Abstract: Background. Normal wound healing results from a complex set of reactions between blood cells, skin cells, and biochemical mediators including pro- and anti-inflammatory molecules, growth factors, cytokines, hormones, and vitamins. As this cascade of reactions is ultimately regulated by the coordinated expression and silencing of numerous genes, the gene expression analysis of hypertrophic and keloid scarring (HS and KS, respectively) should provide important information and improve our understanding of HS and KS pathophysiology. Microarray is a new tool that can shed light on the complex genetic background that regulates pathologic scarring. This review will describe basic principles of microarray technique for wound care professionals and explain how this technology is contributing to a better understanding of HS and KS biology. Methods. A brief review of the literature on microarray in HS and KS over the last 7 years was conducted. Results. The inter-experiment comparisons are somewhat difficult because of differences in the probes used, diverse source of samples, different time points of wound healing, and in-vivo or ex-vivo analysis. Wound healing gene expression must be studied in an environment where all cells and mediators could show how the regulatory network functions. Conclusion. All results confirmed previous findings about HS and KS related to over-expression of collagen or extracellular matrix (ECM) genes. One conclusion after this initial approach is that a standardized animal model, probe, and software for data analyses to compare results would increase the understanding of HS and KS pathophysiology.

Address correspondence to:
Prof. Alfredo Gragnani, PhD
Rua Napoleão dee Barros, 715-4˚ andar
São Paulo, Brazil 04024 002
Phone: 55 11 5576 4118
E-mail: alfredog@ig.com.br

   Hypertrophic scarring (HS) and keloid scarring (KS) are pathologic responses to skin injury induced primarily by burns and deep dermal wounds.¹ Both HS and KS share key pathophysiologic findings such as extracellular matrix (ECM) over deposition, deficit of collagen degradation, and decreased fibroblast apoptosis.²

   Normal wound healing results from a complex set of reactions between blood cells, skin cells, and myriad biochemical mediators including pro- and anti-inflammatory molecules, growth factors, cytokines, hormones, and vitamins.² Although abnormalities at different phases of the scarring process have been described in HS and KS, their significance is not completely understood.³ As this cascade of reactions is ultimately regulated by the coordinated expression and silencing of numerous genes, the gene expression analysis of HS and KS should provide important information and improve understanding of HS and KS pathophysiology.

   Most HS and KS gene expression studies in the literature are based on polymerase chain reaction (PCR) technology^4–7 and in-situ hybridization.⁸ While both processes accurately assess the gene expression of well known genes involved in wound healing, their application is limited by the small number of genes that can be studied at one time.⁹

   The finalization of the Human Genome Project gave rise to a new and rapidly growing technology that enables researchers to determine the relative expression levels of thousands of genes simultaneously, comparing healthy and affected biological samples—the Deoxyribonucleic acid (DNA) microarray, also known as the gene chip or DNA chip.¹⁰

   This review will describe basic principles of microarray technique for wound care professionals and explain how this technology is contributing to a better understanding of HS and KS biology.

Microarray Technique

   RNA extraction, cDNA synthesis, and labeling. Although the nuclear DNA content of any cell from an organism is exactly the same, living cells are different from each other. Where does this difference come from? The answer is on the Ribonucleic acid (RNA).

   Genes are segments of DNA with the information necessary to make a protein, but unless a molecule of messenger RNA (mRNA) takes this information from the nucleus to the ribosomes on the cytoplasm where protein synthesis takes place, nothing happens. Thus, through mRNA and protein synthesis, cells control the way they grow, divide, and die. Therefore, the study of the mRNA provides us with the information about which genes are “turned on” and which genes are “turned off” at a definitive time point.

   Microarray studies produce a “map” of gene expression comparison between two biologic samples, usually one normal (control) and one being studied (pathologic). Moreover, it gives information about the level of expression of the genes. This technique allows thousands of genes to be studied at the same time and provides an overview of the genetic activity underlying specific biological phenomena.

   The first step in a gene microarray experiment is total RNA extraction from the cell population to be studied. The tissue is dissolved in a mixture of organic solvents and while DNA, proteins, and cellular debris are precipitated, RNA remains in solution. The sample at this point contains total RNA, but only mRNA (comprising < 5% of total RNA) reflects gene expression. Methods that exploit its exclusive poly(A)-tail isolate it from the rest of RNA.

   The second step is called reverse transcription and consists of the in-vitro synthesis of DNA molecules that are complementary to mRNA: the complementary DNA (cDNA). During this step, the molecules of cDNA are labeled with mono-Reactive dyes (cy3 and cy5). In a standard microarray experiment, cDNA from normal tissue is labeled with cy3 (green fluorescent dye) and the cDNA from the tissue subject to study is labeled with cy5 (red fluorescent dye). The result of this step is the generation of labeled DNA molecules with the exact same nucleotide sequence of the expressed portions of genes.

   Microarray experiment. Hybridization is a key property of DNA and the basis of microarray concept. Two complementary DNA strands will, in favorable conditions, hybridize to one another, forming a double strand DNA molecule.

   In its simplest form, a microarray is any array (a large number of ordered objects) of biological material that is printed on a solid substrate in a “micro” format, which allows many objects to share a relatively small area. It can be a glass, plastic, or silicon microscope slide (approximately 1 in x 3 in) with tens of thousands of specific pieces of genes printed on it, placed on the substrate by a robot that can deposit very small volumes of material into discrete spots within the array. A single spot contains many copies of the same gene. A computerized database lists which genes are contained in each spot.

   The experiment consists of adding the single strand labeled cDNA to the microarray chip. cDNA molecules will hybridize to their complementary DNA strands on the microarray. Molecules with no complementarity are washed away. The chip is scanned with a green and red laser, which generates colored images that are sent to a computer program. The microarray scanner merges both images (from control and test samples) and displays the composite picture. Green spots represent a gene underexpressed in the test sample when compared to the control. Red spots represent overexpression of genes when compared to controls, and yellow spots correspond to genes equally expressed between the two samples. A black spot means that no reaction occurred, and therefore, the gene was not expressed in any of the samples.

   Clustering and data analyses. This composite picture provides a significant amount of information about gene expression in both samples and must be organized and compared. A natural basis for organizing gene expression data is to group together genes with similar expression patterns. Although various clustering methods can conveniently organize tables of gene expression measurements, the results still represent a massive collection of numbers that is difficult to assimilate. Therefore, a representation of the primary data with the red/yellow/green and black spots is always combined with the clustering information. The result is a representation of gene expression data that, through statistical organization and graphical display, allows biologists to assimilate and explore data in an intuitive manner.¹¹ Such an amount of newly-generated information, since the advent of DNA microarray technology, demands standardization of data analysis in a way that the results could be interpreted unambiguously and be comparable among different laboratories.

   Pursuing this aim, the Microarray Gene Expression Data (MGED) Society, an international organization of biologists, computer scientists, and data analysts formed in 1999, proposed the Minimum Information about a Microarray Experiment (MIAME). Their purpose was to establish a standard for recording and reporting microarray-based gene expression data in public repositories. MIAME is now widely accepted by microarray investigators and specialized journals.¹²

   Gene regulatory networks and gene ontology. The next step is to understand gene regulatory networks (GRN), the on/off switches, and rheostats of a cell operating at the gene level. The GRN dynamically orchestrate the level of expression for each gene in the genome by controlling whether and how vigorously that gene will be transcribed into RNA. Recently developed software that assembles data from the available biological literature helps predict the involved pathways based on gene expression data and in doing so, facilitates GRN identification.

Methods

   A brief review of the literature on microarray in hypertrophic and keloid scarring over the last 7 years was conducted. PubMed was searched using the keywords microarray, keloid, and hypertrophic scarring. The primary methods and genes related with this technique are described.

   Although HS and KS are distinct pathological entities, clinical and pathologic features, as familial pattern, affected parts of the body, and excessive dermal fibrosis, are shared by both. In this article, the terms HS and KS are used indiscriminately as models of fibroproliferative scarring.

Discussion

   Despite the accumulated knowledge about the complex wound healing process, it remains neither possible to predict the development of HS and KS nor to effectively treat affected patients. Microarray is a new tool that can shed light on the complex genetic background that regulates pathologic scarring. Before the advent of microarray, studies on gene expression would focus on a small number of genes; microarray experiments provide information on the expression of thousands of genes simultaneously, like capturing a panoramic photo of gene activity instead of a close-up.

   The ethical limitations for studying scarring in humans have generated intense interest in finding a good animal model for HS and KS. For many years, the lack of such a suitable model hindered the study of these conditions.¹³ However, the recently described red Duroc female pig shares many similarities with human HS such as clinical appearance, time to healing, histopathology, nerve count, and decorin, versican, TGF-b1 and IGF-1 expression, making it the most appropriate model described to date.^14–22

   The wound healing process must be evaluated at different time points. One major advantage of DNA microarray (with an adequate animal model) is that it allows one to study scarring from initial wounding to the final outcome.

   As a rule in microarray studies, the genetic expression of two samples (one affected and one control) is compared. The research discussed in this review draw comparisons between normal skin or wounds not considered fibroproliferative (control samples) and HS and KS (test samples). Over and under differentially expressed genes were referred considering test samples compared to control samples.

   It is well known that over-deposition of collagen and ECM by fibroblasts is a central event in HS. In-vitro studies of cultured fibroblasts were used to test the effect of phenytoin, interleukins, and radiation therapy over gene expression in HCFB and NF.^23–27 While a great amount of information was obtained from these studies, they are the result of gene expression of a single cell type under culture conditions where wound healing was most likely a result of interactions among all cell types in the skin.²⁸

   In ex-vivo studies, biopsies of biological samples are the source of RNA and as a result, the healing microenvironment is preserved. Authors typically use reverse transcriptase PCR (RT-PCR) to validate overexpressed genes. They studied scars ranging from 3- to 168-months-old—an evaluation of the entire wound healing process at a glance.^13,29–31 Only Wu et al¹ studied the gene expression of scar contracture in humans over time at 3, 6, 9, and 12 months (not from the same patient) and noticed cytoskeletal gene overexpression at all time points in HS when compared to normal skin with a peak in the early process and later, a gradual decrease (Table 1).

   Growth factor overexpression was rarely observed when analyzed. Chen et al²⁹ described overexpression of TGF-β1 and NGF in 41 studied growth factors.

   Naitoh et al¹³ found overexpression of the chondrogenesis and osteogenesis related genes, and suggested that the disordered differentiation from a dermal pattern to a chondrocytic-osteogenic lineage may be involved in KS etiology.

   DNA microarray of HS and KS also revealed downregulation of pro-apoptotic genes, however, Satish et al²⁴ described one anti-apoptotic gene downregulated, suggestive of an imbalance between them.

   Naitoh et al¹³ and Satish et al²⁴ described overexpression of tumor related genes (P311, FAP-aTCTP, RPS18, and PPS10) and speculate whether this explains the hyperproliferation and invasion of adjacent skin tissue attributed to fibroblasts in KS.

   In-vitro studies that confirm findings regarding HS- and KS-related to overexpression of collagen or ECM genes are grouped in Table 2. All results confirmed previous findings about HS and KS related to overexpression of collagen or ECM genes.^13,24

   Table 3 shows differentially overexpressed genes in HS and KS. Inter-experiment comparisons are somewhat difficult because of differences in the probes used, the diversity of sample sources, different time points of wound healing, and whether it was an in-vivo or ex-vivo analysis. One conclusion, after this initial approach, is that it would be useful to standardize an animal model, probes, and software used for data analyses. Wound healing gene expression must be studied in an environment where all cells and mediators could show how the regulatory network functions.

   The use of a porcine model of fibroproliferative scarring^14–22 associated with high through-put experiments can help clarify pathophysiological mechanisms and can serve as a platform for therapeutic tests, which could lead to development³² of efficacious treatments or preventative measures for HS and KS.³³

Selected animations that describe and explain RNA extraction, DNA transcription, labeling, hybridization, and microarray technique.

http://www.digizyme.com/competition/examples/genechip.swf

http://multimedia.mcb.harvard.edu/

http://www.affymetrix.com/support/learning/index.affx

http://vcell.ndsu.nodak.edu/animations/

http://learn.genetics.utah.edu/units/biotech/microarray/

http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

http://imagecyte.com/animations/array2.html

http://www.unsolvedmysteries.oregonstate.edu/microarray_07