Human Keloid Research Articles

Tumor-Like Stem Cells Derived from Human Keloid Are Governed by the Inflammatory Niche Driven by IL-17/IL-6 Axis

BackgroundAlterations in the stem cell niche are likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we use keloid, an exuberant fibroproliferative dermal growth unique to human skin, as a model to characterize benign tumor-like stem cells and delineate the role of their “pathological” niche in the development of the benign tumor.Methods and FindingsSubclonal assay, flow cytometric and multipotent differentiation analyses demonstrate that keloid contains a new population of stem cells, named keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. KPCs display elevated telomerase activity and an inherently upregulated proliferation capability as compared to their peripheral normal skin counterparts. A robust elevation of IL-6 and IL-17 expression in keloid is confirmed by cytokine array, western blot and ELISA analyses. The altered biological functions are tightly regulated by the inflammatory niche mediated by an autocrine/paracrine cytokine IL-17/IL-6 axis. Utilizing KPCs transplanted subcutaneously in immunocompromised mice we generate for the first time a human keloid-like tumor model that is driven by the in vivo inflammatory niche and allows testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, specifically IL-6 and IL-17.Conclusions/SignificanceThese findings support our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of KPCs characterized by the uncontrolled self-renewal and increased proliferation, supporting the rationale for in vivo modification of the “pathological” stem cell niche as a novel therapy for keloid and other mesenchymal benign tumors.

Telomere shortening may be associated with human keloids

BackgroundKeloids 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.MethodsWe 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.ResultsIn 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.ConclusionTelomere 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.

Equine lumican (LUM) cDNA sequence and spatio‐temporal expression in an experimental model of normal and pathological wound healing

The development of exuberant granulation tissue, a situation that in some ways resembles the human keloid, compromises both the aesthetic and functional outcomes of wound repair in horses. To help elucidate the underlying molecular mechanisms the spatio-temporal expression of lumican (LUM) mRNA and protein for their potential contributions to tissue remodelling of body and limb wounds, was examined in an established experimental model. Expression was studied in intact skin and in samples of 1-, 2-, 3-, 4- and 6-week-old wounds of the body and forelimb. Temporal gene expression was determined by reverse transcriptase polymerase chain reaction, and protein expression was mapped immunohistochemically. A significant increase in LUM mRNA expression was observed in response to wounding at both anatomical locations, and a significantly higher mRNA level was recorded in thoracic than in limb wounds at weeks 1, 3 and 6 of repair. The immunohistochemical observations partially corroborated the mRNA data. To the authors' knowledge this study is the first to document that the cDNA for LUM is expressed over the different phases of wound repair in horses and suggests that LUM might be involved in both inflammation and remodelling in response to dermal injury. Further studies are now required to verify and quantify the temporal expression of this protein to provide the basis for targeted therapies that might prevent the development of exuberant granulation tissue in horse wound repair.

Differential apoptosis markers in human keloids and hypertrophic scars fibroblasts

Keloids are benign skin tumors and are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are characterized by formation of excess scar tissue beyond the boundaries of the wound. Keloids are often confused with hypertrophic scars because of an apparent lack of morphologic differences. The molecular distinction between scars and keloid is still controversial and, until today, there is no appropriate treatment yet for keloid disease. In this study, we have found, for the first time, p53 mutations in both hypertrophic scar and keloids fibroblasts from cultured cells to various extents. Since p53 plays a central role in the DNA damage response by inducing cell cycle arrest and/or apoptotic cell death, we also set up time course experiments making cell cultures at different times to investigate the phenomenon of apoptosis and its involvement in the process of pathological scarring in both hypertrophic scars and keloids. The extent of apoptosis in this study was investigated by DNA fragmentation and MTT assays, propidium iodide staining, p53 expression, and subcellular distribution. Moreover, the correlation of apoptosis and ROS levels in keloid and hypertrophic scars fibroblasts was assessed. Understanding the molecular mechanisms that determine the regulation of apoptosis during wound healing might allow us to therapeutically modulate these pathways so that apoptotic cell death is reactivated in dysregulated and hypertrophic cells.

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.

Effects of recombinant adenovirus-mediated double suicide genes on implanted human keloid: experiment with athymic mice

To detect the effects of the recombinant adenovirus-mediated double suicide genes constructed by Escherichia coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) and herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV)-CDglyTK on implanted human keloids and mechanisms thereof. Twenty nude mice were implanted with human keloid obtained during operation so as to establish mouse keloid models and then were randomly divided into 4 equal groups: Group A, injected with normal saline (NS) into the keloid once per 3 days for 18 days totally, Group B injected with NS into the keloid and injected intraperitoneally with 5-Fc and GCV; Group C injected with CDglyTK into the keloid, and Group D injected with CDglyTK into the keloid and 5-Fc and GCV injected intraperitoneally. The volume of the implanted keloid tissue was measured 2, 7, 14, 21, 28, 35, and 42 days after operation. On day 42 the keloid tissues were removed to undergo morphological examination, TUNEL method was used to examine the apoptosis of the fibroblasts, and the expression of Bcl-2 and BAX, products of apoptosis-related genes, were detected by immunohistochemistry. Compared to those before treatment the volume of the implanted keloid of Group D began to decrease since 14 days after treatment time-dependently (all P < 0.05), and the volumes of the other 3 groups continued to increase and peaked on days 21, 14, or 7 respectively (all P < 0.05). Microscopy showed infiltration of a larger quantity of histiocyte in the keloid tissue, and more obvious collagen disorganization and apoptosis of fibroblasts in Group D than in the other 3 groups. The protein expression of Bcl-2 was more remarkable and the protein expression of BAX was less remarkable in Group D than in the other 3 groups. The recombinant adenovirus-mediated double suicide gene therapy is effective on the implanted keloid tissue. The main mechanism may be induction of apoptosis in the keloid fibroblasts.

Construction and identification of replication recombinent adenovirus vector of human TGF-β1 shRNA

Objective To construct the recombinant adenovirus vector with human gene TGF-β1 (shRNA,short hairpin RNA)by Cre/lox P system.Methods Human U6 promoters following TGF-β1 shRNA(U6shTGF-β1)were obtained with PCR.Then the promoters were ligated to T-vector.After plasmids pT-U6shTGF-β1 being formed.these plasmids were transformed into E.coli JM109.The target gene fragment was subcloned into a shuttle plasmid PDC316 to construct recombinant shuttle plasmid PDC3 16-TGF-β1.Then the combinant shuttle plasmid and adenovirus genomic plasmid pBHGlox△E1 and 3Cre were cotransfected into 293T cell to construct recombinant adenovirus AdTGF-β1,which was then identified by infection test and PCR amplification.Results U6shTGF-β1 was obtained.The sequence of cloned target fragment was completely consistent with designed sequence.After purification and concentration,the titer of AdTGF-β1 reached 107.3 TCID50/ml.Ad TGF-β1 could transfect 293T cell.Both adenovirus and TGF-β1 target gene fragment could be amplified from rAdTGF-β1 by PCR.Conclusions The recombinant adenovirus expression vector of TGF-β1 and control gene are successfully constructed.This study establishes a foundation for further study on TGF-β1 RNAi vaccines and new gene therapy for human keloid and hypertrophic scar. Key words: Transforming growth factor-β1; Adenovirus vector; RNA interference; Gene therapy; Cicatrix

Equine ANXA2 and MMP1 expression analyses in an experimental model of normal and pathological wound repair

Wounds on horse limbs can develop exuberant granulation tissue which resembles the human keloid. Clues gained from the study of over-scarring in horses might help control fibro-proliferative disorders. The aim of the present study was to clone full-length equine ANXA2 cDNA then to study spatio-temporal expression of ANXA2 and MMP1 mRNA and protein, potential contributors to remodeling, during repair of body (normal) and limb (fibro-proliferative) wounds in an established horse wound model. Cloning of ANXA2 was achieved by screening size-selected cDNA libraries. Expression was studied in intact skin and in biopsies of 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal gene expression was determined by semi-quantitative RT-PCR while protein expression was mapped immunohistochemically. ANXA2 mRNA was up-regulated only in body wounds, corroborating the superior and prompt tissue turnover at this location. Immunohistochemistry partially substantiated the mRNA data in that increased staining for ANXA2 protein was detected in neo-epidermis which formed more rapidly and completely in body wounds. MMP1 mRNA levels in body wounds significantly surpassed those of limb wounds in week one biopsies. The protein was abundant in migrating epithelium of limb wounds at weeks two and four; conversely, body wounds in which epithelialization was near complete showed diminished staining of MMP1. We conclude that ANXA2 and MMP1 might participate in remodeling during wound healing in the horse, and that differences in expression may contribute to the excessive proliferative response seen in the limb.

Pharmakologische Keloidtherapie im Tiermodell

In spite of the high incidence of dermal fibroproliferative disorders, there is no agreement about the treatment of choice. Due to the inability of animals to produce keloid tissue, a standardised model to study the effects of different treatment modalities in vivo is lacking. Therefore, a comparative study on the effect of three pharmacological agents was conducted with human keloid implants in an athymic mouse model. Cubic keloid tissue blocks from 10 human volunteers were implanted in 54 male, athymic, homozygotic mice. The animals were divided into 4 groups, including an untreated control group. Members of each section received either colchicine, nicardipine or triamcinolone applied transdermally into the keloid tissue or into the peritoneum. The tissue specimens of 5 mice each were explanted according to a predetermined time schedule on days 28, 42 and 56 post-implantation and examined using various histological techniques including standard dye and immune histochemistry. The freeze-dried and moist weights of the keloid tissue were determined and analysed. Statistically significant changes regarding declining weight parameters were seen in the colchicines-treated group. Moreover, the densities of fibroblasts and endothelial cells were significantly reduced through colchicines treatment when compared to the control group and the groups treated with the other agents. The triamcinolone group also showed partially significant changes of weight compared to the control group, whereas no statistically significant effect of nicardipine on any parameter was found. Any influence of the host organism could be excluded as there were no signs of rejection or lymphocytic infiltration. Our study represents a successful attempt to create a standardised model for a comparative investigation on keloid tissue in vivo. The effect of colchicine was demonstrated in the light of an inhibitory effect on fibroblastic proliferative activity. The studied model allows a direct comparison of implanted, vascularised keloid tissue and its reactivity to various agents without being biased by numerous unknown variables in humans.

Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair.

BackgroundWound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to CTNNB1 (coding for β-catenin) and PECAM1, genes potentially contributing to the proliferative phase of repair, were previously identified in a mRNA expression study as being up-regulated in 7 day wound biopsies from horses. The aim of the present study was to clone full-length equine CTNNB1 and PECAM1 cDNAs and to study the spatio-temporal expression of mRNAs and corresponding proteins during repair of body and limb wounds in a horse model.ResultsThe temporal pattern of the two genes was similar; except for CTNNB1 in limb wounds, wounding caused up-regulation of mRNA which did not return to baseline by the end of the study. Relative over-expression of both CTNNB1 and PECAM1 mRNA was noted in body wounds compared to limb wounds. Immunostaining for both β-catenin and PECAM1 was principally observed in endothelial cells and fibroblasts and was especially pronounced in wounds having developed exuberant granulation tissue.ConclusionThis study is the first to characterize equine cDNA for CTNNB1 and PECAM1 and to document that these genes are expressed during wound repair in horses. It appears that β-catenin may be regulated in a post-transcriptional manner while PECAM1 might help thoracic wounds mount an efficient inflammatory response in contrast to what is observed in limb wounds. Furthermore, data from this study suggest that β-catenin and PECAM1 might interact to modulate endothelial cell and fibroblast proliferation during wound repair in the horse.

Nondestructive Imaging of Live Human Keloid and Facial Tissue Using Multiphoton Microscopy

To use multiphoton microscopy to image collagen fibers and matrix structure in nonfixed human keloid tissue and normal human facial skin obtained following surgery and to compare the findings to existing knowledge of normal skin and keloid morphology to determine if this technology is a suitable adjunct for conventional histology. Epidermis was removed to expose the fibroblast-rich dermal layer that was then imaged using a multiphoton confocal microscope (Zeiss-Meta 510; Carl Zeiss, Jena, Germany). An 800-nm tunable titanium/sapphire femtosecond laser (Mai-Tai; Newport Co Spectra-Physics, Mountain View, California) was used to excite the tissue; second harmonic generation between 397 and 408 nm and autofluorescent signals were collected. Images were obtained using a Plan-Neofluar x40 oil immersion objective lens and a Plan-Apochromat x63 oil immersion lens. Compared with normal skin, keloids showed disorganized collagen fibers arranged in complex swirls and bundles 20 to 30 microm in diameter. Normal tissue showed collagen fibers as distinct, straight strands less than 10 microm in diameter. Differences between normal and keloid tissue were subtle but apparent. The value of imaging living tissue is a significant benefit. Because keloids and hypertrophic scars result from altered collagen metabolism, the development of clinical multiphoton microscopy systems may allow examination of wound healing dynamics in vivo and potentially provides a means to monitor therapy without the need for biopsy or the risk of injury to tissue.

Differential p63 and p53 Expression in Human Keloid Fibroblasts and Hypertrophic Scar Fibroblasts

The p63 gene belongs to the p53 gene family and encodes for sequence-specific transcription factors. p63 has been characterized primarily in the context of epidermis where is implicated in the establishment of keratinocyte cell fate and in maintenance of epithelial self-renewal. DeltaNp63 isoform has been showed to be involved in several kinds of human tumors of epidermal origin, even nonmalignant, for the neoplastic and proliferative potential. Here, we report the differential expression and the cellular localization of the DeltaNp63 isoform in fibroblasts isolated from human keloids and hypertrophic scars compared to normal skin. Differently from hypertrophic scar, our results show that DeltaNp63 has a nuclear localization and is overexpressed only in keloid fibroblasts, suggesting an essential role of DeltaNp63 in vivo in human keloids. Consistent with our results, we hypothesize that DeltaNp63 overexpression may be oncogenic because of its ability to block the activity of p53 since p53 is underexpressed in fibroblasts from keloids.

Green Tea Extract and (−)-Epigallocatechin-3-Gallate Inhibit Mast Cell-Stimulated Type I Collagen Expression in Keloid Fibroblasts via Blocking PI-3K/Akt Signaling Pathways

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs), and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green tea extract (GTE) and its major catechin, (-)-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phosphatidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic diseases.

Bioenergetics in human Keloid and normal fibroblasts

Bioenergetics in human Keloid and normal fibroblasts

Assessment of Morphological and Immunohistological Alterations in Long-Term Keloid Skin Explants

One of the major impediments in keloid research is the lack of a keloid animal model that can mimic human keloid. This imposes investigative constraints on studying cellular interactions and biochemical processes that normally occur in vivo.Our main objective is to establish an in vitro model for maintaining long-term viable keloid dermal explants as a tool for investigating the pathogenesis of keloid scar formation. Explants of adult keloid scars were cultured in vitro by embedding them in enriched collagen gel matrix and maintaining them for up to 6 weeks, whereupon changes in tissue morphology and cellular differentiation were examined. The effects of medium enrichment, air versus liquid submersion, and different substrates on the explants were examined. Our results indicated that keloid explants embedded in a collagen gel matrix were morphologically better preserved than explants placed on a plastic substrate. Explants with epidermis at the air-liquid interface had better morphology than collagen-submerged explants, and there were no differences between serum-free and serum-supplemented explant cultures. Immunohistochemical and apoptotic analyses were performed to assess cellular viability and differentiation. In situ hybridization confirmed that keloid fibroblasts had sustained collagen type I gene expression throughout the 6 weeks in culture, thus validating the integrity of a long-term keloid culture system. In conclusion, the collagen-embedded skin explant system demonstrates that keloid tissues could be maintained for up to 6 weeks for long-term in vitro studies.

Accumulation of glucose in keloids with FDG-PET

Glucose metabolism has not been investigated in human (in vivo) keloids. In the present study, we performed positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (FDG) to examine glucose metabolism in keloids. Five patients (2 men and 3 women) with typical keloids having a thickness of more than 5 mm were studied. HEADTOME-IV SET-1400W-10 (Shimadzu, Tokyo, Japan) was employed for PET studies. Transmission scanning was performed on each patient. After fasting for more than 4 hours, the patients were injected intravenously with FDG 185-370 (MBq) following each transmission scan. Emission scans were performed 40-55 min after injection. For quantitative evaluation, the regions of interest (Circles ROIs: 6 mm in diameter) were placed on all the keloid lesions and surrounding tissues, and then their standardized uptake value (SUV = the tissue concentration/the activity injected per body weight) was calculated. FDG was defined as showing the accumulation in keloids when its uptake was relatively higher in the keloid than that in the surrounding tissue. The SUV of the keloids ranged from 1.0 to 2.74, with a mean of 1.79. FDG-PET was performed in 5 patients with keloids and low-grade accumulation of FDG was observed in all lesions. This indicated that glucose metabolism was accelerated in keloids.

Gene expression in human keloids is altered from dermal to chondrocytic and osteogenic lineage

Keloids are a dermal fibrotic disease whose etiology remains totally unknown and for which there is no successful treatment. Here, we employed cDNA microarray analysis to examine gene expression in keloid lesions and control skin. We found that 32 genes among the 9000 tested were strongly up-regulated in keloid lesions, of which 21 were confirmed by Northern blotting. These included at least seven chondrocyte/osteoblast marker genes, and RT-PCR analysis revealed that transcription factors specific for these genes, SOX9 and CBFA1, were induced. Immunostaining and in situ hybridization further supported that these markers are expressed in keloid lesions. Intriguingly, scleraxis, a transcription factor known as a marker of tendons and ligaments, was also induced in keloid fibroblasts. We propose that reprogramming of gene expression or disordered differentiation from a dermal pattern to that of a chondrocytic/osteogenic lineage, probably closer to that of tendon/ligament lineage, may be involved in the etiology of keloids.

Hamster (Mesocricetus auratus) cheek pouch as an experimental model to investigate human skin and keloid heterologous graft

To describe the integration process of grafts of total human skin and keloid in hamster (Mesocricetus auratus) cheek pouch, whose sub-epithelium is naturally an "Immunologically Privileged Site". Fragments of human normal skin and keloid from the breast region of mulatto female patients were transplanted into the cheek pouch subepithelium in situ. Surgical procedure and grafted pouches for microscopic exam at several time points of the transplantation were standardized. The integration of grafted fragments of human skin and keloid was seen in late periods (84 days) since the microscopic assessment showed the presence of blood vases within the conjunctive tissue of grafted fragments. It was also possible to see among the grafted fragments the epithelium, the appearing of early cellular infiltrated, epithelial secretion of keratin, the presence of melanocytes, and delayed changes on the aspect of collagen fibers of conjunctive tissue. Pooled results allow to define hamster cheek pouch sub-epithelium as an experimental model to investigating heterologous graft physiology of human total skin and keloid with epithelium.

ROLE OF PHENERGAN IN ABNORMAL SCARS AND KELOIDS

Hypertrophic scar and keloids have affected patients and frustrated physicians for centuries. Hypertrophic scar (HSc) and keloids are a major problem for patients who survive extensive thermal and traumatic skin injuries. HSc and other fibroproliferative disorders are associated with excessive accumulation of collagen and extracellular matrix proteins due to an imbalance between synthesis and degradation. The therapeutic management of hypertrophic scars and keloids include occlusive dressings, compression therapy, intralesional corticosteroid injections, cryosurgery, excision, radiation therapy, laser therapy, interferon therapy and other promising lesser known therapies directed at collagen synthesis. In this study we investigated the effect of phenergan (promethazine hydrochloride) as one of the most potent histamine antagonists on cell proliferation, DNA synthesis and collagen production in fibroblast isolated from human post burn hypertrophic scar, keloids and normal skin. The proliferation of normal skin fibroblast was slightly decreased but hypertrophic scar and keloids showed significant (p&lt;0.001) level of decrease after 72 hours of phenergan (750 μM) treatment. The results of DNA synthesis also significantly (p&lt;0.001) decreased in hypertrophic scar and keloid fibroblasts. Phenergan (1.5 mM) decreased the collagen synthesis upto 61% and 66% in HSc and keloids in comparison to normal skin fibroblast, which showed reduction of 38% after 72 hours. Improved understanding of such regulatory mechanisms may eventually be of therapeutic significance in the control of hypertrophic scar and keloids.

Molecular characterization and expression of p63 isoforms in human keloids.

Keloids are benign skin tumors that develop following wounding. A cDNA product from human keloid specimens was identified using the differential display technique. The full-length cDNA was cloned by RT-PCR using human keloid mRNA as template. The predicted product of the cDNA was found to be 99% identical to the DeltaN-p63 gamma isotype of p63, a transcription factor that belongs to the family that includes the structurally related tumor suppressor p53 and p73. The DeltaN-p63 isotype lacks the acidic N terminal region corresponding to the transactivation domain of p53. Since this can potentially block p53-mediated target gene transactivation, it may serve as a dominant-negative isoform. Real-Time RT-PCR analysis of RNAs from normal skin tissue and keloids showed that the DeltaN-p63 isotype is specifically expressed in keloids, but is virtually undetectable in normal skin. Immunostaining of p63 in normal skin revealed that only basal cells of the epithelium expressed the protein, while in keloid tissues the antigen was detected in the nuclei of cells scattered through all layers of the epithelium and in fibroblast-like cells in the dermis. These results may indicate that aberrant p63 expression plays a role not only in malignant tumors but also in benign skin diseases that show hyperproliferation of epidermal cells in vivo. Moreover, this isoform of p63 could serve as a specific molecular marker for this human disease.