A Methylome and Transcriptome Analysis of Normal Human Scar Cells Reveals a Role for FOXF2 in Scar Maintenance.

Scars are maintained for life and increase in size during periods of growth such as puberty. Epigenetic changes in fibroblasts after injury may underpin the maintenance and growth of scars. In this study, we combined methylome and transcriptome data from normotrophic mature scar and contralateral uninjured normal skin fibroblasts to identify potential regulators of scar maintenance. In total, 219 significantly differentially expressed and 1,199 significantly differentially methylated promoters were identified, of which there were 12 genes both significantly differentially methylated and expressed. Of these, the two transcription factors, FOXF2 and MKX, were selected for further analysis. Immunocytochemistry and qPCR suggested that FOXF2 but not MKX had elevated expression in scar fibroblasts. Using RNA sequencing, FOXF2 knockdown was shown to significantly reduce the expression of extracellular matrix‒related genes, whereas MKX did not appear to affect similar pathways. Finally, FOXF2 knockdown was also shown to significantly decrease collagen I production in scar and keloid fibroblasts. This study provides insights into the maintenance of normotrophic scar, suggesting that FOXF2 is an important regulator of this process. Targeting genes responsible for maintenance of scar phenotype may ameliorate scar appearance and improve patient outcomes in the future.

Secreted Factors from Keloid Keratinocytes Modulate Collagen Deposition by Fibroblasts from Normal and Fibrotic Tissue: A Pilot Study.

Interactions between keratinocytes and fibroblasts in the skin layers are crucial in normal tissue development, wound healing, and scarring. This study has investigated the role of keloid keratinocytes in regulating collagen production by primary fibroblasts in vitro. Keloid cells were obtained from removed patients' tissue whereas normal skin cells were discarded tissue obtained from elective surgery procedures. Fibroblasts and keratinocytes were isolated, cultured, and a transwell co-culture system were used to investigate the effect of keratinocytes on collagen production using a 'scar-in-a-jar' model. Keloid fibroblasts produced significantly more collagen than normal skin fibroblasts in monoculture at the RNA, secreted protein, and stable fibrillar protein level. When keloid keratinocytes were added to normal skin fibroblasts, expression of collagen was significantly upregulated in most samples, but when added to keloid fibroblasts, collagen I production was significantly reduced. Interestingly, keloid keratinocytes appear to decrease collagen production by keloid fibroblasts. This suggests that signaling in both keratinocytes and fibroblasts is disrupted in keloid pathology.

Identification of Differentially Methylated CpG Sites in Fibroblasts from Keloid Scars.

As a part of an abnormal healing process of dermal injuries and irritation, keloid scars arise on the skin as benign fibroproliferative tumors. Although the etiology of keloid scarring remains unsettled, considerable recent evidence suggested that keloidogenesis may be driven by epigenetic changes, particularly, DNA methylation. Therefore, genome-wide scanning of methylated cytosine-phosphoguanine (CpG) sites in extracted DNA from 12 keloid scar fibroblasts (KF) and 12 control skin fibroblasts (CF) (six normal skin fibroblasts and six normotrophic fibroblasts) was conducted using the Illumina Human Methylation 450K BeadChip in two replicates for each sample. Comparing KF and CF used a Linear Models for Microarray Data (Limma) model revealed 100,000 differentially methylated (DM) CpG sites, 20,695 of which were found to be hypomethylated and 79,305 were hypermethylated. The top DM CpG sites were associated with TNKS2, FAM45B, LOC723972, GAS7, RHBDD2 and CAMKK1. Subsequently, the most functionally enriched genes with the top 100 DM CpG sites were significantly (p ≤ 0.05) associated with SH2 domain binding, regulation of transcription, DNA-templated, nucleus, positive regulation of protein targeting to mitochondrion, nucleoplasm, Swr1 complex, histone exchange, and cellular response to organic substance. In addition, NLK, CAMKK1, LPAR2, CASP1, and NHS showed to be the most common regulators in the signaling network analysis. Taken together, these findings shed light on the methylation status of keloids that could be implicated in the underlying mechanism of keloid scars formation and remission.

Genetic influence on scar height and pliability after burn injury in individuals of European ancestry: A prospective cohort study.

After similar extent of injury there is considerable variability in scarring between individuals, in part due to genetic factors. This study aimed to identify genetic variants associated with scar height and pliability after burn injury. An exome-wide array association study and gene pathway analysis were performed on a prospective cohort of 665 patients treated for burn injury. Outcomes were scar height (SH) and scar pliability (SP) sub-scores of the modified Vancouver Scar Scale (mVSS). DNA was genotyped using the Infinium® HumanCoreExome-24 BeadChip. Associations between genetic variants (single nucleotide polymorphisms) and SH and SP were estimated using an additive genetic model adjusting for age, sex, number of surgical procedures and % total body surface area of burn in subjects of European ancestry. No individual genetic variants achieved the cut-off threshold of significance. Gene regions were analysed for spatially correlated single nucleotide polymorphisms and significant regions identified using comb-p software. This gene list was subject to gene pathway analysis to find which biological process terms were over-represented. Using this approach biological processes related to the nervous system and cell adhesion were the predominant gene pathways associated with both SH and SP. This study suggests genes associated with innervation may be important in scar fibrosis. Further studies using similar and larger datasets will be essential to validate these findings.

Loss of Type A neuronal cells in the dorsal root ganglion after a non-severe full-thickness burn injury in a rodent model.

Burn scars can be associated with significant loss of cutaneous sensation, paresthesia and chronic pain. Long-term systemic changes in cutaneous innervation may contribute to these symptoms and dorsal root ganglia have been implicated in the development of chronic neuropathic pain. Therefore we hypothesized that changes in cutaneous innervation after burn injury may be mediated at the level of the dorsal root ganglia. Burn group rats (n=20) were subjected to a unilateral burn injury while 12 control rats underwent sham procedure. The DRGs dermatomally related to the site of burn (Thoracic 13, lumbar 1 and lumbar 2), ipsilateral and contralateral to the injury, were compared for Type A, Type B and total cell number with sham control DRGs, at 4 and 6 weeks after injury. There was a significant decrease in Type A cell count (cell bodies of nerve fibres mediating touch-pressure-vibration sensation) in the 4 week time-point group (p=0.0124) ipsilateral to the burn injury. Total DRG cell count and Type B DRG cell count (cell bodies of fibres mediating pain and itch) on the ipsilateral side was not significantly altered. On the side contralateral to the burn injury, there was no statistically significant change in the total cell count, Type A cell count or Type B cell count. This data showed a decrease in Type A cell number in DRGs after a burn injury, suggesting cell death may mediate some changes observed in cutaneous innervation after a burn. Type B cells constituted a greater proportion of the viable cell population in the ipsilateral DRG after a burn injury. This change may be important in the induction of signalling related to pain and itch and has important implications for the restoration of normal cutaneous innervation after burn injury. Investigating whether neuro-protective or neuro-restorative approaches can ameliorate damage to the DRG will be important to improve sensory outcomes for burn patients.

Identification of factors predicting scar outcome after burn injury in children: a prospective case-control study.

BACKGROUND: There is a lack of rigorous research investigating the factors that influence scar outcome in children. Improved clinical decision-making to reduce the health burden due to post-burn scarring in children will be guided by evidence on risk factors and risk stratification. This study aimed to examine the association between selected patient, injury and clinical factors and the development of raised scar after burn injury. Novel patient factors were investigated including selected immunological co-morbidities (asthma, eczema and diabetes type 1 and type 2) and skin pigmentation (Fitzpatrick skin type). METHODS: A prospective case-control study was conducted among 186 children who sustained a burn injury in Western Australia. Logistic regression was used to explore the relationship between explanatory variables and a defined outcome measure: scar height measured by a modified Vancouver Scar Scale (mVSS). RESULTS: The overall correct prediction rate of the model was 80.6%; 80.9% for children with raised scars (>1 mm) and 80.4% for children without raised scars (≤1 mm). After adjustment for other variables, each 1% increase in % total body surface area (%TBSA) of burn increased the odds of raised scar by 15.8% (95% CI = 4.4-28.5%). Raised scar was also predicted by time to healing of longer than 14 days (OR = 11.621; 95% CI = 3.727-36.234) and multiple surgical procedures (OR = 11.521; 1.994-66.566). CONCLUSIONS: Greater burn surface area, time to healing of longer than 14 days, and multiple operations are independently associated with raised scar in children after burn injury. Scar prevention strategies should be targeted to children with these risk factors.

Identification of factors predicting scar outcome after burn in adults: A prospective case-control study.

This study examined influences on scarring after burn in a prospective study using a defined outcome measure: scar height measured by a modified Vancouver Scar Scale (mVSS). A prospective case-control study was conducted among 616 adult subjects who sustained a burn in Western Australia. Patient factors influencing scar outcome including gender, Fitzpatrick skin type and selected co-morbidities were explored, as well as injury and clinical factors. A logistic regression model for raised scar after burn was developed which achieved an overall correct prediction rate of 81.1%; 74.8% for those with raised scar and 86.0% for those without raised scar. From this study, injury and clinical predictors for raised scar after adjustment for other variables are: increasing %TBSA, greater burn depth as indicated by level of surgical intervention, wound complications and prolonged hospital stay. Intrinsic patient predictors for raised scar in patients with comparable injuries are: young age (≤30 years), female gender and Fitzpatrick skin types 4-6. The strength of association statistics (odds ratios and 95% confidence intervals) reported will be of practical benefit for clinical decision-making and counselling of patients, and plausible biological explanations for the findings support the validity of the results.

Verapamil is Less Effective than Triamcinolone for Prevention of Keloid Scar Recurrence After Excision in a Randomized Controlled Trial.

A double-blind randomized controlled trial with a paired split-scar design compared verapamil, an L-type Ca2+ channel antagonist, and triamcinolone for prevention of keloid recurrence after excision. Ca2+ channel blocking activity of verapamil in keloid cells was explored. One keloid was excised per subject and each wound half randomized to receive intralesional injections of triamcinolone (10 mg/ml) or verapamil (2.5 mg/ml) at monthly intervals (4 doses). Interim analysis was performed after 14 subjects were completed. Survival analysis demonstrated significantly higher keloid recurrence with verapamil compared to triamcinolone 12 months post-surgery (log-rank test, p = 0.01) and higher overall risk of recurrence with verapamil (hazard ratio 8.44, 95% CI 1.62-44.05). The study was terminated early according to the stopping guideline (p < 0.05). Verapamil is safe but not as effective as triamcinolone in preventing keloid recurrence after excision. Further study is necessary to determine if clinical response to verapamil is linked to modulation of intracellular Ca2+.

Descriptive Epidemiology of Unintentional Burn Injuries Admitted to a Tertiary-Level Government Hospital in Nepal: Gender-Specific Patterns.

This study describes the epidemiology of unintentional adult burn injury admissions in a tertiary hospital in Nepal, from 2002 to 2013, focusing on gender-specific patterns. Chi-square tests and Wilcoxon Rank Sum tests were performed. There were 819 unintentional burn admissions: 52% were male and 58% younger than 35 years. The median percentage total body surface area burned (interquartile range) was greater in females than in males (P < .001): 28% (17-40) versus 20% (12-35), and female mortality was higher (32% vs 11%). A higher proportion females were illiterate than males (48% vs 17%). Burns occurred at home (67%), work (28%), and public places (5%); gender-specific patterns were observed. Flame burns accounted for 77%, electricity 13%, and scalds 8%. Kerosene (31%) and biomass (27%) were the major fuels. Cooking, heating, and lighting were the main activities associated with burn injury. Results support interventions to reduce the use of open fires and kerosene and to promote electrical safety.

Intentional burns in Nepal: a comparative study.

AIMS: Intentional burns injuries are associated with high mortality rates, and for survivors, high levels of physical and psychological morbidity. This study provides a comprehensive assessment of intentional burn admissions to the adult Burns Unit at Bir Hospital, Kathmandu, Nepal, during the period 2002-2013. METHODS: A secondary data analysis of de-identified data of patients hospitalized at Bir Hospital, Kathmandu, with a burn during the period of 1 January 2002 to 31 August 2013. Socio-demographic, injury and psychosocial factors of patients with intentional and unintentional burns are described and compared. Chi-square tests, Fisher's exact test and Wilcoxon rank sum tests were used to determine statistical significance. RESULTS: There were a total of 1148 burn admissions of which 329 (29%) were for intentional burn, 293 (26%) were self-inflicted and 36 (3%) were due to assault. Mortality rates for intentional burns were approximately three times those for unintentional burns (60 vs. 22%). When compared to unintentional burns, patients with intentional burns were more likely to be female (79 vs. 48%), married (84 vs. 67%), younger (25 vs. 30 years), have more extensive burns (total body surface area, %: 55 vs. 25) and higher mortality (60 vs. 22%). Intentional burns were more likely to occur at home (95 vs. 67%), be caused by fire (96 vs. 77%), and kerosene was the most common accelerant (91 vs. 31%). A primary psychosocial risk factor was identified in the majority of intentional burn cases, with 60% experiencing adjustment problems/interpersonal conflict and 32% with evidence of a pre-existing psychological condition. A record of alcohol/substance abuse related to the patient or other was associated with a greater proportion of intentional burns when compared with unintentional burns (17 vs. 4%). CONCLUSIONS: The majority of intentional burn patients were female. Almost all intentional burns occurred in the home and were caused by fire, with kerosene the most common accelerant used. Underlying psychosocial risk factors were identified in most cases. Intentional burns resulted in severe burns with high mortality. Intentional burns are not only a serious medical issue; they represent significant public health and gender issues in Nepal.

Determinants of burn first aid knowledge: Cross-sectional study.

INTRODUCTION: This study investigated demographic factors, experience of burn/care and first aid course attendance as factors influencing burn first aid knowledge. METHODS: A cross-sectional study was undertaken using convenience sampling of members of sporting and recreation clubs. The main outcome measure was the proportion of correct responses to multiple-choice questions relating to four burn scenarios: (1) scald, (2) contact burn, (3) ignited clothing, and (4) chemical burn. RESULTS: A total of 2602 responses were obtained. Large gaps (30-50% incorrect answers) were identified in burn first aid knowledge across all scenarios. 15% more individuals gave correct answers if they had attended a first aid course compared to those who had not (p<0.0001); this proportion increased if the course was undertaken within the previous five years (p<0.0001) or contained a burns-specific component (p<0.0001). Males and younger (≤25 years) and older (≥65 years) age-groups had relatively lower levels of burn first aid knowledge. Gender and age were significant predictors of first aid course attendance, with males and younger (≤25 years) and older (≥65 years) age-groups less likely to have attended a first aid course. CONCLUSION: In this sample, first aid training undertaken within the last 5 years with a specific burns component was associated with enhanced burn first aid knowledge.

Changes in the plasma cytokine and growth factor profile are associated with impaired healing in pediatric patients treated with INTEGRA® for reconstructive procedures.

BACKGROUND: Large full thickness skin defects caused by trauma or surgery require skin grafting, often in conjunction with dermal scaffolds such as INTEGRA(®). Due to the size and severity of these procedures, complications such as infection may occur. This can lead to poor healing outcomes. OBJECTIVE: To identify early biomarkers of complications during INTEGRA(®) healing. METHODS: Levels of EGF, TGF-ß1, FGF-2, VEGF, IFN-α, GM-CSF, IL-4 and IL-8 were measured pre-surgery and at days 1, 7 and 25 post-surgery in peripheral blood of 15 pediatric patients treated with INTEGRA(®) for reconstructive procedures. The levels of these molecules were analysed with respect to the occurrence of complications. RESULTS: Complications (local infection) occurred in a group of 4 patients. This resulted in a reduced INTEGRA(®) take rate comparing to the group without complications (71.5±5.4% vs. 98.1±0.7%). In cases with complications there were significantly higher plasma concentrations of IL-4 and FGF-2 on day 7 (p=0.037 and p=0.008 respectively). Other markers were not significantly different between groups or at very low level at all time-points. WCC and CRP remained within normal ranges at all time-points. CONCLUSIONS: This data suggests that elevated levels of IL-4 and FGF-2 at early time-points after surgery may predict the development of complications in patients with INTEGRA(®). This may enable early interventions to prevent complications in procedures involving the use of INTEGRA(®).

Oral doxycycline for the treatment of chronic leg ulceration.

This pilot study investigated oral doxycycline as an adjunct to compression therapy for non-healing venous leg ulcers. Ten patients received doxycycline 20 mg twice daily (low-dose doxycycline) and ten patients received doxycycline 100 mg twice daily (high-dose doxycycline). Utilising a pre-test post-test study design, ulcer area was measured and wound fluid was collected before and after 4 weeks of treatment. In the high-dose doxycycline group, the reduction in median ulcer area was 48% (p = 0.1) and there was a significant reduction in wound fluid total matrix metalloprotease-1 (p = 0.02). These effects were not observed with low-dose doxycycline. There were no significant changes in wound fluid tumour necrosis factor-α or quantitative bacteriology following treatment with low-dose or high-dose doxycycline. There was no significant relationship between change in ulcer area and matrix metalloprotease-1, -8 or -9 activities in wound fluid at the end of treatment. Median wound fluid doxycycline concentrations after 4 weeks of treatment were 0.2 mg/L(0.45 lM) and 2.3 mg/L (5.18 lM) [DOSAGE ERROR CORRECTED] in the low-dose and high-dose groups, respectively, which are lower than that previously shown to inhibit matrix metalloproteases and tumour necrosis factor-α. Our study suggests that doxycycline 100 mg twice daily may improve the healing rate of recalcitrant leg ulcers, however the mechanism remains unclear.

Human pilot studies reveal the potential of a vitronectin: growth factor complex as a treatment for chronic wounds.

Several different advanced treatments have been used to improve healing in chronic wounds, but none have shown sustained success. The application of topical growth factors (GFs) has displayed some potential, but the varying results, high doses and high costs have limited their widespread adoption. Many treatments have ignored the evidence that wound healing is driven by interactions between extracellular matrix proteins and GFs, not just GFs alone. We report herein that a clinical Good Manufacturing Practice-grade vitronectin:growth factor (cVN:GF) complex is able to stimulate functions relevant to wound repair in vitro, such as enhanced cellular proliferation and migration. Furthermore, we assessed this complex as a topical wound healing agent in a single-arm pilot study using venous leg ulcers, as well as several 'difficult to heal' case studies. The cVN:GF complex was safe and re-epithelialisation was observed in all but 1 of the 30 patients in the pilot study. In addition, the case studies show that this complex may be applied to several ulcer aetiologies, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers. These findings suggest that further evaluation is warranted to determine whether the cVN:GF complex may be an effective topical treatment for chronic wounds.

Characterization of tumor necrosis factor-α block haplotypes associated with susceptibility to chronic venous leg ulcers in Caucasian patients.

Polymorphisms in the central major histocompatibility complex (MHC) are associated with several immunopathologic and inflammatory diseases, including chronic venous leg ulcers (CVLU). Because of strong linkage disequilibrium, identification of loci affecting disease susceptibility must be based on comparisons between haplotypes. Here we examine the association of conserved tumor necrosis factor (TNF) block haplotypes with CVLU susceptibility. A total of 171 Caucasian patients with CVLU were compared with 173 age-/gender-matched controls, excluding individuals with type 1 diabetes or rheumatoid arthritis. A total of 194 healthy subjects formed a separate population-based control group. Samples were typed for 38 tumor necrosis factor (TNF) block single nucleotide polymorphisms (SNP), human leukocyte antigen (HLA)-B and HLA-DRB1 alleles. TNF haplotypes were derived using the PHASE algorithm and assigned numbers (FVx) defined previously. The patients and matched controls shared 16 TNF block haplotypes. The patients had increased carriage of FV16 and alleles of the 8.1 and 60.3 MHC ancestral haplotypes (AH). CVLU risk is modulated by alleles within FV16 (e.g., TNF-308A and BAT1intron10 C insertion) or near FV16 in the 8.1AH. CVLU risk may also be mediated by unidentified alleles (not in FV22) marked by HLA-B40 and HLA-DR13. FV16 appears to be the best MHC and TNF block marker of susceptibility. After disease onset, an individual's TNF block haplotype does not modulate CVLU severity.

High-frequency ultrasound measurement for assessing post-thrombotic syndrome and monitoring compression therapy in chronic venous disease.

BACKGROUND: The purpose of this study was to validate high-frequency ultrasound (HFU) measurement of dermal thickness for quantification of edema in patients with different severities of chronic venous disease. METHODS: HFU measurements of dermal thickness were made with a 17-MHz probe (Philips iU22 Ultrasound scanner, Bothell, Wash) or a 20-MHz medium-focus probe (DermaScan-C, Cortex Technology, Denmark), 7.5 cm above the medial malleolus. For validation, 20 patients with venous leg ulcers who were not receiving compression therapy, 20 patients with previous deep vein thrombosis (DVT) and symptoms of post-thrombotic syndrome (PTS) without ulceration, and 31 age-matched healthy controls were measured on a single occasion. To investigate the effect of compression on dermal thickness, the leg ulcer patients from the validation study were treated with compression therapy for 7 weeks and measured after 1, 3, 5, and 7 weeks. The association between dermal thickness and the clinical (C) component of the CEAP classification was examined in a cross-sectional analysis of 157 patients with a confirmed history of DVT >or=3 years ago. RESULTS: Dermal thickness in patients with venous leg ulcers before compression therapy (median, 2.56 mm; interquartile range [IQR], 2.31-2.82 mm) was significantly greater (P = .002) than that in patients with symptoms of PTS without ulceration (median, 2.16 mm; IQR, 1.90-2.36 mm). Dermal thickness in both groups was significantly greater (P < .0001) than the control group (median, 1.34 mm; IQR, 1.29-1.44 mm). Compression therapy caused a steady and significant decrease in dermal thickness during the first 5 weeks until normal control levels were achieved. Dermal thickness increased with increasing CEAP category. In 121 patients with a positive diagnosis of DVT >or=3 years ago from Radiology Department records, a hypothetical test cutoff of 1.985 mm for the prediction of severe PTS noted as C(4b), C(5), and C(6) (lipodermatosclerosis or leg ulceration) had a positive predictive value of 46.9% and a negative predictive value of 90.3%. CONCLUSION: HFU measurement of dermal thickness enables the monitoring of edema reduction by compression therapy. A prospective study is required to determine the temporal dynamics of dermal thickness changes after DVT and the relationship to the development of PTS. This test has the potential to be beneficial in the follow-up of patients after a DVT and provide clinical evidence for using graduated elastic compression stockings to control edema and prevent the development of more advanced skin changes.

Induction of MMP-1, MMP-3 and TIMP-1 in normal dermal fibroblasts by chronic venous leg ulcer wound fluid*.

In the wound bed of chronic venous leg ulcers, an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) may cause excessive proteolysis and impair wound granulation. Soluble mediators in the wound environment may be responsible for this imbalance. The in vitro effect of wound fluid from venous leg ulcers on dermal fibroblast production of MMP-1, MMP-3 and TIMP-1 was compared with the effect of acute wound fluid from two different sources: fluid from post-mastectomy axillary drains and fluid from skin graft donor sites. Significantly higher MMP-1 and MMP-3 levels were induced by chronic venous leg ulcer wound fluid compared with both types of acute wound fluid (P < 0.005). Chronic venous ulcer wound fluid reduced TIMP-1 protein levels significantly more than acute graft fluid (P < 0.05). Venous ulcer wound fluid significantly increased MMP-1 and MMP-3 production in dermal fibroblasts and reduced TIMP-1 production, confirming that mediators in the leg ulcer microenvironment can potentially induce excessive proteolysis in the ulcer dermis by altering the balance between MMPs and TIMPs. Inflammatory mediators including interleukin-1beta and tumour necrosis factor-alpha can induce these MMPs. Further work is required to confirm the factors responsible for the induction of a high MMP and low TIMP profile in fibroblasts by venous ulcer wound fluid.