Stimulation of steroid-suppressed cutaneous healing by repeated topical application of IGF-I: different mechanisms of action based upon the mode of IGF-I delivery.

BACKGROUND: Insulin-like growth factor-I (IGF-I) is accepted as a potent stimulus of wound healing when applied in combination with its binding proteins. However, there is only one study published that has investigated the effect of repeated topical application of unbound IGF-I on ischemic wound healing. The aim of this study was to show the effect of daily topical IGF-I therapy on cutaneous ulcer healing in a steroid-suppressed wound model. MATERIALS AND METHODS: Full-thickness wounds were created on the back of 40 male Sprague-Dawley rats. Before surgery, animals received depot-steroids subcutaneously. Wounds were treated daily with either a standard hydrogel dressing (control), topical IGF-I dissolved in 0.2% methylcellulose gel (IGF-I gel), or a hydrogel dressing containing IGF-I (IGF-I dressing). After 7 days of treatment, wounds were excised and measured by photoplanimetry. SMA- and PCNA-expression as well as the formation of granulation tissue were assessed in tissue sections. Results are given as median(min-max). Differences between groups were calculated by the Mann-Whitney U test. RESULTS: Subcutaneous injection of depot-steroids induced a significant delay in healing, as shown by an enlarged wound size [44(33-65) versus 25(20-35)] mm(2); P = 0.001). In steroid-treated rats, both IGF-I gel and IGF-I dressing enhanced excisional healing, as shown by a significant reduction in wound size (P = 0.0001), with IGF-I released from the dressing being even more effective than IGF-I gel (P = 0.03). However, in these animals only IGF-I released from the hydrogel dressing stimulated SMA- (P = 0.03) as well as PCNA-expression (P = 0.001) and increased granulation tissue formation (P = 0.018). CONCLUSIONS: Our data indicate that a repeated application of topical IGF-I enhances cutaneous ulcer healing. In addition, only the controlled release of IGF-I from the hydrogel dressing is capable of reversing the steroid-induced delay of healing, suggesting different mechanisms of action with respect to the mode of IGF-I delivery.

Lactate stimulates endothelial cell migration.

The significance of the high lactate levels that characterize healing wounds is not fully understood. Lactate has been shown to enhance collagen synthesis by fibroblasts and vascular endothelial growth factor (VEGF) production by macrophages and endothelial cells. VEGF has been shown to induce endothelial cell migration. However, it has not been shown whether accumulated lactate correlates with the biological activity of VEGF. Therefore, we investigated the effect of lactate on migration of endothelial cells. Human umbilical vein endothelial cells and human microvascular endothelial cells were cultured to subconfluent monolayers in standard six-well tissue culture plates. Following a 24-hour serum starvation, cells were treated with the indicated concentrations of l-lactate. Cell migration was assessed using a modified Boyden chamber. VEGF protein in the cell culture supernatant was measured by enzyme-linked immunoassay. Lactate-enhanced VEGF protein synthesis in a time- and dose-dependent manner. Lactate added into the bottom well did not stimulate cellular migration from the upper well. However, lactate when added together with endothelial cells to the bottom well of the Boyden chamber increased cellular migration in a dose-dependent manner. This effect was blocked by anti-VEGF and by cycloheximide. Lactate enhances VEGF production in endothelial cells, although lactate, itself, is not a chemoattractant. We conclude that the lactate-mediated increase in cellular migration is regulated by VEGF.

IGF-I-induced VEGF expression in HUVEC involves phosphorylation and inhibition of poly(ADP-ribose)polymerase.

Insulin-like growth factor-I (IGF-I) has been shown to promote angiogenesis by enhancing vascular endothelial growth factor (VEGF) expression. However, how IGF-I-induces VEGF expression is not yet fully understood. With this investigation, we propose a new possible mechanism involving downregulation of poly(ADP-ribosyl)ation (pADPR). We first demonstrated that IGF-I increased VEGF protein expression in endothelial cells. Inhibitors of mitogen activated kinase (PD 98059), phosphatidyl-3-inositol-kinase (LY 294002), and protein kinase C (staurosporine) diminished the IGF-I effect suggesting the involvement of signal transduction. Since there is an established link between pADPR and transcriptional activity, we focused on a possible role of poly(ADP-ribose)polymerase (PARP). The inhibition of PARP by 3-aminobenzamide or nicotinamide enhanced VEGF expression. Additionally, IGF-I markedly decreased PARP activity. Furthermore, the IGF-I-mediated inhibition of PARP could be demonstrated as a result of protein phosphorylation since phosphorylation of PARP decreased its activity in vitro and IGF-I treatment of endothelial cells induced PARP phosphorylation. The IGF-I-mediated phosphorylation and inhibition of PARP represent a novel mechanism of VEGF protein expression.

TGF-beta3 inhibits pentagastrin-stimulated gastric acid secretion in rats.

BACKGROUND: Transforming growth factor beta3 (TGF-beta3) has been shown to accelerate gastric ulcer healing in rats. However, little is known about the mechanism. In this study we investigated the influence of TGF-beta3 on gastric acid secretion, since gastric hyperacidity is a major cause of gastroduodenal ulcer disease. MATERIAL/METHODS: Male Sprague Dawley rats were equipped with gastric Thomas cannulas and jugular vein catheters. The acute effect of either intravenous TGF-beta3 (400 and 1200 pg/kg/h) or saline (0.15 M) on pentagastrin-stimulated (10 pg/kg/h) gastric acid secretion was evaluated by gastric acid back-titration after 5 days of recovery. Additionally, pentagastrin-stimulated gastric acid secretion was assessed after 48 hours following TGF-beta3 (1200 microg/kg/h) or saline treatment. RESULTS: Pentagastrin significantly increased gastric acid production. TGF-beta3 significantly reduced pentagastrin-stimulated gastric acid secretion in a dose-dependent manner as early as 15 minutes after application (saline: 124.9+/-14.9 microEq H+/15 min, TGF-beta3: 97.7+/-13.1 9 microEq H+/15 min, p<0.002). Additionally, pretreatment with TGF-beta3 abolished the effect of pentagastrin on gastric acid production. This effect lasted throughout the entire recording period of 48 hours. However, baseline physiological gastric acid production was not altered by TGF-beta3. CONCLUSIONS: TGF-beta3 inhibits gastric acid secretion when given prior to as well as after pentagastrin treatment. This implicates both a preventive and a therapeutic role of TGF-beta3 in gastroduodenal ulcer disease.

Growth hormone enhances gastric ulcer healing in rats.

BACKGROUND: Gastric ulcer healing requires the reconstitution of epithelial structures and underlying connective tissue through cellular proliferation, migration, and differentiation. The systemic application of growth hormone (GH) has shown anabolic effects in postoperative and burn therapy by increasing protein synthesis and attenuating protein catabolism. There is also evidence that GH stimulates cell proliferation and differentiation. In this study we evaluated the impact of GH on gastric ulcer healing. MATERIAL/METHODS: Gastric ulcers were induced with a cryoprobe in male Wistar rats (285+/-11 g). The first group of rodents (n=10) received a daily subcutaneous dose of 2.5 mg/kg growth hormone for seven days. The second group (n=10) received only vehicle. After 7 days, ulcer size was determined photoplanimetrically. Cell proliferation and new vessel growth in the ulcer margin were evaluated by quantitative immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and smooth muscle actin (SMA), respectively. RESULTS: The systemic application of GH caused a significant increase in body weight (332+/-7 g vs. 289+/-13 g; p=0.007). Ulcer size was also reduced significantly compared with controls (5.6+/-0.8 mm2 vs. 9.9+/-1 mm2; p=0.005). Immunohistochemical analysis revealed a significant increase in cell proliferation (79.7+/-0.9% pos. cells vs. 64.7+/-1.9% pos. cells; p=0.0001) as shown by PCNA expression, and a significant increase in new vessel growth as demonstrated by SMA expression (1762+/-124 cells/mm2 vs. 1067+/-77 cells/mm2; p=0.0001). CONCLUSIONS: Growth hormone accelerates gastric ulcer healing by stimulating cell proliferation and angiogenesis.