The role of androgens on hypoxia-inducible factor (HIF)-1α-induced angiogenesis and on the survival of ischemically challenged skin flaps in a rat model

Effects of androgens on angiogenesis are controversial. Hypoxia-inducible factor (HIF)-1α promotes expression of vascular endothelial growth factor (VEGF) that stimulates angiogenesis.

Effects of local continuous release of brain derived neurotrophic factor (BDNF) on peripheral nerve regeneration in a rat model

The purpose of this study was to evaluate the effect of continuously released BDNF on peripheral nerve regeneration in a rat model. Initial in vitro evaluation of calcium alginate prolonged-release-capsules (PRC) proved a consistent release of BDNF for a minimum of 8 weeks. In vivo, a worst case scenario was created by surgical removal of a 20-mm section of the sciatic nerve of the rat. Twenty-four autologous fascia tubes were filled with calcium alginate spheres and sutured to the epineurium of both nerve ends. The animals were divided into 3 groups. In group 1, the fascial tube contained plain calcium alginate spheres. In groups 2 and 3, the fascial tube contained calcium alginate spheres with BDNF alone or BDNF stabilized with bovine serum albumin, respectively. The autocannibalization of the operated extremity was clinically assessed and documented in 12 additional rats. The regeneration was evaluated histologically at 4 weeks and 10 weeks in a blinded manner. The length of nerve fibers and the numbers of axons formed in the tube was measured. Over a 10-week period, axons have grown significantly faster in groups 2 and 3 with continuously released BDNF compared to the control. The rats treated with BDNF (groups 2 and 3) demonstrated significantly less autocannibalization than the control group (group 1). These results suggest that BDNF may not only stimulate faster peripheral nerve regeneration provided there is an ideal, biodegradable continuous delivery system but that it significantly reduces the neuropathic pain in the rat model.

Effects of Delta12-prostaglandin J2 on bone regeneration and growth factor expression in rats

OBJECTIVES: Cyclopentenone prostaglandins have been shown to promote osteoblast differentiation in vitro. The aim of this study was to examine in a rat model the effects of local delivery of Delta(12)-prostaglandin J(2) (Delta(12)-PGJ(2)) on new bone formation and growth factor expression in (i) cortical defects and (ii) around titanium implants. MATERIAL AND METHODS: Standardized transcortical defects were prepared bilaterally in the femur of 28 male Wistar rats. Ten microliters of Delta(12)-PGJ(2) at 4 concentrations (10(-9), 10(-7), 10(-5) and 10(-3) mol/l) in a collagen vehicle were delivered inside a half-cylindrical titanium chamber fixed over the defect. Contralateral defects served as vehicle controls. Ten days after surgery, the amount of new bone formation in the cortical defect area was determined by histomorphometry and expression of platelet-derived growth factor (PDGF)-A and -B, insulin-like growth factor (IGF)-I/II, bone morphogenetic protein (BMP)-2 and -6 was examined by immunohistochemistry. In an additional six rats, 24 titanium implants were inserted into the femur. Five microliters of carboxymethylcellulose alone (control) or with Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) were delivered into surgically prepared beds prior to implant installation. RESULTS: Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) significantly enhanced new bone formation (33%, P<0.05) compared with control cortical defects. Delivery of Delta(12)-PGJ(2) at 10(-3) mol/l significantly increased PDGF-A and -B and BMP-2 and -6 protein expression (P<0.05) compared with control defects. No significant difference was found in IGF-I/II expression compared with controls. Administration of Delta(12)-PGJ(2) also significantly increased endosteal new bone formation around implants compared with controls. CONCLUSION: Local delivery of Delta(12)-PGJ(2) promoted new bone formation in the cortical defect area and around titanium implants. Enhanced expression of BMP-2 and -6 as well as PDGF-A and -B may be involved in Delta(12)-PGJ(2)-induced new bone formation.

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Granulocyte colony-stimulating factor supports liver regeneration in a small-for-size liver remnant mouse model

Experimental partial hepatectomy of more than 80% of the liver weight bears an increased mortality in rodents, due to impaired hepatic regeneration in small-for-size liver remnants. Granulocyte colony-stimulating factor (G-CSF) promotes progenitor cell expansion and mobilization and also has immunomodulatory properties. The aim of this study was to determine the effect of systemically administered G-CSF on liver regeneration and animal survival in a small-for-size liver remnant mouse model. Mice were preconditioned daily for 5 days with subcutaneous injections of 5 microg G-CSF or aqua ad injectabile. Subsequently, 83% partial hepatectomy was performed by resecting the median, the left, the caudate, and the right inferior hepatic lobes in all animals. Daily sham or G-CSF injection was continued. Survival was significantly better in G-CSF-treated animals (P < 0.0001). At 36 and 48 h after microsurgical hepatic resection, markers of hepatic proliferation (Ki67, BrdU) were elevated in G-CSF-treated mice compared to sham injected control animals (P < 0.0001) and dry liver weight was increased (P < 0.05). G-CSF conditioning might prove to be useful in patients with small-for-size liver remnants after extended hepatic resections due to primary or secondary liver tumors or in the setting of split liver transplantation.

Adenovirus-mediated transforming growth factor-beta ameliorates ischemic necrosis of epigastric skin flaps in a rat model

BACKGROUND: Gene therapy has been recently introduced as a novel approach to treat ischemic tissues by using the angiogenic potential of certain growth factors. We investigated the effect of adenovirus-mediated gene therapy with transforming growth factor-beta (TGF-beta) delivered into the subdermal space to treat ischemically challenged epigastric skin flaps in a rat model. MATERIAL AND METHODS: A pilot study was conducted in a group of 5 animals pretreated with Ad-GFP and expression of green fluorescent protein in the skin flap sections was demonstrated under fluorescence microscopy at 2, 4, and 7 days after the treatment, indicating a successful transfection of the skin flaps following subdermal gene therapy. Next, 30 male Sprague Dawley rats were divided into 3 groups of 10 rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received subdermal injections of adenovirus encoding TGF-beta (Ad-TGF-beta) or green fluorescent protein (Ad-GFP) as treatment control. The third group (n = 10) received saline and served as a control group. A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Just prior to flap elevation, the injections were given subdermally in the left upper corner of the flap. The flap was then sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. RESULTS: There was a significant increase in mean percent surviving area between the Ad-TGF-beta group and the two other control groups (P < 0.05). (Ad-TGF-beta: 90.3 +/- 4.0% versus Ad-GFP: 82.2 +/- 8.7% and saline group: 82.6 +/- 4.3%.) CONCLUSIONS: In this study, the authors were able to demonstrate that adenovirus-mediated gene therapy using TGF-beta ameliorated ischemic necrosis in an epigastric skin flap model, as confirmed by significant reduction in the necrotic zones of the flap. The results of this study raise the possibility of using adenovirus-mediated TGF-beta gene therapy to promote perfusion in random portion of skin flaps, especially in high-risk patients.

Insulin-like growth factor I improves aspects of mycophenolate mofetil-impaired anastomotic healing in an experimental model

BACKGROUND: Patients taking immunosuppressants after transplantation may require intestinal surgery. Mycophenolate mofetil (MMF) has been found to impair the healing of colonic anastomoses in rats. This study examined whether insulin-like growth factor (IGF) I prevents MMF impairment of anastomotic healing. METHODS: Sixty-three rats were divided into three groups (MMF, MMF/IGF and control). Animals underwent a sigmoid colon anastomosis with a 6/0 suture, and were killed on days 2, 4 and 6 after surgery. Investigations included bursting pressure measurement, morphometric analysis, and assessment of mucosal proliferation by 5-bromo-2'-deoxyuridine and Ki67 immunohistochemistry of the anastomoses. RESULTS: The leak rate was three of 21, one of 20 and two of 20 in the MMF, MMF/IGF-I and control groups respectively. Anastomotic bursting pressures were significantly lower in the MMF group than in the control group on days 2 and 4, but there was no significant difference by day 6. Values in the MMF/IGF-I and control groups were similar. Colonic crypt depth was significantly reduced in MMF-treated animals on days 2 and 4, but this impairment was attenuated by IGF-I on day 4. Similarly, IGF-I reduced the negative impact of MMF on mucosal proliferation on days 2 and 6. CONCLUSION: Exogenous IGF-I improves some aspects of MMF-impaired anastomotic healing.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer

The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer.

The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results demonstrate a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five out of the eight cell lines (IC50 2-43nM). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biological functions, such as cellular morphology, MET-dependent cell motility and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET mutated variants. Animals were randomized for the treatment with EMD1214063 (50mg/kg/day) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, while tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small molecule inhibitor with selective activity towards mutated MET variants.

Immunogenomic analysis of insect bite hypersensitivity in a model horse population.

Equine insect bite hypersensitivity (IBH) is a seasonal IgE-mediated dermatosis caused by bites of insects of the genus Culicoides. A familial predisposition for the disease has been shown but, except for the MHC, the genes involved have not been identified so far. An immunogenomic analysis of IBH was performed in a model population of Old Kladruby horses, all living in the same environment. Clinical signs of IBH were used as phenotypic manifestation of IBH. Furthermore, total serum IgE levels were determined in the sera of these horses and used as an independent phenotypic marker for the immunogenetic analysis. Single nucleotide polymorphisms (SNPs) in candidate immunity-related genes were used for association analyses. Genotypes composed of two to five genes encoding interferon gamma -IFNG, transforming growth factor beta 1 -TGFB1, Janus kinase 2 -JAK2, thymic stromal lymphopoietin -TSLP, and involucrin -IVL were associated with IBH, indicating a role of the genes in the pathogenesis of IBH. These findings were supported by analysis of gene expression in skin biopsies of 15 affected and 15 unaffected horses. Two markers associated with IBH, IFNG and TGFB1, showed differences in mRNA expression in skin biopsies from IBH-affected and non-affected horses (p<0.05). Expression of the gene coding for the CD14 receptor molecule -CD14 was different in skin biopsies at p<0.06. When total IgE levels were treated as binary traits, genotypes of IGHE, ELA-DRA, and IL10/b were associated with this trait. When treated as a continuous trait, total IgE levels were associated with genes IGHE, FCER1A, IL4, IL4R, IL10, IL1RA, and JAK2. This first report on non-MHC genes associated with IBH in horses is thus supported by differences in expression of genes known to play a role in allergy and immunity.

Rumination mediates the prospective effect of low self-esteem on depression: A five-wave longitudinal study

Previous research supports the vulnerability model of low self-esteem and depression, which states that low self-esteem operates as a prospective risk factor for depression. However, it is unclear which processes mediate the effect of low self-esteem. To test for the mediating effect of rumination, the authors used longitudinal mediation models, which included exclusively prospective effects and controlled for autoregressive effects of the constructs. Data came from 663 individuals (aged 16 to 62 years), who were assessed 5 times over an 8-month period. The results indicated that low self-esteem predicted subsequent rumination, which in turn predicted subsequent depression, and that rumination partially mediated the prospective effect of low self-esteem on depression. These findings held for both men and women, and for both affective-cognitive and somatic symptoms of depression. Future studies should test for the mediating effects of additional intrapersonal and interpersonal processes.