Keloid Disease Research Articles

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

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.

Potent Dual Inhibitors of TORC1 and TORC2 Complexes (KU-0063794 and KU-0068650) Demonstrate In Vitro and Ex Vivo Anti-Keloid Scar Activity

Mammalian target of rapamycin (mTOR) is essential in controlling several cellular functions. This pathway is dysregulated in keloid disease (KD). KD is a common fibroproliferative dermal lesion with an ill-defined treatment strategy. KD demonstrates excessive matrix deposition, angiogenesis, and inflammatory cell infiltration. In KD, both total and phosphorylated forms of mTOR and p70S6K(Thr421/Ser424) are upregulated. Therefore, the aim of this study was to investigate adenosine triphosphate–competitive inhibitors of mTOR kinase previously unreported in keloid and their comparative efficacy with Rapamycin. Here, we present two mTOR kinase inhibitors, KU-0063794 and KU-0068650, that target both mTORC1 and mTORC2 signaling. Treatment with either KU-0063794 or KU-0068650 resulted in complete suppression of Akt, mTORC1, and mTORC2, and inhibition of keloid cell spreading, proliferation, migration, and invasive properties at a very low concentration (2.5 μmol l−1). Both KU-0063794 and KU-0068650 significantly (P<0.05) inhibited cell cycle regulation and HIF1-α expression compared with that achieved with Rapamycin alone. In addition, both compounds induced shrinkage and growth arrest in KD, associated with the inhibition of angiogenesis, induction of apoptosis, and reduction in keloid phenotype–associated markers. In contrast, Rapamycin induced minimal antitumor activity. In conclusion, potent dual mTORC1 and mTORC2 inhibitors display therapeutic potential for the treatment of KD.

Altered expression of hyaluronan synthase and hyaluronidase mRNA may affect hyaluronic acid distribution in keloid disease compared with normal skin

Keloid disease (KD) is a fibroproliferative disorder characterised partly by an altered extracellular matrix (ECM) profile. In fetal scarring, hyaluronic acid (HA) expression is increased, but is reduced in KD tissue compared with normal skin (NS). The expression of Hyaluronan Synthase (HAS) and hyaluronidase (HYAL) in KD and NS tissue were investigated for the first time using a range of techniques. Hyaluronan synthase and HYAL mRNA expression were significantly increased in NS tissue compared with KD tissue (P < 0.05). Immunohistological analysis of tissue indicated an accumulation of HAS and HYAL protein expression in KD compared with NS due to the thicker epidermis. No differences were observed in mRNA or protein expression in KD and NS fibroblasts. Reduced expression of HAS and HYAL may alter HA synthesis, degradation and accumulation in KD. Better understanding of the role of HA in KD may lead to novel therapeutic approaches to address the resulting ECM imbalance.

Identification of steroid sensitive responders versus non-responders in the treatment of keloid disease

Intralesional corticosteroid injection is a well-recognised treatment modality for keloid disease (KD). Approximately 50% of KD cases are considered non-responders (or steroid resistant) with no consensus or indicators in detecting steroid-sensitive cases. In view of the undesirable side effects, uncertainty in timing and regularity of steroid treatment, we planned to identify responders and non-responders to target treatment more effectively. A scar injection proforma was developed capturing a detailed history focusing on symptoms and signs (redness, appearance, contour, texture, distortion and severity) associated with KD. The cause, site, number of keloid scars and scar recurrence were recorded as the lesions were injected on a monthly basis. A detailed description of response to steroid injection was documented and photographs were taken. Demographic data were collected on 65 patients (11 to 74 years with mean age 34.7 years, 60% were females, 50% were Caucasian). 77% (n = 50) were classified as steroid responders and showed improvement in symptoms and signs within 3 months. There was a statistically significant correlation between patients with higher contour scores of KD prior to treatment (p = 0.013) and frequency of injections (p = 0.003). Thus, the odds of being a responder were greater for patients with more than one injection and with higher contour scores. This preliminary case series has provided early evidence in enabling identification of steroid responders versus non-responders within a 3-month period. Selection of KD non-responders to steroid treatment can avoid potentially painful injection, its subsequent side effects and unnecessary continuation of redundant therapy and follow-up.

Up-Regulation of Tension-Related Proteins in Keloids

Keloid disease is a fibroproliferative disorder, with an ill-defined treatment that is characterized by excessive extracellular matrix deposition. Mechanical tension promotes deposition of extracellular matrix and overexpression of tension-related proteins, which is associated with keloid disease. The aim of this study was to investigate the effect of tension-related proteins on extracellular matrix steady-state synthesis in primary keloid fibroblasts. Keloid fibroblasts (n = 10) and normal skin (n = 4) fibroblast cultures were established from passages 0 to 3. A panel of 21 tension-related genes from microarray data were assessed at mRNA (quantitative reverse-transcriptase polymerase chain reaction) and protein (in-cell Western blotting) levels. Three genes were significantly altered in keloid tissue and fibroblasts, and their functional role was assessed using siRNA knockdown. Hsp27, α2β1-integrin, and PAI-2 were significantly up-regulated (p < 0.05)in keloid tissue and fibroblasts compared with normal skin. Hsp27, α2β1-integrin, and PAI-2 expression was inhibited by RNA interference. Both the mRNA and protein levels of Hsp27, α2β1-integrin, and PAI-2 significantly decreased (p < 0.05) in keloid fibroblasts at 48 hours after transfection. After down-regulation of Hsp27, α2β1-integrin, and PAI-2, the expression of intracellular extracellular matrix was significantly reduced (p < 0.05). Water-soluble tetrazolium salt-1 assay showed that transfection of Hsp27, α2β1-integrin, and PAI-2 did not influence the viability/metabolic activity of keloid fibroblasts. This study demonstrates overexpression of key tension-related proteins in keloid tissue and keloid fibroblasts. Knockdown of Hsp27, PAI-2, and α2β1-integrin by RNA interference attenuates the expression of mRNA and protein levels and certain other extracellular matrix molecules.

Superior effect of combination vs. single steroid therapy in keloid disease: A comparative in vitro analysis of glucocorticoids

Keloid disease (KD) is a fibroproliferative disorder of unknown etiology. Current use of corticosteroid injection is partially beneficial with 80% recurrence rate. Additionally, the efficacy of different steroids, alone or in combination as opposed to monotherapy, in treating KD remains unclear. Here, we compared the single and combined efficacy of glucocorticoids-dexamethasone (Dex), triamcinolone (TAC), and methylprednisolone (Medrol)-on primary keloid fibroblasts (KFs) (n = 27) and normal skin (n = 19) fibroblasts at cellular, protein, and messenger RNA levels in vitro. Our results demonstrated that cytotoxicity to steroids was dose dependent. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced by Medrol and TAC. Migration and invasion properties of KF were inhibited significantly (p < 0.05) by Medrol and TAC compared with Dex. At both protein and messenger RNA levels, keloid-associated fibrotic markers were significantly (p < 0.05) decreased by Medrol and TAC compared with Dex. However, vascular endothelial growth factor expression was significantly (p = 0.01) decreased by Dex compared with TAC and Medrol. Medrol and TAC caused significant (p < 0.04) apoptosis, whereas Dex inhibited the UV-induced apoptosis and up-regulated survivin. Blocking of glucocorticoid receptor by RU486 inhibited cytoprotective property of Dex and apoptotic properties of TAC and Medrol. Double treatment with Dex + TAC and Dex + Medrol significantly (p < 0.05) induced apoptosis. In conclusion, this is the first study to report the efficacy of three well-known steroids on KF and suggest that combination may be superior than using a single steroid in treating KD.

Photodynamic therapy: an innovative approach to the treatment of keloid disease evaluated using subjective and objective non-invasive tools

Optimal management for keloid disease (KD) is ill defined, with surgical excision resulting in recurrence rates over 50%. Photodynamic therapy (PDT) uses light to activate a photosensitiser localised in diseased tissues. Two recent case studies and in vitro studies on keloid-derived fibroblasts indicate potential use of PDT in treating KD. Therefore, we hypothesized that there may be a role for PDT in the treatment of KD. Twenty KD patients were divided into three groups; existing keloid scar, post-surgical debulking and post-total surgical excision. Patients underwent three treatments of PDT at weekly intervals. Methyl aminolevulinate photosensitiser applied 3h prior to PDT, administered at 37J/cm(2). Non-invasive measures provided quantitative data for pliability, haemoglobin, melanin, collagen and flux. Pain and pruritus scores were measured and patients' were monitored for KD recurrence. All patients had reduced pain and pruritus scores. Haemoglobin flux (p=0.032), collagen (p=0.066) and haemoglobin levels (p=0.060) decreased from week 1 to 3 in all except one patient where measurements were taken and pliability increased significantly (p=0.001). Increases in pliability were significantly related to decreases in flux (p=0.001). Only one patient with a keloid in a stress-prone anatomical location experienced recurrence of KD. All other patients had no recurrence at 9-month follow-up. Minimal side effects were reported. In conclusion, PDT reduces scar formation in KD evidenced by decreased blood flow, increased pliability, decreased collagen and haemoglobin levels. These findings indicate potential utility of PDT in the treatment of KD.

Site-specific immunophenotyping of keloid disease demonstrates immune upregulation and the presence of lymphoid aggregates

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. 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. 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. 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). 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.

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, GSK3β, 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.

Notch signaling pathway in keloid disease: Enhanced fibroblast activity in a Jagged‐1 peptide‐dependent manner in lesional vs. extralesional fibroblasts

Keloid disease (KD) is a fibroproliferative disorder of unknown etiopathogenesis with ill-defined treatment. There is increasing evidence to suggest that aberrant Notch signaling may contribute directly to skin pathogenesis and altered expression of Notch receptors identified in KD. Therefore, the aim of this study was to investigate the Notch signaling pathway in KD compared to normal skin (NS). In this study, we employed in vitro primary cell culture models to elucidate the role of Notch signaling in 44 tissue samples from patients with KD split into keloid and extralesional (EL) samples (internal control) from the same patients, and six NS tissue samples (external control). We show the presence of a significant (p < 0.05) up-regulation of Notch receptors and ligand Jagged-1 (JAG-1) in KD compared to EL and NS tissue samples. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced in JAG-1 antisense-treated primary dermal fibroblasts isolated from KD and treated with γ-secretase inhibitor (blocks proteolytic cleavage and activation of Notch), evaluated by real-time cell analyzer (RTCA) on a microelectronic sensory array. In contrast, extralesional skin fibroblasts (ELF) treated with recombinant human JAG-1 (rh-JAG-1) peptide showed significant (p < 0.05) enhancement of cell spreading, attachment, and proliferation in RTCA. Activation/inhibition of JAG-1 and Notch signaling significantly (p < 0.05) altered the behavior of primary keloid fibroblasts and ELF, in cell migration (using a scratch wound assay), invasion (using a 3D invasion assay), and angiogenesis (in vitro coculture tube formation assay). In conclusion, this is the first study to demonstrate a potential role for the Notch signaling pathway in KD progression and that targeting this pathway may provide a novel strategy for treatment of KD.

Chéloïdes palmaire et plantaire chez un Noir africain

The keloid scar is a fibrous skin tumor, intradermal, and exuberant. It is commonly found on the glabrous skin. The keloid of the palms and soles are rare. Small series are reported in English literature. The authors report a case of large keloids located on both palms and soles, within a context of keloid disease, in a man of 37 years. On the left hand, the keloid scar caused a partial syndactyly IV-V. Large keloid tumors occupied the inner edge and, weight-bearing areas of both feet. These tumors rendered wearing of shoes impossible and interfered with walking. The treatment consisted of total excision of palmar and plantar keloid tumors. The residual defects were covered by a total skin graft taken from the suprapubic region. The results were satisfactory aesthetically, functionally and psychosocially.

Differential cytotoxic response in keloid fibroblasts exposed to photodynamic therapy is dependent on photosensitiser precursor, fluence and location of fibroblasts within the lesion

Treatment of keloid disease (KD) is ill-defined and remains challenging. We previously reported successful clinical application of photodynamic therapy (PDT) in KD. The aim here was to evaluate cytotoxic effect of PDT using methyl aminolevulinate (M-ALA) and 5-aminolevulinic acid (5-ALA) on keloid fibroblasts (KF) (n = 8) from different lesional sites (top, middle and margin) as compared to normal skin fibroblasts (n = 3). The effect of protoporphyrin IX (PpIX) precursors was evaluated by fluorescence emission, LDH and WST-1 assay, reactive oxygen species (ROS) generation and qRT-PCR analysis. Apoptosis/necrosis differentiation and senescence were studied by fluorometric staining with Hoechst 33258/propidium iodide and β-galactosidase activity, respectively. Three hours post incubation with 4 mM precursors of photosensitisers, PpIX accumulation was site specific and higher with M-ALA. Cytotoxicity was also site specific (higher in fibroblasts from middle of the keloid as compared to cells from other sites) and increased proportionately to fluence rates post-PDT. Additionally, cytoproliferation was significantly decreased post-PDT depending on the light energy. Fluorescence analysis revealed that M-ALA instigated higher KF cytotoxicity at lower fluence (≤20 J/cm(2)) while 5-ALA instigated higher KF cytotoxicity at higher fluence, except in cells derived from middle of the keloid. ROS-mediated cytotoxicity was light energy dependent. Senescence was not observed at higher light energies (>10 J/cm(2)). Compared to other sites, fibroblasts from the middle were more prone to cell death post 5-ALA treatment. We conclude that cytotoxicity post-PDT in KD fibroblasts is dependent on the lesional site, precursor of intracellular photosensitiser and fluence. Thus, PDT may be used for site-targeted therapy of KD.

Keloid Scarring: Understanding the Genetic Basis, Advances, and Prospects

Keloid disease is a fibroproliferative dermal tumor with an unknown etiology that occurs after a skin injury in genetically susceptible individuals. Increased familial aggregation, a higher prevalence in certain races, parallelism in identical twins, and alteration in gene expression all favor a remarkable genetic contribution to keloid pathology. It seems that the environment triggers the disease in genetically susceptible individuals. Several genes have been implicated in the etiology of keloid disease, but no single gene mutation has thus far been found to be responsible. Therefore, a combination of methods such as association, gene-gene interaction, epigenetics, linkage, gene expression, and protein analysis should be applied to determine keloid etiology.

Long‐term organ culture of keloid disease tissue

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.

Analyses of CDC2L1 gene mutations in keloid tissue

Keloid disease is the result of a deregulated wound-healing process. Loss of heterozygosity on chromosome 1p36 has been shown to be associated with keloid formation in humans. The cell division cycle 2-like 1 (CDC2L1) gene is known to be essential for eukaryotic cell-cycle control, and has also been mapped to 1p36. To verify the possible association between keloid disease and somatic mutation of the CDC2L1 gene on chromosome 1p36. Mutations of the CDC2L1 gene in keloid and healthy skin tissues were screened by denaturing high-performance liquid chromatography, and confirmed by DNA sequencing analysis. Of the 27 patients with keloid assessed, 21 had mutations. The most prevalent exon affected was exon 7, with 15 patients affected: 10 patients (37%) had a base G deletion at codon 247, and 12 patients (44.4%) had a base A insertion at codon 267 (6 patients (25.9%) had both mutations). The remaining six patients had mutations in exons 11 (codon 433; n = 3) and 14 (codon 520; n = 3). Comparing the keloid skin tissues with the healthy control skin tissues, significant differences were seen between the groups for the base G deletion at codon 247 and the base A insertion at codon 267. We have identified a correlation between two exon 7 mutations of the CDC2L1 gene and keloid disease. A further study of protein-kinase activity should be conducted to confirm the functionality of the CDC2L1 gene in the prevention of scar formation.

Comparative genomic hybridisation analysis of keloid tissue in Caucasians suggests possible involvement of HLA-DRB5 in disease pathogenesis

Keloid disease (KD) is a common fibroproliferative disorder that can occur following cutaneous injury in genetically susceptible individuals. Familial predisposition, high prevalence in certain populations and occurrence in twins suggest a strong genetic component to KD. However, to date no single causative gene has been identified. Copy number variations (CNVs) in genes have been associated with several human diseases including common skin disorders. The objective of this study was, therefore to determine if CNVs in the human genome may contribute to the development of KD. Agilent SurePrint G3 one Million microarrays were used to detect DNA copy number differences in keloid scars of four Caucasian females and compared to commercial reference DNA samples. Subsequent validation was performed with quantitative polymerase chain reactions (qPCR) using 15 KD cases and 27 Caucasian controls. Further validation using a second cohort was carried out with an additional 11 Caucasian controls and 10 KD cases developed from minor skin puncture wounds (caused by acne, vaccination or chickenpox). HLA-DRB1*15 was typed using allele-specific primers in PCR. Five CNV regions located at chromosome (chr) 6p21.32, chr11q11, chr17q12, chr8p23.1, chr22q13.1, chr19p13.1 and chr2q14.3 were selected for validation with qPCR. When comparing controls to subgroups of KD, according to their cause of scarring, chr6p21.32 (primer design targetting HLA-DRB5) was significantly (P<0.005) associated with KD developed from minor skin puncture wounds, which is further validated using a larger sample size (P<0.001). The presence of HLA-DRB5 was associated with HLA-DRB1*15 status; 18 out of 19 individuals were positive for both HLA-DRB5 and HLA-DRB1*15 allele. In conclusion, these preliminary findings further support the possible contribution of the HLA genes in KD pathogenesis.

Addition of novel degenerate electrical waveform stimulation with photodynamic therapy significantly enhances its cytotoxic effect in keloid fibroblasts: First report of a potential combination therapy

We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts. In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm(2) and 10J/cm(2) of red light (633 ± 3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts. The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed. PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL-PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P<0.05). The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy.

Extracellular matrix molecules implicated in hypertrophic and keloid scarring

Tissue regeneration repairs the fabric of the skin to maintain homeostasis after injury. The expression and proliferation of extracellular matrix (ECM) molecules in the dermis, mediated by a range of growth factors and cytokines, is a fundamental element of wound repair. Previous work focused on how these complex molecular mechanisms relate to the formation of raised dermal scars, including keloid and hypertrophic scars, characterized by excessive deposition of ECM molecules. However, the mechanisms in the wound repair pathway which lead to the differential expression and organization of ECM molecules observed in different types of scar tissue are not fully understood. To summarize what is known about the expression and composition of ECM molecules in abnormal scarring, an extensive search of the literature was conducted, focusing on keywords connected to skin scarring, hypertrophic scars and keloid disease. The transcription and translation of collagen I and III, fibronectin, laminin, periostin and tenascin are all increased in raised dermal scar tissue. However, hyaluronic acid, dermatopontin and decorin are decreased, and the expression and localisation of fibrillin and elastin fibres in the dermis are altered compared with normal skin and scars. Recent whole genome profiling and proteomic studies have led to the identification of regulatory elements with different expression profiles in hypertrophic and keloid tissue. If the mechanisms of raised dermal scar formation are to be elucidated and effective therapeutic treatments developed, an integrated approach to research is required, focussing on the interactions between ECM molecules, regulatory elements and pathways.

Assessment of the influence of HLA class I and class II loci on the prevalence of keloid disease in Jamaican Afro-Caribbeans

Keloid disease (KD) is a common abnormal cutaneous fibrotic disorder of unknown aetiopathogenesis. KD is reported to have a strong genetic component as it is often familial and has a high incidence in certain ethnicities, in particular those of Afro-Caribbean origin. Genetic risk factors combined with aberrant lesional inflammatory responses point to the human leukocyte antigen (HLA) system as a viable target for investigating disease aetiology. Sequence specific primer polymerase chain reaction with allele sequencing was used to determine HLA-DQA1 and DQB1 allele frequencies (AF) for 165 KD patients and 119 healthy controls of black Jamaican Afro-Caribbean origin. HLA class I alleles A*01, A*03, A*25, B*07 and Cw*08:02, previously identified as KD associated in a different ethnicity, were also analysed. Allele sequencing confirmed typing accuracy but no statistically significant differences in AF were identified between KD patients and controls. Furthermore, KD subgroups including patient gender, family history and multiple- or single-site scarring did not show significant allele-disease associations.

A Clinical Characterization of Familial Keloid Disease in Unique African Tribes Reveals Distinct Keloid Phenotypes

This study is a clinical characterization of keloid scars in an African population comprising three rural tribes with familial keloids. Site distribution, morphologic features, and other characteristics of the scars were studied to assess whether each tribe had a specific scar phenotype. Keloid scar clinics were set up at Soba Hospital in Khartoum, Sudan, for patient recruitment and management. In addition, familial keloid cases were recruited from rural tribal populations during field trips. A database including clinical and demographic data and digital photographs of all keloid cases was established. Statistical analysis was conducted using SPSS and SAS software. One hundred eleven individuals with keloid scarring (67 male subjects and 44 female subjects) were recruited. Patients were predominantly from three multigenerational pedigrees (total of 38 nuclear families) afflicted with keloid scars residing in different rural regions of Sudan. Two distinct morphologic phenotypes of keloid scarring were observed. The first phenotype has been designated "superficial spreading" (horizontal) keloid and the second has been designated "raised" (vertical) keloid. Clinically significant features and statistically measurable morphologic parameters were compared among these phenotypes (p = 0.001). Furthermore, linear claw-like extensions of keloid (transgression) were noted to be significantly higher in the superficial spreading keloid phenotype (p = 0.03). There is strong evidence of different phenotypes of keloid scarring. Two distinct phenotypes have been observed, described, and statistically verified. Each tribe demonstrated one particular phenotype, with two being superficial spreading and one being raised. Other significant clinical characteristics have been described. This is of significance in understanding both the clinical basis and the genetic basis of keloid scarring.