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1 Department of Surgery, University of Texas Medical Branch and Shriners Hospitals for Children, Galveston 77550; 3 Department of Human Genetics and Biochemistry, University of Texas Medical Branch, Galveston, Texas 77555; and 2 Department of Psychiatry, Washington University, St. Louis, Missouri 63110
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
gene expression profile; microarray; wound healing; skin; rat burn model
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THERMAL INJURY IS ONE of the most severe forms of trauma that affects the organism both locally and systemically. Localized burns affect the skin and impair its ability to act both as a protective barrier to the environment and as an immune organ (1). Physiological responses after a thermal injury include changes in cellular protection mechanisms, local and systemic inflammation, and reperfusion injury, which are all a major consequence of abrupt cellular energy depletion and cell damage (19, 22, 27). Initial local events may result in a cascade of responses, such as hypermetabolism, prolonged catabolism, organ dysfunction, and prolonged immunodeficiency (5, 7, 12, 21, 28).
Burn wound healing is a complex process consisting of an early phase of abrupt energy depletion and necrosis, followed by a two-stage inflammatory phase, delayed cell death, formation of granulation tissue, matrix formation, and remodeling (11, 18, 24). Inflammation-mediated delayed cell death occurs at the wound borders and in surrounding tissue. Increased accumulation of macrophages and fibroblasts at the wound site leads to extracellular matrix deposition and angioneogenesis to form granulation tissue. Surrounding epithelial cells proliferate and migrate to gradually cover the open wound surface. Increased extracellular matrix deposition, especially collagen, and remodeling of the newly formed connective tissue persist for several months after injury (11, 18, 24). In the burn wound, this cascade of events not only takes place locally at the wound site, but also systemically via release of inflammatory mediators (14, 16).
DNA microarray analysis has changed the way biological events are investigated. Now systematic and global approaches are applied to investigate physiological mechanisms in health and disease. Differing from the traditional approach of sequentially studying individual gene products, a complete set of activated genes is now analyzed in toto. The major advantage of this application lies in reducing confounding events and circumstances and allows the characterization of interactions among different cellular pathways that so far are considered separately. Analyses at the genomic level indicate that events involved in biological processes can only be understood in the context of the activity of several thousand genes and only a composite assessment can determine the true significance of signaling events and structural entities (6).
The focus of the present study is to identify local responses to a thermal injury and the initial cellular responses through gene expression patterns in the burn wound in an effort to define the associated genomic events.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Adult male Sprague-Dawley rats (Harlan Sprague Dawley, Houston, TX), weighing 350-375 g, were housed in wire bottom cages in a temperature-controlled room with a 12:12-h light-dark cycle. Rats were acclimatized to their environment for 7 days. All received a liquid diet of Sustacal (Mead Johnson Nutritionals, Evansville, IN) and water ad libitum throughout the study.
Anesthesia was by intraperitoneal injection of pentobarbital sodium (50-90 mg/kg) and buprenorphine-tartrate (0.1-1.0 mg/kg). Rats were shaved and randomized into burn or sham control groups. The burn group received a 40% total body surface area full-thickness scald burn on the dorsum and ventrum as previously described (14). Burned rats were resuscitated with 60 ml/kg lactated Ringer, placed on a warming pad, and, once awake, returned to their cages. Sham-burned animals received anesthesia and equivalent handling without a scald burn. Six rats in each group were killed at 2, 6, 24, and 240 h after burn. Skin (~1 cm2) was harvested from the wound borders or corresponding sites in sham-burned animals and immediately frozen in liquid nitrogen and stored at
70°C for analysis.
This study was approved by the Animal Care and Use Committee of the
University of Texas Medical Branch (UTMB), Galveston, TX, and followed
the guidelines established by the National Research Council.
Total RNA extraction.
Total RNA was isolated from skin samples by acid guanidinium
thiocyanate-phenol-chloroform extraction using TRI Reagent (Molecular Research Center, Cincinnati, OH). This method was based on the single-step method of RNA isolation described by Chomczynski and Sacchi
(3). Samples were homogenized in TRI Reagent on ice, and
total RNA was extracted following the manufacturers' instructions. Purified RNA was quantified by UV absorbance at 260 and 280 nm and
stored in 25-µg aliquots at 70°C for DNA microarray analyses.
Gene array analysis. Twenty-five micrograms of purified total RNA were transcribed into cRNA, purified, and then used as templates for in vitro transcription of biotin-labeled antisense RNA. All protocols followed the recommendations of the manufacturer (Affymetrix, Santa Clara, CA). Twenty micrograms of biotinylated antisense RNA preparation was fragmented, assessed by gel electrophoresis, and placed in a hybridization mixture containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre). Samples were hybridized to an identical lot of Affymetrix microarrays for 16 h. Microarrays were washed and stained using the instrument's standard Eucaryotic GE Wash 2' protocol and antibody-mediated signal amplification. The images from the scanned microarrays were processed with Afffymetrix GeneChip Analysis Suite 3.2. Images from each microarray were scaled and adjusted to an average intensity value for all arrays of 1,500. Scaled average difference values and absolute call data from each microarray were exported to text files and used for statistical analysis. In vitro transcription and chip hybridization were performed in collaboration with the UTMB Genomic Core Facility.
Data analysis. The data analysis of genomic data was carried out as microarray validation, cluster analysis of transcription profiles (using Manhattan distances and looking at all genes expressed), identification of genes expressed only in one group (burned or unburned skin), identification of genes expressed in both groups at significantly different levels, and the temporal analysis of expression profiles.
Identification of present and absent genes. The presence or absence of each probe within the group was determined according to the Affymetrix algorithm. A probe was considered present if its absolute call was present for at least two members of the group containing three samples. Otherwise the probe was regarded as absent, and the respective gene was considered absent in that group.
Chip validation.
For microarray validation, data were clustered to detect gross
discrepancies among different array data. Data of an array would be
discarded if only a small fraction of probes was present compared with
the remaining arrays in the group. The degree of similarity or
dissimilarity among transcription profiles was tested using clustering
methods (2, 9, 23, 26). The clustering of arrays was
performed by SPSS software using unsupervised clustering methods
(Pearson, Euclidian, and Manhattan hierarchical clustering) without
artificially imposing their number. This distance is defined between
two points, x = (x1,
x2, . ... xn) and
y = (y1,
y2, . ... yn), in
n-dimensional spaces as
d(x,y) = 
yi), here n = 8,799. All the
above three clustering procedures yielded similar results. To further
assess the significance of outcome, we performed correlation analysis
of all the present probes. If a large deviation in the number of
present calls or if the correlation coefficient among samples within
one group was <0.85, the data set would be discarded (see Fig. 1). All
data sets used for further analysis met these criteria.
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Identification of genes with significantly different expression. Further analysis focused on genes that were present in both burned and unburned groups. The within-group average of the expression was calculated for each group, and comparison was made between groups. This is done by calculating the expression difference for each gene and listing those that show a larger than twofold increase or decrease in activity compared with the unburned control group. Outliers were eliminated using a "jack-knife" procedure in which an entry is discarded if its value falls outside the three standard deviations calculated from the other two remaining entries. Because of the limited sensitivity of Affymetrix chips in detecting low-abundant mRNAs, we applied the t-test on data with present absolute call. Only statistically significant expression differences as determined were retained (8). Considering the small sample number per group, the power of the t-test was computed and results were discarded, even in light of a significant difference, if the power was <0.8. After establishing a list of genes with significantly changed expression, they were organized into categories depending on their physiological function.
Validation of microarray results using RPA.
To determine local mRNA expression of pro- and anti-inflammatory
cytokines, multiple probe RNase protection assays (RPA) (RiboQuant Pharmingen, San Diego, CA) were performed using a multiprobe template set (rCk-1) containing probes for IL-1
, IL-1
, IL-2, IL-3, IL-4, IL-6, IL-10, TNF-, TNF-, and IFN-
. -32P-labeled
antisense RNA probes from standard and rCK-1 templates were generated
and hybridized with the sample (target) RNA. After the digestion of
free probes and other single-stranded RNA with RNases, protected probes
were purified and resolved on denaturing polyacrylamide gels and
developed on film. Identification of expressed mRNA species was
achieved by correlating the presence of bands to the expected fragment
length. Signal intensity of -32P-labeled probes was
measured by phosphorimaging (Molecular Dynamics), and the quantity of
each mRNA species was determined based on the intensity of the
protected probe fragments. Templates for the analysis of L32 and GAPDH
housekeeping genes are included to allow assessment of total RNA levels
for normalizing samples and technique errors. Levels of the cytokine
mRNA expression were normalized to the housekeeping gene L32.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Correlation analysis of the normalized expression levels of 8,799 probe pairs detected after hybridization of three equally treated burn skin samples with three Affymetrix microarrays per group showed correlation coefficients of 0.98 to 0.99 (Fig. 1). This indicates very small interanimal variations and permits the use of average expression values to be used as representative data for the analysis of burn-induced changes in gene expression patterns.
Hierarchical cluster analysis.
Hierarchical cluster analysis of the gene expression profiles of burned
skin at 2, 6, and 24 h after burn differed significantly from
unburned skin. At 240 h, the transcription profiles of burned skin
grouped closer with the profile of unburned skin (Fig.
2).
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Identification of changes in gene expression after burn.
Genes present only in burned skin are listed in Table
1. Gene
expression analysis revealed 85, 48, 120, and 21 genes in burned skin
at 2, 6, 24, 240 h, respectively. The corresponding numbers of
expression sequence tags (ESTs) are indicated (Fig.
4). Twenty genes were expressed at both 2 and 6 h, 34 genes were expressed at 2, 6, and 24 h. Ten genes
were present in burned skin at all time points and included IL-1
#M98820, growth-related oncogene gro #D11445,
tyrosine kinase p72 syk #U21683, cyclin E
#D14015, JAK2 #AJ000557, and Doc2A
#U70779. Thus initial activation of overall gene expression
was followed by increased activation at 6 and 24 h, returning to
basal levels 240 h after injury (Fig. 4). Genes not detected in
burned skin but present in normal skin are indicated in Table 2 and
Fig. 5.
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Validation of microarray results.
The expression of IL-1 #M98820 in burned skin was
confirmed by measuring mRNA using an RPA, which correlated well with the microarray findings. Similarly, IL-6 #M26744 expression at 24 h and its absence at 10 days corresponded to mRNA values detected by RPA (Fig. 7).
IL-1, IL-2, IL-3, IL-4, IL-10, and TNF- could not be detected in
samples by RPA, consistent with our microarray findings.
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Dynamic of gene expression over time. There were 2,703 genes expressed at all times in both burned skin and control groups. We calculated their expression ratios (burn/control) to identify expression patterns over time. Genes that increased or decreased twofold in at least one time point compared with control were selected. This "filter" eliminated 781 genes (including 338 EST), and the remaining 443 genes were used for further analysis.
In burned skin, 85 genes showed above threefold or greater increases and 54 genes showed threefold or greater decreases in expression compared with normal skin. These genes can be grouped according to function and their involvement in physiological processes, such as cell survival and death, inflammation, cell growth, and differentiation (Table 3; Fig. 8). The temporal distribution of these genes is shown in Fig. 9. Four of these genes initially decreased in expression, followed by more than a threefold increase at 10 days.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The sequence of burn wound healing virtually starts with the injury itself. The concept of injury zones in burn wounds described by Jackson (15) consists of three zones with different degrees of tissue damage. The central "zone of coagulation" is the site of irreversible tissue necrosis; adjacent to it is an area with ischemic tissue at risk for necrosis (zone of stasis), and a peripheral zone characterized by reactive vasodilatation and inflammation (zone of hyperemia). From a surgical standpoint, the intermediate region of injury is the one of greatest clinical interest, as it harbors the potential to undergo tissue necrosis as well as full recovery. We therefore took the biopsy for analysis in the region of the wound border (zone of stasis).
Immediately after injury, cascading cellular and humoral events are
activated leading to capillary leakage or local bleeding and
coagulation. The associated deposition of fibrin provides an early
matrix for immigrating cells and entraps platelets, which in turn
secrete growth factors (PDGF, TGF-, etc.), to attract and activate
fibroblasts, endothelial cells, macrophages, and immune-competent cells
(13).
During the early phase of wound healing, platelets secrete a number of
direct or indirect angiogenic substances such as TGF-, PDGF,
TNF-, and basic FGF, all of which stimulate the proliferation and
in-growth of capillaries. In full-thickness wounds, the proliferating and migrating epithelium arises from the wound border. The rate of
epithelial coverage is modulated by growth factors that stimulate proliferation and chemotaxis, and the presence and production of matrix
metalloproteinases and plasminogen activator to facilitate migration
through the ECM degradation (24). About 1 wk after injury,
fibroblasts, attracted by macrophages and the secreted growth factors,
migrate into the wound and begin collagen production (4).
Type I and III collagens are predominantly synthesized to form new
extracellular matrix. With maturing of newly formed scar at the closed
wound site, specific matrix metalloproteinases are produced by
keratinocytes, fibroblasts, granulocytes, and macrophages to remodel
the deposited extracellular matrix and to facilitate further cell migration.
Gene expression changes. Examination of the cDNA microarray data shows small interanimal differences in all the groups examined over time after burn trauma (0.98 < r < 0.99). Thus this reflects not only significance of the measured expression values (P < 0.05), but also high statistical power.
Cluster analyses results were consistent with the known sequence of physiological events associated with burn trauma in that there was a prompt and an early robust response in gene expression. Cluster analyses results at postburn day 10 were consistent with the onset of recovery that signifies a return to baseline values of gene expression. Examination of present vs. absent gene expression over time showed early transient stimulation of cytokines and other genes encoding inflammatory mediators (Table 1); for example, IL-1 #M98820, IL-6 #M26744, inducible nitric oxide
synthase #U03699, IL-1 converting enzyme
#U84410, prostacyclin synthase #U53855, and COX-2
#S67722. Not surprisingly, a large number of cell survival-promoting genes and proliferative regulators were activated within a few hours after burn trauma (Table 1); for example, p53
#X13058, heat shock protein 70 (hsp70) #Z27118,
and N-ras/p21 #X68394. These signal transduction changes
agree with known burn-induced changes in IGF-I and IGF-I binding
proteins after burn trauma (17).
The early activation of cell cycle and proliferation-related genes,
such as cyclin C #D14013 and E #D13015,
osteoprotegerin #U94330 and topoisomerase #D14046
suggests that these are likely to be initiators of the wound healing
process, a response not foreseen. The sheer magnitude of the number of
genes whose expression is affected speaks to the overwhelming nature of
the force of the trauma.
Interestingly, examination of genes whose expression decreases in
skin shortly after burn trauma (Table 2) shows a preponderance of genes
associated with neuronal, neuroendocrine, and metabolic functions.
This suggests that their absence is likely due to functional and
structural loss of neurons and supporting glia, and a delayed reinnervation associated with inflammation and other events required for wound healing.
When taking a closer look at the complex pattern of significantly
increased gene expression over time after burn trauma (Table 3, Fig.
8), it becomes clear that the affected genes play a role in cell growth
and differentiation, cell signaling, stress response, energy
metabolism, immune response, cell survival and migration, inflammation, or protein synthesis or are structural
proteins. As an early response to the inflicted tissue damage and
necrosis, an increased expression of cell stress-related genes (e.g.,
hsp70 #L16764, hsp60 #X54793, metallothioneine 1 and 2 #M11,794, oxidative stress inducible protein tyrosine
phosphatase #S81474), and of genes related to inflammation,
innate immune response, and transcriptional and translational
mechanisms (Complement C1 #X71127, NGF induced factor A
#AF023087, Calprotectin #L18948) was seen within
2 h after injury. At the same time, gene expression of structural
proteins (Collagen #AJ005394, -tropomyosin
#M60666) decreased. Within 2 to 6 h, transcription
factors, immediate early genes, cell proliferation, and cell
survival-associated genes were affected. Their activation, in many
instances, appears to be over by 10 days (Table 3).
Inflammation-related genes and stress response genes remained the
mainstay of upregulated gene expression for the first 24 h in
agreement with earlier reports (10). Genes involved in
cell migration, such as membrane type matrix metalloproteinase
#X83537 and versican V3 #AF072892, appear to be
activated later. By 10 days after burn, the expression pattern shifted
to increased expression of structural, cellular, and extracellular
matrix proteins, genes involved in the specific immune response, and
regulation of cellular function. Not surprisingly, there was a
repression of genes playing significant roles in energy metabolism, such as pyruvate dehydrogenase #U10357,
phosphoglyceromutase #Z17319, or cytochrome oxidase
#U40836, indicating metabolic failure of the involved
tissue, which was resolved in 10 days. This is consistent with reports
showing a central role for neutrophil migration and immune activation
in burns in rats (25).
All the described events of differential gene expression correlated
well with distinct gene families typically identified by gene cluster
analysis. These genes include cellular- and intercellular mediators,
such as inflammatory and anti-inflammatory cytokines, hormones,
stress-response genes, cell-cycle genes, pro- and anti-apoptotic genes, gene products of cellular metabolism, genes coding for structural proteins, and extracellular matrix products and their proteinases. The assignations from DNA microarray analyses typically correspond to known sequelae of cellular and molecular events associated with burns and sequential and coordinated wound healing. However, there were some interesting exceptions that point to an
activation of some clusters of genes at earlier times than previously
thought and a persistence in the expression of genes previously
believed to display transient responses, such as the inflammatory
mediators gro #D11445 and IL-1 #M98820.
The changes in gene expression in the burn wound can be correlated to
known events in the wound healing process after burn. These events are
coordinated and follow a well-defined sequence (Fig.
10). The initial injury leads to energy
depletion and cellular damage, which may lead to tissue necrosis. Acute
stress genes are initially upregulated to prevent further tissue damage
and trigger restoration of tissue homeostasis. At the same time,
inflammation and nonspecific host defense mechanisms are activated. The
activation of restorative mechanisms, such as growth regulatory genes
and genes encoding for the production of structural proteins, occurs at
later times.
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ACKNOWLEDGEMENTS |
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This study was supported by Shriners Hospitals for Children Grants 8660 and 8490 and National Institutes of Health Grants 1P50GM60338-1 and 5RO1GM572903.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. R. K. Dasu, Shriners Hospitals for Children, 815 Market St., Galveston, TX 77550 (E-mail: drmohan{at}utmb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00170.2002
Received 16 March 2002; accepted in final form 14 June 2002.
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TOP
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RESULTS
DISCUSSION
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