A disposable, canister-free negative-pressure wound therapy device proves feasible, but of inferior efficacy to conventional therapies for treating cutaneous wounds in dogs.

OBJECTIVE: To assess the feasibility of a canister-free negative-pressure wound therapy (NPWT) device (PICO™ 1.6, Smith & Nephew Medical Ltd) and evaluate its effect on early phases of wound healing in canine experimental cutaneous wounds. ANIMALS: 5 adult spayed female research Beagles. PROCEDURES: In a pilot experimental study, 1 full-thickness 2-cm X 2-cm cutaneous wound was surgically created on each hemithorax in each dog. Wounds were treated with either NPWT or a conventional wound dressing for 14 days. Bandage changes and wound evaluations were done at 7 time points. First macroscopic appearance of granulation tissue, smoothness of granulation tissue, and percentages of wound contraction and epithelialization were compared between treatments. Wounds were sampled at 3 time points for histopathologic analyses and semiquantitative scoring. RESULTS: NPWT dressings were well tolerated by all dogs. Complete seal of the dressing required the application of adhesive spray, and maintenance of the vacuum lessened over time. Self-limiting skin irritations appeared in all dogs and hampered the attainment of negative pressure. Granulation tissue developed faster and was more abundant in control wounds. Wound contraction, epithelialization, and fibroblast proliferation were greater in control wounds at the end of the study. CLINICAL RELEVANCE: This canister-free NPWT device is feasible but problematic in maintaining a vacuum, requiring frequent revisions of the dressing. Further studies are necessary to evaluate the effect of this device on early phases of wound healing. Its benefits in wound healing remain unknown.

The Horse as a Model for the Study of Cutaneous Wound Healing.

Significance: Cutaneous wounds are a major problem in both human and equine medicine. The economic cost of treating skin wounds and related complications in humans and horses is high, and in both species, particular types of chronic wounds do not respond well to current therapies, leading to suffering and morbidity. Recent Advances: Conventional methods for the treatment of cutaneous wounds are generic and have not changed significantly in decades. However, as more is learned about the mechanisms involved in normal skin wound healing, and how failure of these processes leads to chronic nonhealing wounds, novel therapies targeting the specific pathologies of hard-to-heal wounds are being developed and evaluated. Critical Issues: Physiologically relevant animal models are needed to (1) study the mechanisms involved in normal and impaired skin wound healing and (2) test newly developed therapies. Future Directions: Similarities in normal wound healing in humans and horses, and the natural development of distinct types of hard-to-heal chronic wounds in both species, make the horse a physiologically relevant model for the study of mechanisms involved in wound repair. Horses are also well-suited models to test novel therapies. In addition, studies in horses have the potential to benefit veterinary, as well as human medicine.

Anti-fibrotic Actions of Equine Interleukin-10 on Transforming Growth Factor-Beta1-Stimulated Dermal Fibroblasts Isolated From Limbs of Horses.

Fibroproliferative disorders occur in both humans and horses following skin injury. In horses, wound healing on the limb is often complicated by the formation of fibroproliferative exuberant granulation tissue, characterized by persistent expression of pro-fibrotic transforming growth factor-beta1 (TGF-ß1) and deficient expression of anti-inflammatory interleukin-10 (IL-10). IL-10 has been shown to directly modulate fibrotic gene expression in human fibroblasts, so we hypothesized that equine IL-10 (eIL-10) may exert similar anti-fibrotic effects on equine dermal fibroblasts. Cell-lines were created from the limb skin of six individual horses. Recombinant eIL-10 was produced and purified, and its effects on the cells investigated in the presence and absence of equine TGF-ß1 (eTGF-ß1). Myofibroblast differentiation and collagen production were examined using immunofluorescent cytometry, cell contractility in a collagen gel assay, and fibrotic gene expression using quantitative PCR. In response to eTGF-ß1, fibroblasts increased in contractility and expression of alpha-smooth muscle actin, collagen types 1 and 3, and matrix metalloproteinase 1, 2, and 9. Equine IL-10 limited cell contractility and production of alpha-smooth muscle actin and type 3 collagen, and decreased mRNA levels of eCol3a1 and eMMP9, while increasing that of eMMP1. Opposing effects on eTGF-ßR3 and eIL-10R1 gene expression were also observed, with mRNA levels decreasing following eTGF-ß1 treatment, and increasing with eIL-10 treatment. These findings indicate that eIL-10 limits the pro-fibrotic effects of eTGF-ß1, potentially through the modulation of fibrotic and receptor gene expression. Further investigations are warranted to assess the therapeutic utility of eIL-10 in the treatment of exuberant granulation tissue.

Treatment of limb wounds of horses with orf virus IL-10 and VEGF-E accelerates resolution of exuberant granulation tissue, but does not prevent its development.

Bandaging of limb wounds in horses leads to formation of exuberant granulation tissue (EGT) that retards healing due to protracted inflammation, aberrant vascularisation and delayed epithelialisation. EGT is not observed if wounds are left undressed or when wounds are on the body. A previous study showed that short-term administration of proteins derived from orf virus dampened inflammation and promoted epithelialisation of open wounds in horses. Here, we investigated the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on the development and resolution of EGT. Excisional wounds were created on the forelimb of four horses, and bandages were maintained until full healing to induce EGT formation. Matching body wounds were created to ensure EGT was limited to the limb, and to differentiate the effects of the viral proteins on normal healing and on EGT formation. Viral proteins or the hydrogel vehicle control were administered topically to site-matched wounds at day 1, with repeat administration at day 8. Wound healing and EGT formation were monitored macroscopically. Wound margin samples were harvested at 2, 7 and 14 days, and at full healing, with histology used to observe epithelialisation, immunofluorescence used to detect inflammatory cells, angiogenesis and cell death, and qPCR to measure expression of genes regulating inflammation and angiogenesis. Limb wounds developed EGT, and exhibited slower healing than body wounds. Viral protein treatment did not accelerate healing at either location nor limit EGT formation in limb wounds. Treatment of limb wounds did however increase epithelialisation and angiogenesis, without dampening inflammatory cell infiltration or gene expression. The healed wounds also had less occlusion and death of blood vessels and fewer epidermal rete ridges following viral protein treatment. These findings indicate that the viral protein treatment does not suppress wound inflammation or EGT formation, but does promote vascular and epidermal repair and EGT resolution.

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

BACKGROUND: Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. HYPOTHESIS/OBJECTIVES: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. ANIMALS: Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. METHODS: Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. RESULTS: Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. CONCLUSIONS AND CLINICAL IMPORTANCE: This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses.

Equine exuberant granulation tissue and human keloids: a comparative histopathologic study.

OBJECTIVE: To compare histopathologic features of a fibroproliferative disorder in horses (exuberant granulation tissue-EGT) and people (keloid). SAMPLE POPULATION: Archival tissue samples of EGT (n = 8) and keloid (12). METHODS: After automated hematoxylin and eosin, histochemical (Gomori trichrome, Verhoeff-van Gieson elastin) and immunohistochemical (vimentin, α-smooth muscle actin, CD34, CD68, CD117) stainings, tissue sections were evaluated using a semi-quantitative grading scale for presence or absence of ulceration, keloidal collagen, myofibroblasts, and elastic fibers as well as degree of inflammation, fibrosis, vascularity, and orientation of collagen fibers. RESULTS: Superficial dermis and deep dermis of both horses and people had increased numbers of haphazardly oriented thickened collagen fibers; however, only keloids contained "keloidal" collagen. Fibroblast numbers were markedly increased in both groups but only EGT had myofibroblasts. Minimal vascularity was observed in the deep dermis of both groups. The superficial dermis in EGT was characterized by small vessels within immature granulation tissue. Macrophages and mast cells were infrequently found in both groups but polymorphonuclear cells were markedly increased in EGT. CONCLUSIONS: Humans and horses are the only mammals known to naturally develop excessive granulation during wound healing; however, similarities and differences between fibroblast populations and associated collagen have not been reported. Inflammatory response may contribute to observed differences in the cellular populations, with EGT possessing markedly increased myofibroblasts, small vessels, and acute inflammatory cells compared with keloids. Further work is warranted to develop common treatment strategies for these fibroproliferative conditions.

Aberrant wound healing in the horse: naturally occurring conditions reminiscent of those observed in man.

Impaired wound healing represents an enormous clinical and financial problem for companion animals and humans alike. Unfortunately, most models used to study healing rely on rodents, which have significant differences in the healing and scarring process and rarely develop complications. In order to better simulate impaired healing, the model should strive to reproduce the natural processes of healing and delayed healing. Wounds on the limbs of horses display similarities to wounds in humans in their epithelialization/contraction ratio, genetic influence as well as dysregulated cytokine profile and the spontaneous development of fibroproliferative disorders. Veterinarians have access to advanced wound therapies that are often identical to those provided to human patients. Wound research in large animals has resulted in new wound models as well as a better understanding of the physiology, immunology, and local environmental impact on both normal and aberrant wound healing. One such model reproduces the naturally occurring fibroproliferative disorder of horses known as exuberant granulation tissue. Comparisons between the normally healing and impaired wounds provide insight into the repair process and can facilitate product development. A better understanding of the wound healing physiopathology based on clinically accurate animal models should lead to the development of novel therapies thereby improving outcomes in both human and veterinary patients.

Skin temperature during cutaneous wound healing in an equine model of cutaneous fibroproliferative disorder: kinetics and anatomic-site differences.

OBJECTIVE: To map skin temperature kinetics, and by extension skin blood flow throughout normal or abnormal repair of full-thickness cutaneous wounds created on the horse body and limb, using infrared thermography. STUDY DESIGN: Experimental. ANIMALS: Standardbreds (n = 6), aged 3-4 years. METHODS: Three cutaneous wounds were created on the dorsolateral surface of each metacarpus and on the lateral thoracic wall. Thoracic skin wounds and those on 1 randomly chosen forelimb healed by second intention without a bandage, whereas contralateral limb wounds were bandaged to induce formation of exuberant granulation tissue (EGT). Thermal data were collected from all planned wound sites before the surgical procedure (baseline), and at 24, 48, 96 hours, 1, 2, and 4 weeks after wounding. Data were analyzed using repeated measures ANOVA and a priori contrasts submitted to Bonferroni sequential correction. Level of significance was P < .05. RESULTS: Cutaneous wound temperature (CWT) increased temporally from preoperative period to week 1 postwounding, independently of anatomic location (P < .0001). CWT of limb wounds was significantly less than that of body wounds throughout healing (P < .01). CWT of limb wounds managed with bandages and developing EGT was significantly less than that of unbandaged limb wounds, which did not develop EGT (P ≤ .01). CONCLUSIONS: CWT varied with anatomic location and throughout healing. CWT of wounds developing EGT was significantly less than that of wounds without EGT.

Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1.

BACKGROUND: Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. OBJECTIVE: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or limb respond differently to hypoxia. METHODS: EDFs were isolated and cultured from skin from body or limb under normoxic or hypoxic conditions for up to 7days. RESULTS: Hypoxia significantly stimulated EDF proliferation, but had no effect on cell survival. The hypoxia-mimetic agent CoCl(2) up-regulated COL1A1 expression and down-regulated MMP2 expression, suggesting an increase in ECM synthesis and a decrease in turnover. Both regulatory effects were inhibited by the addition of echinomycin, indicating that they are mediated by the transcriptional regulatory activity of HIF. No differences were observed between EDFs originating from body or limb for any effect of hypoxia or CoCl(2), suggesting that EGT development does not depend on intrinsic properties of limb fibroblasts. CONCLUSIONS: We conclude that hypoxia regulates ECM remodeling via HIF1 in EDFs, and that this may be an important determinant in the pathogenesis of equine EGT.

Constitutive expression of hypoxia-inducible factor-1 α in keratinocytes during the repair of skin wounds in horses.

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia-inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin-dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia-independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.

Regional differences in wound oxygenation during normal healing in an equine model of cutaneous fibroproliferative disorder.

Wound repair in horse limbs is often complicated by the development of exuberant granulation tissue (EGT) and excessive scarring while body wounds tend to repair uneventfully. EGT resembles the human keloid. While the events leading to keloid formation are not fully elucidated, tissue hypoxia has been proposed as a major contributing factor. The objective of this study was to investigate tissue oxygen saturation in healing full-thickness wounds created on the horse limb and body, using near-infrared spectroscopy. Spectroscopic reflectance data were collected from both anatomic sites at specific times following wounding. The oxygen saturation values of limb wounds were significantly inferior to those of body wounds during the early period of healing, indicating a temporary, relative state of hypoxia in the former during the inflammatory phase of repair. Horses present a weak, persistent inflammatory response to wounding, especially at the limb level. The relative hypoxia present acutely in limb wounds of horses may promote a feeble yet prolonged inflammatory response, which could interfere with and retard the subsequent phases of healing. Ongoing low-grade inflammation in horse wounds is accompanied by up-regulation of various inflammatory and profibrotic mediators, which might ultimately promote the development of fibroproliferative disorders such as EGT.

Correlation of signal attenuation-based quantitative magnetic resonance imaging with quantitative computed tomographic measurements of subchondral bone mineral density in metacarpophalangeal joints of horses.

OBJECTIVE: To evaluate the ability of signal attenuation-based quantitative magnetic resonance imaging (QMRI) to estimate subchondral bone mineral density (BMD) as assessed via quantitative computed tomography (QCT) in osteoarthritic joints of horses. SAMPLE POPULATION: 20 metacarpophalangeal joints from 10 horse cadavers. PROCEDURES: Magnetic resonance (MR) images (dorsal and transverse T1-weighted gradient recalled echo [GRE] and dorsal T2*-weighted GRE fast imaging employing steady-state acquisition [T2*-FIESTA]) and transverse single-slice computed tomographic (CT) images of the joints were acquired. Magnetic resonance signal intensity (SI) and CT attenuation were quantified in 6 regions of interest (ROIs) in the subchondral bone of third metacarpal condyles. Separate ROIs were established in the air close to the joint and used to generate corrected ratios and SIs. Computed tomographic attenuation was corrected by use of a calibration phantom to obtain a K(2)HPO(4)-equivalent density of bone. Correlations between QMRI performed with different MR imaging sequences and QCT measurements were evaluated. The intraobserver repeatability of ROI measurements was tested for each modality. RESULTS: Measurement repeatability was excellent for QCT (R(2) = 98.3%) and QMRI (R(2) = 98.8%). Transverse (R(2) = 77%) or dorsal (R(2) = 77%) T1-weighted GRE and QCT BMD measurements were negatively correlated, as were dorsal T2*-FIESTA and QCT (R(2) = 80%) measurements. Decreased bone SI during MR imaging linearly reflected increased BMD. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this ex vivo study suggested that signal attenuation-based QMRI was a reliable, clinically applicable method for indirect estimation of subchondral BMD in osteoarthritic metacarpophalangeal joints of horses.

Histological and biomechanical effects of palatal sclerotherapy in the horse using sodium tetradecyl sulfate.

Palatal sclerotherapy using sodium tetradecyl sulfate has been suggested as a treatment for dorsal displacement of the soft palate in young Standardbred horses. The present study evaluated histological and biomechanical changes in the equine soft palate following trans-endoscopic treatment with a low dose of this compound. Two horses were euthanased and examined at 2 weeks and at 1, 2, 4 and 6 months post-sclerotherapy, while two further horses served as untreated controls. The technique was easily performed in all cases without major complications. On histological examination there was no evidence of palatal necrosis, inflammation or fibrosis in any of the treated or control animals. There was no variation in the density of palatal connective tissue between individuals, and on biomechanical assessment no significant difference in the stiffness of the palatal tissue was found between treated and control horses at any time. The lower dose of sodium tetradecyl sulfate used in this study relative to previous reports, might explain the absence of tissue alterations. This method of sclerotherapy did not alter the morphology or biomechanical properties of normal equine soft palates.

Equine thrombospondin II and secreted protein acidic and cysteine-rich in a model of normal and pathological wound repair.

Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expression was studied in unwounded skin and in samples from 1, 2, 3, 4, and 6 wk old wounds of the body and limb. Temporal gene expression was determined by semiquantitative RT-PCR, while protein expression was mapped immunohistochemically. The temporal pattern of expression for both genes was similar; wounding caused immediate upregulation of mRNA, which did not return to baseline by the end of the study, and overexpression was noted in body relative to limb wounds. Immunostaining for THBS2 and SPARC was induced by wounding, though no differences in stain location or intensity were detected between body and limb wounds. This study is the first to characterize equine cDNA for THBS2 and SPARC and to document mRNA expression over the different phases of repair. THBS2 and SPARC might modulate angiogenesis during wound healing in the horse, which could protect against the disproportionate fibroplasia commonly afflicting limb wounds and leading to the development of exuberant granulation tissue.

Equine lumican (LUM) cDNA sequence and spatio-temporal expression in an experimental model of normal and pathological wound healing.

The development of exuberant granulation tissue, a situation that in some ways resembles the human keloid, compromises both the aesthetic and functional outcomes of wound repair in horses. To help elucidate the underlying molecular mechanisms the spatio-temporal expression of lumican (LUM) mRNA and protein for their potential contributions to tissue remodelling of body and limb wounds, was examined in an established experimental model. Expression was studied in intact skin and in samples of 1-, 2-, 3-, 4- and 6-week-old wounds of the body and forelimb. Temporal gene expression was determined by reverse transcriptase polymerase chain reaction, and protein expression was mapped immunohistochemically. A significant increase in LUM mRNA expression was observed in response to wounding at both anatomical locations, and a significantly higher mRNA level was recorded in thoracic than in limb wounds at weeks 1, 3 and 6 of repair. The immunohistochemical observations partially corroborated the mRNA data. To the authors' knowledge this study is the first to document that the cDNA for LUM is expressed over the different phases of wound repair in horses and suggests that LUM might be involved in both inflammation and remodelling in response to dermal injury. Further studies are now required to verify and quantify the temporal expression of this protein to provide the basis for targeted therapies that might prevent the development of exuberant granulation tissue in horse wound repair.

Effects of platelet-rich plasma on the repair of wounds on the distal aspect of the forelimb in horses.

OBJECTIVE: To evaluate the effect of platelet-rich plasma on wounds on the distal aspect of the forelimb in horses. ANIMALS: 6 mixed-breed 10- to 15-year-old mares. PROCEDURES: 3 wounds were created on metacarpal regions in each of 6 horses (n = 36 wounds total). Eighteen wounds were treated with platelet-rich plasma and bandaged, whereas 18 control wounds were similarly bandaged with no prior topical treatment. Decrease in wound surface area and the required number of excisions of exuberant granulation tissue were recorded until complete healing. Tissue specimens were taken from wounds at 1 week for histologic examination and measurement of transforming growth factor-beta1 concentrations and at closure for histologic examination, biomechanical evaluation, and measurement of collagen type I and type III mRNA. RESULTS: Platelet-rich plasma favored excessive development of granulation tissue and significantly slowed wound healing at 1, 2, and 3 weeks after surgery. Transforming growth factor-beta1 had a 1.6-fold higher concentration in treated wounds, compared with untreated wounds. Histologic, biomechanical, and gene expression data did not differ significantly between treated and control wounds. CONCLUSIONS AND CLINICAL RELEVANCE: Topical application of autologous platelet-rich plasma did not accelerate or improve the quality of repair of small granulating wounds on limbs of horses. This treatment may better suit wounds with massive tissue loss or, alternatively, chronic wounds that would benefit from a fresh source of mediators to accelerate the healing process.

Nucleotide structure and expression of equine pigment epithelium-derived factor during repair of experimentally induced wounds in horses.

OBJECTIVE: To clone full-length equine pigment epithelium-derived factor (PEDF) complementary DNA (cDNA) and to evaluate its temporal expression during repair of wounds in horses. ANIMALS: 4 clinically normal 2-to 3-year-old Standardbred mares. PROCEDURES: Full-length equine PEDF cDNA was cloned by screening size-selected cDNA libraries derived from biopsy specimens obtained from the wound edge 7 days after experimental creation of a 6.25-cm(2) full-thickness wound in the skin of the lateral thoracic wall. Expression was evaluated in normal skin and in biopsy specimens obtained weekly from experimentally induced wounds on the trunk and limbs of horses. Temporal gene expression was determined by use of reverse transcriptase PCR assay. RESULTS: Equine PEDF shared 87% sequence and 88% peptide homology with human PEDF. Wounding caused upregulation of PEDF mRNA, which did not return to baseline by the end of the study in either anatomic location. Relative overexpression was evident in wounds on the trunk, compared with expression for wounds on the limbs. CONCLUSIONS AND CLINICAL RELEVANCE: This study characterized full-length equine cDNA for PEDF and determined that the gene for PEDF appeared to be upregulated in response to dermal wounding. Although the cause of exuberant granulation tissue is probably multifactorial, these data suggested that PEDF, via its potent antiangiogenic capabilities, may contribute to superior healing in wounds on the trunks of horses by protecting such wounds from excessive formation of vascular granulation tissue that characterizes wounds on the limbs of this species.

Laminin receptor 1 is differentially expressed in thoracic and limb wounds in the horse.

Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body and limb wounds in the horse. Cloning was achieved by screening a cDNA library previously derived from 7-day wound biopsies. Expression was studied in unwounded skin and in samples from 1-, 2-, 3-, 4- and 6-week-old wounds of the body and limb. Temporal gene expression was determined by real time polymerase chain reaction (RT-PCR) while protein expression was mapped immunohistochemically. Full-length cDNA for equine LAMR1 was shown to be highly similar to that of other species. The mRNA expression of LAMR1 was significantly up-regulated only in thoracic wounds, 4 and 6 weeks following wounding (upon epithelialization). Cutaneous wounding induced protein expression at both locations. Our data suggest that up-regulation of LAMR1 protein might favour epithelialization during wound healing. However, its interaction with ligands other than laminin complicates data interpretation. Future studies should quantitatively verify the temporal expression of this protein in order to provide the basis for targeted therapies that might enhance epithelialization.

Equine ANXA2 and MMP1 expression analyses in an experimental model of normal and pathological wound repair.

BACKGROUND: Wounds on horse limbs can develop exuberant granulation tissue which resembles the human keloid. Clues gained from the study of over-scarring in horses might help control fibro-proliferative disorders. OBJECTIVES: The aim of the present study was to clone full-length equine ANXA2 cDNA then to study spatio-temporal expression of ANXA2 and MMP1 mRNA and protein, potential contributors to remodeling, during repair of body (normal) and limb (fibro-proliferative) wounds in an established horse wound model. METHODS: Cloning of ANXA2 was achieved by screening size-selected cDNA libraries. Expression was studied in intact skin and in biopsies of 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal gene expression was determined by semi-quantitative RT-PCR while protein expression was mapped immunohistochemically. RESULTS: ANXA2 mRNA was up-regulated only in body wounds, corroborating the superior and prompt tissue turnover at this location. Immunohistochemistry partially substantiated the mRNA data in that increased staining for ANXA2 protein was detected in neo-epidermis which formed more rapidly and completely in body wounds. MMP1 mRNA levels in body wounds significantly surpassed those of limb wounds in week one biopsies. The protein was abundant in migrating epithelium of limb wounds at weeks two and four; conversely, body wounds in which epithelialization was near complete showed diminished staining of MMP1. CONCLUSION: We conclude that ANXA2 and MMP1 might participate in remodeling during wound healing in the horse, and that differences in expression may contribute to the excessive proliferative response seen in the limb.

Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair.

BACKGROUND: Wound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to CTNNB1 (coding for beta-catenin) and PECAM1, genes potentially contributing to the proliferative phase of repair, were previously identified in a mRNA expression study as being up-regulated in 7 day wound biopsies from horses. The aim of the present study was to clone full-length equine CTNNB1 and PECAM1 cDNAs and to study the spatio-temporal expression of mRNAs and corresponding proteins during repair of body and limb wounds in a horse model. RESULTS: The temporal pattern of the two genes was similar; except for CTNNB1 in limb wounds, wounding caused up-regulation of mRNA which did not return to baseline by the end of the study. Relative over-expression of both CTNNB1 and PECAM1 mRNA was noted in body wounds compared to limb wounds. Immunostaining for both beta-catenin and PECAM1 was principally observed in endothelial cells and fibroblasts and was especially pronounced in wounds having developed exuberant granulation tissue. CONCLUSION: This study is the first to characterize equine cDNA for CTNNB1 and PECAM1 and to document that these genes are expressed during wound repair in horses. It appears that beta-catenin may be regulated in a post-transcriptional manner while PECAM1 might help thoracic wounds mount an efficient inflammatory response in contrast to what is observed in limb wounds. Furthermore, data from this study suggest that beta-catenin and PECAM1 might interact to modulate endothelial cell and fibroblast proliferation during wound repair in the horse.