[Show abstract][Hide abstract] ABSTRACT: Keloid disease (KD) is a common fibroproliferative disorder of unknown etiopathogenesis. Its unique occurrence in human skin and lack of animal models pose challenges for KD research. The lack of a suitable model in KD and over-reliance on cell culture has hampered the progress in developing new treatments. Therefore, we evaluated the effect of two promising candidate antifibrotic therapies: (-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) silencing in a long-term human keloid organ culture (OC). Four millimeters of air-liquid interface (ALI) keloid explants on collagen gel matrix in serum-free medium (n=8 cases) were treated with different modalities (EGCG treatment; PAI-1 knockdown by short interfering RNA (siRNA) and application of dexamethasone (DEX) as control). Normal skin (n=6) was used as control (only for D0 keloid-untreated comparison). Besides routine histology and quantitative (immuno-) histomorphometry, the key phenotypic and growth parameters of KD were assessed. Results demonstrated that EGCG reduced keloid volume significantly (40% by week 4), increased apoptosis (≥40% from weeks 1 to 4), and decreased proliferation (≤17% in week 2). EGCG induced epidermal shrinkage, reduced collagen-I and -III at mRNA and protein levels, depleted 98% of keloid-associated mast cells, and reduced the percentage of both cellularity and blood vessel count by week 4. Knockdown of PAI-1 significantly reduced keloid volume by 28% in week 4, respectively, and reduced collagen-I and -III at both mRNA and protein levels. As expected, DEX increased keloid apoptosis, decreased keloid proliferation, and collagen synthesis, but induced connective tissue growth factor overexpression. In conclusion, using keloid OC model, we provide the first functional evidence for testing candidate antifibrotic compounds in KD. We show that EGCG and PAI-1 silencing effectively inhibits growth and induces shrinkage of human keloid tissue in situ. Therefore, the application of EGCG, PAI-1 silencing, and other emerging compounds tested using this model may provide effective treatment and potentially aid in the prevention of recurrence of KD following surgery.Laboratory Investigation advance online publication, 8 July 2013; doi:10.1038/labinvest.2013.82.
[Show abstract][Hide abstract] ABSTRACT: IDENTIFICATION OF A POTENTIAL PATHOLOGICAL DIAGNOSTIC BIOMARKER FOR KELOID DISEASE: SYNDECAN-1 (CD138) IS UNIQUELY OVEREXPRESSED IN KELOID TISSUE
R. A. Bagabir1,2, F. Syed1,2, R. Paus2,3, A. Bayat1,2
1Plastic & Reconstructive Surgery Research, Manchester Institute of Biotechnology, Manchester, United Kingdom;
2Dermatological Sciences, Manchester, United Kingdom; 3Dermatology, Luebeck, Germany
Keloid disease (KD) is a benign fibroproliferative dermal disease, which progres- sively grows beyond the wound boundary. To date, there is no clear understanding of KD pathogenesis. Interestingly, while investigating the presence of plasma cells in KD, we unexpectedly identified an increased expression of Syndecan-1 in KD. Syndecan-1 (CD138) is a cell surface proteoglycan, which is also shed by the expressing cells in the extracellular matrix. Syndecan-1 is expressed in scarred neonatal wound, but its lack of expression in scarless fetal wound may suggest a potential association between Syndecan-1 and fibrosis in scar formation. It is believed that Syndecan-1 has a role in cell proliferation, migration and tumor progression in many human cancers such as breast cancer. Most importantly, its involvement in keratinocytes proliferation in wound healing and regulation of proteolytic balance in wound bed as evident by the low expression in ulcers. Hence, the aim of this work was to assess the expression pattern of Syndican-1 in keloid disease tissue and fibroblasts compared to normal skin, hypertrophic scar and normal skin scar using gene and protein quantitative analyses. Immunohistochem- istry and in-cell Western blotting were performed to assess the expression of Syn- dican-1. Our results showed overexpression of Syndican-1 in 65 keloid tissue samples compared to 11 controls. Interestingly, keloid cross-sections revealed an intensive immune-reactivity of Syndecan-1 within the keloid reticular dermis, whereas the normal skin surrounding the lesion showed lack of expression. Synde- can-1 expression was also significantly (p < 0.05) overexpressed in keloid primary fibroblasts (n = 6) compared to normal skin (n = 6) and normal scar (n = 6) fibro- blasts. In conclusion, this study shows for the first time that Syndican-1 is signifi- cantly overexpressed in keloid tissue, which could be involved in the progression of fibrogenesis in KD. Further studies are underway to examine the impact of Syndecan-1 knockdown in KD pathogenesis.
No preview · Article · Mar 2013 · Wound Repair and Regeneration
[Show abstract][Hide abstract] ABSTRACT: Summary Background Keloid disease (KD) is a common fibroproliferative disorder of unknown aetiology. T cells and macrophages are increased in KD and are thought to contribute to its pathogenesis. However, while a link between inflammation and fibrotic disorders is well known for other disorders, it remains undetermined in KD.
Objectives Systematically to immunophenotype the inflammatory infiltrate of KD in situ in a site-specific manner, and to compare this with normal skin and scar tissue.
Methods Sixty-eight keloid cases were screened for the presence of all three (intralesional, perilesional and extralesional) keloid-associated specific tissue sites. Subsequently, a complete set of 25 keloid biopsies (from different patients) was compared with normal skin (n = 11) and normal scar (n = 11) samples and subjected to systematic, site-specific quantitative immunohistomorphometry and histochemistry, using a range of immunological markers of B cells, T cells, macrophages, mast cells (MCs) and Langerhans cells.
Results T cells, B cells, degranulated and mature MCs (coexpressing OX40 ligand) and alternative macrophages (M2) were all significantly increased in intralesional and perilesional KD sites compared with normal skin and scar tissue (P < 0·05). Additionally, 10 of 68 KD cases (15%) showed the presence of distinctive lymphoid aggregates, which resembled mucosa-associated lymphoid tissue (MALT).
Conclusions The increased number and activity of MCs and M2 may implicate inflammation in the fibrotic process in KD. The distinct KD-associated lymphoid aggregate resembles MALT, for which we propose the term ‘keloid-associated lymphoid tissue’ (KALT). It may perpetuate inflammatory stimuli that promote KD growth. KALT, MCs and M2 are promising novel targets for future KD therapy.
No preview · Article · Nov 2012 · British Journal of Dermatology
[Show abstract][Hide abstract] ABSTRACT: Keloid disease (KD) is a common fibroproliferative disorder of unknown aetiopathogenesis, with highly unsatisfactory treatment. Therefore, it is crucial to have a robust and clinically relevant model for studying KD pathobiology as well as preclinical testing of potential KD therapeutics. However, the unique occurrence of KD in human skin and the corresponding lack of animal models pose a major challenge in KD research. Therefore, we developed a simplified assay for the serum-free, long-term organ culture of KD tissue that facilitates quantitative analyses of major KD read-out parameters. Four millimetre KD punches embedded in a collagen matrix and organ-cultured at the epidermis air-liquid interphase (ALI) in supplemented William's E medium showed optimal tissue, cell and RNA preservation for up to 6 weeks (as measured by H & E and Pyronin Y histochemistry as well as by MTT assay, lactate dehydrogenase release and quantitative Ki67/TUNEL immunohistomorphometry). The keloid phenotype persisted well during this period, as shown by collagen-I and -III synthesis (Herovici's histochemistry staining and ELISA), and analysis of the expression of significant KD markers (CD3, CD20, CD31, CD34, CD56, tryptase, Langerin, vimentin, neutrophil elastase, CTGF and Collagen). To functionally evaluate whether this assay can test the response to candidate therapeutics, dexamethasone, a glucocorticosteroid often used in KD therapy, was administered. Indeed, dexamethasone significantly reduced the keloid volume and cellularity plus induced epidermal shrinkage. Therefore, this novel assay provides a quantitative, clinically relevant model system for studying KD pathobiology and response to treatment.
Full-text · Article · May 2012 · Experimental Dermatology
[Show abstract][Hide abstract] ABSTRACT: SITE-SPECIFIC IMMUNOPHENOTYPING OF KELOID DISEASE REVEALS THE PRESENCE OF KELOID-ASSOCIATED LYMPHOID TISSUE (KALT)
R. A. Bagabir1,3, R. Byers6, I. Chaudhry6, W. Muller5, R. Paus3,4, A. Bayat1,2,3 1Plastic and Reconstructive Surgery Research, Manchester, Manchester, United Kingdom;
2Manchester Academic Health Science Centre, Manchester, Manchester, United Kingdom;
3Inflammation Science Research Group, Manchester, Manchester, United Kingdom;
4Department of Dermatology, Luebeck, Luebeck, Germany;
5Faculty of Life Sciences, Manchester, Manchester, United Kingdom; 6School of Cancer and Enabling Sciences, Manchester, Manchester, United Kingdom
The link between inflammation and fibrotic disorders, such as hypertrophic scars and scleroderma, is already well known. Even though, there is accumulating indirect evidence of the presence of chronic inflammation in keloid disease (KD), the nature of these inflammatory infiltrates has not been previously characterized. Therefore, the aim of this study was to systematically immunophenotype the inflammatory infiltrates of KD in situ in a strictly site-specific manner, and to compare this to normal scar tissue and healthy human skin. KD samples were excised from 68 patients to closely examine the presence of distinctive immune phenomena. Of these, 25 KD samples permitted examination of all three KD sites (intra-, peri-, extralesional). These samples were subjected to systematic, site-specific quantita- tive immunohistochemistry and enzyme-histochemistry to characterize inflamma- tory infiltrates in detail, using a battery of immunological markers. The data were subjected to quantitative immunohistomorphometry and multispectral image analy- sis, and then were compared with healthy human skin and scar tissue. T-cells (CD3+, CD4+, CD8+), B-cells (CD20+) and mast cells (tryptase+/ckit+ and OX40 ligand), and alternative (M2 using CD163, MHC II) macrophages were all signifi- cantly increased (p<0.05) in intra- and perilesional KD sites compared to normal skin and scar. Interestingly, 14.7% of cases (n = 68) showed the presence of distinc- tive lymphoid aggregates (LAs), which resembled tertiary lymphoid tissue. These LAs were not restricted to microhistological or KD lesional sites. In conclusion, we provide the first evidence of a significant increase in mast cells and alternative (M2) macrophages in KD. Moreover, we report distinct KD-associated lymphoid aggregates, for which we propose the term “keloid-associated lymphoid tissue” (KALT). These findings raise the question whether KALT, along with excessive macrophage and mast cell activities, perpetuates pro-inflammatory stimuli that promote KD tumor growth and may, thus, be a novel target for future KD therapy
[Show abstract][Hide abstract] ABSTRACT: A NOVEL LONG-TERM HUMAN ORGAN CULTURE MODEL IN KELOID DISEASE DEMONSTRATES IN-SITU FUNCTIONAL ANALYSIS AND EVALUATES POTENTIAL THERAPEUTIC COMPOUNDS
R. A. Bagabir1,2, F. Syed1,2, R. Paus2,3, A. Bayat1,2,4
1Plastic & Reconstructive Surgery Research, Manchester; 2Inflammation Science Group, School of Translational, Manchester; 3Experimental Dermatology, Luebeck; 4Department of Plastic And Reconstructive Surgery, Manchester
The unique occurrence of keloid in human skin and the corresponding lack of an animal model pose major challenges in keloid research. Therefore, we aimed to develop a novel long-term keloid organ culture (OC), which preserves the native keloid architecture. The functionality of keloid OC as a future in-situ model was assessed using different candidate drugs. Different parameters of keloid explants including size, medium and collagen matrix gel embedding strategy were assessed using histological, immunological and biochemical (in- cluding LDH, MTT assay) techniques to identify the optimum conditions to maintain keloid architecture. The optimised conditions were then applied in keloid OC (n = 10) to test traditional and novel candidate drugs including: dexamethasone (Decadron), green tea extract [(-)-epigallocatechin-3-gallate (EGCG)] and plasminogen activator inhibitor-1(PAI-1) siRNA knockdown. The effect of these drugs on collagen (I and III) production, fibrosis-related gene expression, apoptosis and proliferation were assessed at different time- points using quantitative real-time PCR (QRT-PCR), ELIZA and quantitative immunohistochemistry. In addition, the weight of keloid OC post treatment was measured to evaluate the degree of keloid shrinkage. 4mm air-exposed keloid OC embedded in a supplemented media preserved the keloid architecture and phenotype for up to 6 weeks. Treatment with dexamethasone or EGCG showed significant reduction in keloid volume, cellularity and vascularity, additionally both agents significantly increased epidermal shrinkage (p o 0.05) and keloid apoptosis (p 0.01). EGCG significantly reduced collagen (p o 0.02) levels, fibrosis-related gene expression and keloid-associated mast cell number. Similar results were achieved with dexamethasone. However, dexamethasone significantly increased connective tissue growth factor expres- sion (CTGF) (p 0.01), and decreased collagen only at mRNA expression. The PAI-1 siRNA- knock-down reduced collagen production significantly (p o 0.03). The results obtained from the candidate drugs are in agreement with the published in-vivo results, indicating that Keloid OC provides an in- structive, quantitative and clinically relevant in-situ model to study keloid pathobiology and future potential drugs
[Show abstract][Hide abstract] ABSTRACT: UP-REGULATION OF TOLL-LIKE RECEPTORS (TLR) 6, 7 AND 8 IN KELOID SCARS: DOES KELOID DISEASE REFLECT AN ABNORMAL INNATE IMMUNE RESPONSE TO VIRAL INFECTION?
R. A. Bagabir1,4, F. Syed1,4, R. Rautemaa6, D. A. McGrouther1,2, R. Paus4,5, A. Bayat1,2,4
1Plastic And Reconstructive Surgery Research, Manchester; 2Department of Plastic And Reconstructive Surgery, Manchester; 4Inflammation Science Group, Manchester; 5Experimental Dermatology, Luebeck; 6Respiratory Research Group, Manchester
Keloid is a benign fibroproliferative dermal tumour that develops in genetically susceptible individuals. Keloid commonly occurs following cutaneous trauma, which is likely to facilitate inoculation of infectious agents. It has been repeatedly observed that keloid develops after clinical evidence of viral infection such as chicken pox and zoster. On this basis, Alonso (2008) speculated that keloid is in- duced by a viral infection. However, research has failed to identify certain viral particles such as Human Herpesvirus-8 (HHV8) and Epstin Barr Virus (EBV) in keloid. Therefore, our objective was to find indirect evidence of viral encounter by the skin immune system in keloid. We specifically compared the expression of toll- like receptors known to recognise single or double-stranded RNA and DNA viral particles i.e. TLRs 2, 3 and 6–9. Quantitative real-time RT-PCR (QRT-PCR), followed by fluorescence-immunohistochemistry (F-IHC) were conducted to ex- amine the expression of these receptors at carefully defined sites within and out- side keloid. The relative gene expression of TLRs within the keloid showed significant up regulation of TLR8 (p o 0.05) and TLR9 (p 0.04). However, the morphometric analysis of F-IHC demonstrated significant increase in TLR8 (p o 0.05) immunoreactivity in the dermis, TLR6 (p = 0.01) and TLR7 (p 0.02) in the epidermis in the intra- and peri-lesional sites of keloid corre- sponding with the percentage of positive cells. The up regulation of TLRs known to recognize viral particles i.e. TLR6-8 in keloid lesions demonstrated here for the first time appears to favour the hypothesis, that the skin innate immune system in keloid shows a heightened responsiveness towards virus-infection. However, it is uncertain which viral ligands are responsible for the observed up-regulation of TLR 6, 7 and TLR 8, and it also remains to be further investigated whether non- viral ligands of TLR 6-8 may influence on keloid aetiopathogenesis