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

<p>Abstract</p> <p>Background</p> <p>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 <it>CTNNB1 </it>(coding for β-catenin) and <it>PECAM1</it>, 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 <it>CTNNB1 </it>and <it>PECAM1 </it>cDNAs and to study the spatio-temporal expression of mRNAs and corresponding proteins during repair of body and limb wounds in a horse model.</p> <p>Results</p> <p>The temporal pattern of the two genes was similar; except for <it>CTNNB1 </it>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 <it>CTNNB1 </it>and <it>PECAM1 </it>mRNA was noted in body wounds compared to limb wounds. Immunostaining for both β-catenin and PECAM1 was principally observed in endothelial cells and fibroblasts and was especially pronounced in wounds having developed exuberant granulation tissue.</p> <p>Conclusion</p> <p>This study is the first to characterize equine cDNA for <it>CTNNB1 </it>and <it>PECAM1 </it>and to document that these genes are expressed during wound repair in horses. It appears that β-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 β-catenin and PECAM1 might interact to modulate endothelial cell and fibroblast proliferation during wound repair in the horse.</p

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 cutaneouswounds 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 datawere collected from all plannedwound 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 EGTChristophe J. Celeste, Karine Deschesne, Christopher B. Riley, and Christine L. Theore

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

<div><p>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.</p></div

Inflammation in limb wounds of horses following treatment with viral proteins.

<p>Quantitative PCR was used to measure the expression of (A) equine (e)<i>MCP-1</i>, (B) <i>eIL-6</i>, (C) <i>eIL-10</i>, and (D) <i>eIL-10Rα</i> in wound margin samples taken 2 days after wounding. Expression of mRNA is relative to that of GAPDH and to levels measured in unwounded skin (day 0). (E) Representative photos of skin sections taken 7 days post-wounding stained with DAPI (blue) and an antibody against the inflammatory cell marker calprotectin (MAC387: green). Enlarged images show nucleated inflammatory cells. (F) Number of calprotectin^+ve cells in the granulation tissue and surrounding skin of wounds following administration of vehicle or viral proteins. Values represent mean ± SEM, n = 4. <i>P</i> values indicated were determined using a two-tailed ratio paired <i>t</i>-test.</p