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.

Inhibition of hyaluronan synthase 2 reduces the abnormal migration rate of keloid keratinocytes.

Keloids are fibroproliferative scars that spread beyond the original wound boundary and are very resistant to treatment. Development of highly effective therapies requires a comprehensive understanding of the mechanisms regulating keloid formation. Previous studies indicated that keloid keratinocytes have abnormal expression of genes involved in differentiation and adhesion, and increased migration rates. The objective of the current study was to better understand the role of hyaluronan synthase 2 (HAS2) in keloid keratinocyte migration and gene expression. Keratinocytes were isolated from keloid scars and normal skin. Migration rates of keloid keratinocytes were quantified using an in vitro scratch assay. Expression levels of HAS2, related HAS1, and HAS3 genes, and genes aberrantly expressed in keloid keratinocytes, were quantified using real-time polymerase chain reaction. Treatment with 4-methylumbelliferone (4MU) was used to inhibit hyaluronic acid synthesis. The expression of HAS2 was significantly increased in keloid vs normal keratinocytes. Treatment with 4MU caused a dose-dependent reduction in keloid keratinocyte migration and HAS2 expression; HAS3 expression was moderately inhibited by 4MU and HAS1 was not expressed. Keloid keratinocytes displayed a motile phenotype in vitro, including loose colonies and widely separated refractile cells; this phenotype was normalized by 4MU. Further, 4MU altered gene expression in keloid keratinocytes. The results suggest that HAS2 overexpression contributes to increased migration and altered gene expression in keloid keratinocytes. Abnormal keratinocyte migration may contribute to the overhealing of keloid scars beyond the original wound boundaries. Therefore, inhibition of HAS2 expression using 4MU may represent a novel strategy for treatment of keloid scarring.

Tissue engineering for in vitro analysis of matrix metalloproteinases in the pathogenesis of keloid lesions.

IMPORTANCE: Keloid lesions form because of alterations in the mechanisms that govern cutaneous wound healing. Although matrix metalloproteinases (MMPs) have been implicated in keloid pathophysiology, many questions still remain about their involvement. Our incomplete understanding of keloid pathophysiology has led to high recurrence rates in current treatments. No reliable animal model is available for studying keloids. OBJECTIVE: To gain a better understanding of the disease mechanisms involved in keloid lesions in the hopes of identifying therapeutic options. DESIGN: Fibroblasts derived from keloid tissue were incorporated in either Matrigel or polyethylene glycol diacrylate mixed with type I collagen to create 3-dimensional models to investigate the role MMPs play in keloid formation. The MMP gene expressions were also compared between fibroblasts isolated from different sites within the same keloid lesion. SETTING: The Johns Hopkins School of Medicine, Baltimore, Maryland. PARTICIPANTS: Keloid fibroblasts were received from the Baylor College of Medicine, and additional keloid fibroblasts were enzymatically isolated from the dermal layer of lesions removed from consenting patients at The Johns Hopkins Hospital. RESULTS: In the Matrigel system, MMP9 and MMP13 were observed to be significantly upregulated in keloid fibroblasts. The addition of decorin resulted in a significant decrease of type I collagen and MMP1, MMP9, and MMP13 gene expressions from keloid fibroblasts. Higher MMP gene expressions were observed in fibroblasts isolated from the margins of the original keloid wound. CONCLUSIONS AND RELEVANCE: MMP9 and MMP13 are expressed significantly more in keloid-derived cells, thus making them 2 potential targets for disease modification. Molecules that target organization of the lesion's matrix can be beneficial in downregulating increased markers during the disease. In addition, heterogeneity is observed with the varying MMP gene expressions from site-specific fibroblasts within the same keloid lesion.

Thalidomide inhibits fibronectin production in TGF-ß1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway.

Keloids are characterized by the vigorously continuous production of extracellular matrix protein and aberrant cytokine activity in the dermis. There is a growing body of evidence that thalidomide, α-N-phthalimidoglutarimide, has anti-fibrotic properties. The aims were to examine possible therapeutic effects of thalidomide on fibronectin expression in transforming growth factor-ß1 (TGF-ß1)-treated normal fibroblasts (NFs) and keloid-derived fibroblasts (KFs) and the underlying mechanism of action, especially the involvement of mitogen-activated protein kinase (MAPKs) and Sma- and Mad-related family (Smads) pathways. In surgically removed human tissues, TGF-ß1 and fibronectin immunoreactivity was high in keloid tissue, but barely detectable in normal tissue. TGF-ß1 induced significant fibronectin expression in NFs and KFs and the effect was inhibited by pretreatment with thalidomide. TGF-ß1 also induced phosphorylation of MAPKs (ERK1/2, p38, and JNK) and Smad2/3 and pretreatment with PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor) inhibited TGF-ß1-induced fibronectin expression. Furthermore, pretreatment with thalidomide inhibited the TGF-ß1-induced phosphorylation of p38 and Smad3, but not that of ERK1/2, JNK, and Smad2. In addition, thalidomide pretreatment inhibited the TGF-ß-induced DNA binding activity of AP-1 and Smad3/4, caused fibronectin degradation by increasing the activity of matrix metalloproteinase 9, and decreased production of TGF-ß1 and fibronectin and the number of fibroblasts in an in vivo keloid model. These results show that thalidomide has an antifibrotic effect on keloid fibroblasts that is caused by suppression of TGF-ß1-induced p38 and Smad3 signaling. Our findings indicate that thalidomide may be a potential candidate drug for the treatment and prevention of keloids.

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.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with different types of scars and keloids.

BACKGROUND: Hypertrophic scars and keloids are fibroproliferative skin disorders characterised by progressive deposition of collagen. Our study is designed to investigate the expression and concentration of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in different types of scars and keloids. METHODS: Total RNA from 19 proliferative hypertrophic scar samples of patients with extended burns (total body surface area (TBSA): 21+/-12%), 18 mature hypertrophic scar samples from patients after elective surgery, 14 keloid samples and 18 normotrophic scar samples was, respectively, extracted, and then mRNA was isolated. Besides, biopsies were obtained from non-scarred skin of the patients and extraction of total RNA performed. Relative mRNA expression of MMP 2, MMP 9, TIMP 1 and TIMP 2 was measured with reverse transcriptase polymerase chain reaction (RT-PCR). Serum concentrations of MMP-1, -2, -9, TIMP-1, and -2 were determined using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Patients with extended hypertrophic scars after burn trauma presented a significantly higher TIMP-1 concentration (p<0.05) in their sera than the other patients. The relative expression of MMP 2 was significantly higher in samples of proliferative hypertrophic scars after burn injury. The relative expression of TIMP 1 and TIMP 2 was significantly higher in scar tissue of patients with proliferative and mature hypertrophic scars and keloids than in their regular skin and in scar samples of patients with normotrophic scars. The expression of TIMP 1 was significantly higher in samples of patients with keloids than in patients with hypertrophic scars. CONCLUSIONS: The concentration of TIMP-1 in sera of patients varies depending on the size of the involved fibrotic scar tissue. A decrease in MMP-to-TIMP expression in scar tissue may contribute to increased synthesis and deposition of collagen, leading to a severe fibrotic reaction with pathologic scar formation. The results implicate non-operative therapy options in these patients that not only down-regulate TIMPs but also increase the activity of MMPs.

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).

An in situ and in vitro investigation for the transglutaminase potential in tissue engineering.

Transglutaminases (TGases) constitute a family of enzymes that stabilize protein assemblies by gamma-glutamyl-epsilon-lysine crosslinks. The role of tissue transglutaminase (TGase 2) in several pathophysiologies, wound healing applications, biomaterials functionalization, and drug delivery systems provides grounds for its use in tissue engineering. Herein, we initially studied the endogenous TGase activity and expression under normal (skin, duodenum, colon, and small bowel) and pathophysiological (keloid scar) conditions on cadaveric human tissues. Successful inhibition was achieved using low concentrations of BOC-DON-QIV-OMe (0.1 mM and 1 mM for normal skin and keloid scar, respectively), iodoacetamide (0.1 mM and 1 mM for normal skin and keloid scar, respectively), and cystamine dihydrochloride (1 mM and 10 mM for normal skin and keloid scar, respectively), whilst di-BOC-cystamine was found ineffective even at 100 mM concentration. Secondly, the addition of exogenous guinea pig liver transglutaminase (gpTGase) onto the inhibited tissues and collagen scaffolds was studied, and results presented advocate its use as potential tissue adhesive and drug delivery tool. However, the investigation of its crosslinking extent using second harmonic generation microscopy and differentially scanning calorimetry revealed rather poor stabilization function. Overall, our study indicates that TGase 2 has a role as a biological glue to consolidate various micro-structural components of tissues and biomaterials.

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.