Oxidative and heat stress gene changes in hypertrophic scar fibroblasts stimulated with interleukin-1beta.

INTRODUCTION: Cutaneous wounds that involve loss of tissue heal through a complex process of generating granulation tissue to initially cover the wound, followed by epithelialization, and contraction. Normal healing requires a delicate balance between cellular, matrix, and vascularity build up and breakdown. Defects in the regulation of this balance can alter normal scar formation through fibroblastic hyperproliferation, which is characteristic of hypertrophic scar formation. METHODS: Primary fibroblasts cultures from hypertrophic scar or adjacent normal skin were seeded in growth medium. Half of each was stimulated with interleukin (IL)-1beta for 6 h and half served as control nonstimulated fibroblasts. Supernatants were tested for expressed proteins and the fibroblasts for changes in gene expression. RESULTS: Comparison between normal skin and hypertrophic scar fibroblasts stimulated with IL-1beta indicate that 15 genes increased and 8 genes decreased expression. When normal skin was stimulated with IL-1beta, there were increases in the expression of heat shock transcription factor-1, hsp70, and IL-6 When hypertrophic scar was stimulated with IL-1beta, there was a decrease in nuclear factor-kappaB, GADD45-alpha, p53, p53 binding protein, and Cox-2 genes. These genes may play specific but different roles in controlling the cellular response to cell stress and apoptosis. CONCLUSION: Data presented suggest that oxidative and heat stress proteins, stimulated through IL-1beta, may be important mediators of abnormal scarring.

Gene expression changes with time in skeletal muscle of severely burned children.

OBJECTIVE: The purpose of this study was to identify gene-expression changes in leg muscle for up to 24 months after a severe thermal injury. SUMMARY BACKGROUND DATA: Hypermetabolism associated with severe burns was thought to cease with wound healing and closure. It has been recently shown that hypermetabolism does not completely resolve after healing, and muscle catabolism continues after hospital discharge; however, just how long after discharge has not been established. METHODS: : Six children, admitted to our hospital within 1 week after injury, were studied. Patients ranged in age from 3 to 18 years, with flame or scald burns covering more than 40% of their body surface area. At 1.5, 6, 12, 18, and 24 months postburn, a biopsy of the vastus lateralis muscle was taken and snap frozen at -80 degrees C. Total RNA was isolated and in vitro transcribed and hybridized to HG-U95 Av.2 Affymetrix arrays. The images were scanned and analyzed using Affymetrix GeneChip Analysis Suite 5.2 and dChip programs. Using 1 to 7 days after injury as baseline, comparisons were made of expression profiles at the various time intervals after injury. RESULTS: When comparisons are made to nonburned children, 38 genes were significantly altered at 1.5 months, 10 genes remained altered at 6 months, 4 remained altered at 12 months, and 2 at 18 months. No differences could be shown at 24 months. Western blot analysis of beta-2 microglobulin and myosin light chain was used to corroborate the microarray data. CONCLUSIONS: Gene changes can be identified for up to 18 months after burn but not at 24 months. These gene changes may provide information concerning what genes in skeletal muscle contribute to recovery from burn trauma.

Gene profiling in muscle of severely burned children: age- and sex-dependent changes.

BACKGROUND: Thermal injury is associated with a pronounced catabolic response in skeletal muscle. This study identifies gene expression changes in skeletal muscle of thermally injured girls and boys using high-density oligonucleotide arrays. METHODS: Six burned children with a mean age of 8.3 +/- 1.3 years and TBSA burn size covering 51 +/- 6% admitted to our hospital with in 48 h of injury and six cleft lip and cleft palate patients were studied. Total RNA was isolated, in vitro transcribed, and hybridized to HG-U95 Av.2 Affymetrix arrays. Messenger RNA expression patterns of controls and burn patients were compared using Affymetrix GeneChip Analysis Suite 5.2 and dChip. RESULTS: Statistical analysis of the 12,625 genes on each array showed a significant increase in the expression of 77 genes in burn children and a decrease in 21 genes when compared to controls (P < 0.05). We found three genes in burned males and two genes in burned females with decreased expression in muscle compared to controls. Chromosomes 1, 2, 7, 12, and 16 showed genes with increased expression in muscle from burned children, while chromosomes 3, 7, 8, 19, and 22 had genes with decreased expression. Categories of genes affected were related to metabolism, proliferation, transcription/translation, immune response, stress response, angiogenesis, and signal transduction. CONCLUSIONS: Genes that are differentially expressed in skeletal muscle of burned children, but whose function in muscle is unknown, include those related to various transcription factors and those known to encode proteins involved in signaling pathways. Further analysis is required to achieve the ultimate goal of making functionally relevant conclusions about the molecular pathology of burn injury.

Gene expression profiles from hypertrophic scar fibroblasts before and after IL-6 stimulation.

The structural rearrangement of collagen fibres in hypertrophic scar causes abnormal contracture, low tensile strength, and raised scars, which cause functional impairment and disfigurement. It is hypothesized that changes in the genes of cytokines, extracellular matrix proteins, and proteins regulating programmed cell death are related to hypertrophic scar formation. To test this hypothesis, fibroblasts were cultured from hypertrophic scars and their response to interleukin-6 (IL-6) stimulation was studied by defining their gene expression profiles. Affymetrix gene chip analysis was used to identify up- or down-regulation in the 12 625 genes present in the affymetrix array. RT-PCR and ELISA assays were used to validate microarray expression profiles further. Comparison of gene profiles showed an increase of 12 genes in hypertrophic scar fibroblasts compared with normal skin fibroblasts, while the expression of 14 genes decreased. Thirty-three genes were affected by IL-6 treatment in the hypertrophic scar fibroblasts, while 57 genes were affected in normal skin fibroblasts. Messenger RNA to beta-actin ratios for matrix metalloproteinase-1 (MMP-1) and MMP-3 were increased with IL-6 in normal skin fibroblasts from 2.43 +/- 0.06 to 5.50 +/- 0.45 and from 0.75 +/- 0.09 to 1.98 +/- 0.01, respectively. No change in these matrix metalloproteinases could be shown with IL-6 stimulation in hypertrophic scar fibroblasts. Secreted protein levels of pro-MMP-1 and MMP-3 were elevated in the supernatants from normal skin fibroblasts from 2.00 +/- 0.09 and 1.72 +/- 0.10 ng/ml to 4.60 +/- 0.12 and 3.41 +/- 0.20 ng/ml, respectively, after treatment with IL-6 (p < 0.05). No changes were observed in hypertrophic scar fibroblasts treated with IL-6. Values are means +/- SEM. The absence of any up-regulation of MMP-1 and MMP-3 in hypertrophic scar fibroblasts, in response to IL-6, suggests that suppression of matrix metalloproteinases may play a role in the excessive accumulation of collagen formed in hypertrophic scars. While the pathogenesis of abnormal hypertrophic scars remains poorly understood, the use of gene expression arrays may prove helpful in identifying the mechanisms responsible for this type of abnormal scar formation and in formulating an effective therapeutic protocol.

Genomic analysis of insulin-like growth factor-I gene transfer in thermally injured rats.

Thermal trauma causes tissue damage by membrane destabilization and energy depletion at the cellular level, resulting in tissue necrosis and inflammation leading to delayed cell death. One therapeutic approach is to block the immediate triggering of the inflammatory cascade that results in prolonged hypermetabolic responses and immune dysfunction while promoting the expression of growth factors. In the present study, we determined hepatic gene expression responses to insulin-like growth factors-i (IGF-I) gene transfer to burned rats using high-density DNA microarray assays. The expression of 123 out of approximately 8,800 genes assayed (1.4% of total) were significantly altered. Of these, 42 genes were altered irrespective of treatment by burn trauma (p < 0.05). Changes in gene expression were confirmed by measuring mRNA levels using reverse transcription-polymerase chain reaction and protein levels by Western blot assays. DNA microarray analyses showed two major mechanisms that mediated beneficial outcomes after IGF-I gene transfer in the burned rat livers. These mechanisms were the stimulation of IGF binding protein potentiation of peripheral IGF-I and the inhibition of the burn-augmented pro-apoptotic and oxidative mitochondrial metabolites stimulated by thermal trauma.

Gene expression profiles of livers from thermally injured rats.

The liver plays an important role in a severe thermal injury by modulating immune function, inflammatory processes and the acute phase response, which are an orchestrated attempt to restore homeostasis. Using high-density oligonucleotide arrays, we examined the gene expression profile in the livers of rats between 2 and 240 h after a 40% total body surface area (TBSA) burn. Alterations in gene expression unique to a thermal injury were identified. Approximately 39 genes out of 8700 genes on each array across all the time points showed a significant change in expression patterns. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses verified significant changes in early growth response-1 (Egr-1) messenger RNA (mRNA) and protein levels corresponding to the array data. Significant increases in serum levels of alpha-2-macroglobulin that correspond to changes in its mRNA levels were observed at 6 and 24 h after burn, p<0.05. The genomic pattern for liver in the hypermetabolic phase after the burn injury involves transcription factors, stress and inflammatory responses, cytoskeletal and extracellular matrix modifications, and regulation of cell proliferation and differentiation. During the initial phase of thermal injury gene expression profiles in the liver may provide some insight into how cellular protection mechanisms and systemic hypermetabolism are initiated and controlled. The genome wide changes observed may provide a rational therapeutic strategy to improve burn care.

Alterations in resistin expression after thermal injury.

BACKGROUND: Burn injury, it was hypothesized, may induce changes in resistin expression that contribute to postburn metabolic derangements. This study examined resistin gene expression, serum levels of resistin protein, and glucose levels in burned mice. METHODS: Ten male Balb-c mice were anesthetized and then given a 30% total burn surface area using heated probes. Burned and sham-burned mice were killed at 2, 4, 24, and 48 hours. The total ribonucleic acid from gonadal fat tissues was isolated for the measurement of resistin gene expression using real-time reverse transcriptase-polymerase chain reaction. Serum levels of resistin, insulin, and glucose were measured. Statistical analysis was performed by two-way analysis of variance using Bonferroni's test to find differences between groups. All p values less than 0.05 were considered significant. RESULTS: Increases in resistin gene expression and serum resistin levels were detected in the burned animals, and these correlated with relative insulin resistance. CONCLUSION: The findings suggest a potential role for resistin in the pathophysiology of the metabolic response to injury.

Matrix metalloproteinase expression in cytokine stimulated human dermal fibroblasts.

In this study, we investigated the effect of inflammatory cytokines on matrix metalloproteinase (MMP-1) and TIMP-1 production in human dermal fibroblasts, which play a pivotal role in wound healing, ranging from the synthesis and remodeling of extracellular matrix (ECM) to the synthesis of growth factors. The balance of MMPs and TIMPs is crucial in directing successful wound repair. Human adult dermal fibroblasts were seeded in six well plates (7.5 x 10(4) cells/ml) in complete media. Eighty to ninety percent confluent cells were treated with interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) for 6h in serum free media with suitable controls run in triplicate. Supernatants were assayed for pro-MMP-1 & TIMP-1. Extracted total RNA was used for reverse transcription polymerase chain reaction (RT-PCR) with sequence specific primers for MMP-1, TIMP-1 and beta-actin. Signal intensity was normalized to the internal control (beta-actin). Statistical analysis used ANOVA. MMP-1 and TIMP-1 mRNA expression were markedly increased with IL-6 and TNF-alpha treatment and remains unchanged with IL-1beta. Pro-MMP-1 protein levels are unchanged with TNF-alpha and significantly increased with IL-1beta and IL-6 treatment. However, TNF-alpha significantly increases TIMP-1 protein levels. Data suggests differential regulation of MMP-1 and TIMP-1 protein levels by the cytokines found in stimulated dermal fibroblasts. Further characterization of this response will provide an understanding of the mechanisms of pathogenesis of extracellular matrix (ECM) and the potential role of metalloproteinases in tissue remodeling after injury.

Gene expression profiles and protein balance in skeletal muscle of burned children after beta-adrenergic blockade.

Propranolol, a nonselective beta-blocker, has been shown effective in hypermetabolic burn patients by decreasing cardiac work, protein catabolism, and lipolysis. This study investigates the effect of propranolol on gene and protein expression changes in skeletal muscle of burned children by use of high-density oligonucleotide arrays to establish the genetic profiles and stable isotope technique to quantitate protein synthesis. Thirty-seven children (mean age 9.7 +/- 1.1 yr) were randomized into groups to receive placebo (n = 23) or propranolol (n = 14) titrated to reduce heart rate by 15%. Children had >40% total body surface area burns (mean 43 +/- 5.6%). Protein net balance was determined by stable-isotope infusion technique. Total RNA from muscle biopsies was isolated, labeled, and cRNA hybridized to the HG-U95Av2 Affymetrix array. Mean net balance of protein synthesis and breakdown was -14.3 +/- 12.9 nmol. min-1. 100 ml leg volume-1 for placebo and +69.3 +/- 34.9 nmol. min-1. 100 ml leg volume-1 in the propranolol-treated children (P = 0.012). Comparison of 12,000 genes in burned children receiving placebo showed increased expression of two genes with time, whereas children receiving propranolol showed increased expression of nine genes with a decrease in five genes. We conclude that burned children receiving propranolol showed a significant upregulation in genes involved in muscle metabolism and downregulation of an important enzyme involved in gluconeogenesis and insulin resistance compared with burned children receiving placebo. The upregulation of genes involved in muscle metabolism correlates well with the increase in net protein balance across the leg.

IGF-I gene transfer effects on inflammatory elements present after thermal trauma.

Major thermal injury results in severe prolonged responses with three components: a hypermetabolic response, inflammatory responses, and endogenous wound-healing processes. We showed that use of liposome-mediated gene transfer of the insulin-like growth factor I (IGF-I) reduces burn-induced inflammatory responses and enhances wound healing. In the present study, we found transient increased levels of IGF-I protein in rats exposed to thermal trauma via liposomal gene transfer in an effort to define the transcriptional events that occur after IGF-I delivery at the site of injury. The beneficial effects of IGF-I gene transfer act partly via amelioration of burn-induced inflammatory responses that mediate cell death through caspase-3 activity and Bax expression. IGF-I gene transfer induces selective stimulation of activation protein-1 DNA-binding activity and activation of antiapoptotic, but not inflammatory, NF-kappaB transcription factors. Data were consistent with our hypothesis that the beneficial effects of IGF-I gene transfer on burned rats act in part via activation protein-1 and NF-kappaB transcriptional regulation and the concordance between the results obtained with antiapoptotic, as opposed to the proapoptotic, sequences as well as the corresponding changes in measures of cell death via Bax and caspase-3 mechanisms.

Matrix metalloproteinases and their tissue inhibitors in severely burned children.

Severe burns cause not only skin injury but several marked systemic derangements. During wound healing, matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases play an important role in tissue regeneration and remodeling processes. Therefore, in the present study, we determined the serum levels of MMPs and tissue inhibitor of metalloproteinase-1 in burn patients over time. Serum samples from 12 severely burned children (mean age 7.9 +/- 2.5 years) with >40% total body surface area burns were obtained within 0.5 hours, 3, 7, and 21 days after injury. Pro-MMP-1, MMP-3, MMP-9, and tissue inhibitor of metalloproteinase-1 serum levels were assayed by enzyme-linked immunoassay and compared to normal healthy volunteers. Two-way analysis of variance and Bonferroni's test were used for statistical analysis. Pro-MMP-1 levels in the serum were significantly elevated by the seventh day after burn. MMP-3 and MMP-9 levels showed significant increases by day 3 and 21 compared to normals, respectively. Tissue inhibitor of metalloproteinase-1 levels did not change with time after burn but were significantly higher by 3 days after burn compared to normal serum. In conclusion, changes in MMPs and tissue inhibitor of metalloproteinase-1 occur in burn patients and those changes may be a mechanism beneficial to wound healing.

Gene expression patterns in skeletal muscle of thermally injured children treated with oxandrolone.

OBJECTIVE: To analyze gene expression patterns in skeletal muscle from burned children. SUMMARY BACKGROUND DATA: Analysis of gene expression patterns in skeletal muscle from burned children can help provide a fundamental understanding of muscle wasting at the molecular level. This study is the first to use such an approach in burned children receiving anabolic treatment. METHODS: Children who received 0.1 mg/kg oxandrolone twice a day (n = 7) were compared to placebo (n = 7). Net protein balance was determined before and after treatment with oxandrolone. Total RNA, extracted from muscle biopsies obtained from burned children age 3 to 18 years, was purified, reverse transcribed, and biotinylated cRNA hybridized to the human high-density oligonucleotide array (U95Av2). Western blot analysis verified the mRNA changes at their protein level. RESULTS: DNA microarray analysis showed two genes significantly changed in muscle from burned children receiving placebo, while the expression of 21 genes was altered with oxandrolone. Muscle net protein balance increased with oxandrolone treatment compared to placebo. CONCLUSIONS: DNA microarray technology will help identify molecular changes that can serve as targets for new therapies to attenuate muscle wasting in severely burned children and thus improve recovery and early rehabilitation.

Gene expression analysis in burn wounds of rats.

The events occurring early in the burn wound trigger a sequence of local and systemic responses that influence cell and tissue survival and, consequently, wound healing and recovery. Using high-density oligonucleotide arrays we identified gene expression patterns in skin samples taken from a region of injury in the burn rat model. The associated genomic events include the differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation, and immune response. The functional gene cluster detected and their time appearance matched the time sequence known to occur in burn wound healing.