Lineage-Negative Bone Marrow Cells Protect Against Chronic Renal Failure

Progressive renal failure continues to be a challenge. The use of bone marrow cells represents a means of meeting that challenge. We used lineage-negative (Lin(-)) cells to test the hypothesis that Lin(-) cell treatment decreases renal injury. Syngeneic Fischer 344 rats were divided into four groups: sham ( laparotomy only, untreated); Nx (five-sixth nephrectomy and untreated); NxLC1 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy day 15); and NxLC3 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy days 15, 30, and 45). On postoperative day 16, renal mRNA expression of interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-6 was lower in NxLC rats than in Nx rats. On postnephrectomy day 60, NxLC rats presented less proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells, and protein expression of monocyte chemoattractant protein-1, as well as decreased interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with sham rats, Nx rats presented greater cell proliferation, whereas NxLC1 rats and NxLC3 rats presented less cell proliferation than did Nx rats. Protein expression of the cyclin-dependent kinase inhibitor p21 and of vascular endothelial growth factor increased after nephrectomy and decreased after Lin(-) cell treatment. On postnephrectomy day 120, renal function (inulin clearance) was significantly better in Lin(-) cell-treated rats than in untreated rats. Lin(-) cell treatment significantly improved survival. These data suggest that Lin(-) cell treatment protects against chronic renal failure. STEM CELLS 2009; 27: 682-692

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1 beta (IL-1 beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alphalbeta (CINC-2 alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process. Copyright (c) 2007 John Wiley & Sons, Ltd.

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Cytotoxicity and antiangiogenic activity of grandisin

Objectives The antitumoural properties of grandisin, a tetrahydrofuran neolignan from Piper solmsianum, were investigated by in-vitro and in-vivo assays using the Ehrlich ascites tumoural (EAT) model. Methods Viability of the tumour cells was evaluated by Trypan blue exclusion and MTT methods, after incubation with grandisin (0.017-2.3 mu M). The effects of grandisin on the activity of caspase-3, -6, -8, and -9 were also investigated using colorimetric protease kits. In-vivo studies were performed in EAT-bearing mice treated intraperitoneally with 2.5, 5 or 10 mg/kg grandisin for 10 days. Key findings Grandisin inhibited the growth of EAT cells, by both methods, with IC50 values less than 0.25 mu M. The results showed that the activity of all the caspases studied increased in grandisin-treated cells, when compared with control, non-treated cells. Administering grandisin to EAT-bearing mice increased survival of the animals, in a dose-dependent manner. Simultaneously, we detected a 66.35% reduction of intraperitoneal tumour cell burden in the animals treated with 10 mg/kg grandisin. Additionally, in these animals, the marked increase of vascular endothelial growth factor (VEGF) levels, induced by EAT development, was decreased with treatment with grandisin, resulting in a reduction of 32.1% of VEGF levels in the peritoneal washing supernatant, when compared with the control. Conclusions The results demonstrated that grandisin induced in-vitro cytotoxicity and antiangiogenic effects in mice while it acted against tumour evolution, prolonging host survival.

Expressão do VEGFR-2 em polpas de dentes decíduos e permanentes jovens humanos

O conhecimento dos mecanismos celulares envolvidos nos fenômenos orgânicos fisiológicos e/ou patológicos, poderá conduzir a diagnósticos clínicos mais precisos, tratamentos mais direcionados e prognósticos mais favoráveis. Considerando esta premissa, foi objetivo deste estudo avaliar por meio de reação imunohistoquímica a presença ou ausência do vascular endothelial growth factor receptor-2 (VEGFR-2) em células endoteliais pulpares de dentes decíduos e permanentes jovens humanos, assim como a distribuição da sua imuno-marcação. A amostra foi constituída de nove dentes decíduos e quatro dentes permanentes jovens, selecionados de indivíduos de ambos os sexos, com idade entre 5 e 10 anos e 11 e 14 anos, respectivamente. Os espécimes foram fixados em formol diluído a 10% com tampão fosfato 0,1 M por 24 horas e descalcificados com solução de Ana Morse. Após, os mesmos foram processados e incluídos em parafina. Realizaram-se cortes de 5 µm de espessura. Uma lâmina de cada amostra foi corada pela técnica de hematoxilina e eosina (H&E) para análise histológica e as demais foram submetidas à reação imunohistoquímica enzimática. Nesta, utilizou-se como anticorpo primário o anti-h VEGFR-2 na diluição de 1:100 e anticorpo secundário biotinilado na diluição/proporção pré-estabelecidas pelo fabricante. Após o processamento das lâminas, os campos microscópicos mais significativos foram capturados e analisados qualitativamente quanto à presença ou ausência do VEGFR-2 nas células endoteliais pulpares, assim como à distribuição da imuno-marcação Os resultados observados mostraram que tanto os dentes decíduos quanto os dentes permanentes jovens apresentaram, especificamente, as células endoteliais pulpares imuno-marcadas para o VEGFR-2. Entretanto, verificou-se que nem todas apresentaram a mesma distribuição de marcação, sendo esta nos dentes decíduos mais evidente próxima à região subodontoblástica. As células endoteliais da polpa dos dentes permanentes jovens demonstraram no mesmo espécime estudado, ausência e presença de marcação endotelial, entretanto esta se apresentou de forma mais uniforme. Levando-se em consideração as limitações encontradas neste estudo, sugere-se que os dentes decíduos apresentam uma capacidade de resposta maior das células endoteliais pulpares ao anti-h VEGFR-2 quando comparados aos dentes permanentes.

Exercise training prevents hyperinsulinemia, muscular glycogen loss and muscle atrophy induced by dexamethasone treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Time course of training-induced microcirculatory changes and of vegf expression in skeletal muscles of spontaneously hypertensive female rats

Exercise-induced vessel changes modulate arterial pressure (AP) in male spontaneously hypertensive rats (SHR). Vascular endothelial growth factor (VEGF) is important for angiogenesis of skeletal muscle. The present study evaluated the time course of VEGF and angiogenesis after short- and long-term exercise training of female SHR and Wistar Kyoto (WKY) rats, 8-9 weeks (200-250 g). Rats were allocated to daily training or remained sedentary for 3 days (N = 23) or 13 weeks (N = 23). After training, the carotid artery was catheterized for AP measurements. Locomotor (tibialis anterior and gracilis) and non-locomotor skeletal muscles (temporalis) were harvested and prepared for histologic and protein expression analyses. Training increased treadmill performance by all groups (SHR = 28%, WKY = 64%, 3 days) and (SHR = 141%, WKY = 122%, 13 weeks). SHR had higher values of AP than WKY (174 ± 4 vs 111 ± 2 mmHg) that were not altered by training. Three days of running increased VEGF expression (SHR = 28%, WKY = 36%) simultaneously with an increase in capillary-to-fiber ratio in gracilis muscle (SHR = 19%, WKY = 15%). In contrast, 13 weeks of training increased gracilis capillary-to-fiber ratio (SHR = 18%, WKY = 19%), without simultaneous changes in VEGF expression. Training did not change VEGF expression and capillarity of temporalis muscle. We conclude that training stimulates time- and tissue-dependent VEGF protein expression, independent of pressure levels. VEGF triggers angiogenesis in locomotor skeletal muscle shortly after the exercise starts, but is not involved in the maintenance of capillarity after long-term exercise in female rats.

Análise imuno-histoquímica das proteínas VEGF e HIF-1α na doença periodontal

Periodontal disease is a complex inflammatory and infectious condition that immune host, front of the microbial aggressions, can lead to disease progression, resulting in tissue destruction. The tissue damage induces the release of regulatory molecules, which protective roles and / or destructive, including proteins VEGF (vascular endothelial growth factor vascular) and HIF-1 α (hypoxia-induced factor α -1). Thus, this study investigated, quantitatively and comparatively, the immunohistochemical expression of VEGF (vascular endothelial growth factor) and HIF-1 α (hypoxia induced factor 1-α), proteins involved in inflammation, angiogenesis and hypoxia, in human gingival tissues. Therefore, 75 samples of gingival tissues were examined. Thirty samples were chronic periodontitis, 30 with chronic gingivitis and 15 healthy gingival. After sections analysis, positives and negatives inflammatory and endothelial cells in the connective tissue were counted and converted into percentage. Data were statistically analyzed using Kruskal-Wallis test and Spearman correlation. The results showed that both proteins marked. It was observed higher immunoreactivity for HIF-1 α at chronic gingivitis and periodontitis specimens compared to healthy sites, however, no statistically significant differences were observed among them (p>0.05). The VEGF immunostaining showed similarity among the cases of periodontitis, gingivitis and healthy gingiva. Moderate and positive correlation statistically significant differences were verified for the expressions of VEGF and HIF-1α in gingival health (r = 0,529, p = 0.04). Thus, it can be conclude that possibly the route of HIF-1 α, is activated in periodontal disease may have involvement of the protein in pathogenesis and progression of disease, and that activation of VEGF, can be in addition to being triggered transcription by HIF-1 α may be also influenced by other additional factors such as the action of periodontal microorganisms endotoxins

Imunoexpressão de vegf-c, vegfr-3 e hif-1α e mensuração da densidade linfática em carcinomas epidermóides de lábio inferior metastáticos e não-metastáticos: uma relação com parâmetros clinicopatológicos e prognósticos

Squamous cell carcinoma of the lower lip is among the most common malignant tumors of the oral and maxillofacial region, with good prognosis in more than 90% of patients with 5-year survival. In these carcinomas, the development of lymph node metastasis decreases the prognosis and it has been associated with the formation of new lymphatic vessels. It has been suggested the important role of vascular endothelial growth factor-C (VEGF-C), the receptor type 3 VEGF (VEGFR-3) and hypoxia-induced factor 1 (HIF-1) in this process. The aim of this study was to evaluate the immunoexpression of VEGF-C, VEGFR-3 and HIF-1α and correlate with intra and peritumoral lymphatic density in squamous cell carcinomas of the lower lip metastatic and non-metastatic. The sample consisted of 50 cases of squamous cell carcinoma of lower lip, of which 25 had regional lymph node metastasis and 25, absence of metastasis. The percentages of cells immunostained for VEGF-C, VEGFR-3 and HIF-1α in front of tumor invasion and in the center of tumor were evaluated. Microvessel density lymphatic (MDL) was determined by the counting of lymph microvessels immunostained by the anti-D2-40 in five fields (200×), in an area of evaluation with 0.7386 mm2. The invasion of the lymph vessels by malignant cells was also evaluated. Immunostaining was correlated with the presence and absence of metastasis, TNM clinical stage, local recurrence, disease outcome (remission of injury or patient death) and histological grading. The analysis of intra and peritumoral lymphatic density showed no significant association with clinicopathological parameters and immunoexpressions of VEGF-C, VEGFR-3 and HIF-1α (p > 0,05). There was a weak positive correlation, significant, between intra and peritumoral lymphatic density (r = 0,405; p = 0,004). VEGF-C showed no significant association with clinicopathological and prognosis parameters (p > 0,05). For VEGFR-3, there was scarce membrane staining and intense and homogenous cytoplasmic staining in neoplastic cells. Percentage of positive cytoplasmic VEGFR-3 in center of tumor, exhibited a statistically significant association with metastasis (p = 0,009), patient death (p = 0,008) and histological grades of malignancy proposed by Bryne et al. (1992) (p = 0,002) and World Health Organization (p = 0,003). A low positive correlation was statistically significant between the immunoreactivity of VEGFC and VEGFR-3 cytoplasmic (r = 0,358; p = 0,011) and between the percentage of positive cytoplasmic VEGFR-3 in front of tumor invasion and in the center of the tumor (r = 0,387; p = 0,005) was also demonstrated. There was no association between HIF-1α, clinicopathological and prognosis parameters, and VEGF-C and VEGFR-3. The percentage of nuclear positivity for HIF-1α was significantly higher in cases without invasion of peritumoral lymphatic (p = 0,040). Based on the results we can conclude that most cytoplasmic expression of VEGFR-3 in center of tumor in metastatic cases, high degree of malignancy and poorly differentiated, contributes to poor outcome of squamous cell carcinoma of the lower lip, including patient death. Intra and peritumoral lymphatic density seems to be not associated with lymph node metastasis in these carcinomas

Hypoxic-preconditioning induces neuroprotection against hypoxia-ischemia in newborn piglet brain

Preconditioning-induced ischemic tolerance has been documented in the newborn brain, however, the signaling mechanisms of this preconditioning require further elucidation. The aims of this study were to develop a hypoxic-preconditioning (PC) model of ischemic tolerance in the newborn piglet, which emulates important clinical similarities to human situation of birth asphyxia, and to characterize some of the molecular mechanisms shown to be implicated in PC-induced neuroprotection in rodent models. One day old piglets were subjected to PC (8% O(2)/92% N(2)) for 3 h and 24 h later were exposed to hypoxia-ischemia (HI) produced by a combination of hypoxia (5% FiO(2)) for a period of 30 min and ischemia induced by a period of hypotension (10 min of reduced mean arterial blood pressure; 70% of baseline). Neuropathologic analysis and unbiased stereology, conducted at 24 h, 3 and 7 days of recovery following HI, indicated a substantial reduction in the severity of brain damage in PC piglets compared to non-PC piglets (P<0.05). PC significantly increased the mRNA expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and its target gene, vascular endothelial growth factor (VEGF) at 0 h, 6 h, 24 h, 3 and 7 days of recovery. Immunoblot analysis demonstrated that PC resulted in HIF-1 alpha protein stabilization and accumulation in nuclear extracts of cerebral cortex of newborn piglet brain compared to normoxic controls. Protein levels of VEGF increased in a time-dependent manner in both cortex and hippocampus following PC. Double-immunolabeling indicated that VEGF is mainly expressed in neurons, endothelial cells and astroglia. Our study demonstrates for the first time the protective efficacy of PC against hypoxic-ischemic injury in newborn piglet model, which recapitulates many pathophysiological features of asphyxiated human neonates. Furthermore, as has been shown in rodent models of preconditioning, our results suggest that PC-induced protection in neonatal piglets may involve upregulation of VEGF. (C) 2011 Elsevier B.V. All rights reserved.

Hypoxia Enhances the Angiogenic Potential of Human Dental Pulp Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

VEGF and MVD expression in sinus augmentation with autologous bone and several graft materials

The aim of this study was to assess vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in maxillary sinus augmentation with autogenous bone and different graft materials for evaluating their angiogenic potential.Biopsies were harvested 10 months after sinus augmentation with a combination of autogenous bone and different graft materials: hydroxyapatite (HA, n = 6 patients), demineralized freeze-dried bone allograft (DFDBA, n = 5 patients), calcium phosphate (CP, n = 5 patients), Ricinus communis polymer (n = 5 patients) and control group - autogenous bone only (n = 13 patients).In all the samples, higher intensities of VEGF expression were prevalent in the newly formed bone, while lower intensities of VEGF expression were predominant in the areas of mature bone. The highest intensity of VEGF expression in the newly formed bone was expressed by HA (P < 0.001) and CP in relation to control (P < 0.01) groups. The lowest intensities of VEGF expression in newly formed bone were shown by DFDBA and polymer groups (P < 0.05). When comparing the different grafting materials, higher MVD were found in the newly formed bone around control, HA and CP (P < 0.001).Various graft materials could be successfully used for sinus floor augmentation; however, the interactions between bone formation and angiogenesis remain to be fully characterized.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis.Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05).Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam.Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.

Panretinal photocoagulation versus PRP plus intravitreal bevacizumab for high-risk proliferative diabetic retinopathy (IBeHi study)

Purpose: To evaluate the effects of panretinal photocoagulation (PRP) compared with PRP plus intravitreal bevacizumab on best corrected visual acuity (BCVA) and total area of fluorescein leakage from active new vessels (NVs) in patients with high-risk proliferative diabetic retinopathy (PDR).Methods: We carried out a prospective study of patients with high-risk PDR and no prior laser treatment who were randomly assigned to receive PRP (PRP group) or PRP plus intravitreal injection of 1.5 mg of bevacizumab (PRP-plus group). In all patients, the PRP was administered at two time-points (weeks 1 and 3), with the intravitreal bevacizumab delivered at the end of the second laser episode in the PRP-plus group. Standardized ophthalmic evaluation including Early Treatment Diabetic Retinopathy Study BCVA as well as stereoscopic fundus photography and fluorescein angiography were performed at baseline and at weeks 4, 9 (+/- 1) and 16 (+/- 2). Main outcome measures included changes in BCVA and in total area of fluorescein leakage from active NVs.Results: Twenty-two (n = 30 eyes) consecutive patients completed the 16-week follow-up. There was no significant difference between the PRP and PRP-plus groups with respect to age, gender, type or duration of diabetes, area of fluorescein leakage from active NVs or BCVA. No significant difference in BCVA was observed between the groups throughout the study period. However, the total area of actively leaking NVs was significantly reduced in the PRP-plus group compared with the PRP group at weeks 4, 9 and 16 (p < 0.001). No major adverse events were identified.Conclusions: In the short-term, the adjunctive use of intravitreal bevacizumab with PRP was associated with a greater reduction in the area of active leaking NVs than PRP alone in patients with high-risk PDR.