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.

Development of a computer-based quantification method for immunohistochemically-stained tissues and its application to study mast cells in equine wound healing (proof of concept).

BACKGROUND: There is a growing interest in the scientific community to use computer-based software programs for the quantification of cells during physiological and pathophysiological processes. Drawbacks of computer-based methods currently used to quantify immunohistochemical staining are the complexity of use, expense of software and overly-simplified descriptions of protocol thereby limiting reproducibility. The precise role of mast cells in equine cutaneous wound healing is unknown. Given the contribution of mast cells to the chronic inflammation observed in human keloid, a pathology similar to exuberant granulation tissue (EGT) in horses, mast cells might be present in high numbers in equine limb wounds predisposed to EGT. The main goal of this study was to develop a reliable and reproducible quantification method for immunostained tissues using a computer software that is widely available, at no cost, to the scientific community. A secondary goal was to conduct a proof of concept using the newly-established method to quantify mast cells during wound healing at different anatomical sites (body and limb) in horses to see if a different pattern is observed in limb wounds, which are predisposed to EGT. RESULTS: A good intraclass correlation coefficient (ICC, 0.67 p < 0.05) was found between the computer-based ImageJ method and manual counting. An excellent intra-operator ICC of 0.90 (p < 0.01) was found for the ImageJ quantification method while a good interoperator ICC of 0.69 (p < 0.01) was measured. No significant difference was observed between the variation of the ImageJ and that of the manual counting method. Mast cells were localized below the epidermis, around cutaneous appendages and blood vessels. Mast cell numbers did not differ significantly in relation to anatomical location or time of healing. CONCLUSIONS: The computer-based quantification method developed is reliable, reproducible, available, cost-free and could be used to study different physiological and pathological processes using immunohistochemistry.

Feasibility of a disposable canister-free negative-pressure wound therapy (NPWT) device for treating open wounds in horses.

BACKGROUND: Wounds are among the most common medical conditions affecting horses and have a major economic impact on the horse industry. Wound healing in horses is distinct to that documented in other species, and often results in delayed healing and extensive scarring, with compromised functional and aesthetic outcomes. To date, there is no conventional method objectively proven to accelerate healing or to successfully prevent complications associated with second intention healing. Several effects of Negative Pressure Wound Therapy (NPWT) may be particularly useful to the management of wounds in horses. However, cumbersome designs of classic NPWT devices render them unsuitable for equine practice. A new lightweight, portable and disposable unit of NPWT (PICO®), should facilitate the use of this modality by equine practitioners. The aim of this study was to evaluate the feasibility of using this canister-free system to treat experimental open wounds in horses. RESULTS: No difficulties were encountered with the application or maintenance of the PICO® system during the ex vivo experiment or during the preliminary in vivo experiment conducted on intact skin. All horses readily tolerated the PICO® but difficulties with adhesion and seal prevented the completion of the experimental wound study despite the use of many adjunctive adhesives. CONCLUSION: The current PICO® dressing design is not suitable to be used as a dressing for open wounds in horses though the device is well tolerated by equine patients. A dressing with a wider adhesive edge, a superior adhesive and a more flexible pad would likely be better adapted to enable its future use in equine practice.

Use of next generation sequencing to investigate the microbiota of experimentally induced wounds and the effect of bandaging in horses.

OBJECTIVES: To use next generation sequencing to characterize the microbiota of horses during healing of skin wounds in two anatomical locations (body and limb) known to present different healing patterns; and to investigate the impact of bandaging on bacterial communities of skin wounds located on the limbs of horses. METHODS: Full-thickness skin wounds were created on the distal extremity of both thoracic limbs and on one lateral mid-thoracic wall of four healthy horses. Limb wounds were randomly assigned to bandaging or not. A full-thickness sample was collected with a biopsy punch from intact thorax and limb skin (T0) and from the margin of one wound per site (thorax, unbandaged limb, bandaged limb) 1 week (T1) and 2 weeks (T2) postoperatively, and at full healing (T3). Thoracic skin samples obtained from three healthy horses were included in the analysis as controls. RESULTS: Anatomic location (thorax vs. limb) significantly influenced bacterial composition of equine skin and healing wounds. Fusobacterium and Actinobacillus were strongly associated with limb wounds during the initial phases of healing. Bandaging had a significant impact on the microbiota during the healing process. The skin microbiota after healing was more similar to samples from controls, demonstrating the resilience and stability of the environment. CONCLUSIONS: Equine skin microbiota is a rich and stable environment that is disturbed by wounding, but returns to its previous stage after full healing. Anatomic location significantly influences bacterial composition of the equine skin during wound healing. Bandaging has a significant impact on the skin microbiota of horses during the healing process. Results of this study provide new insight for a better understanding of the contribution of bacteria to wound healing in horses and may facilitate the future development of therapeutic strategies using commensal bacteria.

Choosing the Best Approach to Wound Management and Closure.

This article aims to help the practitioner by providing the tools to decide which type of closure or healing is best in a given situation. An overview of the main criteria and the different approaches to wound closure is presented. Each wound must be considered as a unique problem that requires a clinician to take into account all of its characteristics and limits to determine the best management approach.

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.

A hypoxia response element in the Vegfa promoter is required for basal Vegfa expression in skin and for optimal granulation tissue formation during wound healing in mice.

Hypoxia in skin wounds is thought to contribute to healing through the induction of hypoxia inducible factor-1 (HIF-1). Although HIF-1 can regulate the expression of vascular endothelial growth factor A (Vegfa), whether hypoxia and HIF-1 are required to induce Vegfa expression in the context of wound healing is unknown. To test this hypothesis, we evaluated Vegfa expression and wound healing in mutant mice that lack a functional HIF-1 binding site in the Vegfa promoter. Full-thickness excisional wounds were made using a biopsy punch, left to heal by second intention, and granulation tissue isolated on a time course during healing. mRNA levels of Vegfa and its target genes platelet-derived growth factors B (Pdgfb) and stromal cell-derived factor-1 (Sdf1) were measured by RT-qPCR, and HIF-1alpha and VEGFA protein levels measured by immunoblotting. Lower levels of Vegfa, Pdgf1 and Sdf1 mRNA were found in intact skin of mutant mice relative to wild-type controls (n = 6 mice/genotype), whereas levels in granulation tissue during wound healing were unaltered. VEGFA protein levels were also lower in intact skin of the mutant versus the wild-type mice. Decreased Vegfa mRNA levels in skin of mutant mice could not be attributed to decreased HIF-1alpha protein expression, and were therefore a consequence of the loss of HIF-1 responsiveness of the Vegfa promoter. Comparative histologic analyses of healing wounds in mutant and wild-type mice (n = 8 mice/genotype) revealed significant defects in granulation tissue in the mutant mice, both in terms of quantity and capillary density, although epithelialization and healing rates were unaltered. We conclude that HIF-1 is not a major regulator of Vegfa expression during wound healing; rather, it serves to maintain basal levels of expression of Vegfa and its target genes in intact skin, which are required for optimal granulation tissue formation in response to wounding.

Short-term treatment of equine wounds with orf virus IL-10 and VEGF-E dampens inflammation and promotes repair processes without accelerating closure.

Healing is delayed in limb wounds relative to body wounds of horses, partly because of sustained inflammation and inefficient angiogenesis. In laboratory animals, proteins derived from orf virus modulate these processes and enhance healing. We aimed to compare immune cell trafficking and the inflammatory, vascular, and epidermal responses in body and limb wounds of horses and then to investigate the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on these processes. Standardized excisional wounds were created on the body and forelimb of horses and their progression monitored macroscopically until healed. Tissue samples were harvested to measure the expression of genes regulating inflammation and repair (quantitative polymerase chain reaction) and to observe epithelialization (histology), innate immune cell infiltration, and angiogenesis (immunofluorescence). Delayed healing of limb wounds was characterized by intensified and extended pro-inflammatory signaling and exacerbated innate immune response, concomitant with the absence of anti-inflammatory eIL-10. Blood vessels were initially more permeable and then matured belatedly, concomitant with retarded production of angiogenic factors. Epithelial coverage was achieved belatedly in limb wounds. Viral proteins were administered to wounds of one body and one limb site/horse at days 1-3, while wounds at matching sites served as controls. Treatment dampened pro-inflammatory gene expression and the innate immune response in all wounds. It also improved angiogenic gene expression, but primarily in body wounds, where it altered blood vessel density and myofibroblast persistence. Moreover, the viral proteins increased epithelialization of all wounds. The short-term viral protein therapy did not, however, improve the healing rate of wounds in either location, likely due to suboptimal dosing. In conclusion, we have further detailed the processes contributing to protracted healing in limb wounds of horses and shown that short-term administration of viral proteins exerts several promising though transient effects that, if optimized, may positively influence healing.

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.

Heart rate variability in relation to stress in the Asian elephant (Elephas maximus).

This study describes a safe, reliable, and accessible means to measure heart rate (HR) and HR variability (HRV) and evaluates the use of HRV as a physiological correlate of stress in the Asian elephant. A probabilistic model indicates that HRV measurements may adequately distinguish between stressed and non-stressed elephants.

Variabilité de la fréquence cardiaque en relation avec le stress chez un éléphant d'Asie(Elephas maximus). Cette étude décrit un moyen sûr, fiable et accessible de mesurer la fréquence cardiaque (FC) et la variabilité de la FC (VFC) et évalue l'utilisation de la VFC comme corrélat physiologique du stress chez l'éléphant d'Asie. Un modèle probabiliste indique que les mesures de la VFC peuvent adéquatement distinguer entre des éléphants stressés et non stressés.(Traduit par Isabelle Vallières).

Validation of a novel technique for creating simulated radiographs using computed tomography datasets.

Understanding radiographic anatomy and the effects of varying patient and radiographic tube positioning on image quality can be a challenge for students. The purposes of this study were to develop and validate a novel technique for creating simulated radiographs using computed tomography (CT) datasets. A DICOM viewer (ORS Visual) plug-in was developed with the ability to move and deform cuboidal volumetric CT datasets, and to produce images simulating the effects of tube-patient-detector distance and angulation. Computed tomographic datasets were acquired from two dogs, one cat, and one horse. Simulated radiographs of different body parts (n = 9) were produced using different angles to mimic conventional projections, before actual digital radiographs were obtained using the same projections. These studies (n = 18) were then submitted to 10 board-certified radiologists who were asked to score visualization of anatomical landmarks, depiction of patient positioning, realism of distortion/magnification, and image quality. No significant differences between simulated and actual radiographs were found for anatomic structure visualization and patient positioning in the majority of body parts. For the assessment of radiographic realism, no significant differences were found between simulated and digital radiographs for canine pelvis, equine tarsus, and feline abdomen body parts. Overall, image quality and contrast resolution of simulated radiographs were considered satisfactory. Findings from the current study indicated that radiographs simulated using this new technique are comparable to actual digital radiographs. Further studies are needed to apply this technique in developing interactive tools for teaching radiographic anatomy and the effects of varying patient and tube positioning.

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.

Translating stem cell therapies: the role of companion animals in regenerative medicine.

Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell-based therapies involving hematopoietic stem cells and mesenchymal stem cells, as well as other adult regenerative cells and recently embryonic stem cells and induced pluripotent stem cells. In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the "One Health Initiative," the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored.

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.