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.

mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice.

The mammalian target of rapamycin (Mtor) gene encodes a serine/threonine kinase that acts as a master regulator of processes as diverse as cell growth, protein synthesis, cytoskeleton reorganization, and cell survival. In the testis, physiological roles for Mtor have been proposed in perinatal Sertoli cell proliferation and blood-testis barrier (BTB) remodeling during spermatogenesis, but no in vivo studies of Mtor function have been reported. Here, we used a conditional knockout approach to target Mtor in Sertoli cells. The resulting Mtor(flox/flox); Amhr2(cre/+) mice were characterized by progressive, adult-onset testicular atrophy associated with disorganization of the seminiferous epithelium, loss of Sertoli cell polarity, increased germ cell apoptosis, premature release of germ cells, decreased epididymal sperm counts, increased sperm abnormalities, and infertility. Histopathologic analysis and quantification of the expression of stage-specific markers showed a specific loss of pachytene spermatocytes and spermatids. Although the BTB and the ectoplasmic specializations did not appear to be altered in Mtor(flox/flox);Amhr2(cre/+) mice, a dramatic redistribution of gap junction alpha-1 (GJA1) was detected in their Sertoli cells. Phosphorylation of GJA1 at Ser373, which is associated with its internalization, was increased in the testes of Mtor(flox/flox); Amhr2(cre/+) mice, as was the expression and phosphorylation of AKT, which phosphorylates GJA1 at this site. Together, these results indicate that Mtor expression in Sertoli cells is required for the maintenance of spermatogenesis and the progression of germ cell development through the pachytene spermatocyte stage. One mechanism of mTOR action may be to regulate gap junction dynamics by inhibiting AKT, thereby decreasing GJA1 phosphorylation and internalization. mTOR regulates gap junction alpha-1 protein distribution in Sertoli cells and is necessary for progression through the pachytene spermatocyte stage.

ß-catenin stabilization in gonadotropes impairs FSH synthesis in male mice in vivo.

Although classically considered a WNT signaling intermediary, ß-catenin (CTNNB1) can also mediate GnRH induction of gonadotropin ß-subunit (Fshb and Lhb) transcription in the murine gonadotrope-like cell line LßT2. Here, we assessed CTNNB1's role in gonadotropin synthesis in vivo. We used a Cre/lox approach to introduce both gain- and loss-of-function mutations in the murine Ctnnb1 gene in gonadotrope cells. Gonadotropin production and fertility were normal in Ctnnb1 knockout mice. Similarly, females harboring a deletion of exon 3 of Ctnnb1, which stabilizes the resulting CTNNB1 protein, showed normal fertility and gonadotropin synthesis. Interestingly, males with the activating CTNNB1-Δexon 3 mutation exhibited 50% reductions in FSH synthesis and secretion, without a corresponding change in LH. This selective regulation of FSH suggested an alteration in the activin/inhibin/follistatin system. Indeed, CTNNB1-Δexon 3 males showed a 60% increase in serum inhibin B levels, and in culture, their pituitaries exhibited a greater sensitivity to exogenous inhibin than controls. At the same time, pituitary, but not testicular, follistatin (Fst) expression was increased significantly in these mice. Castration normalized FSH levels in CTNNB1-Δexon 3 males to those seen in castrated controls. Paradoxically, pituitaries from CTNNB1-Δexon 3 males exhibited greater basal and activin-stimulated FSH synthesis in vitro. Similarly, CTNNB1-Δexon 3 overexpression potentiated activin A-induced murine Fshb promoter activity in LßT2 cells. Together, these results indicate that CTNNB1 is dispensable for gonadotropin synthesis in vivo. However, sustained CTNNB1 signaling potentiates activin-induced Fshb expression in gonadotropes, but this effect is overcome in vivo by enhanced inhibin feedback sensitivity and Fst expression.

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.

Palatal sclerotherapy for the treatment of intermittent dorsal displacement of the soft palate in 51 standardbred racehorses.

This retrospective study evaluated the efficacy and side effects of palatal sclerotherapy in standardbred racehorses suspected to have intermittent dorsal displacement of the soft palate (IDDSP). Fifty-one horses were treated with multiple endoscopically guided injections of 3% sodium tetradecyl sulfate in the soft palate. Two groups were identified: those that had respiratory noises during exercise (n = 27) and those that did not (n = 24). Treatment was well-tolerated. Furthermore, horses significantly reduced their racing times for the last 400 m compared with their times before treatment and even when their times were compared to the mean times for horses in the same race. In conclusion, palatal sclerotherapy appears to be a suitable alternative therapeutic option for horses suspected to have IDDSP.

Electrosurgical tenoscopic desmotomy of the accessory ligament of the superficial digital flexor muscle (proximal check ligament) in horses.

OBJECTIVE: To report a tenoscopic technique using monopolar electrosurgery to transect the accessory ligament of superficial digital flexor muscle (AL-SDFM) and outcome in 33 horses. STUDY DESIGN: Case series. ANIMALS: Horses (n=33). METHODS: Medical files and surgery video recordings of horses that had AL-SDFM desmotomy performed by tenoscopy with monopolar electrosurgical electrodes were reviewed. RESULTS: Of 33 horses, 24 were Standardbred racehorses with surgery performed bilaterally for superficial digital flexor tendonitis and 9 horses had flexural deformity. Severe (n=6) and mild (6) intrathecal hemorrhage was the most common intraoperative complication. Large intrathecal vessels including the nutrient artery were successfully electrocoagulated and AL-SDFM transection was completed. Clear/serosanguinous drainage from skin incisions was observed for 4.3±3.3 days (mean, SD). Protracted wound drainage for >4 days occurred in 10 horses, principally in the group treated for flexural deformities (P=.01). CONCLUSIONS: Sixty-four AL-SDFM were transected under tenoscopic observation using monopolar electrodes. Electrocoagulation of large intrathecal vessels, including the nutrient artery, was possible in all cases and allowed completion of desmotomy. Postoperative wound care was similar to routine tenoscopy in most (70%) horses. Aseptic protracted wound drainage was observed in 30% of horses (principally those with flexural deformity), and led to a prolonged hospitalization.

Palatal sclerotherapy: a potentially useful treatment of intermittent dorsal displacement of the soft palate in juvenile standardbred racehorses.

This study was aimed at evaluating the tolerability and the efficacy of palatal sclerotherapy in juvenile standardbred racehorses with easily audible "snoring-like" respiratory noises suspected to be the result of intermittent dorsal displacement of the soft palate. The palate of 8 horses was injected with sodium tetradecyl sulfate under videoendoscopic guidance. Palatal sclerotherapy resulted in resolution of the respiratory noise in 7 horses, improvement of performance in 6 horses, and mild side effects in only 3 horses. This preliminary study suggests that palatal sclerotherapy is a safe, repeatable, inexpensive, and promising technique that should be considered as an alternative to existing treatments of intermittent dorsal displacement of the soft palate.

Effects of continuous oral administration of phenylbutazone on biomarkers of cartilage and bone metabolism in horses.

OBJECTIVE: To evaluate the effects of continuous oral administration of phenylbutazone on serum and synovial fluid biomarkers of skeletal matrix metabolism in horses. ANIMALS: 11 adult female horses without clinical or radiographic evidence of joint disease. PROCEDURES: Horses were randomly assigned to control or treatment groups. Phenylbutazone was administered orally twice daily at a dose of 4.4 mg/kg for 3 days to the treatment group and subsequently at a dose of 2.2 mg/kg for 7 days. Serum and radiocarpal synovial fluid samples were obtained at baseline and thereafter at regular intervals for 4 weeks. Biomarkers of cartilage aggrecan synthesis (chondroitin sulfate 846) and type II collagen synthesis (procollagen type II C-propeptide) and degradation (collagen type II cleavage) were assayed. Biomarkers of bone synthesis (osteocalcin) and resorption (C-terminal telopeptide of type I collagen) were also measured. RESULTS: No significant differences were found between control and treatment groups or temporally for the biomarkers chondroitin sulfate 846, procollagen type II C-propeptide, collagen type II cleavage, and C-terminal telopeptide of type I collagen in serum or synovial fluid. A significant increase in osteocalcin concentration occurred in synovial fluid during treatment in the treated group. No treatment effect was detected for serum osteocalcin concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that continuous phenylbutazone administration at recommended doses altered some biomarkers in healthy equine joints after short periods of administration. Increased osteocalcin concentration may indicate an undetermined anabolic effect of phenylbutazone administration on periarticular bone or transient induction of osteogenesis in articular chondrocytes or a mesenchymal subpopulation of synoviocytes.

Comparative study on microvascular occlusion and apoptosis in body and limb wounds in the horse.

Wound repair in horse limbs is often complicated by exuberant granulation tissue, a condition characterized by excessive fibroplasia and scarring and that resembles hypertrophic scars and keloids in man. The aim of this study was to compare microvascular occlusion and apoptosis in wounds of the limb with those of the body, which heal normally. Five 6.25 cm(2) wounds were created on both forelimbs and on the body of six horses. One limb was bandaged to stimulate excessive fibroplasia. Weekly biopsies were evaluated histologically and immunohistochemically for mutant p53 protein by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling to localize and quantify apoptosis, and by electron microscopy to measure microvessel luminal diameters. Histologic examination revealed protracted inflammation as well as slowed epithelialization and deficient fibroblast orientation in limb wounds, particularly those with excessive fibroplasia. Microvessels were occluded significantly more often in limb wounds, and the balance of apoptotic signals was altered against apoptosis in the former, although this could not be confirmed quantitatively. Data suggest that microvascular occlusion and a dysregulated apoptotic process may be involved in the excessive accumulation of extracellular matrix within limb wounds. This might provide a basis for the development of targeted therapies to prevent and treat excessive fibroplasia and extensive scarring in horses.

Effect of a silicone-containing dressing on exuberant granulation tissue formation and wound repair in horses.

OBJECTIVE: To determine the effect of a silicone dressing on the rate and quality of repair of limb wounds and compare microvascular occlusion and apoptosis in wounds treated with the silicone dressing and those treated with a conventional dressing in horses. ANIMALS: 5 horses. PROCEDURE: Horses received two 6.25-cm2 wounds on each metacarpus. Ten wounds were treated with a silicone dressing; the other 10 were treated with a control dressing. Quality of repair and wound size were evaluated at each bandage change. Time to healing and the number of excisions of exuberant granulation tissue were recorded. Biopsy specimens taken from healed wounds were evaluated semiquantitatively via histologic examination, p53 immunohistochemical analysis, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) to quantify apoptosis, and electron microscopic examination to measure microvessel luminal diameters. RESULTS: The silicone dressing surpassed the conventional dressing in preventing formation of exuberant granulation tissue and improving tissue quality. Microvessels were occluded significantly more often in wounds dressed with the silicone gel, which also diminished the expression of mutant p53, an indirect inhibitor of apoptosis, although greater apoptosis was not confirmed quantitatively by use of TUNEL. CONCLUSIONS AND CLINICAL RELEVANCE: Because the silicone dressing inhibited the formation of exuberant granulation tissue, it may be integrated in a management strategy designed to improve the repair of limb wounds in horses.

Repeated intraarticular injections of triamcinolone acetonide alter cartilage matrix metabolism measured by biomarkers in synovial fluid.

Although intraarticular (IA) corticosteroids are frequently used to treat joint disease, the effects of their repeated use on articular cartilage remains controversial. The aim of our study was to determine the effects of a clinically recommended dose of IA triamcinolone acetonide (TA), on synovial fluid (SF) biomarkers of cartilage metabolism. Ten adult horses, free of osteoarthritis (OA) in their radiocarpal joints, were studied. One radiocarpal joint of each horse was randomly chosen for treatment and the contralateral anatomically paired joint acted as the control. Aseptic arthrocentesis was performed weekly on both joints for 13 weeks. The initial results from the first 3 weeks of the experimental period established baseline untreated control marker levels for each joint, each being its own control. On weeks 3, 5, and 7, a sterile suspension of 12 mg of TA was injected into the treated joint and an equivalent volume of sterile saline solution (0.9%) was injected into the control joint. SF was immunoassayed for biomarkers of aggrecan turnover (CS 846 & KS), types I and II collagen cleavage (C1,2C) and type II collagen synthesis (CPII). In treated joints, there was a significant increase in CS 846, KS, C1,2C and CPII epitope concentrations following IA TA injections when compared to baseline levels. There was also a significant increase in C1,2C and CPII epitope concentrations in the contralateral control joints following IA TA injections in the treated joint. Significant differences were observed between treated and control joints for all markers except CPII. These findings indicate that TA alters articular cartilage and collagen metabolism in treated and, interestingly, also in control joints, suggesting a systemic effect of the drug. Though intuitively the observed findings would favor the hypothesis that long-term IA TA treatment changes joint metabolism and this may have detrimental effects; further studies would be necessary to confirm this.

Management of equine hoof injuries.

Hoof injuries are common in horses. Some wounds can pose diagnostic and treatment challenges because of the rigid nature of horn,the tissue involved, the deeper underlying structures eventually invaded, or the pattern of healing. By combining knowledge about the anatomy and biomechanical properties of the foot and healing characteristics of the hoof with adapted general principles of wound management, satisfactory clinical outcomes usually result.

Synovial fluid levels and serum pharmacokinetics in a large animal model following treatment with oral glucosamine at clinically relevant doses.

OBJECTIVE: To examine the concentration of glucosamine in the synovial fluid and its pharmacokinetics in serum in a large animal model following dosing with glucosamine HCl at clinically relevant levels. METHODS: Eight adult female horses were studied. After an overnight fast, glucosamine HCl (20 mg/kg of body weight) was administered by either nasogastric (NG) intubation or intravenous (IV) injection. Blood samples were collected before dosing and at 5, 15, 30, 60, 120, 180, 240, 360, 480, and 720 minutes after dosing. Synovial fluid samples were collected from the radiocarpal joints 48 hours before dosing and at 1 and 12 hours after dosing. Glucosamine was assayed by fluorophore-assisted carbohydrate electrophoresis. RESULTS: The maximum concentration of glucosamine in serum reached approximately 300 muM ( approximately 50 microg/ml) following IV dosing and approximately 6 microM (approximately 1 microg/ml) following NG dosing. Synovial fluid concentrations reached 9-15 microM with IV dosing and 0.3-0.7 microM with NG dosing, and remained elevated (range 0.1-0.7 microM) in most animals even at 12 hours after dosing. Following NG dosing, the median serum maximal concentration of 6.1 microM (range 4.38-7.58) was attained between 30 minutes and 4 hours postdose. The mean apparent volume of distribution was 15.4 liters/kg, the mean bioavailability was 5.9%, and the mean elimination half-life was 2.82 hours. CONCLUSION: Clinically relevant dosing of glucosamine HCl in this large monogastric animal model results in serum and synovial fluid concentrations that are at least 500-fold lower than those reported to modify chondrocyte anabolic and catabolic activities in tissue and cell culture experiments. We conclude that the apparent therapeutic benefit of dietary glucosamine on pain and joint space width in humans and animals may be secondary to its effects on nonarticular tissues, such as the intestinal lining, liver, or kidney, since these may be exposed to much high levels of glucosamine following ingestion.