Characterization of the acute temporal changes in excisional murine cutaneous wound inflammation by screening of the wound-edge transcriptome

Abstract

This work represents a maiden effort to systematically screen the transcriptome of the healing wound-edge tissue temporally using high-density GeneChips. Changes during the acute inflammatory phase of murine excisional wounds were characterized histologically. Sets of genes that significantly changed in expression during healing could be segregated into the following five sets: up-early (6–24 h; cytokine-cytokine receptor interaction pathway), up-intermediary (12–96 h; leukocyte-endothelial interaction pathway), up-late (48–96 h; cell-cycle pathway), down-early (6–12 h; purine metabolism) and down-intermediary (12–96 h; oxidative phosphorylation pathway). Results from microarray and real-time PCR analyses were consistent. Results listing all genes that were significantly changed at any specific time point were further mined for cell-type (neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells) specificity. Candidate genes were also clustered on the basis of their functional annotation, linking them to inflammation, angiogenesis, reactive oxygen species (ROS), or extracellular matrix (ECM) categories. Rapid induction of genes encoding NADPH oxidase subunits and downregulation of catalase in response to wounding is consistent with the fact that low levels of endogenous H₂O₂ is required for wound healing. Angiogenic genes, previously not connected to cutaneous wound healing, that were induced in the healing wound-edge included adiponectin, epiregulin, angiomotin, Nogo, and VEGF-B. This study provides a digested database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of cutaneous wound healing comprehensively.

impaired wound healing states in the elderly lead to substantial morbidity and mortality and a cost to the health services of over 9 billion dollars per annum. Recently we have developed a novel approach to specifically laser capture blood vessels from standard 3 mm human wound biopsies such that the captured blood vessel tissue element would lend itself to genomic screening as well as to verification of candidate genes using quantitative PCR (65). In humans, we have also identified stress-sensitive transcripts in wound site neutrophils (63). While results from clinical material are of extraordinary interest, they often pose limitations in the design of studies aimed at gaining mechanistic insight.

Our current understanding of the mechanisms underlying cutaneous wound healing is primarily derived from the study of experimental animal models, which lend themselves to more detailed scrutiny. Murine models are commonly studied because of the availability of a large number of genetically modified mice that help address pointed hypotheses (13). Wound healing represents the outcome of a large number of interrelated biological events that are orchestrated over a temporal sequence in response to injury and its microenvironment. Previous reports have characterized global patterns of gene expression in the burn wound (19). This work represents a maiden effort to screen the transcriptome of the healing wound-edge tissue on a temporal basis using high-density GeneChips. Focus of the study was directed toward examining acute changes in the wound-edge tissue during the inflammatory phase, which is viewed as setting the stage for the consequent phases.

MATERIALS AND METHODS

Secondary-intention excisional dermal wound model.

Young (8-wk old) male C57BL/6 mice were used for this study. Two 8 × 16 mm full-thickness excisional wounds (71) were placed on the dorsal skin, equidistant from the midline and adjacent to the four limbs (Fig. 1A, inset). The animals were killed at the indicated time points after wounding and wound-edge (1–2 mm, Fig. 1B) tissues were harvested. All animal studies were performed in accordance with protocols approved by the Laboratory Animal Care and Use Committee of the Ohio State University. During the wounding procedure, mice were anesthetized by low-dose isoflurane inhalation for 5–10 min per standard recommendation (41). While there are no direct data on how 5–10 min isoflurane exposure may influence cutaneous injury following acute excision, it is generally known that isoflurane exposure for 30 min or more may influence hemodynamics and leukocyte rolling velocities in the mesenteric microcirculation during lipopolysaccharide-induced inflammation (30).

Fig. 1.

Secondary-intention excisional dermal wound closure in mice. A: two 16 × 8 mm full-thickness rectangular (inset) excisional wounds were placed on the dorsal skin, equidistant from the midline and adjacent to the 4 limbs. These wounds were left to heal by secondary intention. Wound closure is shown as percentage of area of initial wound determined on the indicated day after wounding. The shaded area indicates time period selected for the study of gene expression profile in wound edge tissue postinjury. Data are means ± SD, n = 4. *P < 0.05; **P < 0.001 compared with 0 h time point postwound. B: definition of wound edge tissue used for microarray analysis. The dotted area represents the wound edge tissue. Tattooing was performed on 4 corners (filled circles) of the wound site to keep track of the edges. To minimize the possible interfering effects of tattoo-related trauma, the procedure was performed 1 wk ahead of the wounding. The tissue containing tattoo dots was excluded from analyses.

Determination of wound area.

Imaging of wounds was performed with a digital camera (Canon PowerShot G6). The wound area was determined using WoundMatrix software as described previously (71).

Histology.

Formalin-fixed paraffin-embedded or OCT-embedded frozen wound-edge specimens were sectioned. The paraffin sections (4 μm) were deparaffinized and stained with hematoxylin & eosin by standard procedures. Immunohistochemical staining of paraffin or frozen sections was performed as described earlier (60) using the following primary antibodies: anti-neutrophil (1:200; AbD Serotec, Raleigh, NC), F4/80 (1:100, AbD Serotec) or CD31 (1:200; BD Pharmingen, San Diego, CA). Secondary antibody detection and counterstaining were performed as described previously (60).

GeneChip probe array analyses.

To identify sets of gene differentially expressed during the temporal course of healing, we utilized the GeneChip approach (59, 61–63, 65). Total RNA was extracted (TRIzol, GIBCO BRL) from wound-edge tissue 0, 6, 12, 24, 48, and 96 h after wounding. Further clean-up of RNA was performed using the RNeasy kit (Qiagen). The quality of RNA thus obtained was examined using the Agilent 2100 Bioanalyzer. Targets were prepared for microarray hybridization according to previously described protocols (59, 61, 62, 64). To assess the quality of hybridization, samples were hybridized for 16 h at 45°C to GeneChip test arrays. Satisfactory samples were hybridized with the Murine Genome Array U74Av2 for the screening of >12,000 probe sets. The arrays were washed, stained with streptavidin-phycoerythrin, and were then scanned with the GeneArray scanner (Affymetrix) in our own facilities as described earlier (59, 61, 65). Data were collected from four mice (n = 4) in each group representing a time point. Tissue from both wounds of each mice were pooled to represent one sample. Each such sample was analyzed using a separate GeneChip resulting in four separate data sets from each group.

Data analyses.

Data acquisition and image processing were performed using GCOS (Gene Chip Operating Software, Affymetrix). Raw data were collected and analyzed using Stratagene ArrayAssist Expression Software v. 5.1 (Stratagene). Additional processing of data was performed using dChip software (v. 1.3, Harvard University) (59). Data normalization and background corrections were performed using GC-RMA. Differentially expressed genes were identified using a two-class t-test where significance level was set at P < 0.05 with Benjamin-Hochberg false discovery rate correction (59, 86). Genes that were >2.0 fold up- or downregulated compared with 0 h intact skin samples were selected. A detailed analysis scheme has been illustrated in Fig. 3. All genes that were significantly changed at any single time point compared with 0 h samples were subjected to hierarchical clustering. Major clusters of genes that changed during the course of healing were identified. The genes from the cluster were subjected to further functional analysis using DAVID (Database for Annotation, Visualization and Integrated Discovery; NIAID, NIH). Major known pathways [Kyoto Encyclopedia of Genes and Genomes (KEGG)] identified in each of the cluster have been shown as supplemental figures.1 In addition, all genes that were significantly changed at any single time point were subjected to specific filters for wound cell-type or processes (see Figs. 7 and 8). Select differentially expressed candidate genes were verified with quantitative real-time PCR.

Reverse transcription and quantitative real-time PCR.

Tissue mRNA was quantified by real-time PCR assay using double-stranded DNA binding dye SYBR Green-I as described previously (62, 65). The primer set used for the individual genes are listed in Supplemental Table S1. GAPDH was used as a reference housekeeping gene.

Statistics.

Results are presented as means ± SD. In these cases, difference between means was tested using Students t-test. Microarray data processing is described above under GeneChip probe array analyses.

RESULTS

To avoid dilution of the effects at the wound-edge, only 1 mm of the margin was collected as wound-edge tissue (Fig. 1). To maximize the yield of wound-edge tissue we studied a relatively large excisional wound (Fig. 1) as described previously (60, 70, 71). In this model, complete closure is achieved in 2 wk (Fig. 1). In the interest of tight focus, the emphasis of this study was directed at the first 96 h of the repair process primarily covering the acute inflammatory phase (Fig. 1B shaded area, and Fig. 2). During this 96 h period, a modest but statistically significant decrease of wound size was observed. Before profiling the transcriptome of the wound-edge tissue at specific time points after wounding, we sought to histologically characterize the wound-edge tissue at those time points. The Mason trichrome stain (Fig. 2A) provided a reasonable appreciation of cellularity and advancement of the epidermal edge. At 6 h after wounding, the wound-edge tissue was characterized by the presence of abruptly discontinuous epidermal edge at the perimeter of the defect caused by wounding (Fig. 2A, 6 h). At this time point, infiltration of a few neutrophils and macrophages to the wound site was noted (Fig. 2B, 6 h). Overall cellularity in the granulation tissue was low, and CD31+ endothelial cells were not detected (Fig. 2B, 6 h). An additional 6 h later, i.e., 12 h after wounding, the epidermal cells did not advance substantially but were noted to orient themselves for downward advancement along the perimeters of the injured skin (Fig. 2A). Compared with the 6 h wound edge, a larger number of neutrophils were noted at the wound site while the count of macrophages remained unchanged (Fig. 2B, 12 h). Consistent with the sharply increased abundance of neutrophils at the wound site, the cellularity of the 12 h granulation tissue was higher but devoid of endothelial cells (Fig. 2B, 12 h). An additional 12 h later, i.e., 24 h after wounding, cellular proliferation as well as downward advancement of the epidermal cells was noted (Fig. 2A, 24 h). While the abundance of neutrophils subsided, there was a clear increase in the presence of macrophages (Fig. 2B, 24 h). For the first time, a few isolated endothelial cells were noted in the granulation tissue (Fig. 2B, 24 h). One day later, i.e., 48 h after wounding, changes in the epidermal layer of tissue were prominent. In addition to the downward migration and proliferation noted on the previous day, the epidermal layer of cells migrated laterally across the wound to cover the defect caused by wounding (Fig. 2A, 48 h). Remaining neutrophils started getting pushed upward toward the eschar tissue, while the number of macrophages that were noted in day 1 after wounding continued to be present (Fig. 2B, 48 h). On this day 2 after wounding, the presence of both isolated as well as clusters of endothelial cells in the granulation tissue were evident (Fig. 2B, 48 h). On day 4 after wounding, the hyperproliferative epithelium was clearly evident as arching over the defect (Fig. 2A, 96 h). The layer of tissue advanced from both ends of the defect as nascent tissue that is several layers of cell in thickness (not shown). Very few neutrophils were noted, while a significant number of macrophages continued to exist in the granulation tissue (Fig. 2B, 96 h). At this time point, the abundance of endothelial cells in the granulation tissue sharply increased, but very few of such cells seem to encircle a lumen and be a part of functional blood vessel.

Fig. 2.

Histological characterization of healing wound tissue used for gene expression profiling studies. Wound (Fig. 1) tissues from excisional wounds were collected at indicated time postinjury. Formalin-fixed paraffin sections or OCT-embedded frozen sections were stained using Masson Trichrome procedure (A). This procedure results in blue-black nuclei, blue collagen and cytoplasm. Epidermal cells are stained in red. i: Low magnification (×1.25) images showing cross-section of entire wounds at 6 and 96 h postwounding. Boxed area shows the wound edge tissue shown in ii and iii. Wound-edge tissue imaged with ×5 (ii) or ×20 (iii) objectives. Scale bar = 200 μm (for ii) and 50 μm (for iii). D, dermis; EGT, early granulation tissue; Es, Eschar tissue; FP, fibrin plug; HE, hyperproliferative epithelium. The newly forming epithelial tip is shown with an arrow. Alternatively, sections were immunostained as shown in B. B, i: Antineutrophil antibody (brown, shown with black arrow) that recognizes a polymorphic 40 kDa antigen expressed by polymorphonuclear cells. Counterstaining was performed using hematoxylin (blue); ii: F4/80 antibody (brown, shown with black arrow) that recognizes the murine F4/80 antigen, a 160 kDa cell surface glycoprotein expressed on a wide range of mature tissue macrophages. Counterstaining was performed using hematoxylin (blue); iii: Cd31 antibody (red fluorescence, shown with white arrow) that recognizes the mouse CD31, a 140 kDa cell surface glycoprotein that is expressed at high levels on endothelial cells. Counterstaining was performed using DAPI (blue, fluorescence). Scale bar, 50 μm. iv: Relative quantification (arbitrary units) of neutrophils, macrophages, and CD31+ endothelial cells from tissue sections obtained 6–96 h postwound was performed using a image processing tool kit. Data are means ± SD (n = 3). *P < 0.05 compared with 6 h time point postwounding.

The microarray study and data analysis design is illustrated in Fig. 3 and was modeled on the basis of our previously published studies (57, 59, 65). A total of 12,489 probe sets were screened. Six hours after wounding, the expression of very few genes in the wound-edge tissue was significantly changed. Beyond that time point, a comparable number of genes were significantly changed in response to wounding. Taken together, the upregulated (filled) and downregulated (open) genes accounted for 4–5% of the total number of probe sets screened (Fig. 4). When analyzed temporally, the sets of genes that significantly changed in expression during the healing process could be segregated into the following five sets (Fig. 5): up-early (6–24h), up-intermediary (12–96 h), up-late (48–96 h), down-early (6–12 h), and down-intermediary (12–96 h). Functional categories of each of these sets of genes are itemized in Table 1. The specific genes and their signaling pathways are illustrated in Supplemental Figs S1–S5. Up-early: The genes upregulated in early phase as shown in Fig. 5 were subjected to functional analysis using DAVID (NIAID, NIH). KEGG-based analysis identified the cytokine-cytokine receptor interaction pathway as being primarily affected (Supplemental Fig. S1). Up-intermediary: Using the approach outlined above, we noted the leukocyte-endothelial interaction pathway to be primarily affected at this temporal phase (Supplemental Fig. S2). Up-late: The cell-cycle pathway related genes were most affected at this phase (Supplemental Fig. S3). Down-early: Genes related to purine metabolism were rapidly and transiently downregulated following wounding (Supplemental Fig. S4). Down-intermediary: The expression of genes functionally associated with oxidative phosphorylation was downregulated in the inflammatory phase of healing (Supplemental Fig. S5).

Fig. 3.

GeneChip data analysis scheme used to identify kinetics of differentially expressed genes in dermal wounds. Image acquisition and processing were performed using GCOS (GeneChip operating software, Affymetrix). GC-RMA was applied for data normalization and background correction. ArrayAssist v5.1 software was used to identify significant (P < 0.05; false discovery rate corrected) differentially expressed genes in wound tissue compared with skin (0 h) from the site where wound was created. Details of software and other resources for data analysis have been provided in methods.

Fig. 4.

Number of differentially expressed genes in course of healing. Total number of genes differentially expressed (significant, P < 0.05) at a specific time postwounding. Gray shaded area in each bar shows number of downregulated vs. the solid area that shows number of upregulated genes.

Fig. 5.

Cluster of genes showing specific pattern of expression in wound tissue during healing. All genes that were significantly changed at any single time point were subjected to hierarchical clustering. Five major clusters of genes that change during the course of healing were identified. Major functional categories in each of these clusters were identified and are presented in Table 1.

Table 1.

Functional cluster analysis of data presented in Figure 5. Major clusters at indicated time points are shown

upregulated at 48–96 h (up-late, 299 probe sets)

• Keratinization

• Cell cycle*

• Histone core

• Cytoskeleton

• DNA replication

• ATP binding

• Proteolysis

• Hydrolase

• Ion transport

upregulated at 12–96 h (up-intermediary, 219 probe sets)

• Apoptosis

• Proteolysis

• Cytoskeleton

• Immune response*

• Kinase

• Rac GTPase

• Glucose metabolism

• ATPase activity

• Signal transduction/NADPH oxidase

• Transcription factor

upregulated at 6–24 h (up-early, 208 probe sets)

• Chemotaxis*

• Defense response*

• Cytokine-receptor interaction*

• Transcription factor

• Kinase

downregulated at 6–12 h (down-early, 32 probe sets)

• Cellular metabolism

downregulated at 12 h–96 h (down-intermediary, 416 probe sets)

• Oxidoreductase*

• Muscle protein

• Mitochondrion

• Calcium ion binding

• Mitochondrial membrane

• Glucose metabolism

• cytoskeleton

• Lim domain

• Ion transport

• Glycoprotein

• Ubiquitin cycle

• Protein modification

• Protein kinase

See Supplemental Figures S1–S4 for pathways and individual genes involved.

Arranged in the order of fold-change, the top 50 genes that were statistically significantly upregulated or downregulated in the wound-edge tissue during the five time points (6, 12, 24, 48, and 96 h) studied are listed in Supplemental Tables S2–S6. Select genes from these lists were picked for verification of results using quantitative real-time PCR (Fig. 6). Results of all time points obtained from microarray and real-time PCR approaches tightly correlated (r values indicated in each panel of Fig. 6), validating the data mining approach adopted in the current study. Findings related to commonly studied genes are consistent with the literature. The proinflammatory cytokine interleukin-1 beta (IL-1β) was acutely induced (Fig. 6) as previously reported (5). Immune response gene-1 (Irg1) is found in LPS-induced macrophages (3). Acute and transient induction of Irg1 was noted in response to open wound that lends itself to bacterial colonization. JunB is a component of the AP1 transcription factors that regulates epidermal regeneration, wound inflammation, as well as contraction (21, 81). Rapid and transient induction of JunB was observed in the wound-edge tissue. Ras-related associated with diabetes or Rrad is a prototypical member of a family of novel Ras-related GTPases. After muscle injury, Rrad is expressed within the myogenic progenitor cell population during the early phases of regeneration (29). Our observation represents first evidence demonstrating that high levels of the Rrad gene are found in the wound-edge tissue as early as 6 h after wounding. High levels of Rrad mRNA were noted during the entire inflammatory phase (Fig. 6). Similar temporal response was also noted for CXCL5. ELR(+) CXC chemokines such as CXCL5 attract and activate neutrophils, amplify the inflammatory cascade, and stimulate local production of cytokines that have negative inotropic and proapoptotic effects (9). Uridine phosphorylase (Upp) is a cytokine-inducible enzyme that converts the pyrimidine nucleoside uridine into uracil. Upon availability of ribose-1-phosphate, Upp can also catalyze the formation of nucleosides from uracil as well as from 5-fluorouracil, therefore involved in fluoropyrimidine metabolism (6). We observed sustained induction of Upp1 in the wound-edge tissue (Fig. 6). The functional significance of this response remains unclear at this time. Chronic granulomatous disease, a condition associated with impaired wound healing, results from defects in the phagocyte NADPH oxidase, central to which is the membrane-bound cytochrome b-245. The alpha polypeptide of cytochrome b-245 (Cyba) encodes the NADPH oxidase-light chain p22-phox (8), a NADPH oxidase component with significant vascular functions (27). Adipocytes represent a significant source of serum amyloid A3 (Saa3), which is now known to play a role in monocyte recruitment and inflammation (26). Saa3 is known to be an inducible acute-phase protein in the skin (80). We noted early induction of Saa3 in the wound-edge tissue. The induction intensified with time. Keratins are intermediate filament-forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells during wound repair (90). The type II keratin 6 is known to be expressed in the wound edge (43). We observed progressive induction of keratin 6B (Krt6B) in the wound-edge tissue as early as in the inflammatory phase (Fig. 6). Stromelysin-3 (ST3) is a member of the matrix metalloproteinase (MMP) family, MMP-11, which is expressed in the skin during wound healing (89). Typically associated with cutaneous scar formation, we observed that MMP11 was induced in the wound-edge tissue in the latter half of the inflammatory phase (Fig. 6).

Fig. 6.

Real-time PCR validation of GeneChip microarray expression analysis. Expression levels of selected genes identified (Tables S2–S6) using GeneChip analysis were independently determined using real-time PCR. The correlation coefficient (r) of data obtained from microarray analysis vs. real-time PCR is shown for each of the gene. The regression is derived from 4 pairs of data at each time point for a total number of 24. PCR data represent means ± SD, n = 5. The animals used for real-time PCR were a set that was independent of the animals used for microarray analysis. Irg-1, immunoresponsive gene 1; IL-1β, interleukin 1 beta; JunB, Jun-B oncogene; Rrad, Ras-related associated with diabetes; CXCL5, chemokine (C-X-C motif) ligand 5; Upp1, uridine phosphorylase 1; Cyba, cytochrome b-245, alpha polypeptide (p22 phox); Saa3, serum amyloid A 3; Krtb6, keratin 6B; MMP11, matrix metallopeptidase 11.

The design of the current study was aimed at examining cell-specific gene expression in the wound-edge tissue as a function of time. All genes that were significantly changed at any specific time point (as shown in Fig. 5) were subjected to further mining for cell-type specific genes. This mining was based on annotation of the genes in the Affymetrix database. The following five major wound-associated types of cells were targeted: neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells. Based on the temporal nature of the expression pattern in response to wounding, subsets of genes were identified within the cell-specific clusters (Fig. 7). The annotations for each cell-specific cluster are listed in Tables 2, A–E. The abundance of neutrophil-specific genes was low in the intact skin. Wounding increased the presence of such genes. The response was clearly noted at 6 h after wounding and peaked at 12 h followed by gradual lowering of the abundance of neutrophil-specific genes at the wound-edge (Fig. 7Ai). These results are consistent with the histological evidence demonstrating a comparable temporal response for neutrophil recruitment into the wound-edge tissue (Fig. 2B). The kinetics of enrichment of macrophage specific genes in the wound-edge tissue was somewhat delayed compared with the pattern for neutrophil-specific genes (Fig. 7, Ai vs. Bii). This result is consistent with the histological observations presented in Fig. 2.

Fig. 7.

Visualization of the expression pattern of candidate genes representing wound-specific cell types in a healing wound. All genes that were significantly changed at any single time point were subjected to specific filtration for wound cell type specific genes. The following 5 major wound cell types were targeted: neutrophils (A), macrophages (B), endothelial cells (C), fibroblasts (D), and pluripotent stem cells (E). Further subsets based on patterns of kinetics within each of the cell type were identified and indicated on margins of each cluster. Annotations of each cell type-specific subclusters (e.g., i and ii) are presented in Tables 2, A–E.

Table 2.

A. Annotation of neutrophil specific gene cluster shown in Figure 7A

Probe ID	Gene Description	Symbol
102914_s_at	B-cell leukemia/lymphoma 2 related protein A1a	Bcl2a1a
102424_at	chemokine (C-C motif) ligand 3	Ccl3
104388_at	chemokine (C-C motif) ligand 9	Ccl9
102718_at	chemokine (C-C motif) receptor 5	Ccr5
102719_f_at	chemokine (C-C motif) receptor 5	Ccr5
161968_f_at	chemokine (C-C motif) receptor 5	Ccr5
101160_at	chemokine (C-X-C motif) ligand 2	Cxcl2
101436_at	chemokine (C-X-C motif) ligand 9	Cxcl9
101728_at	complement component 5a receptor 1	C5ar1
162181_f_at	Fc receptor, IgE, high affinity I, gamma polypeptide	Fcer1 g
101793_at	Fc receptor, IgG, high affinity I	Fcgr1
102879_s_at	Fc receptor, IgG, high affinity I	Fcgr1
101800_at	formyl peptide receptor, related sequence 2	Fpr-rs2
102884_at	inositol polyphosphate-5-phosphatase D	Inpp5d
102353_at	integrin beta 2	Itgb2
103486_at	interleukin 1 beta	Il1b
102218_at	interleukin 6	Il6
103266_at	leukotriene B4 receptor 1	Ltb4r1
160564_at	lipocalin 2	Lcn2
102957_at	lymphocyte cytosolic protein 2	Lcp2
102430_at	myeloid differentiation primary response gene 88	Myd88
102326_at	neutrophil cytosolic factor 2	Ncf2
103662_at	neutrophil cytosolic factor 4	Ncf4
101554_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
104149_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
100328_s_at	paired-Ig-like receptor A1	Lilrb3
162475_f_at	peptidoglycan recognition protein 1	Pglyrp1
102663_at	plasminogen activator, urokinase receptor	Plaur
103579_at	RAS-related C3 botulinum substrate 2	Rac2
103448_at	S100 calcium binding protein A8 (calgranulin A)	S100a8
103887_at	S100 calcium binding protein A9 (calgranulin B)	S100a9
161650_at	secretory leukocyte peptidase inhibitor	Slpi
104692_at	selectin, platelet	Selp
103488_at	selectin, platelet (p-selectin) ligand	Selplg
102641_at	SFFV proviral integration 1	Sfpi1
103839_at	sphingosine kinase 1	Sphk1
100425_at	spleen tyrosine kinase	Syk
162206_f_at	suppressor of cytokine signaling 3	Socs3
92232_at	suppressor of cytokine signaling 3	Socs3
104601_at	thrombomodulin	Thbd
160469_at	thrombospondin 1 /// similar to thrombospondin 1	Thbs1
101464_at	tissue inhibitor of metalloproteinase 1	Timp1
92369_at	transforming growth factor alpha	Tgfa
100397_at	TYRO protein tyrosine kinase binding protein	Tyrobp

B. Annotation of macrophage specific gene cluster shown in Figure 7B
Probe Set ID	Gene Title	Gene Symbol
	Sub-cluster i (up-regulated, 6–12 h post wounding)
101160_at	chemokine (C-X-C motif) ligand 2	Cxcl2
101450_at	colony stimulating factor 1 (macrophage)	Csf1
101554_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
101996_at	protein tyrosine phosphatase, nonreceptor type 2	Ptpn2
102218_at	interleukin 6	Il6
102239_at	B-cell leukemia/lymphoma 3	BclIII
102362_i_at	Jun-B oncogene	Junb
102363_r_at	Jun-B oncogene	Junb
102424_at	chemokine (C-C motif) ligand 3	Ccl3
102430_at	myeloid differentiation primary response gene 88	Myd88
102736_at	chemokine (C-C motif) ligand 2	Ccl2
103066_at	thymidylate kinase family LPS-inducible member	Tyki
103486_at	interleukin 1 beta	Il1b
103839_at	sphingosine kinase 1	Sphk1
104149_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
104155_f_at	activating transcription factor 3	Atf3
104156_r_at	activating transcription factor 3	Atf3
104388_at	chemokine (C-C motif) ligand 9	Ccl9
104406_at	prostaglandin E synthase	Ptges
104533_at	proviral integration site 1	Pim1
104538_at	prostaglandin I2 (prostacyclin) synthase	Ptgis
104647_at	prostaglandin-endoperoxide synthase 2	Ptgs2
104712_at	myelocytomatosis oncogene	Myc
160894_at	CCAAT/enhancer binding protein (C/EBP), delta	Cebpd
160933_at	interferon gamma induced GTPase	Igtp
162206_f_at	suppressor of cytokine signaling 3	Socs3
92232_at	suppressor of cytokine signaling 3	Socs3
92562_at	nuclear factor, erythroid derived 2, like 2	Nfe2l2
92694_at	chitinase 3-like 3	Chi3l3
92731_at	pentraxin related gene	Ptx3
92793_at	tumor necrosis factor receptor superfamily, member 1a	Tnfrsf1a
92830_s_at	zinc finger protein 36	Zfp36
92849_at	chemokine (C-C motif) ligand 6	Ccl6
92925_at	CCAAT/enhancer binding protein (C/EBP), beta	Cebpb
93328_at	histidine decarboxylase	Hdc
93858_at	chemokine (C-X-C motif) ligand 10	Cxcl10
93871_at	interleukin 1 receptor antagonist	Il1rn
93914_at	interleukin 1 receptor, type I	Il1r1
93956_at	interferon-induced protein with tetratricopeptide repeats 3	Ifit3
94140_at	macrophage scavenger receptor 1	Msr1
94142_at	colony stimulating factor 3 (granulocyte)	Csf3
94146_at	chemokine (C-C motif) ligand 4	Ccl4
94224_s_at	interferon activated gene 203	Ifi203
94246_at	E26 avian leukemia oncogene 2, 3′ domain	Ets2
94747_at	colony stimulating factor 2 receptor, beta 1, low-affinity (granulocyte-macrophage)	Csf2rb1
94748_g_at	colony stimulating factor 2 receptor, beta 1, low-affinity (granulocyte-macrophage)	Csf2rb1
94755_at	interleukin 1 alpha	Il1a
94761_at	chemokine (C-C motif) ligand 7	Ccl7
94929_at	protein tyrosine phosphatase, nonreceptor type 1	Ptpn1
95348_at	chemokine (C-X-C motif) ligand 1	Cxcl1
95349_g_at	chemokine (C-X-C motif) ligand 1	Cxcl1
96551_at	C-type lectin domain family 4, member e	Clec4e
96752_at	intercellular adhesion molecule	Icam1
97106_at	mitogen activated protein kinase kinase kinase 8	Map3k8
98088_at	CD14 antigen	Cd14
98417_at	myxovirus (influenza virus) resistance 1	Mx1
98500_at	interleukin 1 receptor-like 1	Il1rl1
98501_at	interleukin 1 receptor-like 1	Il1rl1
98579_at	early growth response 1	Egr1
98773_s_at	immunoresponsive gene 1	Irg1
98774_at	immunoresponsive gene 1	Irg1
99099_at	signal transducer and activator of transcription 3	Stat3
99100_at	signal transducer and activator of transcription 3	Stat3
99413_at	chemokine (C-C motif) receptor 1	Ccr1
	Sub-cluster ii (up-regulated, 24–96 h post wounding)
102330_at	allograft inflammatory factor 1	Aif1
93097_at	arginase 1, liver	Arg1
101521_at	baculoviral IAP repeat-containing 5	Birc5
102914_s_at	B-cell leukemia/lymphoma 2 related protein A1a	Bcl2a1a/
93869_s_at	B-cell leukemia/lymphoma 2 related protein A1a	Bcl2a1a
100988_at	BCL2-like 11 (apoptosis facilitator)	Bcl2l11
104023_at	Cd300D antigen	Cd300a
101878_at	CD72 antigen	Cd72
93454_at	CD93 antigen	Cd93
160815_at	cDNA sequence BC032204	BC032204
98406_at	chemokine (C-C motif) ligand 5	Ccl5
93397_at	chemokine (C-C motif) receptor 2	Ccr2
102718_at	chemokine (C-C motif) receptor 5	Ccr5
102719_f_at	chemokine (C-C motif) receptor 5	Ccr5
161968_f_at	chemokine (C-C motif) receptor 5	Ccr5
103210_at	colony stimulating factor 2 receptor, beta 2	Csf2rb2
101728_at	complement component 5a receptor 1	C5ar1
100300_at	cytochrome b-245, beta polypeptide	Cybb
103518_at	cytotoxic T lymphocyte-associated protein 2 beta	Ctla2b
102896_at	docking protein 1	Dok1
160901_at	FBJ osteosarcoma oncogene	Fos
162181_f_at	Fc receptor, IgE, high affinity I, gamma polypeptide	Fcer1 g
101793_at	Fc receptor, IgG, high affinity I	Fcgr1
102879_s_at	Fc receptor, IgG, high affinity I	Fcgr1
92188_s_at	feline sarcoma oncogene	Fes
94697_at	Gardner-Rasheed feline sarcoma viral (Fgr) oncogene	Fgr
97384_at	glia maturation factor, gamma	Gmfg
99597_at	guanine nucleotide binding protein, alpha inhibiting 2	Gnai2
99598_g_at	guanine nucleotide binding protein, alpha inhibiting 2	Gnai2
93483_at	hemopoietic cell kinase	Hck
100511_at	histocompatibility (minor) HA-1	Hmha1
102884_at	inositol polyphosphate-5-phosphatase D	Inpp5d
98828_at	integrin alpha M	Itgam
104308_at	integrin alpha X	Itgax
102353_at	integrin beta 2	Itgb2
104750_at	interferon gamma inducible protein 47	Ifi47
93425_at	interferon regulatory factor 5	Irf5
104669_at	interferon regulatory factor 7	Irf7
98002_at	interferon regulatory factor 8	Irf8
103639_at	interferon-induced protein with tetratricopeptide repeats 2	Ifit2
100428_at	laminin, gamma 2	Lamc2
97507_at	lectin, galactoside-binding, soluble, 3 binding protein	Lgals3bp
98003_at	leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 3	Lilrb3
94278_at	lymphocyte cytosolic protein 1	Lcp1
102957_at	lymphocyte cytosolic protein 2	Lcp2
99071_at	macrophage expressed gene 1 /// similar to macrophage expressed gene 1	Mpeg1
94792_at	macrophage scavenger receptor 1	Msr1
97203_at	MARCKS-like 1	Marcksl1
99957_at	matrix metallopeptidase 9	Mmp9
97763_at	neutrophil cytosolic factor 1	Ncf1
103614_at	nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100	Nfkb2
100328_s_at	paired-Ig-like receptor A1	Lilrb3
103866_at	phosphatidylinositol 3-kinase catalytic delta polypeptide	Pik3cd
103299_at	phospholipase D family, member 4	Pld4
160801_at	PQ loop repeat containing 1	Pqlc1
101881_g_at	procollagen, type XVIII, alpha 1	Col18a1
99638_at	procollagen, type XVIII, alpha 1	Col18a1
102851_s_at	protein tyrosine phosphatase, nonreceptor type 6	Ptpn6
101048_at	protein tyrosine phosphatase, receptor type, C	Ptprc
103451_at	PTK2 protein tyrosine kinase 2 beta	Ptk2b
103579_at	RAS-related C3 botulinum substrate 2	Rac2
95612_at	replication factor C (activator 1) 5	Rfc5
102649_s_at	retinoic acid early transcript 1, alpha	Raet1a
103448_at	S100 calcium binding protein A8 (calgranulin A)	S100a8
103887_at	S100 calcium binding protein A9 (calgranulin B)	S100a9
97519_at	secreted phosphoprotein 1	Spp1
103488_at	selectin, platelet (p-selectin) ligand	Selplg
160836_at	(semaphorin) 4D	Sema4d
102712_at	serum amyloid A 3	Saa3
102641_at	SFFV proviral integration 1	Sfpi1
92975_at	SH3-domain binding protein 2	Sh3bp2
96562_at	solute carrier family 11	Slc11a1
103065_at	solute carrier family 20, member 1	Slc20a1
100425_at	spleen tyrosine kinase	Syk
102012_at	src family associated phosphoprotein 2	Skap2
103205_at	T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 protein A3	Tcirg1
92807_at	thioredoxin 1	Txn1
98304_at	toll-like receptor 6	Tlr6
101918_at	transforming growth factor, beta 1	Tgfb1
94928_at	tumor necrosis factor receptor superfamily, member 1b	Tnfrsf1b
100397_at	TYRO protein tyrosine kinase binding protein	Tyrobp
99564_at	ubiquitin-like, containing PHD and RING finger domains, 1	Uhrf1
99799_at	vav 1 oncogene	Vav1
103349_at	Yamaguchi sarcoma viral (v-yes-1) oncogene homolog	Lyn

C. Annotation of endothelial specific gene cluster shown in Figure 7C
Probe Set ID	Gene Description	Symbol
	Sub-cluster i (down-regulated, 12–96 h post wounding)
102703_s_at	aquaporin 4	Aqp4
102704_at	aquaporin 4	Aqp4
104590_at	myocyte enhancer factor 2C	Mef2c
104591_g_at	myocyte enhancer factor 2C	Mef2c
104592_i_at	myocyte enhancer factor 2C	Mef2c
102720_at	endothelial-specific receptor tyrosine kinase	Tek
102327_at	amine oxidase, copper containing 3	Aoc3
100614_at	myoglobin	Mb
100494_at	fibroblast growth factor 1	Fgf1
103214_at	heat shock protein 2	Hspb2
101178_at	sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C	Sema3c
103001_at	vascular endothelial growth factor B	Vegfb
101990_at	lactate dehydrogenase B	Ldhb
103215_g_at	heat shock protein 2	Hspb2
100629_at	glutathione S-transferase, mu 5	Gstm5
100605_at	tropomyosin 2, beta	Tpm2
100997_at	desmin	Des
101521_at	baculoviral IAP repeat-containing 5	Birc5
100153_at	neural cell adhesion molecule 1	Ncam1
100928_at	fibulin 2	Fbln2
	Sub-cluster ii (up-regulated, 12–96 h post wounding)
101160_at	chemokine (C-X-C motif) ligand 2	Cxcl2
100484_at	matrix metallopeptidase 13	Mmp13
102663_at	plasminogen activator, urokinase receptor	Plaur
101972_at	napsin A aspartic peptidase	Napsa
103024_at	a disintegrin and metallopeptidase domain 8	Adam8
101464_at	tissue inhibitor of metalloproteinase 1	Timp1
102798_at	adrenomedullin	Adm
102025_at	chemokine (C-X-C motif) ligand 13	Cxcl13
102957_at	lymphocyte cytosolic protein 2	Lcp2
102884_at	inositol polyphosphate-5-phosphatase D	Inpp5d
102914_s_at	B-cell leukemia/lymphoma 2 related protein A1a	Bcl2a1a
101728_at	complement component 5a receptor 1	C5ar1
104469_at	podoplanin	Pdpn
104601_at	thrombomodulin	Thbd
103210_at	colony stimulating factor 2 receptor, beta 2, low-affinity (granulocyte-macrophage)	Csf2rb2
100425_at	spleen tyrosine kinase	Syk
103707_at	complement component 3a receptor 1	C3ar1
160101_at	heme oxygenase (decycling) 1	Hmox1
104701_at	basic helix-loop-helix domain containing, class B2	Bhlhb2
103818_at	solute carrier family 7 (cationic amino acid transporter, y+ system), member 7	Slc7a7
100428_at	laminin, gamma 2	Lamc2
103839_at	sphingosine kinase 1	Sphk1
103451_at	PTK2 protein tyrosine kinase 2 beta	Ptk2b
100308_at	procollagen, type VIII, alpha 1	Col8a1
103866_at	phosphatidylinositol 3-kinase catalytic delta polypeptide	Pik3cd
100134_at	endoglin	Eng
100970_at	thymoma viral proto-oncogene 1	Akt1
100016_at	matrix metallopeptidase 11	Mmp11
100915_at	myosin, heavy polypeptide 9, nonmuscle	Myh9
103462_at	dedicator of cyto-kinesis 2	Dock2
101881_g_at	procollagen, type XVIII, alpha 1	Col18a1

D. Annotation of fibroblast specific gene cluster shown in Figure 7D
Probe Set ID	Gene Description	Symbol
	Sub-cluster i (up-regulated, 12–96 h post wounding)
100484_at	matrix metallopeptidase 13	Mmp13
102712_at	serum amyloid A 3	Saa3
101631_at	SRY-box containing gene 11	Sox11
101993_at	tenascin C	Tnc
101464_at	tissue inhibitor of metalloproteinase 1	Timp1
102218_at	interleukin 6	Il6
100016_at	matrix metallopeptidase 11	Mmp11
101918_at	transforming growth factor, beta 1	Tgfb1
102021_at	interleukin 4 receptor, alpha	Il4ra
100884_at	aldo-keto reductase family 1, member B8	Akr1b8
100009_r_at	SRY-box containing gene 2	Sox2
100277_at	inhibin beta-A	Inhba
102204_at	v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (avian)	Mafb
102195_at	mitogen-activated protein kinase kinase kinase kinase 4	Map4k4
101881_g_at	procollagen, type XVIII, alpha 1	Col18a1
100581_at	cystatin B	Cstb
100507_at	nephroblastoma overexpressed gene	Nov
100019_at	versican	Vcan
100420_at	filaggrin	Flg
	Sub-cluster ii (down-regulated, 12–96 h post wounding)
102774_at	epidermal growth factor	Egf
101028_i_at	actin, alpha, cardiac	Actc1
100003_at	ryanodine receptor 1, skeletal muscle	Ryr1
100605_at	tropomyosin 2, beta	Tpm2
102256_at	T-box 15	Tbx15
100494_at	fibroblast growth factor 1	Fgf1
100879_at	actinin alpha 3	Actn3
100068_at	aldehyde dehydrogenase family 1, subfamily A1	Aldh1a1
100997_at	desmin	Des
101892_f_at	thymoma viral proto-oncogene 1 interacting protein	Aktip
101029_f_at	actin, alpha, cardiac	Actc1
100566_at	insulin-like growth factor binding protein 5	Igfbp5

E. Annotation of pluripotent stem cell specific gene cluster shown in Figure 7E
Probe Set ID	Gene Description	Symbol
	Sub-cluster i (down-regulated, 6–96 h post wounding)
92722_f_at	sine oculis-related homeobox 1 homolog (Drosophila)	Six1
	Sub-cluster ii (up-regulated, 6–96 h post wounding)
160069_at	geminin	Gmnn
100009_r_at	SRY-box containing gene 2	Sox2

Footnote to Tables 2A–E: Probe ID, Affymetrix probe identifications.

Wounding is associated with the disruption of vasculature (22). Consistently, we have observed that endothelial cell specific genes which are abundant in the intact skin, sharply disappear in response to wounding (Fig. 7Ci). Although histological visualization does not support the reappearance of endothelial cells in the early phase (6–12 h) after wounding, the more sensitive microarray approach to detect endothelial cell-specific genes demonstrates the appearance of few endothelial cell related genes in the wound-edge tissue during such early phase. Consistent with the histological observations presented in Fig. 2D, the abundance of endothelial cell-specific genes in the wound-edge tissue sharply increased from 24–96h after wounding (Fig. 7Cii).

Dermal fibroblasts represent an integral component of the skin tissue. The abundance of fibroblast specific genes in the intact skin tissue (Fig. 7Dii) was therefore expected. In response to wounding, the abundance of this set of genes in the wound edge tissue was sharply lowered and stayed that way over the 96 h study (Table 2D). Wounding, however, increased the abundance of a distinct set of fibroblast-related genes whose basal abundance in the intact skin tissue is low (Fig. 7Di). The abundance of such genes in the wound-edge tissue increased progressively over time. It remains unknown to what extent induction in the resident cells and infiltration of blood-borne cells following wounding contributes to such increased abundance of this subset of genes (Table 2D).

Based on annotations in the Affymetrix database and on our data mining approach, only three pluripotent stem cell specific genes were noted to change. Six1, implicated in maintenance of the differentiated state of tissues, was noted to be relatively abundant in the intact skin. The abundance of this gene in the wound-edge tissue decreased in response to wounding (Fig. 7Ei, Table 2E). Two other genes in this category, geminin and Sox2, were noted to be present in low abundance in the intact skin but the abundance in the wound-edge tissue increased 48h (up-late, as defined in Fig. 5) after wounding.

To enable appreciation of our results from the temporal analysis of transcriptome profiling in the context of functional processes that are implicated in wound repair, candidate genes identified were clustered on the basis of their annotation linking them to inflammation, angiogenesis, reactive oxygen species (ROS), and extracellular matrix (ECM) as depicted in Fig. 8. In the category of inflammation (Fig. 8A), two subsets of upregulated genes were identified. A larger subset of genes that was up-early in response to wounding (Fig. 8Aii, Table 3A). The second smaller subset of genes was up-late (Fig. 8Ai, Table 3A). The up-early subset of genes included neutrophil-related genes that are consistent with the infiltration of neutrophils to the wound-edge tissue as depicted in Fig. 2. The up-late subset of genes included macrophage-related genes, and the changes may be explained by the temporal kinetics of macrophage recruitment to the wound-edge tissue as noted in Fig. 2. In the functional category of angiogenesis, three subsets of genes were identified that responded to wounding (Fig. 8B). The smallest subcluster of genes in this category represented those that were found at an elevated level in the intact skin but were rapidly downregulated in response to wounding (Fig. 8Bi, Table 3B). The other two subcluster of genes in this functional category represented genes that were up-early (Fig. 8Biii) and up-late (Fig. 8Bii) in response to wounding (Table 3B).

Fig. 8.

Visualization of the expression pattern of candidate genes representing repair-specific functional categories in a healing wound. All genes that were significantly changed at any single time point were subjected to specific filtration for process/factors-specific genes. The following processes/factors that play a major role in wound healing were targeted: inflammation (A), angiogenesis (B), reactive oxygen species (ROS, C), and extracellular matrix (ECM, D). Further subsets based on patterns of kinetics within each of the processes were identified and indicated on margins of each cluster. Annotations of each process-specific subclusters are presented in Tables 3, A–D.

Table 3.

A. Inflammation specific gene cluster shown in Figure 8A

Probe Set ID	Gene Description	Symbol
	Sub-cluster I (down-regulated, 12–96 h post wounding)
160519_at	tissue inhibitor of metalloproteinase 3	Timp3
93451_at	LIM domain only 7	Lmo7
102774_at	epidermal growth factor	Egf
92323_at	mitogen-activated protein kinase 12	Mapk12
	Sub-cluster ii (up-regulated, 12–96 h post wounding)
101024_i_at	small proline-rich protein 2A	Sprr2a
101025_f_at	small proline-rich protein 2A	Sprr2a
102914_s_at	B-cell leukemia/lymphoma 2 related protein A1a	Bcl2a1a
103070_at	signal-regulatory protein alpha	Sirpa
93397_at	chemokine (C-C motif) receptor 2	Ccr2
93869_s_at	B-cell leukemia/lymphoma 2 related protein A1a	Bcl2a1a
98002_at	interferon regulatory factor 8	Irf8
99071_at	macrophage expressed gene 1	Mpeg1
99701_f_at	small proline-rich protein 2B	Sprr2b
99957_at	matrix metallopeptidase 9	Mmp9
	Sub-cluster iii (up-regulated, 6–12 h post wounding)
99979_at	cytochrome P450, family 1b1	Cyp1b1
99413_at	chemokine (C-C motif) receptor 1	Ccr1
99387_at	formyl peptide receptor 1	Fpr1
98988_at	nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta	Nfkbiz
98501_at	interleukin 1 receptor-like 1	Il1rl1
98304_at	toll-like receptor 6	Tlr6
97519_at	secreted phosphoprotein 1	Spp1
96596_at	N-myc downstream regulated gene 1	Ndrg1
96562_at	solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1	Slc11a1
95349_g_at	chemokine (C-X-C motif) ligand 1	Cxcl1
95348_at	chemokine (C-X-C motif) ligand 1	Cxcl1
94769_at	matrix metallopeptidase 8	Mmp8
94761_at	chemokine (C-C motif) ligand 7	Ccl7
94146_at	chemokine (C-C motif) ligand 4	Ccl4
94140_at	macrophage scavenger receptor 1	Msr1
93871_at	interleukin 1 receptor antagonist	Il1rn
93573_at	metallothionein 1	Mt1
93425_at	interferon regulatory factor 5	Irf5
93328_at	histidine decarboxylase	Hdc
92731_at	pentraxin related gene	Ptx3
92694_at	chitinase 3-like 3	Chi3l3
92232_at	suppressor of cytokine signaling 3	Socs3
92217_s_at	glycoprotein 49 A	Gp49a
161968_f_at	chemokine (C-C motif) receptor 5	Ccr5
161650_at	secretory leukocyte peptidase inhibitor	Slpi
160469_at	thrombospondin 1	Thbs1
104647_at	prostaglandin-endoperoxide synthase 2	Ptgs2
104601_at	thrombomodulin	Thbd
103887_at	S100 calcium binding protein A9 (calgranulin B)	S100a9
103486_at	interleukin 1 beta	Il1b
103448_at	S100 calcium binding protein A8 (calgranulin A)	S100a8
102957_at	lymphocyte cytosolic protein 2	Lcp2
102779_at	growth arrest and DNA-damage-inducible 45 beta	Gadd45b
102736_at	chemokine (C-C motif) ligand 2	Ccl2
102719_f_at	chemokine (C-C motif) receptor 5	Ccr5
102718_at	chemokine (C-C motif) receptor 5	Ccr5
102712_at	serum amyloid A 3	Saa3
102663_at	plasminogen activator, urokinase receptor	Plaur
102424_at	chemokine (C-C motif) ligand 3	Ccl3
102218_at	interleukin 6	Il6
102021_at	interleukin 4 receptor, alpha	Il4ra
101561_at	metallothionein 2	Mt2
101554_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
101160_at	chemokine (C-X-C motif) ligand 2	Cxcl2

B. Angiogenesis specific gene cluster shown in Figure 8B
Probe Set ID	Gene Description	Symbol
	Sub-cluster I (down-regulated, 6–96 h post wounding)
99104_at	adiponectin	Adipoq
98802_at	epiregulin	Ereg
95531_at	angiomotin	Amot
160484_at	reticulon 4	Rtn4
103001_at	vascular endothelial growth factor B	Vegfb
102720_at	endothelial-specific receptor tyrosine kinase	Tek
100494_at	fibroblast growth factor 1	fgf1
	Sub-cluster ii (up-regulated, 48–96 h post wounding)
99957_at	matrix metallopeptidase 9	Mmp9
99638_at	procollagen, type XVIII, alpha 1	Col18a1
92676_at	runt related transcription factor 2	Runx2
92558_at	vascular cell adhesion molecule 1	Vcam1
92365_at	c-fos induced growth factor	Figf
92210_at	angiopoietin 2	Angpt2
104154_at	transformation related protein 53	Trp53
101881_g_at	procollagen, type XVIII, alpha 1	Col18a1
	Sub-cluster iii (up-regulated, 6–24 h post wounding)
99100_at	signal transducer and activator of transcription 3	Stat3
99099_at	signal transducer and activator of transcription 3	Stat3
98629_f_at	hypoxia inducible factor 1, alpha subunit	Hif1a
98628_f_at	hypoxia inducible factor 1, alpha subunit	Hif1a
97519_at	secreted phosphoprotein 1	Spp1
96119_s_at	angiopoietin-like 4	Angptl4
94147_at	serine (or cysteine) peptidase inhibitor, clade E, member 1	Serpine1
92777_at	cysteine rich protein 61	Cyr61
92369_at	transforming growth factor alpha	Tgfa
160469_at	thrombospondin 1 /// similar to thrombospondin 1	Thbs1
160101_at	heme oxygenase (decycling) 1	Hmox1
104712_at	myelocytomatosis oncogene	Myc
104647_at	prostaglandin-endoperoxide synthase 2	Ptgs2
104469_at	podoplanin	Pdpn
101464_at	tissue inhibitor of metalloproteinase 1	Timp1
100621_at	ribonuclease/angiogenin inhibitor 1	Rnh1
100134_at	endoglin	Eng

C. ROS-specific gene cluster shown in Figure 8C
Probe Set ID	Gene Description	Symbol
	Sub-cluster I (down-regulated, 12–96 h post wounding)
100614_at	myoglobin	Mb
102382_at	aryl hydrocarbon receptor nuclear translocator-like	Arntl
102720_at	endothelial-specific receptor tyrosine kinase	Tek
160090_f_at	aldolase 1, A isoform	Aldoa
160479_at	catalase	Cat
160481_at	phosphoenolpyruvate carboxykinase 1, cytosolic	Pck1
160547_s_at	thioredoxin interacting protein	Txnip
160913_at	nebulin-related anchoring protein	Nrap
161889_f_at	Aldolase 1, A isoform	Aldoa
93084_at	solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4	Slc25a4
93332_at	CD36 antigen	Cd36
93543_f_at	glutathione S-transferase, mu 1	Gstm1
93836_at	BCL2/adenovirus E1B interacting protein 1, NIP3	Bnip3
93996_at	cytochrome P450, family 2, subfamily e, polypeptide 1	Cyp2e1
95053_s_at	succinate dehydrogenase complex, subunit B, iron sulfur (Ip)	Sdhb
96058_s_at	aldehyde dehydrogenase 2, mitochondrial	Aldh2
98007_at	ribosomal protein S6 kinase, polypeptide 2	Rps6ka2
99104_at	adiponectin, C1Q and collagen domain containing	Adipoq
	Sub-cluster ii (up-regulated, 48–96 h post wounding)
100988_at	BCL2-like 11 (apoptosis facilitator)	Bcl2l11
101160_at	chemokine (C-X-C motif) ligand 2	Cxcl2
101554_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
101561_at	metallothionein 2	Mt2
102292_at	growth arrest and DNA-damage-inducible 45 alpha	Gadd45a
102362_i_at	Jun-B oncogene	Junb
102363_r_at	Jun-B oncogene	Junb
102430_at	myeloid differentiation primary response gene 88	Myd88
102798_at	adrenomedullin	Adm
103887_at	S100 calcium binding protein A9 (calgranulin B)	S100a9
104149_at	nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha	Nfkbia
104692_at	selectin, platelet	Selp
104701_at	basic helix-loop-helix domain containing, class B2	Bhlhb2
104712_at	myelocytomatosis oncogene	Myc
160101_at	heme oxygenase (decycling) 1	Hmox1
92562_at	nuclear factor, erythroid derived 2, like 2	Nfe2l2
92694_at	chitinase 3-like 3	Chi3l3
93573_at	metallothionein 1	Mt1
94146_at	chemokine (C-C motif) ligand 4	Ccl4
94769_at	matrix metallopeptidase 8	Mmp8
94939_at	CD53 antigen	Cd53
95348_at	chemokine (C-X-C motif) ligand 1	Cxcl1
95349_g_at	chemokine (C-X-C motif) ligand 1	Cxcl1
96042_at	superoxide dismutase 2, mitochondrial	Sod2
98010_at	nuclear factor, erythroid derived 2	Nfe2
98088_at	CD14 antigen	Cd14
99985_at	thioredoxin reductase 1	Txnrd1
	Sub-cluster iii (up-regulated, 6–12 h post wounding)
100059_at	cytochrome b-245, alpha polypeptide	Cyba (p22phox)
100300_at	cytochrome b-245, beta polypeptide	Cybb (gp91phox)
102353_at	integrin beta 2	Itgb2
102389_s_at	growth associated protein 43	Gap43
102641_at	SFFV proviral integration 1	Sfpi1
103662_at	neutrophil cytosolic factor 4	Ncf4 (p40 phox)
103707_at	complement component 3a receptor 1	C3ar1
92807_at	thioredoxin 1	Txn1
93483_at	hemopoietic cell kinase	Hck
93536_at	BclII-associated X protein	Bax
94930_at	thrombospondin 2	Thbs2
96085_at	glutathione S-transferase, alpha 4	Gsta4
96562_at	solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1	Slc11a1
97013_f_at	cytochrome b-245, alpha polypeptide	Cyba (p22phox)
97763_at	neutrophil cytosolic factor 1	Ncf1 (p47phox)
98002_at	interferon regulatory factor 8	Irf8
98433_at	BH3 interacting domain death agonist	Bid
98436_s_at	caspase 3	Casp3
98828_at	integrin alpha M	Itgam
99810_at	glutathione peroxidase 2	Gpx2
99915_at	amphiregulin	Areg

D. Extracellular matrix (ECM) specific gene cluster shown in Figure 8D
Probe Set ID	Gene Description	Symbol
	Sub-cluster i (down-regulated, 12–96 h post wounding)
100494_at	fibroblast growth factor 1	Fgf1
100597_at	glycogenin	Gyg
100997_at	desmin	Des
101394_at	sarcoglycan, gamma (dystrophin-associated glycoprotein)	Sgcg
102720_at	endothelial-specific receptor tyrosine kinase	Tek
103243_at	epithelial membrane protein 2	Emp2
103395_at	sarcoglycan, alpha (dystrophin-associated glycoprotein)	Sgca
103721_at	Nephronectin	Npnt
160319_at	SPARC-like 1 (mast9, hevin)	Sparcl1
160519_at	tissue inhibitor of metalloproteinase 3	Timp3
160631_s_at	sarcoglycan, alpha (dystrophin-associated glycoprotein)	Sgca
161907_s_at	tenascin XB	Tnxb
161984_f_at	procollagen, type III, alpha 1	Col3a1
162262_f_at	glycogenin	Gyg
92407_at	myomesin 1	Myom1
92441_at	fibroblast activation protein	Fap
93300_at	transforming growth factor, beta 2	Tgfb2
93563_s_at	nidogen 2	Nid2
94122_at	myocilin	Myoc
	Sub-cluster ii (up-regulated, 48–96 h post wounding)
100019_at	versican	Vcan
100428_at	laminin, gamma 2	Lamc2
100484_at	matrix metallopeptidase 13	Mmp13
101436_at	chemokine (C-X-C motif) ligand 9	Cxcl9
101464_at	tissue inhibitor of metalloproteinase 1	Timp1
101918_at	transforming growth factor, beta 1	Tgfb1
101993_at	tenascin C	Tnc
102362_i_at	Jun-B oncogene	Junb
102363_r_at	Jun-B oncogene	Junb
102641_at	SFFV proviral integration 1	Sfpi1
102663_at	plasminogen activator, urokinase receptor	Plaur
102794_at	chemokine (C-X-C motif) receptor 4	Cxcr4
103005_s_at	CD44 antigen	Cd44
103465_f_at	serum amyloid A 2	Saa2
104647_at	prostaglandin-endoperoxide synthase 2	Ptgs2
104712_at	myelocytomatosis oncogene	Myc
160094_at	actin related protein 2/3 complex, subunit 4	Arpc4
160511_at	chemokine (C-X-C motif) ligand 12	Cxcl12
160774_at	ectonucleoside triphosphate diphosphohydrolase 1	Entpd1
160901_at	FBJ osteosarcoma oncogene	Fos
162362_f_at	tenascin C	Tnc
92365_at	c-fos induced growth factor	Figf
92369_at	transforming growth factor alpha	Tgfa
92562_at	nuclear factor, erythroid derived 2, like 2	Nfe2l2
92676_at	runt related transcription factor 2	Runx2
92731_at	pentraxin related gene	Ptx3
92849_at	chemokine (C-C motif) ligand 6	Ccl6
93793_at	LIM and SH3 protein 1	Lasp1
94085_at	serglycin	Srgn
94140_at	macrophage scavenger receptor 1	Msr1
94147_at	serine (or cysteine) peptidase inhibitor, clade E, member 1	Serpine1
94769_at	matrix metallopeptidase 8	Mmp8
94792_at	macrophage scavenger receptor 1	Msr1
95434_at	actin related protein 2/3 complex, subunit 3	Arpc3
95803_at	signal-regulatory protein alpha	Sirpa
95804_g_at	signal-regulatory protein alpha	Sirpa
96603_at	quiescin Q6	Qscn6
97519_at	secreted phosphoprotein 1	Spp1
97790_s_at	laminin, alpha 3	Lama3
98067_at	cyclin-dependent kinase inhibitor 1A (P21)	Cdkn1a
98433_at	BH3 interacting domain death agonist	Bid
98579_at	early growth response 1	Egr1
99099_at	signal transducer and activator of transcription 3	Stat3
99100_at	signal transducer and activator of transcription 3	Stat3
99957_at	matrix metallopeptidase 9	Mmp9
	Sub-cluster iii (up-regulated, 6–24 h post wounding)
100016_at	matrix metallopeptidase 11	Mmp11
100021_at	cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle)	Chrna1
100308_at	procollagen, type VIII, alpha 1	Col8a1
100928_at	fibulin 2	Fbln2
100986_at	four and a half LIM domains 2	Fhl2
101881_g_at	procollagen, type XVIII, alpha 1	Col18a1
103554_at	a disintegrin and metallopeptidase domain 19 (meltrin beta)	Adam19
161215_at	cholinergic receptor, nicotinic, gamma polypeptide	Chrng
92701_at	bone morphogenetic protein 1	Bmp1
94732_at	repetin	Rptn
94930_at	thrombospondin 2	Thbs2
98623_g_at	insulin-like growth factor 2	Igf2
99327_at	kallikrein related-peptidase 8	Klk8
99638_at	procollagen, type XVIII, alpha 1	Col18a1

Footnote to Tables 3A–D: Probe ID, Affymetrix probe identifications.

At the wound site, ROS are generated at sustained low levels by resident nonphagocytic cells. In the inflammatory phase, copious amounts of ROS are generated by phagocytic cells (60, 69). While excessive ROS may complicate healing, endogenously generated ROS are essential to support the healing process (60). According to annotations in the Affymetrix database ROS-generating, ROS-sensitive, as well as ROS-metabolizing genes are collectively clustered as ROS-related transcripts. Three subsets of ROS-related genes changed significantly in response to wounding. The first subset (Fig. 8Ci, Table 3C), genes abundant in the intact skin were rapidly downregulated in response to wounding. The other two subsets included the up-early (Fig. 8Ciii) and up-late (Fig. 8Cii) genes, the abundance of which in the wound-edge tissue increased in response to wounding (Table 3C). In the functional category of ECM, the subsets of genes represented the same pattern as for the ROS-related and angiogenesis categories. The tables present a complete listing of each of the genes in the subsets described above. Functional aspects of select candidate genes in each category and subset are discussed below.

DISCUSSION

For secondary-intention excisional wounds, advancement of wound edge represents a major mechanism by which a wound defect is closed (91). Changes in the wound-edge tissue are therefore of outstanding interest (20, 66). This work represents the maiden effort temporally characterizing the acute inflammatory phase of the murine excisional cutaneous wound using high-density microarray. In the acute inflammatory phase, chemotaxis is the primary mechanism by which cell movements are directed in response to wounding. Chemotaxis involves a complex cascade of events including formation of signaling complexes via receptor-cytokine interactions (31). In this study, chemotaxis and cytokine-receptor interaction pathways represented the early upregulated genes in response to wounding. Interactions of leukocytes with endothelial cells represent early-intermediate events in acute inflammation wound repair (44). Consistently we noted that components of this pathway were upregulated during the intermediary phase (12–96 h) after wounding. Genes upregulated in response to wounding in the late phase (48–96 h) represented the cell cycle pathway, which is known to be implicated in additional cell supply during the process of tissue repair (14, 68). Cutaneous wound repair is associated with changes in purine metabolism (58). We noted that wounding induced early (6–12 h) downregulation of enzymes of the purine metabolism pathway. Specifically, nucleoside diphosphate kinase and ADP-ribose diphosphatase were downregulated in the wound-edge tissue in response to wounding. Nucleoside diphosphate kinase phosphorylates a nucleoside diphosphate to the corresponding triphosphate at the expense of ATP. Downregulation of this enzyme may be aimed at conserving ATP in the wound-edge tissue for fueling tissue repair. ADP-ribose facilitates neutrophil chemotaxis (54). Downregulation of ADP-ribose diphosphatase would attenuate the hydrolysis of ADP-ribose to AMP, making ADP-ribose available to support wound angiogenesis (12). During the interval 12–96 h after wounding, genes encoding proteins of the oxidative phosphorylation pathway were downregulated. Candidate genes in this category included NADH dehydrogenases, component of the succinate dehydrogenase complex, cytochrome c-1, cytochrome c-oxidase, and ATP synthase. This response may explain wound tissue ATP deficiency that is recognized as a common limiting factor in cutaneous wound healing (7, 11).

Changes in the expression of functionally categorized genes on one hand lent support to currently proposed hypotheses while on the other hand generated novel insight into the acute inflammatory phase of tissue repair. Inflammation-related early upregulated genes included known players such as several chemokine receptors and ligands. Recruitment of leukocyte subtypes to the wound site is tightly regulated by chemokines. Moreover, the presence of chemokine receptors on resident cells (e.g., keratinocytes, endothelial cells) indicates that chemokines also contribute to the regulation of epithelialization, tissue remodeling, and angiogenesis. Thus, chemokines are in an exclusive position to integrate inflammatory events and reparative processes and are important modulators of wound healing (24). Interleukin-6 (IL-6) is a pleiotropic cytokine that is produced by normal constituents of the skin, including epidermal cells, fibroblasts, and dermal endothelial cells. The inflammatory response that occurs after cutaneous wounding is a prerequisite for healing, and inflammatory cytokines such as IL-6 are involved in this process (23). Consistently, we noted that IL-6 represented an early upregulated gene in response to wounding. Metallothioneins (Mt) are a class of ubiquitously occurring low-molecular-weight cysteine- and metal-rich proteins containing sulfur-based metal clusters that are inducible by inflammation (16). Expression of the Mt gene is upregulated in the skin in regions of high mitotic activity. This induction of Mt in the wound margin may reflect its role in promoting cell proliferation and re-epithelialization. The action of Mt in these processes may result from the large number of Zinc-dependent and copper-dependent enzymes required for cell proliferation and matrix remodeling (38). We noted that Mt1 and Mt2 are rapidly induced in response to wounding.

Thrombospondin 1 (TSP1) null mice suffer from impaired healing of cutaneous wounds. Immunohistochemical analyses have demonstrated that the granulation tissue of TSP1-null mice is not well vascularized. TSP1-nulls also exhibit impaired macrophage recruitment to the injury site (2). Our observation that TSP1 is rapidly induced in response to wounding is consistent with these findings. More recent studies have demonstrated that platelets, TSP1, and dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation (35). TSP-1 also promotes proliferative healing through stabilization of PDGF (37). Thrombomodulin is a cell surface anticoagulant that is expressed by endothelial cells and epidermal keratinocytes. In both human and murine wounds, thrombomodulin is absent in keratinocytes at the leading edge of the neoepidermis but is expressed strongly by stratifying keratinocytes within the neoepidermis (55). We observed that thrombomodulin was rapidly induced in response to cutaneous wounding as demonstrated previously.

The inflammation-related genes that were induced 12–96 h after wounding included matrix included MMP9. Previous gelatin zymography studies showed that MMP9 is expressed from day 1 to day 5 after healing (53). Also known as gelatinase B, MMP9 coordinates and effects epithelial regeneration (46). Degradation of the ECM, which is an indispensable step in tissue remodeling processes such as embryonic development and wound healing of the skin, has been attributed to collagenolytic activity of MMPs. Macrophage expressed gene (Mpeg1) represented another gene in this category that has never heretofore been known in the context of wound healing. Mpeg1 expression in mouse cells and cell lines is restricted to mature macrophage and macrophage-like cells, with increased Mpeg1 expression detected as progenitor cells differentiated into macrophages (77).

In the functional category of angiogenesis, the three subsets of genes identified include those that were induced early (6–24 h), induced late (48–96 h), and those that were downregulated in the time frame of 6–96 h. Genes encoding transcriptional regulators such as Stat3 and HIF1α were induced rapidly following wounding. Signal transducer and activator of transcription 3 (Stat3) is one of a family of cytoplasmic proteins that participate in normal cellular responses to cytokines and growth factors as transcription factors. Stat3 modulates various physiological functions including cell survival, cell-cycle regulation, and angiogenesis through regulation of gene expression. Stat3 activation contributes to skin wound healing, keratinocyte migration, and hair follicle growth (67). PDGF induces fibroblast migration under the control of STAT3-SOCS3 (48). Disrupted vasculature at the wound site causes tissue hypoxia and induces the HIF transcriptional machinery (18). The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts (10). Recently we have noted that Cyr61 protein was markedly overexpressed in blood vessels laser captured from the chronic human wound compared with blood vessels obtained from the intact skin (65). Consistent with the observation in patients, Cyr61 was observed to be rapidly induced in the wound-edge tissue following injury. Transforming growth factor-α (TGF-α) plays a significant early role in wound epithelialization in vivo (36). In humans, TGF-α represents a major human serum factor that promotes human keratinocyte migration (40). Our results indicate that TGF-α is rapidly induced in response to cutaneous wounding. The broad role of the TGF-β signaling pathway in vascular development, homeostasis, and repair is well appreciated. Endoglin (CD105) is emerging as a novel modifier of TGF-β receptor signaling at the ligand and receptor activation level. Direct effects of endoglin on cell adhesion and migration are known. The emerging roles for endoglin in the determination of stem cell fate and tissue patterning make it an interesting candidate in the biology of wound healing (4). Although it has been recognized that endoglin plays a crucial role in blood cell-mediated vascular repair (85), its significance in cutaneous wound repair remains to be addressed. This work presents first evidence that cutaneous wounding rapidly induces endoglin in the wound-edge tissue.

Genes related to the angiogenesis functional category that were induced in the late 48–96 h time period primarily represented the extracellular matrix and adhesion molecules. In addition to MMP9, which has been discussed above in the inflammation category, the gene encoding procollagen type XVIII α1 was included in this subset. Loss of this basement membrane proteoglycan enhances angiogenesis and vascular permeability during atherosclerosis by distinct gene dose-dependent mechanisms (47). The significance of this proteoglycan in cutaneous wound healing remains to be understood. The injury-inducible gene angiopoietin-1 (Ang-2) (33) was upregulated in the wound-edge tissue. A ligand for Tie2, Ang-2, is produced by angiogenic vessels and is a chemoattractant for Tie2-expressing monocytes (39). Vascular cell adhesion cell molecule-1 (VCAM1, CD106) was also observed to be induced following 48–96 h of wounding. Injury-activated vascular endothelium expresses VCAM-1, a member of the adhesion molecule superfamily, to which monocytes and lymphocytes can bind. These inflammatory cells can then move through the endothelium by diapedesis and release cytokines and enzymes, important components in the progression of the lesion. The specific significance of VCAM1 in cutaneous wound healing remains to be examined.

Functional angiogenic outcomes do not depend solely on any single gene but on the net balance of angiogenesis-related genes. Thus, an understanding of the genes that are downregulated in response to injury is necessary to appreciate the situation as a whole. This work identified a set of angiogenesis-related genes that were downregulated in response to injury. Although it is known that these gene products are functionally related to angiogenesis, their significance to cutaneous wound healing remains unknown. Adiponectin is an adipocyte-specific adipocytokine with antiatherogenic and antidiabetic properties, and its plasma levels are reduced in association with obesity-linked diseases. Adiponectin stimulates angiogenesis in response to ischemic stress, as known to occur in wounds, by promoting AMP-activated kinase signaling (72). Epiregulin, a member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes (74). Epiregulin plays a critical role in immune/inflammatory-related responses of keratinocytes and macrophages at the barrier from the outside milieu (75). Angiomotin is an angiostatin binding protein that facilitates endothelial cell migration and tube formation. It plays an important role in growth factor-induced migration of endothelial cells (1). Angiomotin is important for endothelial polarization during migration, and it controls Rac1 activity in endothelial and epithelial cells (1). Therapeutic antibodies targeting angiomotin inhibit angiogenesis in vivo (84). As an angiogenic factor, VEGF-A has received most attention, as it is a potent stimulator of vascular growth. Results in clinical trials of VEGF-A as a therapeutic agent have fallen short of high expectations because of serious edematous side effects caused by its activity in promoting vascular permeability. VEGF-B, a related factor, binds some of the VEGF-A receptors but not to VEGF receptor 2, which is implicated in the vascular permeability promoting activity of VEGF-A. Recent data support the use of VEGF-B as a therapeutic agent to promote vascular growth, in part, by potentiating angiogenesis. Furthermore, the lack of vascular permeability activity associated with either transgenic overexpression of the VEGF-B gene in endothelial cells or application of VEGF-B protein to the skin of mice indicates that use of VEGF-B as a therapy should not be associated with edematous side effects (83). Reticulons are a group of integral membrane proteins that have a uniquely conserved COOH-terminal domain named RHD. In mammalian genomes, transcripts are produced from four genes, rtn1 to rtn4, under the regulation of tissue or cell type-specific expression. Although this family exists in almost all eukaryotes, only the rtn4 gene product, Nogo (reticulon 4), has gained relatively more in-depth attention. Despite predominant localization in the endoplasmic reticulum, Nogo on the cell surface appears to play a critical role as an inhibitory molecule for axonal growth and regeneration in humans and rodents. Biological functions of reticulon 4 or Nogo include inhibition of neurite growth from the cell surface via specific receptors, intracellular trafficking, cell division, and apoptosis (87). Significance of the observed downregulation of the reticulon 4 in the wound-edge tissue remains unclear.

The current study provides a comprehensive account of the temporal changes in the expression of genes encoding ECM proteins during the acute inflammatory phase. The three subsets of genes identified include those that were induced early (6–24 h), induced late (48–96 h), and those that were downregulated in the time frame of 12–96 h. While some of the results confirmed previous findings, a majority of the findings represent changes in genes previously never studied in the context of wound healing. The rapidly induced genes are discussed first. MMP11, also known as stromelysin-3, was rapidly induced by injury. MMP11 represents zinc-dependent endopeptidases involved in matrix degradation and tissue remodeling. Despite intense efforts since its first characterization 15 yr ago, its role and target substrates in different diseases remain largely unknown (42). Fibulin-2 is an ECM protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development (76). The role of fibulin-2 in cutaneous wound healing remains to be examined. Repetin was identified as a novel member of the “fused gene” subgroup within the S100 gene family encoding a murine epidermal differentiation protein. The “fused” gene family comprises profilaggrin, trichohyalin, repetin, hornerin, the profilaggrin-related protein, and a protein encoded by c1orf10. Functionally, these proteins are associated with keratin intermediate filaments and partially crosslinked to the cell envelope. Repetin expression is scattered in the normal epidermis but strong in the acrosyringium and in the inner hair root sheath. Ultrastructurally, repetin is a component of cytoplasmic nonmembrane “keratohyalin” F-granules in the stratum granulosum of normal epidermis, similar to profilaggrin (32).

The second wave (48–96 h) of ECM-related genes induced by injury was more numerous than the first (6–24 h) wave. Candidates in this late-responding group were mostly related to cytokines, chemokines, and specific MMPs. The observed induction of TGF-β1 and TIMP1 in the late inflammatory phase is consistent with prior literature (78, 93). MMPs 8, 9, and 13 were induced in this time frame as well. MMP-8 (collagenase-2) is a neutrophil-derived highly effective type I collagenase, recently indicated to be important for acute wound healing (56).MMP9 (gelatinase B) coordinates and effects epithelial regeneration (46). The observed induction of MMP13 in the wound-edge tissue warrants revisiting the hypothesis that cutaneous wound healing is independent of MMP13 (28). Versican is a major hyaluronan-binding component in the dermis that is induced in response to injury. Interestingly, the hyaluronan receptor CD44 is also concurrently induced. The high-molecular-weight polysaccharide hyaluronan is a major component of the ECM between the vital cells of human skin epidermis. The levels of hyaluronan and those of the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican correlate with the aggressiveness of different human carcinomas of epithelial origin (34). However, their significance in wound healing remains unknown.

A small set of genes were observed to be downregulated acutely in response to wounding. The significance of these changes in wound healing remains obscure. Glycogen synthesis in the wound-edge tissue seems to be turned down by lowering of glycogenin expression. Glycogenin initiates glycogen synthesis in an autocatalytic reaction in which individual glucose residues are covalently linked to tyrosine 194 to form a short priming chain of glucose residues that is a substrate for glycogen synthase, which, combined with the branching enzyme, catalyzes the bulk synthesis of glycogen (88). Another gene downregulated in response to injury was nidogen-2. Nidogens represent classical linkers joining laminin and collagen IV networks in basement membranes. Nidogen-2 is equivalent to nidogen-1, and both help develop the basement membrane (50). The significance of lowered nidogen-2 expression in wound healing remains unclear.

ROS, generated at low concentrations during the acute inflammatory phase, are known to be required for wound healing (25, 52, 60, 69, 71). Excess ROS, however, may complicate the healing process. Thus, we sought to functional cluster all ROS-related genes and analyze their changes in response to injury. The three subsets of genes identified include those that were induced early (6–12 h), induced late (48–96 h), and those that were downregulated in the time frame of 12–96 h. These findings are completely novel and provide key insight into the redox regulation of wound healing (73). Of outstanding interest, several components of the NADPH oxidase system such as gp91phox, p22phox, and p47phox were induced rapidly in response to wounding. These components represent critical elements that help drive redox signaling relevant to wound healing (69). Mice deficient in gp91phox and p47phox are known to suffer from impaired healing (60). Defect in p47phox in humans causes chronic granulomatous disease, which is associated with impaired wound healing.

In the 48–96 h period after injury, ROS-sensitive genes were noted to be upregulated. This could be viewed as a consequence of the generation of ROS that is known to occur shortly after wounding (51, 60). ROS-inducible genes in this subset included Mt (49), myc (15), selectin (82), and heme oxygenase (45). Several other products of genes in this list are known to possess antioxidant functions (17, 79, 92). This response may directed toward defending the wound tissue against the threat of excess ROS. The functional significance of the genes downregulated in response to injury remains unclear. Low levels of H₂O₂ are known to be required for wound healing (51, 60, 69). Injury-induced lowering of catalase gene expression may be viewed as an attempt to slow H₂O₂ decomposition at a time when H₂O₂ is required as a second messenger for wound repair.

In summary, the current work presents a detailed analysis of the acute inflammatory phase of murine excisional wound healing from a functional genomics standpoint. While some of the observations confirm previous findings reported in the literature, the vast majority of the results of this study provide new evidence toward novel hypotheses that should help elucidate the biology of cutaneous wound healing comprehensively.

GRANTS

Supported by National Institutes of Health RO1 awards GM-069589, GM-077185, and partly by DK-076566.