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Physiol. Genomics 22: 157-170, 2005. First published May 3, 2005; doi:10.1152/physiolgenomics.00018.2005
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Received 20 January 2005; accepted in final form 26 April 2005.
Physiological Genomics 22:157-170 (2005)
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Profiling of differentially expressed genes in wound margin biopsies of horses using suppression subtractive hybridization

Josiane Lefebvre-Lavoie, Jacques G. Lussier and Christine L. Theoret

Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Quebec, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Disturbed gene expression may disrupt the normal process of repair and lead to pathological situations resulting in excessive scarring. To prevent and treat impaired healing, it is necessary to first define baseline gene expression during normal repair. The objective of this study was to compare gene expression in normal intact skin (IS) and wound margin (WM) biopsies using suppression subtractive hybridization (SSH) to identify genes differentially expressed during wound repair in horses. Tissue samples included both normal IS and biopsies from 7-day-old wounds. IS cDNAs were subtracted from WM cDNAs to establish a subtracted (WM-IS) cDNA library; 226 nonredundant cDNAs were identified. Detection of genes previously shown to be expressed 7 days after trauma, including the pro-{alpha}2-chain of type 1 pro-collagen (COL1A2), annexin A2, the pro-{alpha}3-chain of type 6 pro-collagen, ß-actin, fibroblast growth factor 7, laminin receptor 1, matrix metalloproteinase 1 (MMP1), secreted protein acidic cystein rich, and tissue inhibitor of metalloproteinase 2, supported the validity of the experimental design. A RT-PCR assay confirmed an increase or induction of the cDNAs of specific genes (COL1A2, MMP1, dermatan sulfate proteoglycan 2, cluster differentiation 68, cluster differentiation 163, and disintegrin and metalloproteinase domain 9) within wound biopsies. Among these, COL1A2 and MMP1 had previously been documented in horses; 68.8% of the cDNAs had not previously been attributed a role during wound repair, of which spermidine/spermine-N-acetyltransferase, serin proteinase inhibitor B10, and sorting nexin 9 were highly expressed and whose known functions in other processes made them potential candidates in regulating the proliferative response to wounding. In conclusion, we identified novel genes that are differentially expressed in equine wound biopsies and that may modulate repair. Future experiments must correlate changes in mRNA levels for precise molecules with spatiotemporal protein expression within tissues.

gene profile; wound repair; suppression subtractive hybridization; equine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TRAUMATIC WOUNDS are common in the horse, where primary closure is frequently precluded by considerable tissue loss and contamination, such that repair must occur by second intention. Horses suffer from chronic nonhealing wounds similar in appearance to venous leg ulcers in humans and, conversely, from excessive fibroplasia (also referred to as "proud flesh"), which subsequently compromises epithelialization and contraction. Both conditions ultimately lead to extensive scarring, which may adversely affect function.

Chronic, indolent wounds appear to result, in part, from an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Chronic wound fluid is characterized by elevated levels of proteinases, which lead to excessive protein degradation and the inactivation of critical growth factors (72). Chronic wounds also contain reduced levels of TIMPs, exacerbating the degradative environment.

Equine proud flesh resembles pathological scarring in humans (hypertrophic scar, keloid) in which the normal sequence of repair becomes dysregulated and the evolving scar is trapped in the proliferative phase of repair (24, 31). Several factors have been incriminated in this condition in horses including an inefficient inflammatory response to trauma (78, 79), persistent local upregulation of profibrotic cytokines (69, 70, 73), and a disparity between the synthesis and degradation of collagen (61) as well as microvascular occlusion and deficient apoptosis of the cellular components of granulation tissue (unpublished data). Despite this knowledge, attempts at ameliorating the repair of chronic wounds and preventing the development of keloids in the horse have been disappointing. This no doubt relates to the lack of information pertaining to the molecular mechanisms regulating repair.

Dermal wound repair involves intricate exchanges between multiple cell types, cytokines, and extracellular matrix (ECM) molecules acting locally and in parallel with numerous systemic factors such as platelets, the coagulation cascade, and cellular and humoral components of the immune system (66). Events are conventionally divided into synchronized and interrelated phases including acute inflammation, cellular proliferation, and, finally, matrix synthesis and remodeling with scar formation. The transition between phases requires the activation and/or silencing of many genes, such that a disturbance in gene expression could lead to abnormal scarring.

A handful of studies have analyzed the expression of specific genes during normal or impaired healing, in particular, those regulating ECM molecules and cytokines (56, 9, 62), although only one has been performed in the horse (61). While these investigations have yielded valuable data, they are far from comprehensive. Indeed, given the complexity of the repair process, a thorough outline of all contributing molecules is required if healing is to be positively influenced. Furthermore, before abnormal genetic responses to wounding can be interpreted, the gene expression profile of normal injured skin must be characterized, as has recently been done in humans (12) and rats (66).

The objective of this study was to pinpoint genes that are differentially expressed during the proliferative phase of repair by comparing gene expression in biopsies of 7-day-old wounds [wound margin (WM) biopsies] with that in intact skin (IS). Identification and characterization of gene expression patterns will contribute to a better understanding of the overall repair process and ultimately permit the development of novel diagnostic and therapeutic strategies to resolve wound healing complications.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animal model, tissue collection, and RNA isolation.
Four healthy 2- to 3-yr-old Standardbred mares were used for the experiment, which was conducted in accordance with the rules of the Canadian Council on Animal Care. Protocols were approvd by an internal (Faculty and University) committee. The animals were kept in standing stalls and examined daily for signs of discomfort and systemic illness, and wounds were monitored until complete healing.

Horses were sedated then local anesthesia was performed at the site destined for wounding on one randomly chosen hemithorax per horse. The surgical sites were aseptically prepared, and a full-thickness, 6.25-cm2 wound was created with a scalpel and left to heal by second intention with no dressing. Excised skin from each wound was kept as the normal IS sample. Full-thickness specimens were taken 7 days postoperatively with an 8-mm-diameter biopsy punch. The WMs included a 3- to 4-mm section of the wound margin composed of peripheral skin and the migrating epithelium as well as a 3- to 4-mm section of granulation tissue from the wound center. Biopsies were snap frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNA from IS and WM was extracted as previously described (3). The concentration of total RNA was quantified by measuring optical density at 260 nm, and its quality was evaluated by visualizing the 28S and 18S ribosomal bands after electrophoretic separation on agarose gel with ethidium bromide (58).

Suppression subtractive hybridization.
To counter interanimal variation, identical amounts (1 µg) of total RNA from each horse were pooled within IS and WM groups. The suppression subtractive hybridization (SSH) procedure was validated and is used routinely in our laboratory (8, 17, 36). Briefly, the SMART PCR cDNA synthesis kit was used to generate double-stranded cDNA for both IS and WM samples according to the manufacturer's instruction (user manual PT30411, BD Biosciences Clontech; Mississauga, Ontario, Canada). To produce the first-strand cDNA, 1 µg of total RNA from each pooled group was reverse transcribed in a total volume of 10 µl with the addition of 42 ng of T4 gene 32 protein (Roche Applied Science, Laval, Quebec, Canada) with an oligo-dT30 primer [CDS: 5'-AAGCAGTGGTAACAACGCAGAGTACT(30)(A/C/G/T)(A/G/C)-3'] and PowerScript reverse transcriptase (BD Biosciences Clontech). Second-strand cDNAs were generated with the SMART II 5'-anchored oligonucleotide (5'-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3'), followed by a PCR amplification of 20 cycles for IS samples and 19 cycles for WM samples using Advantage 2 DNA polymerase (BD Biosciences Clontech). The forward (WM-IS) and reverse (IS-WM) reactions were obtained by subtracting, respectively, the IS cDNAs from WM cDNAs and WM cDNAs from IS cDNAs using PCR-Select cDNA subtraction technology (user manual PT1117–1, BD Biosciences Clonetch). To execute this subtraction, WM and IS cDNAs were digested with RsaI to generate blunt-ended cDNA fragments (0.2–2 kb) suitable for optimal subtractive hybridization.

Subtraction efficiency was assessed via PCR amplification using equine gene-specific primers by comparing the abundance of cDNAs before and after subtraction. Two genes were analyzed: one that is constitutively expressed, glyceraldehyde 3-phosphate dehydrogenase (GAPD), (GAPD GenBank Accession No. AF157626, sense: 5'-CAAGTTCCATGGCACAGTCACGG-3'; antisense: 5'-AAAGTGGTCGTTGAGGGCAATGC-3'); and another that is known to be upregulated in wound healing in rodents, matrix metalloproteinase 3 [MMP3 (40, 47)] (MMP3 GenBank Accession No. U62529, sense: 5'- GTTACTATGCGTGGCAGCGTGC-3'; antisense: 5'-GTGTTGGTCGAGTGATAGAGACC-3'). Advantage 2 DNA polymerase (BD Biosciences Clontech) was used to perform PCR amplification, and 5-µl aliquots were removed after 15, 20, 25, and 30 cycles for analysis on agarose gel. Subtraction efficiency was estimated by noting the different number of cycles needed to generate approximately equal amounts of the corresponding PCR product in subtracted and unsubtracted samples.

Cloning of subtracted cDNAs.
The subtracted cDNAs were cloned into the pDrive plasmid (Qiagen PCR cloning kit, Qiagen; Mississauga, Ontario, Canada) in 10-µl ligation reaction to construct the WM-IS subtracted library and used to transform competent TOP10F' Escherichia coli as described previously (36).

Differential hybridization screening.
The subtracted WM-IS cDNA library (950 individual colonies) was used to establish macroarrays for differential screening, as previously described (36, 17). The insert of each cDNA clone was amplified by PCR (27 cycles) in 96-well plates using the PCR-nested primers 1 and 2R as supplied by the manufacturer (BD Biosciences Clontech) along with AmpliTaq DNA polymerase (Roche Molecular Systems). An aliquot of each amplification product was denatured in 0.3 M NaOH containing 5% bromophenol blue, and 15 µl were vacuum transferred onto a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech; Baie d'Urfé, Quebec, Canada) using a 96-well dot-blot apparatus to create cDNA microarrays. Membranes were then cross-linked with 150-mJ ultraviolet light (GS Gene Linker, Bio-Rad; Mississauga, Ontario, Canada). Positive (MMP3) and negative (EquC1, glycoprotein member of the lipocalin superfamily) control cDNAs were transferred onto macroarrays. For each 96-well plate, four replicate cDNA macroarray membranes were generated.

The subtracted (WM-IS and IS-WM) as well as unsubtracted (WM and IS) cDNA pools were used as complex hybridization probes for differential screening of macroarrays of the WM-IS cDNA library. The cDNA probes were labeled by random priming incorporating [{alpha}32P]dCTP (NEN Life Sciences; Boston, MA) as previously described (17). Hybridization and washing conditions of macroarrays were as previously described (17). Equal quantities (in counts/min) of each heat-denatured cDNA probe, subtracted (WM-IS and IS-WM) or unsubtracted (WM and IS), were used to individually hybridize replicate WM-IS macroarray membranes. The membranes were then thoroughly washed and exposed to a phosphor screen for 4 h, and the images were digitized (Storm 840, Amersham-Pharmacia Biotech). DNA sequencing and gene expression analysis were finally used to further characterize the differentially hybridizing cDNA clones.

DNA sequencing and sequence analysis.
PCR amplification of the cDNA clones identified as differentially expressed by the WM-IS subtracted probe was performed on the PCR product generated initially for the macroarrays. The primers used for the 12 PCR cycles were PCR-nested 1 and PCR-nested 2R. Products were purified and visualized on agarose gels to keep those containing a single cDNA band. Sequencing reactions were performed on clones via the dideoxy method (Big Dye Terminator 3.0, ABI Prism, Applied Biosystem; Branchburg, NJ) using the PCR-nested 1 (1.5 mM) or PCR-nested 2R (1.5 mM) primers. An ABI Prism 310 sequencer (Applied Biosystem) was used to analyze the sequencing reactions. Ultimately, 315 clones provided adequate results, and their nucleic acid sequences were analyzed by Basic Local Alignment Search Tool (BLAST) against GenBank databases [NR and expressed sequence tag (EST)]. To be considered homologous to a GenBank sequence, a cDNA sequence was required to have at least 100 bp matched with a probability value of <e–30 for the NR bank and <e–20 for the EST bank. The differentially expressed cDNA clones were then categorized into three groups: 1) genes with known sequence and function, 2) genes with known sequence but unknown function, and 3) sequences with no significant match.

Gene expression analysis.
Semiquantitative RT-PCR was used to confirm the differential expression pattern of selected cDNA clones identified by SSH. The clones were analyzed with RNA from the four horses used in the experiment, and for each horse the IS and WM samples were compared. SMART PCR cDNA synthesis technology (BD Biosciences Clontech) was used to generate cDNAs from 1 µg of total RNA from each IS and WM sample, as described above. Equine gene-specific primers were for MMP1 (MMP1 GenBank Accession No. AF48882, sense: 5'-GACACAGGAGCCCAGTCGTTG-3'; antisense: 5'- GAATGAGAGAGTCCAAGGGAATG-3'), the pro-{alpha}2-chain of type 1 pro-collagen (COL1A2 GenBank Accession No. AB070839, sense: 5'-CGAAACCTGTATCCGGGCTCAAC-3'; antisense: 5'- GTCCAAAAGTGCAATGTCAAGGATG-3'), dermatan sulfate proteoglycan 2 (DSPG2 GenBank Accession No. AF038127, sense: 5'-GACACCACGCTGCTGGACCTG-3'; antisense: 5'-CAAGTGAAGTTCCCTCAAATGAGG-3'), cluster differentiation 68 (CD68 GenBank Accession No. DN625868, sense: 5'-GCAGCGCAGTGGACATTCTTGG-3'; antisense: 5'-GCTCAGAGTGGCTGGTAGGTG-3'), cluster differentiation 163 (CD163 GenBank Accession No. DN625936, sense: 5'-CACTACTTGTTCTGGACGTGTGG-3'; antisense: 5'-GTAAGCAGCTGTCTCTGTCTTCG-3'), disintegrin and metalloproteinase domain 9 (ADAM9 GenBank Accession No. DN625845, sense: 5'-GCTGATCTGGTTTCCAGTTGTCC-3'; antisense: 5'-CAGAGGACTGCTGCACATAGAC-3'), and GAPD and MMP3 as described earlier. The PCR products were separated on a Tris-acetate-EDTA buffer (TAE)-agarose gel with ethidium bromide and visualized with ultraviolet light, and the images were then digitized. Image signal intensity was analyzed by densitometry using NIH Image (Research Services Branch of the National Institutes of Health, http://rsb.info.nih.gov/nih-image/).

Statistical analysis.
Gene-specific signals for RT-PCR analyses were normalized by establishing a ratio with the corresponding GAPD signals for each sample. Homogeneity of variance between IS and WM biopsies was verified by O'Brian and Brown-Forsythe tests (28). Corrected values of gene-specific mRNA levels were compared between IS and WM biopsies by one-tailed paired t-test (SAS version 9.0; Cary, NC). Data are presented as least-square means ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animal model.
Wounds in all horses healed uneventfully. None became infected or developed exuberant granulation tissue (proud flesh).

Identification of differentially expressed genes using SSH.
A cDNA library containing transcripts upregulated 7 days postwounding was obtained by subtracting IS cDNAs from WM cDNAs via SSH (WM-IS). A reverse library was also constructed by subtracting WM cDNAs from IS cDNAs to be used as a control (IS-WM). PCR amplification analysis was used to verify efficiency of the subtraction procedure by comparing the expression of GAPD and MMP3 before and after subtraction. As expected, GAPD showed a marked decrease in the relative abundance of cDNA in the WM-IS sample after SSH. Indeed, GAPD products were detected after only 15 PCR cycles in the WM unsubtracted sample, whereas in the subtracted sample (WM-IS) 20 PCR cycles were necessary for detection on agarose gels (Fig. 1A). To evaluate the enrichment efficiency of genes known to be upregulated during wound repair in rodents, the abundance of MMP3 (40, 47) cDNA was investigated (Fig. 1B). MMP3 PCR products were observed after 25 PCR cycles in both unsubtracted samples, whereas in the subtracted sample (WM-IS) the MMP3 signal was detected after only 20 PCR cycles, revealing that MMP3 cDNA had been efficiently enriched. Conversely, the reverse subtracted sample (IS-WM) presented no signal, indicating a complete depletion of MMP3 cDNA. These results confirm the effectiveness of both the normalization and subtraction steps.



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  		Fig. 1. Evaluation of subtraction efficiency. A: reduction of GAPD cDNA after subtraction in the wound margin (WM)-intact skin (IS) sample. Equine GAPD-specific primers were used to performed PCR on WM-IS subtracted and WM unsubtracted samples. Aliquots were collected at different PCR cycles as indicated. GAPD PCR products (760 pb) were present after only 15 PCR cycles in the WM unsubtracted sample, whereas in the subtracted sample (WM-IS), 20 PCR cycles were necessary for detection. B: enrichment of matrix metalloproteinase 3 (MMP3) cDNA after subtraction in the WM-IS sample. Equine MMP3-specific primers were used to perform PCR on WS-IS and IS-WM subtracted as well as WM and IS unsubtracted samples. Aliquots were collected at different PCR cycles as indicated. MMP3 PCR products (670 pb) were observed after fewer cycles for the WM-IS subtracted sample (20 PCR cycles) than for the WM unsubtracted sample (25 PCR cycles).

 
Subtracted cDNAs were then cloned into a plasmid vector to generate the WM-IS cDNA library. Differential hybridization screening was performed on the 950 randomly selected bacterial colonies to eliminate false-positive clones. Colonies were spotted onto four identical sets of macroarrays, and the subtracted (WM-IS and IS-WM) and unsubtracted (IS and WM) cDNA preparations were, respectively, used as probes to hybridize the macroarrays. Selection of differentially expressed cDNA clones was achieved by comparing signal intensities between the four macroarrays as defined by the following criteria. Positive clones hybridized 1) more strongly with WM-IS subtracted probes than with WM unsubtracted probes, 2) more weakly with IS-WM subtracted probes than with IS unsubtracted probes, and 3) more strongly with WM-IS subtracted probes than with IS-WM subtracted probes. Representative differential screening results are illustrated in Fig. 2.



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  		Fig. 2. Representative differential screening results of macroarrays of the WM-IS cDNA library. Four identical sets of membranes were dot blotted with PCR-amplified cDNA fragments obtained by supression subtracted hybridization. Hybridization of macroarrays was then performed with four different probes: WM-IS subtracted cDNAs (A), WM unsubtracted cDNAs (B), IS-WM subtracted cDNAs (C), and IS unsubtracted cDNAs (D). The dots in the top left corner served as internal controls: 1 indicates MMP3 (positive control) and 2 indicates Equ C1 (negative control). Examples of cDNAs that were preferentially expressed in WM compared with IS are indicated by an arrow [neuroblastoma RAS viral (v-ras) oncogene homolog-related gene] and an arrowhead [pro-{alpha}2-chain of type 1 procollagen (COL1A2)].

 
Of the initial 950 clones, differential screening identified 405 true positives. After visualization on agarose gels, 361 clones were deemed of adequate quality to be analyzed by sequencing. Three hundred and fifteen clones ultimately generated satisfactory sequencing results and were matched against GenBank databases. This comparison revealed that 61.9% (195/315) corresponded to 129 nonredundant known genes; 20.9% (66/315) corresponded to uncharacterized cDNAs (bacterial artificial chromosome or EST clones), of which 47 were nonredundant; and 17.2% (54/315) corresponded to novel sequences, of which 41 were nonredundant (Fig. 3). Table 1 lists all the compared sequences, known genes, and uncharacterized and novel sequences as well as their frequency of identification by differential screening. Furthermore, among the 129 known genes, 31 (24.8%) have already been documented in dermal wound repair, and 8 (6.4%) have been reported in tissue repair, not necessarily specific to skin, whereas the expression of 86 (68.8%) has not previously been described during wound repair (Fig. 3).



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  		Fig. 3. Classification of the sequenced WM-IS cDNA clones. Known genes correspond to genes with known sequences and functions (E < e–30). Uncharacterized sequences correspond to genes with known sequences but unknown functions (bacterial artificial chromosome clones, cDNAs, or expressed sequence tags; E < e–20). Novel sequences correspond to sequences with no significant match in the GenBank database (E > e–20). Known genes were further subdivided into three categories: 1) genes whose presence had previously been documented in dermal wound repair; 2) genes whose presence had previously been reported in tissue repair, not necessarily specific to skin; and 3) genes whose presence had not previously been reported during wound repair.

 

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  		Table 1. Identification of cDNA clones found by suppression subtractive hybridization to be present in equine wound margin biopsies compared with intact skin

 
Gene expression analysis.
To confirm that the genes we identified by SSH are differentially expressed between normal IS and 7-day-old WM, a comparative RT-PCR assay was performed on seven selected genes as described in Table 1. For this procedure, cDNAs were generated from total RNA, and the number of PCR cycles was optimized for each gene. All of the genes analyzed in this manner showed a statistically significant increase in 7-day-old WM compared with normal IS (Fig. 4). The increase was 4-fold for COL1A2, 5-fold for DSPG2, 25-fold for CD68, 14-fold for CD163, 11-fold for ADAM9, and 2-fold for MMP3, whereas MMP1 was induced in WM compared with IS.



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  		Fig. 4. Analysis of mRNA expression by RT-PCR. Total RNA was extracted from IS and from 7-day-old WM from 4 horses used in the experiments. The precise methodology is described in MATERIALS AND METHODS. The control gene, GAPD, showed no significant difference between WM and IS samples. Gene-specific signals were thus normalized with corresponding GAPD mRNA signals for each sample. A: COL1A2 mRNA displayed a fourfold higher expression level in WM than in IS (P < 0.001). B: expression level of MMP1 mRNA was induced in WM (P < 0.003). C: dermatan sulfate proteoglycan 2 (DSPG2) displayed a fivefold higher expression level in WM than in IS (P < 0.001). D: expression of cluster differentiation (CD)68 mRNA was 25-fold higher in WM than in IS (P < 0.001). E: expression of CD163 mRNA was 14-fold higher in WM than in IS (P < 0.001). F: expression of disintegrin and metalloproteinase 9 (ADAM9) mRNA was 11-fold higher in WM (P < 0.003). G: expression of MMP3 mRNA was twofold higher in WM than in IS (P < 0.003). Probability values were obtained by one-tailed paired t-test analyses. Data are presented as least-square means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Disruption in the regulation of gene transcription might favor the development of chronic indolent wounds or fibroproliferative disorders such as proud flesh in the horse. Characterization of the specific genes regulating the proliferative phase of repair should provide valuable information about processes such as angiogenesis, fibroplasia, and epithelialization. Although some key genes are already known, it is clear that many more remain to be identified.

Our biopsies contained multiple distinct cell types, which rendered data interpretation challenging; however, as the interaction between these cells is central to repair, we deemed it crucial to investigate WM samples rather than isolated cells in culture. Because several of the basic events controlling wound repair at the gene level remain obscure, we elected to use SSH rather than more conventional molecular approaches that target candidate genes.

Of the high-throughput molecular techniques currently available, cDNA microarrays are very efficient as they determine the simultaneous expression of thousands of genes. However, gene identification is limited to those present on the microarray, precluding the discovery of new genes. Additionally, at the onset of our study, equine-specific microarrays had not yet been developed (20). An added benefit of SSH over cDNA microarray technology is that PCR amplification of the cDNA pools before hybridization enables SSH to be performed with limited quantities of mRNA, a significant advantage when attempting RNA extraction from tough horse skin.

To verify the differential expression of genes identified by SSH, a RT-PCR assay compared the expression of specific genes in normal IS and in 7-day-old WM biopsies. These genes were selected according to the availability of equine-specific primers as well as their redundancy of expression, their oligonucleotide sequence, and their role. Three had a previously described role in dermal wound repair [COL1A2 (65, 61), DSPG2 (5), and MMP1 (65, 74, 61)], whereas the others were not known to be expressed in healing dermal wounds (CD68, CD163, and ADAM9). All six genes showed a statistically significant increase in WM compared with normal IS, validating both the model and the techniques. Additionally, we identified nine other genes that had previously been detected in 7-day-old wounds in other species: COL1A2 (61), MMP1 (61), TIMP-2 (65), pro-{alpha}3-chain of type 6 pro-collagen (COL6A3) (48), secreted protein acidic cystein rich (SPARC) (53), fibroblast growth factor 7 (FGF-7) (76), annexin A2 (ANXA2) (44), laminin receptor 1 (LAMR1) (49), and ß-actin (ACTB) (4). Their identification further corroborates our methodology.

The COL1A2 gene codes for the pro-{alpha}2-chain of type 1 pro-collagen. While collagen is essential to the structural integrity and mechanical strength of tissues, its abnormal accumulation results in fibrosis. During wound repair, collagen participates in wound contraction via cross-linking and regulates cytokine activity through specific binding. Cytokines, in particular transforming growth factor-ß (TGF-ß), can stimulate collagen type 1 synthesis by fibroblasts (23). Collagen type 1 {alpha}2 mRNA is expressed in equine dermal wounds 7 days after trauma (61). The protein has been shown to accumulate to a greater extent and in a disorganized manner in horse wounds that are predisposed to excessive scarring (61, 78). In the present study, screening of our WM-IS subtracted cDNA library identified the COL1A2 gene 14 times.

DSPGs play a significant role in tissue development and assembly as well as participating in direct and indirect signaling and modulating the cellular response to growth factors (18). For example, DSPGs are thought to increase growth factor-mediated fibroblast migration and proliferation (15). DSPG2 has been previously identified in dermal wound repair 7 days after trauma (5) and was found twice in the present study. Because proud flesh is characterized by an increased density of dermal fibroblasts, it might be interesting to study the implication of DSPG2 in limb wounds predisposed to excessive scarring.

MMP1 is critical to wound repair as it is one of only three collagenases able to degrade interstitial collagens. Reduced expression of MMP1 retards such important processes as cell migration, angiogenesis, and tissue remodeling, thus slowing the repair process. A study (74) defining the pattern of change during the repair of skin wounds in pigs reported no detectable mRNA for MMP1 in IS; however, by 24 h postwounding, levels peaked and then progressively declined until day 35. In the present study, screening of the WM-IS cDNA library confirmed the presence of MMP1 in 7-day-old body wounds in horses. Defective collagenolysis is a feature of hypertrophic scars and keloids in humans, and excess TGF-ß, which inhibits ECM turnover by concurrently inducing TIMP and reducing MMP expression, has been incriminated. While TGF-ß mRNA and protein levels persist over time in repairing equine limb versus body wounds (61, 69), a recent study (61) documented a significantly greater amount of MMP1 mRNA in the body than leg skin of horses before wounding; however, this difference in gene expression disappeared 7 days after trauma (61). These data are unexpected, as it could be anticipated that wounds of the horse limb, predisposed to excessive fibroplasia, would display reduced levels of MMPs during the proliferative phase of repair. We plan to verify this data using SSH to compare biopsies from 7-day-old body and leg wounds in horses.

While CD68, CD163, and ADAM9 have not specifically been associated with the repair of skin wounds, these genes are known to regulate certain responses to trauma. Human CD68 and its mouse homolog macrosialin are transmembrane proteins found almost exclusively in macrophages and macrophage-like cells (27). Although the exact function of CD68/macrosialin proteins remains to be elucidated, in practice CD68 is often used to evaluate the importance of monocytes/macrophages in tissues (22). We report an increase in CD68 expression in 7-day-old WM biopsies compared with IS, suggesting the presence of monocytes/macrophages in the tissues, as previously shown histologically (69). Interestingly, wound repair in the horse is characterized by a weak but protracted inflammatory phase (78), which may perpetuate the release of tissue-damaging lysosomal enzymes as well as mediators such as TGF-ß, which overstimulate fibroplasia leading to the formation of exuberant granulation tissue (13). It would be interesting to investigate whether CD68 mRNA is differentially expressed in equine limb wounds predisposed to the development of proud flesh versus the normally repairing body wounds investigated herein.

CD163 is exclusively expressed on peripheral blood monocytes and tissue macrophages (32). Binding of haptoglobin- hemoglobin complexes to CD163-bearing cells allows degradation of the ligand and metabolism of heme, which appears to minimize inflammation. Moreover, shedding of CD163 generates substantial amounts of soluble receptor in plasma (43), which actively inhibits lymphocyte proliferation, thereby modulating the cells' response to an inflammatory stimulus (71). It has recently been shown that TGF-ß markedly reduces the expression of CD163, via transcriptional regulation (51). The protective effect of CD163 against inflammation may be relevant in wound repair. In the present study, we documented a significant increase of CD163 in 7-day-old wounds compared with IS. Interestingly, an increased expression of CD163 was also documented in chemically induced skin blisters in humans, concomitant with differentiation of blood monocytes into macrophages (50). In view of the prolonged inflammation (78) and persistent expression of TGF-ß within repairing equine limb wounds compared with body wounds (61, 69), we hypothesize that CD163 gene expression might be diminished in limb wounds in horses.

ADAM9 contains both metalloproteinase and disintegrin domains and is probably implicated in the transition from wound bed provisional matrix to collagenous scar. The disintegrin domain supports integrin-mediated cell adhesion and may thus play a role in regulating cell motility and proliferation. Furthermore, ADAMs have emerged as major sheddases in that they enable release of cell surface proteins including cytokines, their receptors, and cell adhesion molecules, with a subsequent impact on extracellular signaling (30). The ability of ADAM9 to interact with integrins and ECM proteins as well as play a decisive role in signaling events suggests that it could modulate wound repair (77). Wounds on the limbs of horses may become trapped in the proliferative phase of repair, triggering subsequent delays in wound contraction and epithelialization. Given the roles of ADAM9, a deficiency might impede the replacement of fibronectin by proteoglycans and collagen, sustaining immature granulation tissue within the wound bed. This is a feature of chronic wounds, as are delays in epithelial migration, also a potential effect of insufficient ADAM9. Conversely, excess ADAM9 could stimulate overproliferation of fibroblasts with attending hypergranulation, characteristic of limb wounds of horses. This might be compounded by the release of certain cytokines, exerting chemotactic and mitogenic effects. It would be interesting to study the spatiotemporal expression of ADAM9 during the proliferative phase of repair in horses given its likely role in both normal and pathological repair.

Of the 31 genes previously shown by others to be expressed during dermal repair, 9 genes had been documented specifically 7 days after trauma (mentioned above), whereas the presence of 22 genes had been shown in dermal wound repair but not specifically during the proliferative phase (Table 2). We also identified eight cDNAs corresponding to genes documented in the repair of bone [cathepsin K (29) and calumenin (45)], retina [serine proteinase inhibitor (SERPIN), clade F, member 1 (68, 60)], cornea [lumican (57) and cadherin 11 (42)], or nervous tissue [prosaposin (21) and lipocortin 1, lumican and ferritin light chain (46)].


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  		Table 2. Classification of 22 genes previously reported to be present in dermal wound repair but not known to be expressed specifically during the proliferative phase

 
The remaining 68.8% cDNAs identified by SSH screening had not previously been attributed a role in wound repair. Among these, we believe that spermidine/spermine-N-acetyltransferase (SSAT), SERPIN B10, and sorting nexin 9 (SNX9) in particular could be interesting novel candidate genes associated with the proliferative phase of dermal wound repair in horses.

Catabolism of the polyamines spermidine and spermine is achieved by the collaborative effort of two enzymes, cytosolic SSAT and a polyamine oxidase (25). Spermidine and spermine appear crucial to the proliferation of mammalian cells via the promotion of cell growth or the induction of apoptosis when they occur in excess (75), whereas it appears that activation of polyamine catabolism, via SSAT, is more closely related to an antiproliferative action (25). It has recently been shown that overexpression of SSAT enhances integrin-mediated migration of leukocytes and epithelial and endothelial cells (11). Thus SSAT could potentially be involved in cell migration and/or apoptosis, which are both critical to wound repair. Specifically, it would be interesting to investigate the role of SSAT during angiogenesis and epithelialization as well as during chronic inflammation when leukocyte migration is excessive and persistent.

The SERPINs are a superfamily of proteins that trap their targets by undergoing a conformational rearrangement to protect cells from proteinase-mediated injury. SERPIN B10, formerly known as proteinase inhibitor 10 or BOMAPIN, belongs to the ovalbumin/serpin clade B and is a competitive inhibitor of thrombin and trypsin. It possesses a 45% amino acid identity with plasminogen activator inhibitor 2, human leukocyte elastase inhibitor, and cytoplasmic antiproteinase (54). It has been attributed a role in the regulation of cell growth or differentiation and angiogenesis as well as tumor cell invasiveness. It is thus possible that SERPIN B10 is involved in both the inflammatory and proliferative phases of repair, where it may modulate clot formation and/or alter the formation of new blood vessels within granulation tissue.

SNX9 belongs to a group of proteins believed to participate in sorting processes in the cell. It has been found to cooperate with activated Cdc42-associated kinase-2 (Ack2) through an interaction occurring between a proline-rich domain of Ack2 and the Src homology 3 domain of SNX9. In mammalian cells, Ack2 interacts with the receptor for EGF, with interaction stabilized by SNX9. Upon stimulation with EGF, SNX9 becomes tyrosine phosphorylated and is suggested to play a role together with Ack2 in the degradation and recycling of the receptor for EGF, leading to reduced levels in cells (38). Because EGF modulates numerous cellular activities during the healing process, depletion of its receptor through the action of SNX9 must be tightly regulated or could lead to aberrant repair including, among others, delays in epithelialization.

It is worthwhile to note that this study profiled gene expression at one specific time point, while wound repair is a dynamic process. Indeed, one would expect progressive changes in the expression pattern of a gene over time. Future studies have been planned to address this issue; specifically, we intend to map the spatiotemporal expression of selected genes in both normal wounds and those healing aberrantly.

In conclusion, we have succeeded, through the use of SSH, in sketching a partial blueprint of the baseline gene expression profile during the proliferative phase of normal dermal wound repair in horses. It is apparent from this study and others that many genes may be active players in the normal transcriptional response to injury. Although the immediate significance of some gene sequences identified by SSH may not be readily apparent, we did identify several others that are highly expressed after injury and may have an unappreciated role in regulating the proliferative response to wounding. This initial step serves as a precursor to elucidating abnormal genetic responses to trauma to eventually predict which wounds may be predisposed to a chronic inflammatory response or excessive fibroplasia and extensive scarring. Moreover, the data generated herein enable the design of hypothesis-driven studies that will describe the function of key genes in biological processes. Future experiments must correlate changes in mRNA levels for precise molecules with spatiotemporal protein expression within the tissues.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Research was funded by the Natural Sciences and Engineering Research Council of Canada as well as the Fonds Québécois de Recherche en Nature et Technologie of Québec.


   ACKNOWLEDGMENTS
 
Bandage materials were generously donated by Smith-Nephew Canada. The authors thank Élodie Lepault, Christophe Céleste, Marilys Ducharme-Desjarlais, and Tania Fayad for technical assistance as well as Marco Langlois for help with the figures.


   FOOTNOTES
 
Address for reprint requests and other correspondence: C. L. Theoret, Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, 3200, rue Sicotte, St-Hyacinthe, Québec, Canada J2S 7C6 (E-mail: christine.theoret{at}umontreal.ca).

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/physiolgenomics.00018.2005


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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