Interleukin-6 serum level and gene polymorphism in keloid patients.

The formation of keloid is associated with accumulation of extracellular matrix (ECM) formed mainly of collagen and fibronectin. Persistent deregulated IL-6 synthesis causes the development of various diseases. This study aim to investigate interleukin 6 (IL-6) serum level and gene polymorphism in a sample of Egyptian patients having keloid. This study was carried out on 90 subjects; 60 patients with keloid, and 30 age and sex matched apparently healthy control. All subjects underwent full history taking, clinical examinations, weight and length measuring to calculate BMI, dermatological examination, analysis of IL6-572 gene polymorphism using REFLP- PCR and IL-6 serum level using ELISA.IL-6 serum levels were significantly higher in keloid patients than control group (75.54±39.18) vs (19.17±6.06), (p <0.001). The higher serum levels of IL-6 were associated with GG genotype (104.84±19.12) followed by CG (57.64±35.38) genotype (P<0.001). GG genotype was significantly higher in keloid patients and increased the risk for keloid development by nearly14 folds (p<0.001, OR (95%CI) =13.81).  CG genotype was significantly observed in keloid patients and increased the risk for keloid development by about 4 times (p=0.010, OR (95%CI) =4.27). G Allele significantly increased the risk for keloid development by about 5 folds (P <0.001 OR =5.11). In conclusion, there was a great association between IL-6 572 gene polymorphism and its serum level in patients with keloid specifically who have family history.

Keloid patients have higher peripheral blood endothelial progenitor cell counts and CD34+ cells with normal vasculogenic and angiogenic function that overexpress vascular endothelial growth factor and interleukin-8.

BACKGROUND: One suggested reason for aberrant wound healing in keloid scars is chronic inflammation of the dermis. We hypothesized that excessive blood vessel formation and high capillary density in keloid tissue is caused by dysfunction of endothelial progenitor cells. METHODS: We compared the number of circulating endothelial progenitor cells and vasculogenic and angiogenic capacity, as well as secretory function, of circulating CD34+ cells in keloid patients and healthy individuals. RESULTS: Compared to mononuclear cell cultures from healthy donors, cultures of peripheral blood mononuclear cells obtained from keloid patients showed a more than twofold increase in the number of peripheral blood EPCs (fibronectin-adhering cells that phagocytized acetylated low-density lipoprotein and bound Ulex europaeus agglutinin-I lectin). However, there was no difference in colony-forming ability and participation in in vitro angiogenesis between circulating CD34+ cells isolated from keloid patients and healthy individuals. This means that circulating CD34+ /endothelial progenitor cells in keloid patients have normal vasculogenic and angiogenic function. However, CD34+ cells derived from keloid patients demonstrated a more than sevenfold expression of the interleukin-8 gene and a more than fivefold expression of the vascular endothelial growth factor gene than CD34+ cells derived from healthy individuals. CONCLUSIONS: These results support the role of vascular endothelial growth factor and interleukin-8 in increased recruitment of endothelial progenitor cells in keloid patients.

Evaluation of the Role of High-mobility Group Box 1 Protein in Patients With Keloids: A Case Control Study.

BACKGROUND: Keloids cause cosmetic problems, pain, and pruritus. Different modes of therapy are limited by their efficacy and side effects. High-mobility group box 1 protein (HMGB1) may play a role in keloid pathogenesis; therefore, the therapeutic potential of box A1, an antireceptor of advanced glycation end products antibody, and other inhibitors of HMGB1 may play a role in the treatment of keloids. OBJECTIVE: This study evaluates the role of HMGB1 in patients with keloids by comparing their serum level with healthy controls. MATERIALS AND METHODS: Forty patients with keloids and 40 controls were enrolled in this study. Detailed history and clinical evaluation were performed. A 3-mL sample of whole blood was obtained from both patient groups and centrifuged immediately. The resultant supernatant serum was frozen at -20°C for the detection and quantification of HMGB1 using enzyme-linked immunosorbent assay. RESULTS: There was a statistically significant elevation in the mean value of HMGB1 in keloid cases (74.38 ± 40.16) compared with the mean value of the controls (52.00 ± 5.41; P = .001). Mean value of HMGB1 was positively correlated with keloid severity. CONCLUSIONS: High-mobility group box 1 was found to be elevated in patients with keloids compared with their controls, suggesting its role in excessive scarring and the role of its antagonists in therapy.

Monocytes co-cultured with reconstructed keloid and normal skin models skew towards M2 macrophage phenotype.

Several abnormalities have been reported in the peripheral blood mononuclear cells of keloid-forming patients and particularly in the monocyte cell fraction. The goal of this in vitro study was to determine whether monocytes from keloid-prone patients contribute to the keloid phenotype in early developing keloids, and whether monocyte differentiation is affected by the keloid microenvironment. Therefore, keloid-derived keratinocytes and fibroblasts were used to reconstruct a full thickness, human, in vitro keloid scar model. The reconstructed keloid was co-cultured with monocytes from keloid-forming patients and compared to reconstructed normal skin co-cultured with monocytes from non-keloid-formers. The reconstructed keloid showed increased contraction, dermal thickness (trend) and α-SMA+ staining, but co-culture with monocytes did not further enhance the keloid phenotype. After 2-week culture, all monocytes switched from a CD11chigh/CD14high/CD68low to a CD11chigh/CD14low/CD68high phenotype. However, only monocytes co-cultured with either reconstructed keloid scar or normal skin models skewed towards the more fibrotic M2-macrophage phenotype. There was negligible fibroblast and fibrocyte differentiation in mono- and co-cultured monocytes. These results indicate that monocytes differentiate into M2 macrophages when in the vicinity of early regenerating and repairing tissue, independent of whether the individual is prone to normal or keloid scar formation.

Three-dimensional poly lactic-co-glycolic acid scaffold containing autologous platelet-rich plasma supports keloid fibroblast growth and contributes to keloid formation in a nude mouse model.

BACKGROUND: There is a lack of proper animal models to study keloid formation. AIM: To create three-dimensional poly lactic-co-glycolic acid (PLGA) scaffolds containing autologous platelet-rich plasma (PRP) as an in vitro culture environment for keloid fibroblasts (KL), and to study their implantation into nude mice to mimic the process of keloid formation. METHODS: Normal fibroblasts (FB) and KL cells were isolated from surgical specimens and transduced with lentivirus loaded with green fluorescent protein (GFP) and luciferase genes. The FB and KL cells were three-dimensionally cultured for 14-18days in PLGA scaffolds containing PRP. Ten mice were implanted with KL cells in their left forelimbs(KL), and FB-scaffolds (FB+PLGA) in their right forelimbs. An additional ten mice were implanted with PLGA scaffolds without cells (PLGA) in their left forelimbs, and KL-scaffolds (KL+PLGA) in their right forelimbs. Graft volume and collagen content were analyzed 120days after the implantation. RESULTS: in vivo luminescence cell imaging showed that the FB cells proliferated in the PLGA scaffolds within 60days after implantation, and reached a plateau afterwards until 120days after implantation. The KL cells continuously proliferated in the PLGA scaffolds until 120days after implantation. The KL+PLGA group showed higher graft volumes than the FB+PLGA group 120days after the implantation (median volume, 166.95 vs. 63.34mm3); however, the difference is not statistically significant (P=0.743), due to a large variation of the graft volume within each group. Furthermore, Sirius red staining revealed increased collagen I deposition, and immunohistochemistry showed large-scale accumulation of α-smooth muscle actin (α-SMA), collagen I, and collagen III in the KL+PLGA grafts. CONCLUSION: The three-dimensional PLGA scaffold containing PRP supports keloid fibroblast growth and contributes to keloid formation in a nude mouse model.

Bilateral breast keloids in an elderly woman associated with bilateral breast cancers and high concentration of serum tumor growth factor-ß.

We report a case of bilateral annular breast keloids in a 72-year-old woman who had been suffering from bilateral breast cancers. Histopathologically, the keloids showed unique distribution of α-SMA+, CD34- myofibroblasts and α-SMA-, CD34+ fibroblasts depending on the region. High serum levels of tumor growth factor-ß were detected at 6 months after the development of the breast keloids, but not at 10 months. CD163-positive cells were abundantly detected in the skin of the elevated portion of the keloids. In contrast, these cells were considerably less numerous in the skin of the central healing portion compared with the skin of the elevated expanding portion. One interesting idea based on these results is that high levels of tumor growth factor-ß released from CD163-positive cells played a crucial role in the formation of breast keloids through active induction of fibroblast differentiation into myofibroblasts. The present case strongly supports the previously proposed idea that keloids can form as a paraneoplastic phenomenon in breast cancer patients with keloid constitution.

Association of interleukin-6 gene polymorphisms and circulating levels with keloid scars in a Chinese Han population.

The aim of the present study is to explore the effect of IL-6 gene polymorphisms on the development of keloid scar (KS) in the Chinese Han population. Genotyping of IL-6 was performed by the polymerase chain reaction (PCR), followed by restriction fragment length polymorphism assays (PCR-RFLP). Serum level of IL-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results indicated that when the IL-6 -572 CC homozygote genotype was used as the reference group, the GG genotype was found to be associated with a significantly increased risk of KS (GG vs CC: OR = 2.097, 95%CI  = 1.100-3.995, P  =  0.025). When the IL-6 -572 C allele was used as the reference group, the G allele was found to be associated with significantly increased risk of KS (G vs C: OR =  1.317, 95%CI  = 1.002-1.730, P = 0.048). Furthermore, we observed a marked increase in serum IL-6 levels in KS patients with GG genotypes when compared to KS patients harboring the CC genotype. In conclusion, our results suggest that IL-6 gene polymorphism was associated with keloid scars in the southeastern Chinese Han population.

Association of plasminogen activator inhibitor-1 and vitamin D receptor expression with the risk of keloid disease in a Chinese population.

Keloid disease (KD) is a benign fibroproliferative scarring condition of unknown etiopathogenesis. Plasminogen activator inhibitor-1 (PAI-1) and vitamin D receptor (VDR) have been shown to play important roles in the progression of tissue fibrosis; therefore, both these genes are potential susceptibility genes for KD. We aimed to determine whether the gene expression levels of PAI-1 and VDR are altered in Chinese KD patients. We measured the expression of PAI and VDR in human peripheral blood lymphocytes in 236 patients with keloid and 219 age- and sex-matched healthy controls by quantitative real-time polymerase chain reaction. We found that PAI-1 expression in peripheral blood lymphocytes was significantly higher in patients with KD than in control individuals (p < 0.0001), while VDR expression was significantly lower in KD patients than in control individuals (p < 0.0001). High levels of PAI-1 and low levels of VDR expression were significantly associated with an increased risk for KD. PAI-1 and VDR might play important roles in keloid development. Gene expression levels of PAI-1 and VDR may, therefore, be used as potential markers for the prediction of keloid development after scarring.

Serum miRNAs Signature Plays an Important Role in Keloid Disease.

The molecular mechanism underlying the pathogenesis of keloid is largely unknown. MicroRNA (miRNA) is a class of small regulatory RNA that has emerged as a group of posttranscriptional gene repressors, participating in diverse pathophysiological processes of skin diseases. We investigated the expression profiles of miRNAs in the sera of patients to decipher the complicated factors involved in the development of keloid disease. MiRNA expression profiling in the sera from 9 keloid patients and 7 normal controls were characterized using a miRNA microarray containing established human mature and precursor miRNA sequences. Quantitative real-time PCR was performed to confirm the expression of miRNAs. The putative targets of differentially expressed miRNAs were functionally annotated by bioinformatics. MiRNA microarray analysis identified 37 differentially expressed miRNAs (17 upregulated and 20 downregulated) in keloid patients, compared to the healthy controls. Functional annotations revealed that the targets of those differentially expressed miRNAs were enriched in signaling pathways essential for scar formation and wound healing. The expression profiling of miRNAs is altered in the keloid, providing a clue for the molecular mechanisms underlying its initiation and progression. MiRNAs may partly contribute to the etiology of keloids by affecting the critical signaling pathways relevant to keloid pathogenesis.

Altered circulating endothelial progenitor cells in patients with keloid.

Evidence has suggested that vascular endothelial growth factor (VEGF), a crucial growth factor in regulating endothelial progenitor cells (EPCs), plays a central role in keloid formation. However, the levels of circulating EPCs in patients with keloid have not yet been explored. The aim of this study was to determine the number of circulating EPCs in patients with keloid. Circulating EPCs (defined as CD45- CD34+CD133+VEGFR2+cells) and VEGF levels from 39 patients with keloid and 22 healthy controls (HCs) were assessed by flow cytometry and ELISA, respectively. EPCs were detectable in the peripheral blood of patients with keloid. The number of circulating EPCs and the levels of plasma VEGF were significantly higher in patients with keloid than in HCs. However, no correlation was found between the number of circulating EPCs and the serum VEGF levels. This study provides the first evidence that EPCs are increased in the peripheral blood of patients with keloid. Understanding the roles of EPCs in keloid fromation may lead to the development of novel therapeutic strategies for keloid.

Contribution of hyperammonemia and inflammatory factors to cognitive impairment in minimal hepatic encephalopathy.

To assess the contribution of hyperammonemia and inflammation to induction of mild cognitive impairment (or MHE). We analyzed the presence of mild cognitive impairment (CI) by using the PHES battery of psychometric tests and measured the levels of ammonia and of the inflammatory cytokines IL-6 and IL-18 in blood of patients with different types of liver or dermatological diseases resulting in different grades of hyperammonemia and/or inflammation. The study included patients with 1) liver cirrhosis, showing hyperammonemia and inflammation; 2) non-alcoholic fatty liver disease (NAFLD) showing inflammation but not hyperammonemia; 3) non-alcoholic steatohepatitis (NASH) showing inflammation and very mild hyperammonemia; 4) psoriasis, showing inflammation but not hyperammonemia; 5) keloids, showing both inflammation and hyperammonemia and 6) controls without inflammation or hyperammonemia. The data reported show that in patients with liver diseases, cognitive impairment may appear before progression to cirrhosis if hyperammonemia and inflammation are high enough. Five out of 11 patients with NASH, without liver cirrhosis, showed cognitive impairment associated with hyperammonemia and inflammation. Patients with keloids showed cognitive impairment associated with hyperammonemia and inflammation, in the absence of liver disease. Hyperammonemia or inflammation alone did not induce CI but the combination of certain levels of hyperammonemia and inflammation is enough to induce CI, even without liver disease.

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.

[Establishment of immortalized lymphoblastoid cell bank of a keloid pedigree].

OBJECTIVE: To investigate a method for establishing immortalized lymphoblastoid cell bank of keloid pedigree so as to provide a long-term source of specimens for keloid research. METHODS: With Epstein-Barr virus transformation, fresh and frozen blood samples collected from all members of the keloid pedigree were used respectively to establish the immortalized lymphoblastoid cell lines of B lymphocytes. RESULTS: Twenty-seven immortalized lymphoblastoid cell lines of the keloid pedigree were obtained successfully, and all cell lines survived cryopreservation in liquid nitrogen. CONCLUSIONS: The immortalized lymphoblastoid cell bank of the keloid pedigree can preserve the whole gene information and provide long-term DNA sources for keloid research. Preparation of the cell lines with fresh blood is more efficient than that with frozen blood.

[Establishment of immortal lymphoblastoid cell bank of keloids pedigree].

OBJECTIVE: To provide perpetual research materials for long term studies by establishing immortal lymphoblastoid cell bank of keloids pedigree. METHODS: The immortal lymphoblastoid cell lines of keloids pedigree were established by Epstein-Barr virus transformation of peripheral blood B lymphocytes. RESULTS: 27 immortal lymphoblastoid cell lines of keloids pedigree were obtained successfully, all of the immortal lymphoblastoid cell lines were successfully revivificated after been frozen in liquid nitrogen. CONCLUSIONS: It is important to establish immortal lymphoblastoid cell bank of keloids pedigree and provide long-term DNA materials for deep study of keloids in the future.

Genetic susceptibility to keloid disease and hypertrophic scarring: transforming growth factor beta1 common polymorphisms and plasma levels.

Keloid disease and hypertrophic scars are dermal tumors that are often familial and typically occur in certain races. Their exact etiology is still unknown. Transforming growth factor beta1 (TGF-beta1) plays a central role in wound healing and fibrosis and has been implicated in the pathogenesis of keloid disease and hypertrophic scar. The aims of this study were to measure the plasma level of TGF-beta1 in patients compared with controls, and to investigate the association of five common single nucleotide polymorphisms in TGF-beta1 with the risk of keloid disease and hypertrophic scar formation. Platelet-poor plasma levels of TGF-beta1 in 60 patients (15 with hypertrophic scar and 45 with keloid disease) and 18 controls were measured using an enzyme-linked immunoabsorbent assay technique. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-beta1 polymorphisms. DNA samples from 133 patients (101 with keloid disease and 32 with hypertrophic scar) and 200 controls were examined. All patients and controls were Caucasians of Northern European extraction. There was no statistically significant difference in TGF-beta1 plasma levels between patients with keloid disease and hypertrophic scar and controls. There was also no statistically significant difference in genotype or allele frequency distributions between patients and controls for codons 10, 25, and 263 and for -509 and -800 single nucleotide polymorphisms of the TGF-beta1 gene. These results suggest that TGF-beta1 plasma levels and common polymorphisms are not associated with a risk of keloid disease and hypertrophic scar formation. This lack of association may be significant in view of the importance attached to the role of TGF-beta1 in dermal scarring. To the authors' knowledge, this is the first report of a case-control association study in keloid disease and hypertrophic scars using any single nucleotide polymorphisms.

Keloids in rural black South Africans. Part 2: dietary fatty acid intake and total phospholipid fatty acid profile in the blood of keloid patients.

In the second part of this study, emphasis is placed on nutritional intakes (fatty acids and micronutrients) and fatty acid intake and metabolism in the blood, respectively, according to a combined 24 h recall and standardized food frequency questionnaire analyses of keloid prone patients (n=10), compared with normal black South Africans (n=80), and total phospholipid blood (plasma and red blood cell ) analyses of keloid patients (n=20), compared with normal individuals (n=20). Lipid extraction and fractionation by standard procedures, total phospholipid (TPL) separation with thin layer chromatography, and fatty acid methyl ester analyses with gas liquid chromatography techniques were used. Since nutrition may play a role in several disease disorders, the purpose of this study was to confirm or refute a role for essential fatty acids (EFAs) in the hypothesis of keloid formations stated in part 1 of this study. (1)According to the Canadian recommendation (1991), we observed that in keloid patients linoleic acid (LA) and arachidonic acid (AA) dietary intakes, as EFAs of the omega-6-series, are higher than the recommended 7-11 g/d. However, the a-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) dietary intakes, as EFAs of the omega-3 series, are lower than the recommendation of 1.1-1.5 g/d. This was also the case in the control group, where a higher dietary intake of the omega-6 fatty acids and a slightly lower dietary intake of the omega-3 fatty acids occurred. Thus, we confirm a high dietary intake of LA (as a product of organ meats, diary products and many vegetable oils) and AA (as a product of meats and egg yolks), as well as lower dietary intakes of ALA (as a product of grains, green leafy vegetables, soy oil, rapeseed oil and linseed), and EPA and DHA (as products of marine oils). Lower micronutrient intakes than the recommended dietary allowances were observed in the keloid group that may influence EFA metabolism and/or collagen synthesis. Of cardinal importance may be the lower intake of calcium in the keloid patients that may contribute to abnormal cell signal transduction in fibroblasts and consequent collagen overproduction, and the lower copper intake that may influence the immune system, or perhaps even the high magnesium intake that stimulates metabolic activity. Micronutrient deficiencies also occurred in the diets of the normal black South Africans that served as a control group. In the case of plasma TPLs, deficiency of the omega-3 EFA series (ALA, EPA and DHA) occurred, and this is in accordance with the apparent lower omega-3 EFA intake in the diets of these patients. In the case of the red blood cell TPLs, as a true and reliable source of dietary fatty acid intake and metabolism, sufficient EFAs of the omega-6 series (LA and AA) and the omega-3 series (ALA, EPA and DHA) occurred. For this study group a relative deficiency of nutritional omega-3 EFA intake apparently did occur, but was probably compensated for by blood fatty acid metabolism.