Silvia Passos Andrade

Angiotensin-(1-7): an update.

The renin-angiotensin system is a major physiological regulator of arterial pressure and hydro-electrolyte balance. Evidence has now been accumulated that in addition to angiotensin (Ang) II other Ang peptides [Ang III, Ang IV and Ang-(1-7)], formed in the limited proteolysis processing of angiotensinogen, are importantly involved in mediating several actions of the RAS. In this article we will review our knowledge of the biological actions of Ang-(1-7) with focus on the puzzling aspects of the mediation of its effects and the interaction Ang-(1-7)-kinins. In addition, we will attempt to summarize the evidence that Ang-(1-7) takes an important part of the mechanisms aimed to counteract the vasoconstrictor and proliferative effects of Ang II.

Opposing actions of angiotensins on angiogenesis.

Using the murine sponge model of angiogenesis, associated to functional and morphological parameters we have demonstrated opposing actions of angiotensin II (Ang II) and angiotensin-(1-7;Ang-1-7) in modulating fibrovascular tissue growth. Angiogenesis in the implants was assessed at day 7 postimplantation by extracting the hemoglobin content, by determining the outflow rate of sodium fluorescein applied intraimplant and by histological analysis. Furthermore, the proliferative activity of control and angiotensin-treated implants was established using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4 -sulfonyl)2H-tetrazolium)assay. The hemoglobin content in the control implants was 2.4 +/- 0.14 (microg/mg wet weight) versus 3.6 +/- 0.27(Ang II;100 ng) and 0.86 +/- 0.07 Ang-(1-7); 20 ng. Blood flow in the implants as determined by t1/2 values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation) showed that Ang II stimulated angiogenesis, whereas Ang-(1-7) inhibited it. The proliferative activity of the sponge-induced fibrovascular tissue was enhanced by Ang II and diminished by Ang-(1-7). These results show the pro-versus anti-angiogenic effects of these angiotensin molecules, providing evidence for their opposing effects on vascular tissue growth and wound healing in vivo.

Effects of the PAF receptor antagonist UK74505 on local and remote reperfusion injuries following ischaemia of the superior mesenteric artery in the rat

The effects of the long lasting and potent PAF receptor antagonist UK74505 were assessed on the local and remote injuries following ischaemia and reperfusion (I/R) of the superior mesenteric artery (SMA) in rats. In a severe model of ischaemia (120 min) and reperfusion (120) injury, in addition to the local and remote increases in vascular permeability and neutrophil accumulation, there was significant tissue haemorrhage, blood neutropenia, systemic hypotension and elevated local and systemic TNF‐α levels. Post‐ischaemic treatment with the selectin blocker fucoidin (10 mg kg−1) prevented neutrophil accumulation in tissue and, in consequence, all the local and systemic injuries following severe I/R. Treatment with an optimal dose of UK74505 (1 mg kg−1) also reversed local and remote neutrophil accumulation, increases in vascular permeability and intestinal haemorrhage. UK74505 partially inhibited blood neutropenia and reperfusion‐induced hypotension. Interestingly, both fucoidin and UK74505 prevented the local, but not systemic, increases of TNF‐α levels following severe I/R injury, demonstrating an important role of migrating cells for the local production of TNF‐α. However, the results do not support a role for PAF as an intermediate molecule in the production of systemic TNF‐α. The beneficial effects of UK74505 and other PAF receptor antagonists in models of I/R injury in animals and the safety of UK74505 use in man warrant further investigations of the use of this drug as preventive measure for I/R injury in humans.

Atorvastatin inhibits inflammatory angiogenesis in mice through down regulation of VEGF, TNF-α and TGF-β1

While compelling evidence indicates beneficial effects of statins on inflammatory processes, besides their cholesterol-lowering activities, the actions on angiogenesis are less clear-cut. Our aim was to investigate the effects of atorvastatin on key components of inflammatory angiogenesis in the murine sponge model. Polyester-polyurethane sponges, used as a framework for fibrovascular tissue growth, were implanted in Swiss mice. Atorvastatin (0.6, 3 mg/kg/day) was given orally for 8 days in drinking water. The implants collected at day 9 postimplantation were processed for the assessment of hemoglobin, myeloperoxidase (MPO), N-acetylglucosaminidase (NAG) and collagen. Relevant inflammatory, angiogenic and fibrogenic cytokines were also determined. Atorvastatin treatment resulted in significant decrease in sponge vascularization (Hb content) and in VEGF levels at both doses. Neutrophil influx (MPO activity) was not affected by the compound whereas macrophage recruitment (NAG activity) was inhibited, suggesting a degree of selectivity by atorvastatin for this cell population. The level of CCL2 (MCP1-JE) was decreased only with 0.6 mg/kg. Atorvastatin was also able to reduce collagen deposition and the levels of transforming growth factor (TGF-beta1) intraimplant, dose-dependently. The inhibitory function of atorvastatin on multiple parameters of main components of inflammatory angiogenesis revealed in this study is clearly associated with the modulatory effects of HMG-CoA reductase on VEGF, TNF-alpha and TGF-beta1 production.

Glucose-induced inhibition of angiogenesis in the rat sponge granuloma is prevented by aminoguanidine

Angiogenesis and granulation tissue formation that occur following subcutaneous implantation of sponge implants in nondiabetic rats were inhibited by topical administration of D-glucose (22 mM). The healing impairment induced by glucose was analogous to healing failures associated with diabetes. Angiogenesis has been determined by measuring hemoglobin content in the implants, correlated with histological evidence of cellular infiltration and granulation tissue formation. The amount of hemoglobin detected in the glucose-treated implants was significantly lower (0.06+/-0.005 g/dl) than the amount in the controls that received glucose 5 mM (0.12+/-0.012 g/dl), saline (0.10+/-0.006 g/dl) or mannitol (0.086+/-0.007 g/dl). Parallel histological studies corroborated the biochemical findings. Daily intraperitoneal injection of aminoguanidine (AG, 50 mg/kg) prevented glucose-induced inhibition of neovascularization and cellular infiltration in the sponge granuloma. Our results show the direct inhibitory effect of high glucose in the development of granulation tissue and indicate that it may be associated with nonenzymatic glycation of key components of the healing process in the rat sponge granuloma.

Angiotensin-ll-lnduced Angiogenesis in Sponge Implants in Mice

Stimulators of angiogenesis hold potential in promoting the development of collateral circulation in ischaemic tissue and accelerating would healing, but promote pathological vasoformation in angiogenesis-dependent diseases (solid tumours, atherosclerosis). The renin-angiotensin system is implicated in both beneficial angiogenesis and pathological vascular growth. We investigated the angiogenic activity of angiotensin II (AII) in a sponge implant model in mice; this peptide enhanced angiogenesis, as well as glycosaminoglycan (GAG, chondroitin sulfate proteoglycan) and protein synthesis in sponge matrix in mice in a dose-dependent fashion. Extensive angiogenesis was achieved with AII (1 microgram), which gave no significant increase in wet weight and protein and only a small effect on GAG. In the implants treated with AII (2 micrograms) no further increase in angiogenesis was observed, whereas a marked effect was shown in wet weight (326 +/- 15 vs. 424 +/- 27 mg), total protein (18 +/- 1 vs. 25 +/- 1 micrograms/ww) and GAG (98 +/- 10 vs. 160 +/- 13 ng/ww). The local blood flow has been determined by measuring the washout rate of 133Xe injected into the implants, correlated with histological evidence of vessel growth. This model of angiogenesis has allowed sequential studies of fibrovascular tissue infiltration simultaneously with histological and biochemical parameters of angiogenesis.

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.

Mechanisms of wound healing responses in lupus-prone New Zealand White mouse strain

Host responses to synthetic implants are analogous to healing, the process of repair that follows injury. Normally, the processes of wound healing follow well‐established patterns but conditions such as autoimmune diseases profoundly affect tissue repair. We have analyzed sponge‐induced wound healing responses in lupus‐prone New Zealand White and control (Balb/c) mouse strains by measuring inflammation, extracellular matrix deposition, angiogenesis, and cytokine production in polyether–polyurethane sponge implanted subcutaneously in male mice of these two strains. Although there was no difference in the gross appearance of the implants, further analysis of the wound healing responses, induced from 7 to 21 days post implantation, disclosed important differences between the New Zealand White and Balb/c strains. The intensity of inflammation (circulating tumor necrosis factor‐α and inflammatory leukocytes levels) was lower but implant fibrosis (collagen and transforming growth factor‐β1) was higher in New Zealand White, compared with Balb/c mice. Angiogenesis (hemoglobin, vascular endothelial growth factor, and vascularity) in New Zealand White implants peaked earlier than in Balb/c mice. In conclusion, we have shown that wound healing responses are clearly different in this strain of lupus‐prone mice and suggest that this pattern of repair was critically influenced by impaired inflammation and accelerated angiogenesis in the New Zealand White strain.

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.