Daniel Escorsim Machado

Higher expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and metalloproteinase-9 (MMP-9) in a rat model of peritoneal endometriosis is similar to cancer diseases

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.

A selective cyclooxygenase-2 inhibitor suppresses the growth of endometriosis with an antiangiogenic effect in a rat model

OBJECTIVE To analyze the antiangiogenic effects of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib on the growth of endometrial implants in a rat model of peritoneal endometriosis. DESIGN Pharmacologic interventions in an experimental model of peritoneal endometriosis. SETTING Research laboratory in the Federal University of Rio de Janeiro. ANIMAL(S) Twenty female Sprague-Dawley rats with experimentally induced endometriosis. INTERVENTION(S) After implantation and establishment of autologous endometrium onto the peritoneum abdominal wall, rats were randomized into groups and treated with parecoxib or the vehicle by IM injection for 30 days. MAIN OUTCOME MEASURE(S) Vascular density, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1, the distribution of activated macrophages, the expression of COX-2, and the prostaglandin concentration in the endometriotic lesions treated with parecoxib were analyzed. RESULT(S) The treatment significantly decreased the implant size, and histologic examination indicated mostly atrophy and regression. A reduction in microvessel density and in the number of macrophages, associated with decreased expression of VEGF and Flk-1, also were observed. The treatment group showed a low concentration of prostaglandin E(2). CONCLUSION(S) These results suggest that the use of COX-2 selective inhibitors could be effective to suppress the establishment and growth of endometriosis, partially through their antiangiogenic activity.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model

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, flow‐cytometric 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.

Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 μg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 μg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.

Topical application of Acheflan on rat skin injury accelerates wound healing: a histopathological, immunohistochemical and biochemical study

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.

Extracellular matrix secreted by reactive stroma is a main inducer of pro-tumorigenic features on LNCaP prostate cancer cells

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.

Comparison of biodistribution profile of monoclonal antibodies nanoparticles and aptamers in rats with breast cancer

Abstract The use of monoclonal antibodies and aptamers is growing every single day, as the use of nanoparticle systems. Although most of the products are under investigation, there are a few commercialized products available at the market, for human consume. In this study, we have compared three formulations (aptamer anti-MUC1, monoclonal antibody – Trastuzumab and monoclonal antibodies nanoparticles – PLA/PVA/MMT trastuzumab) to identify their profile as also to understand their behavior into an alive biological system. In this direction the radiolabeling of the products were done and they were all tested in animals (in vivo) in two conditions: healthy rats and breast cancer induced animals. The results showed that the nanoparticle has the better biodistribution profile, followed by the aptamer. We conclude that more studies and a global effort to elucidate the biological behavior of drugs and especially nano-drugs are necessary.

Euterpe oleracea Extract (Açaí) Is a Promising Novel Pharmacological Therapeutic Treatment for Experimental Endometriosis.

This study investigated the therapeutic potential of Euterpe oleracea extract (açaí) on the growth and survival of endometriotic lesions using an experimental model. Twenty female Sprague-Dawley rats were randomized into two groups after the implantation and establishment of autologous endometrium onto the peritoneum abdominal wall and treated with 200 mg/kg hydroalcoholic solution extract from açaí stone or vehicle via gastric tube for 30 consecutive days. Body weight, lesion surface areas, histological and immunohistochemistry analyses of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2) and F4-80 were performed. Levels of VEGF, VEGFR-2, MMP-9 and COX-2 mRNA were measured. Flow cytometry of F4-80 was performed, and ELISA immunoassays measured prostaglandin E2 (PGE2), VEGF and nitric oxide (NO) and concentrations. Macrophage cell line J774.G8 was treated with 10, 20, and 40 μg/mL of açaí for 24, 48 and 72 h, and cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Açaí treatment significantly decreased the implant size, and histological examination indicated atrophy and regression. A reduction in immunostaining and mRNA expression of VEGF, MMP-9 and COX-2 was observed, and F4-80 was lower in the treated group than the control group. The treated group also exhibited lower concentrations of PGE2, VEGF and NO compared to the control group. Macrophages cells treated with 20 and 40 μg/ml of açaí reduced cell viability in about 50% after 24, 48 and 72 h. Our results suggest that açaí effectively suppressed the establishment and growth of endometriotic lesions, and this agent is a promising novel pharmacological therapeutic treatment for endometriosis.

Composition of sulfated glycosaminoglycans and immunodistribution of chondroitin sulfate in deeply infiltrating endometriosis affecting the rectosigmoid

The composition of sulfated glycosaminoglycans (GAGs) and the tissue distribution of chondroitin sulfate (CS) were analyzed in deeply infiltrating endometriosis (DIE) of rectosigmoid, using metachromatic staining, and biochemical analysis employing electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against CS. The sulfated GAGs were characterized as dermatan sulfate (DS), heparan sulfate (HS) and CS; and DS strongly predominated compared to HS and CS. Immunostaining procedures showed that CS was concentrated in the endometriosis foci, distributed throughout the stroma around the glands. This is the first report describing the composition of sulfated GAGs and the tissue location of CS in DIE by means of histochemical, biochemical and immunohistochemical analyses. These results confirmed that in DIE of rectosigmoid, as in eutopic endometrium [Nasciutti, L.E., Ferrari, R., Berardo, P.T., Souza, M.L.S., Takiya, C.M., Borojevic, R., Abrao, M.S., Silva, L.C.F., 2006. Distribution of chondroitin sulfate in human endometrium. Micron 37, 544-550], CS was the dominant sulfated GAG in stroma of the lesion foci.

Combined effect of vascular endothelial growth factor and its receptor polymorphisms in endometriosis: a case-control study

OBJECTIVE Endometriosis is a multifactorial gynecological disease, whose pathogenesis is crucially dependent on angiogenesis, which is signaled via vascular endothelial growth factor (VEGF) and its receptor (VEGFR2). We hypothesize that single nucleotide polymorphisms (SNPs) in VEGF and VEGFR2 genes may influence the onset and/or the progression of endometriosis. The main aim of this study was to investigate the contribution of VEGF and VEGFR2 SNPs as risk factors for endometriosis, as well as their association with endometriosis symptoms. STUDY DESIGN A case-control study was conducted, involving 293 endometriosis patients and 223 controls, who were submitted to laparoscopic or laparotomy surgery at hospitals from the Brazilian public health system. Genotyping of VEGF (-2578C>A, -460T>C, -1154G>A, +405G>C and +936C>T) and VEGFR2 (-604T>C, 1192C>T) SNPs was performed by TaqMan real-time polymerase chain reaction. The association between SNPs and endometriosis, deep infiltrating endometriosis (DIE) or endometriosis symptoms was estimated by odds ratios (OR) with their 95% confidence intervals (CI), which were calculated using multivariate logistic regression models. RESULTS VEGF variant alleles -2578A and -1154A were associated with increased endometriosis risk (OR: 1.39, 95% CI: 1.04-1.87 and OR: 1.63, 95% CI: 1.12-2.37, respectively), whereas VEGF 405C and VEGFR2 1192T were associated with lower risk of endometriosis (OR: 0.66, 95% CI: 0.43-1.00 and OR: 0.58, 95% CI: 0.40-0.84, respectively). The combination of wild-type genotypes of both VEGF -2578C>A and -1154G>A with variant genotypes of both VEGF +405G>C and VEGFR2 1192C>T showed the best protective effect against the development of endometriosis, either considering all cases (OR: 0.33, 95% CI: 0.12-0.89) or only DIE (OR: 0.30, 95% CI: 0.10-0.87). The combination of variant genotypes of VEGF -2578C>A, -1154G>A, +405G>C and VEGFR2 1192C>T was also protective against DIE (OR: 0.67, 95% CI: 0.46-0.96). VEGFR2 1192C>T were associated with reduced cyclical urinary complaints (OR: 0.40, 95% CI: 0.18-0.88). CONCLUSIONS Our results indicate that VEGF SNPs -2578C>A and -1154G>A increase endometriosis risk, whereas VEGF +405G>C and VEGFR2 1192C>T are protective against disease development, with VEGFR2 1192C>T also reducing cyclical urinary symptoms. The combined analysis of VEGF-VEGFR2 genotypes suggests a gene-gene interaction in endometriosis susceptibility.