Mohan R. K. Dasu

Improved Net Protein Balance, Lean Mass, and Gene Expression Changes with Oxandrolone Treatment in the Severely Burned

ObjectiveTo determine the effects of the anabolic agent oxandrolone on muscle protein and gene expression in severely burned children. Summary Background DataThe authors previously showed that oxandrolone increased net muscle protein synthesis in emaciated burned patients receiving delayed treatment for severe burns. They hypothesized that similar effects would be seen in those treated early after burn. MethodsThirty-two severely burned children were enrolled in a prospective randomized trial. Subjects underwent studies to assess leg protein net balance 5 days after the first excision and grafting procedure. Immediately after these studies, treatment with placebo (n = 18) or 0.1 mg/kg oxandrolone (n = 14) twice a day was started. One week after this, another net balance study was performed in each subject. Body weights and total body potassium counting were used to determine body compositional changes. Muscle biopsies were taken 1 week after treatment in oxandrolone subjects to examine gene expression changes with gene array (12,600 genes). ResultsProtein net balance did not change in the placebo group, while oxandrolone-treated subjects had a significant improvement. Body weights and fat free mass significantly decreased in the placebo group, while no changes were found in the oxandrolone-treated subjects. Expression changes were seen in 14 genes in the oxandrolone group compared to placebo. Some of these included myosin light chain (+2.7-fold change), tubulin (+2.3), calmodulin (−2.3), and protein phosphatase I inhibitor (−2.8). ConclusionsOxandrolone improves protein net balance and lean mass in the severely burned. These changes are associated with increased gene expression for functional muscle proteins.

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 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 β‐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. Copyright

The use of beta-adrenergic blockade in preventing trauma-induced hepatomegaly.

Objective:The objective of this study was to test the hypothesis that hepatomegaly in burned children can be attenuated or reversed by blocking lipolysis and reducing free fatty acids delivered to the liver. Summary Background Data:Accelerated lipolysis in severely burned children has been shown to play an important role in the accumulation of hepatic TGs. Severely burned children who survive 10 days or more after injury commonly have enlarged livers often twice or more normal size for their sex, age, and weight. Methods:Ninety-eight children, 2 to 18 years of age, with burns covering more than 40% of their body surface and who received either propranolol (β-adrenergic blockade) or placebo were studied. Liver weights were measured by ultrasonic scanning. Body composition changes were identified by dual-image x-ray absorptiometry and validated by whole-body potassium-40 scintillation counting. Discarded abdominal cutaneous adipose tissue was collected before and after propranolol or placebo for microarray analysis. Results:In 80% of severely burned children studied not receiving propranolol, liver sizes increased by 100% or more while 86% of burned children receiving propranolol showed a decrease or no change in liver size over the same period of time after injury. Gene expression patterns of adipose tissue after propranolol treatment showed that all of the identified genes related to lipid metabolism were down-regulated. Conclusions:Data reported here support the hypothesis that β-adrenergic blockade can reduce delivery of fatty acids to the liver and hepatic congestion commonly found in severely burned children by inhibiting lipolysis and reducing hepatic blood flow.

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

ObjectiveTo analyze gene expression patterns in skeletal muscle from burned children. Summary Background DataAnalysis 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. MethodsChildren 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. ResultsDNA 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. ConclusionsDNA 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.

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 Bonferronis 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. (WOUND REP REG 2003;11:177–180)

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 ~8,800 genes assyed (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 giant hairy naevi

Background: Congenital neomelanocytic naevi appear in nearly 1% of newborns. Giant hairy naevi (GHN) are uncommon lesions covering large areas of the body. They are of concern because they have the potential to transform into malignant melanomas. Aims: To describe gene expression profiles of GHN and nearby normal skin from patients with GHN and normal control skin (from patients with cleft lip/palate). Methods: Tissues from three patients with GHN and two normal controls were studied for differences in gene expression profiles. Total RNA was isolated from normal skin near the hairy naevus, GHN, and skin from normal controls. The RNA samples were subjected to probe labelling, hybridisation to chips, and image acquisition according to the standard Affymetrix protocol. Results: There were 227 genes affected across all samples, as determined by DNA microarray analysis. There was increased expression of 22 genes in GHN compared with nearby normal skin. Decreased expression was noted in 73 genes. In addition, there was increased expression of 36 genes in normal skin near GHN compared with normal control skin, and decreased expression of five genes. Categories of genes affected were those encoding structural proteins, proteins related to developmental processes, cell death associated proteins, transcription factors, growth factors, stress response modulators, and collagen associated proteins. Changes in mRNA expression were checked by reverse transcription polymerase chain reaction. Conclusions: Genetic profiles of GHN may provide insight into their pathogenesis, including their potential for malignant transformation. Such information may be useful in improving the understanding and management of these lesions.

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°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 β-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.

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 Bonferronis 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.