Andrezza Vilaça Belo

Murine Chemokine CXCL2/KC Is a Surrogate Marker for Angiogenic Activity in the Inflammatory Granulation Tissue

Objectives: A wide range of compounds inhibit formation of new blood vessels in a variety of models, accompanied by decreases in pro‐angiogenic cytokines. The authors sought a surrogate marker for the complex process of neovascularization by correlating inhibition of cytokine production with anti‐angiogenic effect.

Vasodilator effect of angiotensin-(1-7) in mature and sponge-induced neovasculature.

Angiotensin-(1-7) (Ang-(1-7)), a peptide constituent of the renin-angiotensin system, has been shown to act as a vasodilator mediator in pre-existing (skin) and newly formed vasculatures (14-day-old sponge implants). Blood flow was determined by the outflow rate of sodium fluorescein applied intradermally or intraimplant and the results were expressed in t(1/2) values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation). We showed that the t(1/2) value was significantly lower (4.1+/-0.46) in the implants compared with the cutaneous vasculature (5.7+/-0.5). Ang-(1-7) 20 ng was able to decrease t(1/2) values in both vasculatures. The specific receptor antagonist, D-Ala7-Ang-(1-7) (A-779), prevented Ang-(1-7)-induced vasodilation and altered the basal vascular tone of the implants. The vasodilator effect was also abolished by nitric oxide (NO) synthase inhibitors in both vasculatures and by indomethacin in the implant. Selective AT(1) and AT(2) receptor antagonists did not alter the vasodilation induced by the peptide. These results establish the vasodilator effect of Ang-(1-7) in the cutaneous and implant vasculature and that the peptide is produced endogenously by the fibrovascular tissue, and suggest that this peptide contributes for the vasodilation found in newly formed vascular beds (wound healing, chronic inflammatory processes and tumors).

Differential Effects of Antiangiogenic Compounds in Neovascularization, Leukocyte Recruitment, VEGF Production, and Tumor Growth in Mice

Angiogenesis and inflammation play critical roles in tumor growth. Using an in vivo tumor model, we report that thalidomide (100 mg kg− 1 day− 1) or clotrimazole (120 mg kg− 1 day− 1), inhibit blood vessel formation (determined by hemoglobin content), leukocyte recruitment [myeloperoxidase (MPO) activity; N-acetylglucosaminidase (NAG) activity], and vascular endothelial growth factor production. Inhibition of angiogenesis ranged from 35% to 65%. Clotrimazole was the most potent antiangiogenic compound and the agent capable of inhibiting tumor growth. Thalidomide was able to reduce the inflammatory reaction (MPO and NAG activities) by 50% to 70%, but was unable to delay tumor development. These results suggest that for this type of solid tumor the degree of neovascularization, rather than inhibition of inflammatory cell recruitment, is a determinant factor in tumor development. As the contribution of angiogenesis and inflammation to cancer progression vary markedly among different tumor types, it may be relevant to consider these factors in cancer therapy using antiangiogenesis/antiinflammatory approaches.

Differential effects of thalidomide on angiogenesis and tumor growth in mice.

Thalidomide, clinically used as an antiinflammatory and antitumoral drug, inhibited sponge-induced angiogenesis when administered systemically (100 mg/kg−1) in mice. However, it failed to inhibit solid Ehrlich tumor in the same mouse strain. We have used functional, biochemical and histological parameters to assess neovascularization and fibrovascular tissue infiltration of the mice sponge granuloma. The neovascularization growth as detected by development of blood flow and hemoglobin content extracted from the implants showed that thalidomide inhibited fibrovascular tissue formation by 40%. The functional and biochemical parameters correlated well with the histological study. Thalidomide had no inhibitory effect in the development of Ehrlich tumor. The detection of this selective action using the same animal strain bearing two different processes, supports the hypothesis that rather than species specificity, thalidomide is tissue specific. This approach may be used to identify the specificity of other therapeutic agents against distinct angiogenesis-dependent diseases.

Inflammatory Response Patterns in ICSI Patients: A Comparative Study Between Chronic Anovulating and Normally Ovulating Women

The aim of this study was to evaluate inflammatory response in chronic anovulating infertility women undergoing intracytoplasmic sperm injection. Thirteen infertile women with chronic anovulation and 23 normally ovulating women were prospectively evaluated. N-acetylglucosaminidase (NAG), myeloperoxidase (MPO), monocyte chemoattractant protein 1 (MCP-1), and C-reactive protein (CRP) concentrations were evaluated in serum and follicular fluid. Women with chronic anovulation presented higher NAG and MPO activity in follicular fluid when compared with normally ovulating women. Serum MPO activity was higher in the control group compared to the chronic anovulation group. Both serum and follicular fluid CRP concentrations were higher in women with chronic anovulation in comparison with the control group. Higher MCP-1 follicular fluid concentrations and serum levels of CRP were associated with the occurrence of ovarian hyperstimulation syndrome. Patients with chronic anovulation exhibited significantly higher follicle macrophage/neutrophil activation as well as unspecific inflammatory response by comparison with normally ovulating women.

Evaluation of N‐acetilglucosaminidase and myeloperoxidase activity in patients with endometriosis‐related infertility undergoing intracytoplasmic sperm injection

Aim:  Inflammation is as an important factor in ovulation with the active participation of leucocytes and their inflammatory mediators. The present study was performed to compare the activity of the inflammatory enzymes myeloperoxidase (MPO) and N‐acetylglucosaminidase (NAG) in patients with endometriosis‐related infertility and in normally ovulating women undergoing intracytoplasmic sperm injection (ICSI).

Identification of local angiogenic and inflammatory markers in the menstrual blood of women with endometriosis.

The aim of this study was to evaluate the presence of myeloperoxidase (MPO), N-acetyl-β-D-glucosaminidase (NAG), tumor necrosis factor alpha (TNF-α) and vascular endothelial growth factor (VEGF) in peripheral and menstrual blood in women with (n=10) and without (n=7) endometriosis. NAG and MPO activities were evaluated by enzymatic methods, whereas TNF-α and VEGF by immunoassay. No significant differences were found for these markers, neither in menstrual nor in peripheral blood between groups. Menstrual blood NAG (P=0.039) and MPO (P=0.0117) activities in the endometriosis group were significantly higher than in peripheral blood. NAG and MPO presented positive linear correlation in peripheral (P=0.07; r=0.641) and menstrual blood (P=0.01; r=0.603). These findings point to the existence of an increased local inflammatory activity in women with endometriosis.

Clotrimazole is an inhibitor of inflammatory angiogenesis and the metabolic activity in sponge granuloma.

Clotrimazole (CLT), clinically used as an antifungal drug, inhibited sponge-induced angiogenesis and granulation tissue metabolic activity when administered systemically (120 mg/kg) in rats. We have used functional, biochemical and histological parameters to assess neovascularization and fibrovascular tissue infiltration of the rat sponge granuloma. The sequential development of local blood flow as determined by the outflow rate of sodium fluorescein applied intraimplant, showed that the t 1/2 values for the fluorescence peak in the bloodstream decreased in the control group from an initial value of 11 ± 0.87 min (avascular implants, day 1) to 7.6 ±1.5 min at day 7 postimplantation. By contrast t 1/2 values in the CLT-treated group remained stable during the 7-day period. The hemoglobin content extracted from the control implants was 2.7 ± 0.14 μgHb/w.w vs. 1.8 ± 0.18 μgHb/w.w in the treated group. The functional and biochemical parameters correlated well with the histological study. Furthermore, the metabolic activity of the sponge-induced granulomas was inhibited by CLT. Because CLT is an inhibitor of signal transduction interfering with the ionic fluxes across the cell membranes, our results suggest that the onset and maintenance of inflammatory angiogenesis induced by subcutaneous implantation of sponge matrix may be regulated by ionic fluxes.

N-acetylglucosaminidase, myeloperoxidase and vascular endothelial growth factor serum levels in breast cancer patients

Inflammatory cells surround breast carcinomas and may act promoting tumor development or stimulating anti-tumor immunity. N-acetylglucosaminidase (NAG) has been employed to detect macrophage accumulation/activation. Myeloperoxidase (MPO) is considered a marker for neutrophils activity/accumulation. Vascular Endothelial Growth Factor (VEGF) is as strong pro-angiogenic cytokine. The aim of this study was to measure the systemic inflammatory response by measuring serum levels of NAG, MPO and VEGF in women diagnosed with breast cancer and associate this response to the peritumoral inflammatory infiltrate and to prognostic factors. Serum samples obtained from women with no evidence of disease (n=31) and with breast cancer (n=68) were analyzed for the activities of NAG, MPO and VEGF by enzymatic assay. Serum levels of NAG and VEGF were higher in healthy volunteers (P<0.0001) and serum levels of MPO were higher in patients with breast cancer (P=0.002). Serum levels of NAG were positively correlated to serum levels of MPO and VEGF (P<0.0001 and P=0.0012, respectively) and MPO and VEGF serum levels had also a positive correlation (P=0.0018). The inflammatory infiltrate was not associated to serum levels of the inflammatory markers, and higher levels of MPO were associated to lymphovascular invasion negativity (P=0.0175).

Distinct pattern of inflammatory enzyme activities in human ovarian cancer and benign myoma

Objective. Inflammatory cells and their products are significant components of malignancies. This study was performed to determine the activity of inflammatory enzymes m yeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) in ascitic fluid, sera or peritoneal lavage fluid from patients with epithelial ovarian cancer (EOC). Methods. Eighteen patients age ranged from 25 to 79 years (54.6 ±2.9 years) with epithelial ovarian cancer submitted to surgical treatment (EOC group) and 17 patients with uterine myoma (Myoma group) submitted to abdominal hysterectomy (control group) were prospectively studied. MPO and NAG activities were evaluated colorimetrically in sera, ascites or peritoneal lavage fluid obtained from the patients at the time of laparotomy. Results. In a total of 18 EOC, there were stage I in 1 case (5.5%), II in 3 (16.7%), III in 11 (61.1%) and IV in 3 cases (16.7%). MPO activity in sera of EOC was higher than in the ascitic fluid from the same patients. Conversely, MPO activity was similar in sera from both EOC and myoma-bearing patients. Comparison between NAG activities in sera from both groups showed much higher values in the OEC patients. Furthermore, inflammatory enzyme activities were overall associated with the stage of the disease. Conclusions . Our results show that inflammation has been positively correlated with cancer and that the pattern of a systemic inflammatory response induced by EOC differs quantitatively from that of a typical benign pelvic condition.