Angiopreventive versus angiopromoting effects of allopurinol in the murine sponge model.

Recent data has indicated that, besides its classical therapeutic indication in hyperurecemia and gout, xanthine oxidase inhibitors can be used to various forms of ischemia and other types of tissue and vascular injuries. We tested the hypothesis that allopurinol, an inhibitor of xanthine oxidase (XO), might modulate acute and/or chronic inflammatory angiogenesis induced by subcutaneous implantation of synthetic matrix in mice. C57/BL6 male mice (6-7 weeks) were implanted with polyether-polyurethane sponge discs. The animals received by oral gavage 1.0mg/kg of allopurinol for six consecutive days in two treatment regimen. In the first series of experiments, the treatment was initiated 24h post-implantation and the implants were removed at day 7 post-implantation. For the assessment of the effect of the compound on chronic inflammation, the treatment was initiated at day 8 post-implantation and the implants removed 14days post-implantation. Angiogenesis as determined by hemoglobin content, VEGF levels and number of vessels intraimplant, and inflammation (myeloperoxidase -MPO, n-acetyl-ß-d-glucosaminidase -NAG, TNF-α and CCL2 levels) were reduced by allopurinol treatment in acute phase. Similarly, the treatment inhibited nitric oxide and H2O2 production. However, fibrogenesis determined by collagen deposition and levels of TGF-ß1 increased in the implants after allopurinol treatment. In marked contrast with the effects when the treatment initiated 24h post-implantation, allopurinol increased angiogenesis and inflammation but reduced collagen and TGF-ß1 levels intra-implant, when the treatment was started during the chronic inflammatory process. The dual effects of allopurinol described here, extend its range of actions as a potential agent able to modulate the components of the fibrovascular tissue present in both physiological (healing processes) as well as in chronic fibroproliferative diseases. These modulatory effects depended on the phase at which the treatment was initiated.

Distinct types of tumors exhibit differential grade of inflammation and angiogenesis in mice.

Inflammation, angiogenesis and cytokine production are common features of almost, if not all tumors. However, the extent of these processes induced by different types of tumors has not been evaluated. We investigated the growth pattern of the experimental metastatic tumors, B16F10 melanoma, CT26.WT colon and 4T1 mammary cells inoculated in the flank of syngeneic mice and determined the degree of inflammation, angiogenesis, and production level of pro-inflammatory and pro-angiogenic cytokines within the tumors. In addition, we have analyzed vascular changes in the interface between the tumors and the adjacent cutaneous tissue and levels of relevant pro-inflammatory and pro-angiogenic cytokines systemically. The weight of tumors 15 days post-inoculation of 10(6) cells was markedly different. Melanomas were 2 and 10-fold heavier than colon and mammary tumors, respectively. Locally, CT26.WT tumor cells induced more vessels in cutaneous tissue adjacent to the tumors but systemically, the plasma levels of VEGF were higher (approximately 2-fold) in 4T1 tumor-bearing mice compared with the other two tumors. Mammary tumors presented the most prominent inflammatory content as assessed by a range of markers (inflammatory enzymes and cytokines). The vascular index, as determined by the intra-tumor amount of hemoglobin and number of vessels in hot spot areas, was also higher (approximately 2-fold) in melanomas compared with the other two tumors. These findings showing that distinct tumor types determine differential grade of inflammation, angiogenesis and host interaction in mice may provide new insights to tailor differential therapeutic approach based on the status of tumor biomarkers.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor.

BACKGROUND AND PURPOSE: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.

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.

Atorvastatin inhibits inflammatory angiogenesis in mice through down regulation of VEGF, TNF-alpha and TGF-beta1.

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.

Tumor growth, angiogenesis and inflammation in mice lacking receptors for platelet activating factor (PAF).

Tumor growth is associated with angiogenesis and inflammation and the endogenous lipid, platelet activating factor (PAF), is a pro-inflammatory and pro-angiogenic mediator. We therefore measured tumor growth, angiogenesis and inflammation in normal (WT) mice and those lacking the receptor for PAF, through gene deletion (PAFR-KO). Growth of solid tumors derived from colon 26 cells was not altered but that from Ehrlich cells was markedly (5-fold) increased in the PAFR-KO mice, relative to the WT strain. Angiogenesis, as tumor content of VEGF or hemoglobin, was increased in both tumors from the mutant strain. Inflammation, as neutrophil and macrophage accumulation and chemokine (CXCL2 and CCL2) content of tumors, was decreased or unchanged in the tumors implying an overall decrease in the inflammatory response in the PAFR-KO strain. We also assessed growth of the Ehrlich tumor in its ascites form, after i.p. injection. Here growth (ascites volume) was inhibited by about 30%, but neutrophil and macrophage numbers were increased in the ascites fluid from the PAFR-KO mice. Angiogenesis in the peritoneal wall, which is not invaded by the tumor cells, was increased but leukocyte infiltration decreased in the mutant strain. Our results show, unexpectedly, that tumor-induced angiogenesis was increased in mice lacking response to PAF, from which we infer that in normal (WT) mice, PAF is anti-angiogenic. Further, although growth was still associated with angiogenesis in PAFR-KO mice, growth was not correlated with inflammation (leukocyte accumulation).

Angiogenesis and inflammation in skeletal muscle in response to ascites tumor in mice.

This study addresses the interaction between Ehrlich ascites tumor and skeletal abdominal muscle, presenting quantitative analysis of ascites-induced angiogenesis and inflammation in this tissue of mice bearing-tumor. Time-dependent changes in the muscle (cellular activity, angiogenesis, inflammation and cytokines production) were assessed by morphometric, functional, and biochemical parameters at days 1, 4 and 8 after i.p. inoculation of Ehrlich tumor cells (2.5 x 10(7)). The number of cells stained with AgNOR technique (argyrophilic nucleolar organizer region) in the muscle, together with MTS assay used as markers of cellular activity increased progressively in parallel with the out flow rate of sodium fluorescein (blood flow index), hemoglobin content (vascular index) and VEGF production. Likewise, the inflammatory process in the muscle, as assessed by myeloperoxidase (MPO) and n-acethylglucosaminidase (NAG) activities and the levels of the chemokines, keratinocyte-derived chemokine (CXC1-3/KC) and macrophage-chemoattractant protein (CCL2/MCP-1) increased with tumor development. The combination of techniques used to describe angiogenesis and inflammation in a muscle model system has proved to be suited for quantitative measurements of microvascular changes and cellular infiltration occurring in the abdominal muscle wall of ascites-bearing mice. This study holds potential for investigating events and mechanisms associated with skeletal muscle response to neoplasic stimulus.

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.

Impaired inflammatory angiogenesis, but not leukocyte influx, in mice lacking TNFR1.

The majority of biological responses classically attributed to tumor necrosis factor alpha (TNF-alpha) is mediated by p55 receptor (TNFR1). Here, we aimed to clarify the biological role of TNFR1-mediated signals in an in vivo inflammatory angiogenesis model. Polyester-polyurethane sponges, used as a framework for tissue growth, were implanted in C57Bl/6 mice. These implants were collected at days 1, 7, and 14 post-implant for enzyme-linked immunosorbent assay or at days 7 and 14 for hemoglobin, myeloperoxidase, and N-acetylglucosaminidase measurements, used as indexes for angiogenesis, neutrophil, and macrophage accumulation, respectively. In TNFR1-deficient C57Bl/6 mice, there was a significant decrease in sponge vascularization but not in late inflammatory cell influx. It is interesting that levels of vascular endothelial growth factor were significantly lower in TNFR1-deficient than in wild-type mice at days 1 and 7. Levels of angiogenic chemokines, CC chemokine ligand 2/murine homologue of monocyte chemoattractant protein-1 and CXC chemokine ligand 1-3/keratinocyte-derived chemokine, were significantly lower in TNFR1-deficient mice at days 1 and 7 after implantation, respectively. These observations suggest that TNFR1-mediated signals have a critical role in sponge-induced angiogenesis, possibly by influencing the effector state of inflammatory cells and hence, modulating the angiogenic molecular network.

Production and in vivo effects of chemokines CXCL1-3/KC and CCL2/JE in a model of inflammatory angiogenesis in mice.

OBJECTIVE: Using the murine sponge model, we investigated the temporal relationship between angiogenesis, leukocyte accumulation and endogenous generation of the pro-inflammatory chemokines CXCL1-3/KC and CCL2/JE. Furthermore, the effects of exogenous administration of these chemokines were studied. METHODS: Angiogenesis in the implants was assessed by measuring the hemoglobin content (vascular index) and leukocyte accumulation quantified by evaluating MPO and NAG enzyme activities. RESULTS: A progressive increase in hemoglobin content and in enzymatic activities was observed during the whole period. The levels of CXCL1-3/KC and CCL2/JE in the implants peaked at days 7 and 1, respectively. Exogenous administration of CXCL1-3/KC (100 ng/day intra-implant) applied at days 1-3 resulted in increased neovascularization and macrophage accumulation. Intra-implant injections of CCL2/JE (100 ng/day) also resulted in increased angiogenesis and macrophage accumulation. CONCLUSIONS: These results demonstrated that the chemokines, CXCL1-3/KC and CCL2/JE, are generated within the sponge compartment and that neovascularization and inflammatory cells influx can be modulated by exogenous administration of the chemokines.

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.

Mechanisms of angiotensin-(1-7)-induced inhibition of angiogenesis.

Angiotensin-(1-7) [ANG-(1-7)], an endogenous bioactive peptide constituent of the renin-angiotensin system, acts as an inhibitory growth factor in vitro and in vivo. In this study, we evaluated whether the antiangiogenic effect of ANG-(1-7) in the mouse sponge model of angiogenesis might be receptor mediated and involved in the release of nitric oxide (NO). The hemoglobin content (microg/mg wet tissue) of 7-day-old sponge implants was used as an index of the vascularization and showed that daily injections of ANG-(1-7) (20 ng) inhibited significantly the angiogenesis in the implants relative to the saline-treated group. The specific receptor antagonist D-Ala(7)-ANG-(1-7); A-779 prevented ANG-(1-7)-induced inhibition of angiogenesis. The antiangiogenic effect was also abolished by pretreatment with NO synthase inhibitors aminoguanidine (1 mg/ml) or N(G)-nitro-L-arginine methyl ester (0.3 mg/ml). Selective AT1 and AT2 angiotensin-receptor antagonists and an angiotensin-converting enzyme inhibitor, in combination with ANG-(1-7) or alone, did not alter angiogenesis in the implants. These results establish that the regulation of the vascular tissue growth by ANG-(1-7) is associated with NO release by activation of an angiotensin receptor distinct from AT1 and AT2.

Sponge implant model of angiogenesis.

The process of capillary growth, angiogenesis, is an integral part of wound healing and repair mechanisms. When it occurs during these conditions, it is tightly controlled and strictly delimited. However, in tumor growth and in a variety of vascular diseases, unrestrained angiogenesis can contribute significantly to the pathology and persistence of these manifestations (1). Research on angiogenesis was initiated with the development of several bioassays that have permitted direct observations of the microvasculature in the living animal. The bioassays have been used for a variety of purposes; for example, to detect angiogenesis activity in malignant and normal cells and tissue, to screen purified test substances for angiogenic activity, and to elucidate the cellular events that accompany vessel growth. The response observed after the introduction of an appropriate stimulus such as mechanical injury or injection of neoplastic tissue implants has allowed the cataloging of the main events of the angiogenic cascade as well as the characterization of pro- and antiangiogenic factors.

Assessment of angiogenesis and tumor growth in conscious mice by a fluorimetric method.

Angiogenesis and tumor growth in conscious mice have been determined using the kinetics of appearance of a fluorescent tracer in the bloodstream after application to subcutaneously implanted sponges bearing tumor cells. The functional parameter expressed in terms of half-time (t(1/2); time taken for the fluorescence to reach 50% of the peak in the systemic circulation), which is inversely proportional to blood flow, showed that in the tumor-free implants t(1/2) values decreased from 11.55 +/- 1 min at day 1 to 5.7 +/- 0. 44 min by day 14. In the tumor-bearing implants, this process was accelerated and maximum vascularization was achieved by day 7 (3 days after tumor cell inoculation). Increases in t(1/2) values were observed at days 10 and 14, which paralleled the tumor growth as indicated by wet weight. The hemoglobin content (microg Hb/mg wet weight) in the tumor-free group increased during the 14-day period. In contrast, in the tumor-bearing implants. Hb concentration decreased per unit of tissue weight. Dexamethasone treatment for 13 days prevented fibrovascular tissue infiltration in tumor-free implants, but was unable to delay tumor growth, indicating that this procedure can be used to exclude the inflammatory reaction induced by the implantation technique, thus allowing tumor angiogenesis to be studied without the confounding influence of the host inflammatory cells. The results of our experimental observation indicate the suitability of this combination of techniques for analyzing angiogenesis induced by tumor cells and several hemodynamic features of Ehrlich tumor growth in awake animals.

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-alpha 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-alpha levels following severe I/R injury, demonstrating an important role of migrating cells for the local production of TNF-alpha. However, the results do not support a role for PAF as an intermediate molecule in the production of systemic TNF-alpha. 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.

Immunization by subcutaneous implants of polyester-polyurethane sponges coupled with antigen

A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 µg. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 µg in Al(OH)3 or 100 µg in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.

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.

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.