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.

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.

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.

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.

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.