Metformin inhibits inflammatory angiogenesis in a murine sponge model.

To investigate the effects of metformin on angiogenesis, on inflammatory cell accumulation and on production of endogenous cytokines in sponge implant in mice. Polyester-polyurethane sponges were implanted in Swiss mice and metformin (40 or 400mg/kg/day) was given orally for six days. The implants collected at day 7 postimplantation were processed for the assessment of hemoglobin (Hb), myeloperoxidase (MPO), N-acetylglucosaminidase (NAG) e collagen used as indexes for angiogenesis, neutrophil and macrophage accumulation and extracellular matrix deposition, respectively. Relevant inflammatory, angiogenic and fibrogenic cytokines were also determined. Metformin treatment attenuated the main components of the fibrovascular tissue, wet weight, vascularization (Hb content), macrophage recruitment (NAG activity), collagen deposition and the levels of transforming growth factor (TGF-beta1) intraimplant. A regulatory function of metformin on multiple parameters of main components of inflammatory angiogenesis has been revealed giving insight into the potential therapeutic underlying the actions of metformin.

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. METHODS: Three anti-angiogenic compounds, clotrimazole (120 mg kg(-1) day(-1)), thalidomide (100 mg kg(-1) day(-1)), and rosiglitazone (10 mg kg(-1) day(-1)), were used to inhibit angiogenesis developing over 9 days, in sponges implanted subcutaneously in Swiss mice. Angiogenesis was assessed by hemoglobin content and by histology. Content of cytokines in implants was measured by specific immunoassays and accumulation of neutrophils or macrophages in implants by measuring myeloperoxidase or N-acetylglucosaminidase activity, respectively. RESULTS: These compounds caused equal inhibition of angiogenesis (about 40%). However, implant levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF-alpha) or the macrophage chemoattractant cytokine, CCL2/MCP-1/JE, and accumulation of macrophages were more variably inhibited. Only the neutrophil chemokine, CXCL2/KC, was inhibited equally by the three compounds, in this model. CONCLUSIONS: Anti-angiogenic effect was most clearly and closely correlated with levels of the chemokine KC. Thus, measurement of the chemokine KC might provide an adequate surrogate marker for the functional process of neovascularization in our model.

Inhibition of inflammatory angiogenesis by distant subcutaneous tumor in mice.

We investigated angiogenesis, inflammatory cells accumulation and endogenous production of cytokines in sponge implants of tumor-bearing mice. Seven days after inoculation of Ehrlich tumor cells (2.5 x 10(6)), sponge discs were implanted subcutaneously in the dorsa of mice to induce the formation of fibrovascular tissue. The implants of tumor-bearing and non tumor-bearing animals were assessed for neovascularization and leukocyte accumulation, together with levels of relevant cytokines, vascular endothelial growth factor VEGF), tumor necrosis factor alpha (TNF-alpha), CXCL1-3/KC and CCL2/JE. In the implants of tumor-bearing animals angiogenesis (assessed by hemoglobin content and VEGF levels in the implants) and leukocyte accumulation (assessed by myeloperoxidase -MPO- and N- acetylglucosaminidase-NAG-enzyme activities) were all significantly less than those in the implants of non tumor-bearing animals. Although the chemokine CXCL1-3/KC was lower in the implants of tumor-bearing animals, the chemokine CCL2/JE was increased in this group. The production of TNF-alpha in the implants was not modified by the presence of the subcutaneous tumor. The combination of the methodologies used in this study has provided a novel approach to investigate the interaction between two distinct proliferating tissues that share common features (angiogenesis, cell recruitment, inflammation) and has shown that the predominant inhibitory effect of a tumor mass over repair process is associated with altered cytokine production.

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 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.

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 t1/2 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 t1/2 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 microgramsHb/w.w vs. 1.8 +/- 0.18 microgramsHb/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.

Sponge-induced angiogenesis in mice and the pharmacological reactivity of the neovasculature quantitated by a fluorimetric method.

Sponge-induced angiogenesis in mice and pharmacological reactivity of the neovasculature have been determined by a fluorimetric method. Pharmacokinetic studies following subcutaneous, intradermal, and intraimplant administration of sodium fluorescein resulted in a biphasic curve from which estimation of t1/2 for absorption and elimination of the dye were possible. Following topical injection of the dye at days 1, 4, 7, 10, and 14 postimplantation, measurement of fluorchrome generated emission in the systemic circulation reflected the development of blood flow in and around the implants and the interaction of the angiogenic site with the systemic circulation. The t1/2 values for the fluorescence peak in the bloodstream decreased steadily from an initial value of 6.41 +/- 0.28 min (avascular implant) to 2.78 +/- 0.23 min in fully vascularized implants (day 14). The reactivity of the neovasculature to ET-1 was dose-dependent and similar to the skin vasculature. By contrast, no reactivity to histamine was detected in the implant blood vessels, whereas it was present in the skin. These results show that the pharmacological response of the neovasculature differs from the response of mature blood vessels. The angiogenic stimulus (bFGF, 300 ng daily) decreased t1/2 for the fluorescence peak, whereas dexamethasone (1 mg/kg) increased it. Parallel histological studies corroborated the functional findings. These observations indicate the suitability of this assay to study angiogenesis, functional and pharmacological characterization of the neovasculature, and the interaction of the angiogenic site with the systemic circulation.