Matrigel cytometry: a novel method for quantifying angiogenesis in vivo.

Many of the current in vivo methods to evaluate angiogenesis are poorly quantifiable. Recently, the Matrigel plug assay has become the method of choice in many studies involving in vivo testing for angiogenesis. When known angiogenic factors are mixed with Matrigel and injected subcutaneously into mice, endothelial cells migrate into the gel plug. These endothelial cells form vessel-like structures, a process that mimics the formation of capillary networks. Here, we present a modification of the traditional Matrigel assay with improved method to quantify the amount of endothelial cells that incorporate into the plug. The removed plugs were subjected to a mild protease treatment, yielding intact cells. The liberated cells were then stained using an endothelial cell-specific markers, and counted by flow cytometry. This novel combination of FACS analysis with the traditional Matrigel assay improves the ability to quantify in vivo angiogenesis, and for the first time enables to determine the number of migrating and proliferating endothelial cells which reflects the angiogenesis rate.

Mechanism of action of thalidomide and 3-aminothalidomide in multiple myeloma.

We have explored the mechanism of the antiangiogenic effects of thalidomide by structure-activity studies. These investigations revealed that angiogenesis inhibition correlates with teratogenicity but not with tumor necrosis factor-alpha (TFA-alpha) inhibition. Additionally, one analog of thalidomide, 3-aminothalidomide, exhibited an unusual capacity to directly inhibit myeloma cell proliferation. This activity did not correlate with TNF-alpha inhibition. Thus 3-aminothalidomide was found to inhibit multiple myeloma through effects on both the tumor and vascular compartment. The effects of an inhibitor of both the tumor and vascular compartments of a tumor on tumor growth may be synergistic.

Rapid ocular angiogenic control via naked DNA delivery to cornea.

PURPOSE: To determine the efficacy and safety of naked plasmid gene therapy to the corneal stroma and epithelium. METHODS: Naked plasmid DNA was injected under pressure into the cornea of mice. The expression of genes coding for beta galactosidase (beta-gal), enhanced green fluorescent protein (EGFP), vascular endothelial growth factor (VEGF), and soluble Flt-1 (s-Flt) was recorded and measured with regard to dose, time course, and bioactivity. RESULTS: LacZ gene expression of the protein beta-gal was demonstrated as early as 1 hour, with expression persisting for 10 days. Plasmid-injected corneas remained clear and free of inflammation. EGFP was bicistronically expressed with VEGF to demonstrate the practicality of simultaneous in vivo analysis of gene expression and growth factor bioactivity. Corneal injection of a plasmid containing VEGF cDNA induced corneal and anterior chamber neovascularization. Moreover, corneal injection of plasmid containing the cDNA for the soluble form of the VEGF receptor Flt-1 effectively prevented corneal neovascularization. CONCLUSIONS: The cornea is readily accessible for gene therapy in the laboratory and in the clinic. The method described is safe, effective, titratable, and easily monitored. Naked DNA delivery to the cornea has the potential to alter the treatment of a wide variety of corneal and anterior segment diseases.

Strain-dependent anterior segment neovascularization following intravitreal gene transfer of basic fibroblast growth factor (bFGF).

BACKGROUND: A promising strategy for delaying death of photoreceptor cells in retinal degenerative disease is to support survival of these cells through intraocular delivery of growth/neurotrophic factors. One factor that has received great attention is basic fibroblast growth factor (bFGF; fgf-2), a known stimulator of angiogenesis. We evaluated the potential for neovascularization induced by adenovirus-mediated intravitreal delivery of bFGF. METHODS: Recombinant adenoviruses carrying the low molecular weight (18 kD) or the high molecular weight (22, 23 and 24 kD) forms of human bFGF, driven by the cytomegalovirus (CMV) promoter/enhancer, were prepared. Viruses were delivered to eyes of different strains of mice and rats through intravitreal injection. Contralateral eyes were injected with control virus carrying a reporter gene [green fluorescent protein (GFP) or lacZ]. Transgene expression was assessed by Western analysis and by immunohistochemistry. Neovascularization was evaluated in vivo and histologically at termination of the experiment. RESULTS: Adenovirus-mediated delivery of the 18 kD form of bFGF resulted in anterior segment neovascularization in a strain-dependent fashion. Generation of new blood vessels was not observed after injection of the higher molecular weight forms of bFGF or of control solutions. CONCLUSION: The low molecular weight form (18 kD) (but not the high molecular weight forms) of bFGF drives angiogenic response in the anterior segment of specific strains of mice. Genetic modifiers may contribute to and/or prevent neovascularization induced by bFGF.

Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium.

Choroidal neovascularization in age-related macular degeneration is a frequent and poorly treatable cause of vision loss in elderly Caucasians. This choroidal neovascularization has been associated with the expression of vascular endothelial growth factor (VEGF). In current animal models choroidal neovascularization is induced by subretinal injection of growth factors or vectors encoding growth factors such as VEGF, or by disruption of the Bruch's membrane/retinal pigment epithelium complex with laser treatment. We wished to establish a transgenic murine model of age-related macular degeneration, in which the overexpression of VEGF by the retinal pigment epithelium induces choroidal neovascularization. A construct consisting of a tissue-specific murine retinal pigment epithelium promoter (RPE(65) promoter) coupled to murine VEGF(164) cDNA with a rabbit beta-globin-3' UTR was introduced into the genome of albino mice. Transgene mRNA was expressed in the retinal pigment epithelium at all ages peaking at 4 months. The expression of VEGF protein was increased in both the retinal pigment epithelium and choroid. An increase of intravascular adherent leukocytes and vessel leakage was observed. Histopathology revealed intrachoroidal neovascularization that did not penetrate through an intact Bruch's membrane. These results support the hypothesis that additional insults to the integrity of Bruch's membrane are required to induce growth of choroidal vessels into the subretinal space as seen in age-related macular degeneration. This model may be useful to screen for inhibitors of choroidal vessel growth.

Cytochalasin E, an epoxide containing Aspergillus-derived fungal metabolite, inhibits angiogenesis and tumor growth.

Several previously identified inhibitors of angiogenesis have been epoxide-containing fungus-derived metabolites. We therefore hypothesized that novel epoxide-containing low molecular weight compounds structurally resembling known antiangiogenic agents may also exhibit antiangiogenic activity. Cytochalasin E was found to be a potent and selective inhibitor of bovine capillary endothelial (BCE) cell proliferation. Cytochalasin E differed from other cytochalasins by the presence of an epoxide. The epoxide was required for activity, because acid-catalyzed hydrolysis of the epoxide abrogated the specificity and potency of cytochalasin E. Phalloidin staining indicated that disruption of actin stress fibers by cytochalasin E occurred only at relatively high concentrations. Lower concentrations of cytochalasin E preferentially inhibited BCE cell proliferation without disrupting actin stress fibers. In vivo, cytochalasin E inhibited angiogenesis induced by basic fibroblast growth factor by 40% to 50% in the mouse cornea assay and inhibited the growth of Lewis lung tumors by approximately 72%. Cytochalasin E is a potent antiangiogenic agent that may hold promise for the treatment of cancer and other types of pathologic angiogenesis.

Genetic heterogeneity of angiogenesis in mice.

Many diseases, including cancer, are dependent on the growth of new blood vessels, a process known as angiogenesis. Differences in an individual's ability to grow new blood vessels may influence the rate of progression of these diseases. Here we show that different strains of inbred mice have an approximately 10-fold range of response to growth factor-stimulated angiogenesis in the corneal micropocket assay. The in vitro migratory activity of endothelial cells from aortic rings of selected strains correlated with the in vivo responsiveness. Further, a differential sensitivity to angiogenesis inhibitors was seen between strains, with one strain demonstrating resistance to both TNP-470 and thalidomide. These results suggest the presence of genetic factors that control individual angiogenic potential.

Treatment of the Kasabach-Merritt syndrome with pegylated recombinant human megakaryocyte growth and development factor in mice: elevated platelet counts, prolonged survival, and tumor growth inhibition.

Kasabach-Merritt Syndrome (KMS) is seen in children with large vascular tumors. KMS is characterized by very low platelet counts and a consumption of coagulation factors causing life-threatening complications. It has been proposed that thrombopenia in these patients is caused by intratumoral trapping of platelets. The truncated form of the cMpl-receptor ligand thrombopoietin, pegylated human megakaryocyte growth and development factor (Peg-rHuMGDF), is an agent that stimulates platelet production. We hypothesized that stimulation of the platelet production would prevent the life-threatening complications of patients with KMS owing to low platelet counts. In a mouse model of KMS, with tumors derived from a hemangioendothelioma cell line, we studied the effect of Peg-rHuMGDF. Treatment with Peg-rHuMGDF (10 microg/kg/day intraperitoneally) increased platelet counts by 7-8-fold compared with control tumor-bearing mice after 11 d of treatment (p < 0.001, n = 8). Survival was significantly increased, with 50% of treated animals alive at 1 mo versus 0% in untreated controls. Interestingly, we also observed an inhibition of tumor growth by 75% (p < 0.001, n = 8). Hematoxylin and eosin staining showed fresh fibrin clots in the treated tumors, suggesting that higher platelet counts caused intravascular thrombosis of tumor vessels. We conclude that increased platelet production in this model of KMS resulted in an antivascular tumor effect via platelet trapping. Further, we propose that thrombopoietin may be of critical value in preventing life-threatening complications from KMS.

Combination oral antiangiogenic therapy with thalidomide and sulindac inhibits tumour growth in rabbits.

Neovascularization facilitates tumour growth and metastasis formation. In our laboratory, we attempt to identify clinically available oral efficacious drugs for antiangiogenic activity. Here, we report which non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit corneal neovascularization, induced by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). This antiangiogenic activity may contribute to the known effects of NSAIDs on gastric ulcers, polyps and tumours. We found that sulindac was one of the most potent antiangiogenic NSAIDs, inhibiting bFGF-induced neovascularization by 50% and VEGF-induced neovascularization by 55%. Previously, we reported that thalidomide inhibited growth factor-induced corneal neovascularization. When we combined sulindac with thalidomide, we found a significantly increased inhibition of bFGF- or VEGF-induced corneal neovascularization (by 63% or 74% respectively) compared with either agent alone (P < 0.01). Because of this strong antiangiogenic effect, we tested the oral combination of thalidomide and sulindac for its ability to inhibit the growth of V2 carcinoma in rabbits. Oral treatment of thalidomide or sulindac alone inhibited tumour growth by 55% and 35% respectively. When given together, the growth of the V2 carcinoma was inhibited by 75%. Our results indicated that oral antiangiogenic combination therapy with thalidomide and sulindac may be a useful non-toxic treatment for cancer.

The antiangiogenic agents TNP-470 and 2-methoxyestradiol inhibit the growth of angiosarcoma in mice.

BACKGROUND: Endothelial malignancies, such as angiosarcoma and hemangioendothelioma, are often resistant to chemotherapy and surgery, and may result in death. Improved means of therapy are needed for these disorders. OBJECTIVE: We wanted to determine whether angiosarcoma can be treated with angiogenesis inhibitors in mice. METHODS: Mice were inoculated with a cell line that gives rise to angiosarcoma and were treated with the angiogenesis inhibitors 2-methoxyestradiol and TNP-470. Response to therapy was monitored by measurement of tumors. RESULTS: TNP-470 caused an 84% reduction in tumor size, and 2-methoxyestradiol caused a 68% reduction in tumor size. CONCLUSION: Angiogenesis inhibitors are highly effective in treatment of angiosarcoma in mice. Clinical trials of these agents in humans with angiosarcoma and hemangioendothelioma are warranted.

Long-term remission of Crohn's disease treated with thalidomide: a seminal case report.

A 31-year-old female with severe Crohn's disease for 15 years who had been treated with corticosteroids and 6-mercaptopurine, was treated with thalidomide initially for erythema nodosum. While on thalidomide all symptoms of Crohn's disease disappeared and she was able to discontinue all other drugs. At this writing she has been on thalidomide as sole therapy for over 4 years with the exception of a 5-week hiatus, during which time her symptoms recurred, but again disappeared after resumption of thalidomide therapy. This case suggests that thalidomide may be a useful therapy for Crohn's disease and provides impetus for a clinical trial of thalidomide for Crohn's disease.

Involvement of platelets in tumour angiogenesis?

Preclinical and clinical research show that tumour growth is dependent on angiogenesis. Activation of the coagulation cascade is commonly found in patients with cancer. We propose that platelets contribute to tumour-induced angiogenesis. The basis of our hypothesis is that platelets are a rich source of stimulators and inhibitors of angiogenesis and their interaction with the endothelium. Presumably, the antithrombotic state of normal endothelium is disturbed by endothelial stimuli derived from tumour cells. This hypothesis may explain the suggested clinical benefits of anticoagulants in cancer and implies that targeting of platelet interaction with tumour vasculature will inhibit angiogenesis.

Regulation of vascular endothelial growth factor expression by insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) levels are correlated with retinal ischemia-associated intraocular neovascularization in humans. Since VEGF is required for iris and retinal neovascularization in animal models of retinal ischemia, we tested whether IGF-I could act as an indirect angiogenic factor by increasing VEGF gene expression. IGF-I increased retinal pigment epithelial (RPE) cell VEGF mRNA in a concentration-dependent manner with an EC50 of 7 nmol/1 (53.6 ng/ml). RPE and bovine smooth muscle cells exposed to 50 nmol/l (383 ng/m1) IGF-I achieved peak VEGF mRNA expression within 2 h. IGF-I-treated RPE cells increased VEGF protein levels in conditioned media and stimulated capillary endothelial cell proliferation. Blockade of the IGF-I receptor with a neutralizing antibody abrogated the VEGF increases in RPE cells. Further, hypoxia-mediated and IGF-I-mediated increases in VEGF mRNA and protein levels were additive in RPE cells, and the hypoxia-induced VEGF increases were independent of endogenous IGF-I. VEGF promoter activity was enhanced by IGF-I in RPE cells, but VEGF transcript half-life was unaltered. In summary, the supplementation of RPE and smooth muscle cell cultures with IGF-I at 5-100 nmol/l increased VEGF mRNA and secreted protein levels. The VEGF increases in RPE cells occurred primarily through enhanced transcription of the VEGF gene and via the IGF-I receptor. Elevated IGF-I levels may promote neovascularization through increased retinal VEGF gene expression.

Effects of thalidomide and related metabolites in a mouse corneal model of neovascularization.

Thalidomide, when administered orally, is an inhibitor of angiogenesis in the basic fibroblast growth factor (bFGF)-induced rabbit cornea micropocket assay. We now show in the mouse that thalidomide given intraperitoneally but not orally significantly inhibits bFGF-induced and vascular endothelial growth factor (VEGF)-induced corneal neovascularization. We further demonstrate that this inhibition is independent from thalidomide's ability to suppress tumor necrosis factor-alpha (TNF-alpha) production. Experiments examining thalidomide's enantiomers reveal-that the S(-)-enantiomer has the strongest antiangiogenic activity in VEGF-induced and bFGF-induced corneal neovascularization. Structure activity studies suggest that thalidomide's anti-angiogenic activity is related to the open ring metabolites resulting from hydrolysis. Together these data support a correlation between thalidomide's antiangiogenic and teratogenic activities.

Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470.

Angiogenesis, the growth of new blood vessels, occurs normally in female reproductive organs. We tested the hypothesis that angiogenesis inhibition may affect fertility by studying the reproductive system in either pregnant or nonpregnant cycling mice after treatment with the angiogenesis inhibitor AGM-1470. Administration of AGM-1470 to pregnant mice resulted in complete failure of embryonic growth due to interference with decidualization, placental and yolk sac formation, and embryonic vascular development. When nonpregnant cycling female mice were chronically treated with AGM-1470, inhibition of endometrial maturation and corpora lutea was observed. These data suggest that processes in reproduction can be controlled through angiogenesis inhibition.

Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite which disrupts microtubule function, has been shown to inhibit proliferating cells in vitro and suppress certain murine tumors in vivo. In vitro screening has determined that breast cancer cell lines are most sensitive to inhibition by 2-ME. Additionally, 2-ME has been shown to inhibit angiogenesis in vitro. We tested whether 2-ME suppresses cytokine-induced angiogenesis in vivo and inhibits growth of a human breast carcinoma in severe combined immunodeficient mice. A model of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-induced corneal neovascularization in C57BL/6 mice was used to evaluate the antiangiogenic effects of 2-ME and other microtubule inhibitors such as Taxol, vincristine, and colchicine. 2-ME (150 mg/kg p.o., n = 20) inhibited bFGF and VEGF-induced neovascularization by 39% and 54%, respectively. Taxol (6 mg/kg i.p., n = 17) inhibited bFGF and VEGF-induced neovascularization by 45% and 37%, respectively. Vincristine (0.2 mg/kg i.p., n = 8) and colchicine (0.25 mg/kg i.p., n = 8) had no effect. Treatment with 2-ME (75 mg/kg p.o., n = 9) for 1 month suppressed the growth of a human breast carcinoma in mice by 60% without toxicity. Recognition of the antiangiogenic and antitumor properties of 2-ME and Taxol may be crucial in planning clinical applications to angiogenesis-dependent diseases.

New activity of spironolactone. Inhibition of angiogenesis in vitro and in vivo.

BACKGROUND: The formation of new blood vessels (angiogenesis) is a critical component in a variety of pathological settings, including solid tumor growth, macular degeneration, and atherosclerosis. METHODS AND RESULTS: We have found that orally administered spironolactone inhibited the area of angiogenesis induced by basic fibroblast growth factor (bFGF) in a rabbit corneal micropocket assay. Additionally, spironolactone inhibited bFGF- and vascular endothelial growth factor-stimulated capillary endothelial cell proliferation in vitro, inhibited bFGF-stimulated capillary endothelial cell chemotaxis in vitro, and caused avascular zones when placed on the chick chorioallantoic membrane. Experiments analyzing spironolactone metabolites revealed that the major human metabolites 6 beta-hydroxy-7 alpha-thiomethyl spironolactone and canrenoic acid retained antiangiogenic activity. The antiangiogenic activity appears to be unrelated to the antiandrogenic and antimineralocorticoid effects of spironolactone. CONCLUSIONS: These experiments hold promise for the potential use of spironolactone as an orally administered drug for the treatment of many diverse diseases dependent on angiogenesis.

Experimental corneal neovascularisation using sucralfate and basic fibroblast growth factor.

PURPOSE: To develop a non-inflammatory model of both acute and chronic angiogenesis in the rabbit cornea using a known directly angiogenic cytokine. METHODS: Pellets made of the slow-release polymer Hydron (polyhydroxyethylmethacrylate) and containing sucralfate and/or basic fibroblast growth factor (basic-FGF) were implanted into rabbit corneas. The neovascular response to the implantation of pellets containing basic-FGF alone, sucralfate alone or a titration of basic-FGF in the presence of a constant amount of sucralfate was measured. The role of inflammation in the neovascular response was also investigated. RESULTS: The addition of sucralfate to the pellets led to the sustained release of basic-FGF resulting in a predictable and aggressive neovascular response with a low dose of basic-FGF that by itself was unable to elicit neovascularisation. At a dose of 500 ng per pellet, approximately one-third of the surface area of the cornea was vascularised within eight days of implantation. Minimal or no vascularisation occurred with the same dose of basic-FGF without sucralfate. While this dose of basic-FGF induced corneal oedema, only minimal inflammation was observed and the response was unaffected by ionising radiation. A less aggressive though still robust neovascular response with no or only minimal oedema was observed when the dose was lowered to 50 ng of basic-FGF per pellet. Some induced vessels persisted for more than three months. CONCLUSION: This is an inexpensive in vivo model of angiogenesis with the advantages of the neovascularisation being aggressive, predictable, persistent, unassociated with an obvious inflammatory response and induced by the sustained release of an agent known to have a direct stimulatory action on endothelial cells.

A model of angiogenesis in the mouse cornea.

PURPOSE: The study of angiogenesis depends on reliable and reproducible models for the stimulation of a neovascular response. The purpose of this research was to develop such a model of angiogenesis in the mouse cornea. METHODS: Uniformly sized Hydron pellets containing either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) and sucralfate were prepared and implanted into the stroma mouse cornea adjacent to the temporal limbus. RESULTS: Neovascularization of the corneal stroma began on day 3 and was sustained through day 8. The bFGF-induced neovascularization was consistent and dose dependent in C57B1/6, as well as in severe combined immune deficient, beige natural killer cell-deficient, and nude mouse strains. Biomicroscopic and histologic examination of bFGF- and VEGF-induced angiogenesis was notable for the absence of corneal edema or substantial inflammation. CONCLUSIONS: This noninflammatory model of corneal neovascularization is especially advantageous because it is reproducible, economical, and facilitates investigation of angiogenesis in various murine tumor models as well as in genetically defined murine strains.

Interactions of 2-methoxyestradiol, an endogenous mammalian metabolite, with unpolymerized tubulin and with tubulin polymers.

2-Methoxyestradiol (2ME) is an endogenous mammalian catabolite of estradiol with antimitotic activity. Although it is a competitive inhibitor of the binding of colchicine to tubulin, it has unusual effects on glutamate-induced tubulin polymerization. Polymer that was little changed in morphology assembled at a reduced rate and was relatively cold stable. We have now examined interactions of [4-3H]-2ME with unpolymerized tubulin and polymer. The [3H]2ME binds avidly to tubulin even on ice, and it is readily displaced by other colchicine site drugs. An association rate constant on ice of 1.9 x 10(2) M-1s-1 was obtained. Scatchard analysis indicated a single class of binding site and an association equilibrium constant of 5.7 x 10(5) M-1. These values lead to a calculated dissociation rate constant of 3.3 x 10(-4) s-1. In glutamate-induced tubulin assembly, a reaction that requires GTP and leads to the formation of sheets of parallel protofilaments, increasing amounts of [3H]2ME were incorporated into polymer, reaching near-stoichiometry with tubulin at 100 microM 2ME. Equivalent binding of [3H]2ME occurred when the drug was added to preformed polymer, but binding of [3H]2ME to polymer was not readily inhibited by colchicine site drugs. Significant amounts of [3H]2ME were also incorporated into microtubule polymer formed with microtubule-associated proteins, glycerol, or 4-morpholineethanesulfonate buffer, but the stoichiometry was substantially lower than that in the sheet polymer induced by either glutamate or 1,4-piperazineethanesulfonate buffer. The structural differences between the microtubule and sheet polymers leading to these differences in apparent affinity for 2ME are unknown, but presumably interaction of the estrogen metabolite with cellular microtubules has functional significance related to the antimitotic properties of the compound.