Mônica Alves Neves Diniz Ferreira

Aqueous Extract of Brazilian Green Propolis: Primary Components, Evaluation of Inflammation and Wound Healing by Using Subcutaneous Implanted Sponges

Propolis is a chemically complex resinous bee product which has gained worldwide popularity as a means to improve health condition and prevent diseases. The main constituents of an aqueous extract of a sample of green propolis from Southeast Brazil were shown by high performance liquid chromatography/mass spectroscopy/mass spectroscopy to be mono- and di-O-caffeoylquinic acids; phenylpropanoids known as important constituents of alcohol extracts of green propolis, such as artepillin C and drupanin were also detected in low amounts in the aqueous extract. The anti-inflammatory activity of this extract was evaluated by determination of wound healing parameters. Female Swiss mice were implanted subcutaneously with polyesther-polyurethane sponge discs to induce wound healing responses, and administered orally with green propolis (500 mg kg−1). At 4, 7 and 14 days post-implantation, the fibrovascular stroma and deposition of extracellular matrix were evaluated by histopathologic and morphometric analyses. In the propolis-treated group at Days 4 and 7 the inflammatory process in the sponge was reduced in comparison with control. A progressive increase in cell influx and collagen deposition was observed in control and propolis-treated groups during the whole period. However, these effects were attenuated in the propolis-treated group at Days 4 and 7, indicating that key factors of the wound healing process are modulated by propolis constituents.

Sponge‐induced angiogenesis and inflammation in PAF receptor‐deficient mice (PAFR‐KO)

To determine biological functions of platelet‐activating factor (PAF) in chronic inflammation, we have investigated the kinetics of angiogenesis, inflammatory cells recruitment and cytokine production in sponge‐induced granuloma in wild type and PAF receptor‐deficient mice (PAFR‐KO). Angiogenesis as determined by morphometric analysis and hemoglobin content was significantly higher in the implants of PAFR‐KO mice at all time points. Treatment with PAF receptor antagonist UK74505 (30 mg kg−1) also increased angiogenesis in sponge implants. Neutrophils and macrophages accumulation, as determined by myeloperoxidase and N‐acetylglucosaminidase activities in the supernatant of implanted sponges were markedly decreased in PAFR‐KO mice. Surprisingly, the levels of the proinflammatory chemokines, keratinocyte‐derived chemokine and chemokine monocyte chemoattractant protein 1 were higher in the implants of the transgenic animals. We have shown that angiogenesis was stimulated in PAFR‐KO mice whereas inflammation was decreased, indicating that PAF is an endogenous regulator of new blood vessels formation in the inflammatory microenvironment induced by the sponge implant.

Genetic background determines mouse strain differences in inflammatory angiogenesis.

Inflammation and angiogenesis are key components of fibrovascular tissue growth, a biological event underlying both physiological (wound healing) and pathological conditions (tumor development, chronic inflammation). We investigated these components in three frequently used mouse strains (Swiss, Balb/c and C57BL/6J) to verify the influence of genetic background on the kinetics of inflammatory cell recruitment/activation, neovascularization, extracellular matrix deposition, and cytokine production in polyether-polyurethane sponge implanted subcutaneously in male mice of these strains. The kinetics of neutrophil recruitment/activation as assessed by myeloperoxidase (MPO) activity was 2- and 3-fold higher in Balb/c implants at day 1 compared with Swiss and C57BL/6J implants, respectively. Macrophage accumulation/activation as NAG (n-acetyl β-glucosaminidase) activity was higher in Swiss implants. The levels the monocyte chemoattractant protein 1 (CCL2(MCP-1)) peaked at day 10 in the three types of implants but was produced more by C57BL/6J mice. Angiogenesis (hemoglobin, vascular endothelial growth factor-VEGF, and number of vessels) differed among the strains. Swiss implants had the highest hemoglobin content but the lowest VEGF levels. In contrast, Balb/c implants had higher VEGF levels but lower hemoglobin. Collagen deposition and transforming growth factor β-1; TGFβ-1 levels also varied among the groups. Swiss and Balb/c implants had progressive increase in TGFβ-1 from 4 to 14 days, while C57BL/6J implants achieved the peak at day 10 and fell at day 14. These findings emphasize the major contribution of genetic background in the temporal pattern and intensity of inflammatory angiogenesis components that may have functional consequences in physiological and pathological conditions where these processes co-exist.

Brazilian Green Propolis Inhibits Inflammatory Angiogenesis in a Murine Sponge Model

Angiogenesis and inflammation are persistent features of several pathological conditions. Propolis, a sticky material that honeybees collect from living plants, has been reported to have multiple biological effects including anti-inflammatory and anti-neoplasic activities. Here, we investigated the effects of water extract of green propolis (WEP) on angiogenesis, inflammatory cell accumulation and endogenous production of cytokines in sponge implants of mice over a 14-day period. Blood vessel formation as assessed by hemoglobin content and by morphometric analysis of the implants was reduced by WEP (500 mg kg−1 orally) compared to the untreated group. The levels of vascular endothelial growth factor (VEGF) increased progressively in the treated group but decreased after Day 10 in the control group. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activities, respectively. Neutrophil accumulation was unaffected by propolis, but NAG activity was reduced by the treatment at Day 14. The levels TGF-β1 intra-implant increased progressively in both groups but were higher (40%) at Day 14 in the control implants. The pro-inflammatory levels of TNF-α peaked at Day 7 in the control implants, and at Day 14 in the propolis-treated group. Our results indicate that the anti-inflammatory/anti-angiogenic effects of propolis are associated with cytokine modulation.

Differential effects of rolipram on chronic subcutaneous inflammatory angiogenesis and on peritoneal adhesion in mice.

The specific PDE4 inhibitor (rolipram) has been shown to attenuate excessive accumulation/activation of inflammatory cells and fibroblasts and cytokine production in several pathological conditions through cyclic nucleotide modulation. Here, using the murine sponge model to induce chronic subcutaneous inflammatory response and to elicit the formation of intraperitoneal adhesions we explored the hypothesis that rolipram would exert beneficial effects on decreasing key components of both processes (inflammatory cell recruitment, angiogenesis, and deposition of extracellular matrix component). Two doses of rolipram (0.2 or 2 mg/kg/day) were administered orally for 7 days in groups of mice bearing either subcutaneous or intraperitoneal polyether-polyurethane implants. Rolipram was effective in inhibiting angiogenesis as assessed by hemoglobin content and VEGF levels in subcutaneous implants (about 40% with both doses) but failed to exert this activity in intraperitoneal implants. Conversely, accumulation of neutrophils and macrophages determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities intraimplant, respectively, was attenuated only in intraperitoneal implants by the treatment. Levels of TNF-alpha and MCP-1 were also determined and rolipram at both doses decreased the production of both cytokines in intraperitoneal implants. The levels of MCP-1 in the subcutaneous implants were not affected by the treatment. Fibrosis was evaluated by determining the amount of collagen and production of TGF-beta1 intraimplant. Both parameters were attenuated by rolipram. These results have shown differential sensitivity of proliferating tissues to PDE4 inhibitor indicating that this agent may be used to target inflammatory angiogenesis selectively.

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.

Thalidomide attenuates mammary cancer associated-inflammation, angiogenesis and tumor growth in mice.

Thalidomide has proven to exert anti-inflammatory, anti-proliferative and anti-angiogenic activities in both neoplastic and non-neoplastic conditions. We investigated the effects of this compound on key components (blood vessel formation, inflammatory cell recruitment/activation, cytokine production) of 4T1 mammary tumor in mice. In addition, tumor growth and lung metastasis were evaluated. 4T1 cells were injected subcutaneously into Balb/c mice. After tumor engraftment (5days), thalidomide (150mg/kg) was administered to the treated group for 7days. Tumors of control (saline) and treated groups were sized regularly, removed 12days after inoculation and processed for biochemical and immunohistological parameters to assess neovascularization, inflammation and proliferative activity. Daily oral dose of thalidomide was able to reduce in 46% the tumor volume. The number of metastasis in the lungs was less in the thalidomide-treated group compared with the control animals. Assessment of tumor vascularization revealed a significant decrease in blood vessels formation by thalidomide. Likewise, the expression of FGF-1 showed weaker cytoplasmic positivity in the group treated with thalidomide compared with the control group. The levels of two cytokines, VEGF (pro-angiogenic) and TNF-α (pro-inflammatory) were decreased in tumor samples of thalidomide-treated group compared with the control group. Accumulation of neutrophils or macrophages in the 4T1 tumor measured by the activities of inflammatory enzymes, myeloperoxidase (MPO) for neutrophils and N-acetyl-β-D-glucosaminidase (NAG) for macrophages was inhibited by the treatment. By targeting key components of 4T1 tumor simultaneously, thalidomide was effective in attenuating tumor growth and metastasis. This approach, suppression of inflammation and angiogenesis may provide further insights for both prevention and treatment of cancer.

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).

Kinetics of implant-induced inflammatory angiogenesis in abdominal muscle wall in mice.

Injury of skeletal abdominal muscle wall is a common medical condition and implantation of synthetic or biological material is a procedure to repair musculofascial defects. We proposed to characterize the dynamics of inflammatory cell recruitment, newly formed blood vessels, cytokine production and fibrogenesis in the abdominal skeletal muscle in response to polyether-polyurethane sponge implants in mice. At 2, 4, 7 and 10days after implantation the muscle tissue underneath the sponge matrix was removed for the assessment of the angiogenic response (hemoglobin content, vascular endothelial growth factor and morphometric analysis of the number of vessels) and inflammation (myeloperoxidase and n-acethyl-B-d-glucosaminidase activities, cytokines). In addition, muscle fibrogenesis was determined by the levels of TGF-β1 and collagen deposition. Hemoglobin content, wash out rate of sodium fluorescein (indicative of blood flow) and the number of vessels increased in the abdominal muscle bearing the synthetic matrix in comparison with the intact muscle. Neutrophil recruitment peaked in the muscle at day 2, followed by macrophage accumulation at day 4 post-injury. The levels of the cytokines, VEGF, TNF-α, CCL-2/MCP-1 were higher in the injured muscle compared with the intact muscle and peaked soon after muscle injury (days 2 to 4). Collagen levels were higher in sponge-bearing muscle compared with the non-bearing tissue soon after injury (day 2). The implantation technique together with the inflammatory and vascular parameters used in this study revealed inflammatory, angiogenic and fibrogenic events and mechanisms associated with skeletal muscle responses to synthetic implanted materials.

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.