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Summary. Background: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. Objectives: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. Methods and results: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flowcytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Conclusion: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

BackgroundEndometriosis is a common disease characterized by the presence of a functional endometrium outside the uterine cavity, causing pelvic pain, dysmenorrheal, and infertility. This disease has been associated to development of different types of malignancies; therefore new blood vessels are essential for the survival of the endometrial implant. Our previous observations on humans showed that angiogenesis is predominantly found in rectosigmoid endometriosis, a deeply infiltrating disease. In this study, we have established the experimental model of rat peritoneal endometriosis to evaluate the process of angiogenesis and to compare with eutopic endometrium.MethodsWe have investigated the morphological characteristics of these lesions and the vascular density, VEGF and its receptor Flk-1 and MMP-9 expression, and activated macrophage distribution, using immunohistochemistry and RT-PCR.ResultsAs expected, the auto-transplantation of endometrium pieces into the peritoneal cavity is a well-established method for endometriosis induction in rats. The lesions were cystic and vascularized, and demonstrated histological hallmarks of human pathology, such as endometrial glands and stroma. The vascular density and the presence of VEGF and Flk-1 and MMP-9 were significantly higher in endometriotic lesions than in eutopic endometrium, and confirmed the angiogenic potential of these lesions. We also observed an increase in the number of activated macrophages (ED-1 positive cells) in the endometriotic lesions, showing a positive correlation with VEGF.ConclusionThe present endometriosis model would be useful for investigation of the mechanisms of angiogenesis process involved in the peritoneal attachment of endometrial cells, as well as of the effects of therapeutic drugs, particularly with antiangiogenic activity.

BackgroundEndometriosis is regarded as a complex and heterogeneous disease in which genetic and environmental factors contribute to the phenotype. The Vascular Endothelial Growth Factor (VEGF) plays important roles in the pathogenesis of endometriosis. The present study was aimed at investigating the contribution of VEGF polymorphisms as risk factors for the development of endometriosis. This is the first study to evaluate the combined influence of the five most common VEGF polymorphisms.MethodsThis study was conducted at two hospitals from the Brazilian public health system, and comprised 294 women submitted to laparoscopic or laparotomy surgery: 182 patients had a histologically confirmed diagnosis of endometriosis (cases), whereas 112 had no evidence of the disease (controls). The VEGF polymorphisms were determined by TaqMan real-time polymerase chain reaction. The odds ratio (OR) with their 95% confidence intervals (CI) were calculated using an unconditional logistic regression model.ResultsEndometriosis patients and controls did not differ regarding age distribution, whereas the body mass index was significantly lower in endometriosis patients, when compared with controls (23.1 ± 3.9 versus 27.3 ± 5.9, P < 0.001). The evaluation of gynecological symptoms, including dysmenorrhea, non-cyclic chronic pelvic pain, dyspareunia and infertility, indicates significantly higher prevalences among endometriosis cases. The variant allele -1154A was significantly associated with endometriosis, either considering all cases (OR: 1.90, 95% CI: 1.23–2.97), deep infiltrating endometriosis (DIE) (OR: 1.83, 95% CI: 1.16-2.90) or moderate and severe endometriosis (stages III-IV) (OR: 1.97, 95% CI: 1.21-3.19). No significant differences were found in allele or genotype distributions of the –2578C > A, -460 T > C, +405G > C and +936C > T polymorphisms between endometriosis cases and controls. A total of six haplotypes were inferred derived from four polymorphisms (-2578C > A, -460 T > C, -1154G > A and +405G > C). There was a protective association between CCGG haplotype and endometriosis, either considering all cases (OR: 0.36, 95% CI: 0.15–0.86), DIE (OR: 0.37 95% CI: 0.15 – 0.90) or stages III-IV (OR: 0.35 95% CI: 0.13 – 0.95).ConclusionsThe present results indicate a positive association between VEGF -1154G > A and the risk of developing endometriosis, whereas the CCGG haplotype may be protective against the development of disease.

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BackgroundTendinopathy pathogenesis is associated with inflammation. Regulatory T (Treg) cells contribute to early tissue repair through an anti-inflammatory action, with the forkhead box P3 (FOXP3) transcription factor being essential for Treg function, and the FC-receptor-like 3 (FCRL3) possibly negatively regulating Treg function. FCRL3 –169T>C and FOXP3 –2383C>T polymorphisms are located near elements that regulate respective genes expression, thus it was deemed relevant to evaluate these polymorphisms as risk factors for tendinopathy development in athletes.MethodsThis case-control study included 271 volleyball athletes (146 tendinopathy cases and 125 controls) recruited from the Brazilian Volleyball Federation. Genotyping analyses were performed using TaqMan assays, and the association of the polymorphisms with tendinopathy evaluated by multivariate logistic regression.ResultsTendinopathy frequency was 63% patellar, 22% rotator cuff and 15% Achilles tendons respectively. Tendinopathy was more common in men (OR = 2.87; 95% CI = 1.67–4.93). Higher age (OR = 8.75; 95% CI = 4.33–17.69) and more years of volleyball practice (OR = 8.38; 95% CI = 3.56–19.73) were risk factors for tendinopathy. The FCRL3 –169T>C frequency was significantly different between cases and controls. After adjustment for potential confounding factors, the FCRL3 –169C polymorphism was associated with increased tendinopathy risk (OR = 1.44; 95% CI = 1.02–2.04), either considering athletes playing with tendon pain (OR = 1.98; 95% CI = 1.30–3.01) or unable to train due to pain (OR = 1.89; 95% CI = 1.01–3.53). The combined variant genotypes, FCRL3 –169TC or –169CC and FOXP3 –2383CT or –2383TT, were associated with an increased risk of tendinopathy among athletes with tendon pain (OR = 2.24; 95% CI: 1.14–4.40 and OR = 2.60; 95% CI: 1.11–6.10). The combined analysis of FCRL3 –169T>C and FOXP3 –2383C>T suggests a gene-gene interaction in the susceptibility to tendinopathy.ConclusionsFCRL3 –169C allele may increase the risk of developing tendinopathy, and together with knowledge of potential risk factors (age, gender and years playing) could be used to personalize elite athletes’ training or treatment in combination with other approaches, with the aim of minimizing pathology development risk.

BackgroundDermal wound healing involves a cascade of complex events including angiogenesis and extracellular matrix remodeling. Several groups have focused in the study of the skin wound healing activity of natural products. The phytomedicine Acheflan®, and its main active constituent is the oil from Cordia verbenacea which has known anti-inflammatory, analgesic and antimicrobial activities. To our knowledge, no investigation has evaluated the effect of Acheflan® in an experimental model of skin wound healing.The present study has explored the wound healing property of Acheflan® and has compared it with topical effectiveness of collagenase and fibrinolysin by using Wistar rat cutaneous excision wound model.MethodsAnimals were divided into four groups: untreated animals are negative control (NC), wounds were treated topically every day with Collagenase ointment (TC), with Fibrinolysin ointment (TF) and with cream Acheflan (TAc). Skin samples were collected on zero, 8th and 15th days after wounding. The healing was assessed by hematoxylin-eosin (HE), picrosirius red, hydoxyproline content and immunohistochemical analysis of the vascular endothelial growth factor (VEGF) and matrix metalloprotease-9 (MMP-9). Statistical analysis was done by ANOVA and Student t-test (p < 0.05).ResultsThe histological analysis HE of wound in the TAc group was more efficient because it was possible to observe the complete remodeling of the epidermis indicating the regression of lesions compared with the NC. The evaluation of picrosirius staining has demonstrated a significant increase of collagen distribution in the TC and TAc treatments compared with NC and TF groups. These results are corroborated with hydroxyproline content. Skin TC and TAc treated rats have showed an increase of VEGF and MMP-9 compared with NC and TF groups. All parameters were significant (P < 0.05).ConclusionThe phytomedicine Acheflan® (oil of Cordia verbenacea) and TC possess higher therapeutic properties for wound healing compared with TF. These ointments seem to accelerate wound healing, probably due to their involvement with the increase of angiogenesis and dermal remodeling.

Tumor microenvironment modifications are related to the generation of reactive stroma and to critical events in cancer progression, such as proliferation, migration and apoptosis. In order to clarify these cellular interactions mediated by reactive stroma, we investigated the effects of cell-cell contacts, and the influence of soluble factors and extracellular matrix (ECM) secreted by Benign Prostate Hyperplasia (BPH) reactive stroma over LNCaP prostate tumor cells. Using in vitro functional assays, we demonstrated that ECM strongly stimulated LNCaP cell proliferation and migration, while inhibiting apoptosis, and inducing a deregulated expression pattern of several genes related to prostate cancer (PCa) progression. Conversely, reactive stromal cells per se and their secreted conditioned medium partially modulated these pro-tumorigenic events. These data indicate that secreted ECM in reactive stroma microenvironment contains key molecules that positively modulate important cancer hallmarks.