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.

Selectivity of the parkinsonian neurotoxin MPTP: toxic metabolite MPP+ binds to neuromelanin.

Methylphenyltetrahydropyridine (MPTP) selectively destroys neuronal cell bodies in the melanin-containing substantia nigra of humans and other primates. We show that methylphenylpyridine (MPP+), an active metabolite of MPTP which is accumulated intraneuronally by the catecholamine uptake system, binds with high affinity to melanin and neuromelanin. MPP+ bound intracellularly to neuromelanin may be released gradually, resulting in subsequent damage to the neurons of the substantia nigra.

Thyrotropin-releasing hormone improves cardiovascular function in experimental endotoxic and hemorrhagic shock.

Thyrotropin-releasing hormone significantly improved cardiovascular function when it was injected intravenously into conscious rats subjected to experimental endotoxic or hemorrhagic shock. Because thyrotropin-releasing hormone appears to be a "physiologic: opiate antagonist without effects on pain responsiveness, it may provide therapeutic benefits in the treatment of shock or acute hypotension.

Inhibition of angiogenesis in vivo by interleukin 12.

BACKGROUND: In previous animal studies, interleukin 12 (IL 12) was shown to inhibit the growth of a wide spectrum of tumors in vivo but to have no direct effect on tumor cells in vitro. Also, contrary to the expectation of a T-cell-mediated effect, the antitumor activity of IL 12 was not completely abrogated in tests of T-cell-deficient mice. These observations suggest that IL 12 may possess antiangiogenic properties that account for its tumor-inhibitory effects in vivo. PURPOSE: Our goal was to investigate the hypothesis that IL 12 has antiangiogenic properties. METHODS: A model of basic fibroblast growth factor-induced corneal neovascularization in mice was used to evaluate the effects of IL 12 and interferon gamma (IFN gamma) on angiogenesis in vivo. Different strains of male mice, e.g., immunocompetent C57BL/6 mice, severe combined immune-deficient (SCID) mice, natural killer cell-deficient beige mice, and T-cell-deficient nude mice, were treated with IL 12 (1 microgram/day) intraperitoneally for 5 consecutive days. The extent of neovascularization in response to a basic fibroblast growth factor pellet and the inhibition of neovascularization by IL 12 or IFN gamma were assessed by measuring the maximal vessel length and the corneal circumference involved in new blood vessel formation. The antitumor activities of IL 12 and of the angiogenesis inhibitor AGM-1470 were evaluated in Lewis lung carcinoma-bearing mice. In vitro proliferation studies were performed on bovine capillary endothelial cells, mouse pancreatic islet endothelial cells, and mouse hemangioendothelioma cells. RESULTS: IL 12 treatment almost completely inhibited corneal neovascularization in C57BL/6, SCID, and beige mice. This potent suppression of angiogenesis was prevented by the administration of IFN gamma-neutralizing antibodies, suggesting that the suppression was mediated through IFN gamma. In addition, the administration of IFN gamma reproduced the antiangiogenic effects observed during treatment with IL 12. Treatment with IL 12 and AGM-1470 combined did not increase toxicity and showed a trend toward enhanced antitumor efficacy in Lewis lung carcinoma-bearing mice. CONCLUSIONS: IL 12 strongly inhibits neovascularization. This effect is not mediated by a specific cell type of the immune system. Instead, IL 12 has been shown to induce IFN gamma, which, in turn, appears to play a critical role as a mediator of the antiangiogenic effects of IL 12. IMPLICATIONS: Recognition of the mechanisms of the antiangiogenic properties of IL 12 may be crucial in planning its clinical applications, including a possibility of coadministration with other inhibitors of neovascularization.

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.

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.

MPTP: a neurotoxin relevant to the pathophysiology of Parkinson's disease. The 1985 George C. Cotzias lecture.

MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) elicits selective destruction of nigrostriatal dopamine neurons in humans and animals along with clinical symptoms of parkinsonism. Recent studies clarify mechanisms accounting for this neurotoxicity. MPTP binds with high affinity to monoamine oxidase, which transforms it to the pyridinium MPP+ . MPP+ is selectively concentrated by the dopamine neuronal uptake system. In nigral cells, binding by melanin of MPP+ affords a "depot" release mechanism to maintain prolonged high intracellular concentrations sufficient to destroy cells. PC-12 cells provide a model catecholamine cell culture for screening environmentally occurring substances that may be relevant in the etiology of idiopathic Parkinson's disease.

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.

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.

Naloxazone lacks therapeutic effects in endotoxic shock yet blocks the effects of naloxone.

Cardiovascular effects of the high affinity, irreversible opiate antagonist, naloxazone, were investigated in conscious rats subjected to endotoxic shock. Unlike other less selective opiate antagonists such as naloxone, naloxazone failed to block or reverse the hemodynamic effects of endotoxemia. However, naloxazone pretreatment prevented the usual therapeutic effects of naloxone in endotoxic shock. Results indicate that high affinity (mu 1-site) opiate binding is not involved in the pathophysiological actions of endogenous opiates in shock, and suggest that interactions among opioid receptor subtypes may occur in vivo.

Multiple opioid receptors: evidence for mu-delta binding site interactions in endotoxic shock.

Using antagonists with selectivity for the delta (ICI 154,129) and mu (beta-funaltrexamine) binding sites, evidence was obtained to indicate that delta receptors within the brain mediate the endogenous opioid component of endotoxic hypotension. The therapeutic actions of intravenous ICI 154,129 were dose related, with effective doses between 15-60 mg/kg. Evidence for a functional interaction between mu and delta binding sites was obtained: prior occupancy of the mu binding site by beta-funaltrexamine prevented the usual therapeutic response to the delta antagonist ICI 154,129 in endotoxemic rats. These data indicate that mu and delta binding sites may be a part of the same macromolecular complex which interact through allosteric coupling.

Naloxone or TRH fails to improve neurologic deficits in gerbil models of "stroke".

The effects of naloxone or thyrotropin releasing hormone (TRH) upon neurologic outcome were evaluated in gerbil models of cerebral ischemia. Following temporary bilateral carotid occlusion, hypotension was transiently reversed by these endorphin antagonists. However, neither drug altered time to awaken, time to death, or the severity of neurologic signs (ptosis, movement, retracted paws, circling, righting reflexes, seizures, or opisthotonus) when evaluated by a blinded rater. Hot plate escape and roto-rod performance were also unaltered by naloxone or TRH; TRH, but not naloxone, increased respiratory rates. Thus, the transient improvement of cardiorespiratory function produced by these drugs is unrelated to the morbidity and mortality associated with temporary cerebral ischemia in the gerbil. Additional studies evaluating the effects of naloxone or TRH upon neurologic outcome following permanent unilateral carotid occlusion also failed to show any therapeutic effects of these drugs. Both morphine and TRH exacerbated the effects of ischemia. Of gerbils which developed neurologic impairment, the deficit was usually ipsilateral to the occluded carotid. Collectively, these results indicate that neither naloxone nor TRH prevents ischemic deficits in the gerbil. Further studies with different cerebral ischemia models in other species are required to clarify the possible therapeutic effects of these drugs in experimental stroke.

M 154,129, a putative delta antagonist, reverses endotoxic shock without altering morphine analgesia.

Studies were conducted with the putative delta opiate receptor antagonist M 154,129 to evaluate its potential for reversing circulatory shock without altering nociceptive processes. M 154,129 (30 mg/kg iv) did not alter tail flick or hot plate latencies by itself, nor did it alter the antinociceptive effects of morphine (4 mg/kg iv). Following endotoxic shock hypotension in conscious rats, M 154,129 (30 or 60 mg/kg iv) produced a rapid return of arterial pressure to pretreatment levels. These data indicate that circulatory shock may be mediated at delta opioid receptors. Moreover, delta antagonists may have therapeutic benefit in reversing shock without intensification of pain.

Neuromelanin: a role in MPTP-induced neurotoxicity.

Methylphenyltetrahydropyridine (MPTP) selectively destroys melanin-containing neurons in the substantia nigra of humans and other primates. Methylphenylpyridine (MPP+), an active metabolite of MPTP, which is accumulated intraneuronally by the catecholamine uptake system, binds with high affinity to neuromelanin. MPP+ bound intracellularly to neuromelanin may be released gradually, resulting in damage to the neurons of the substantia nigra. Chloroquine, a drug which blocks MPP+ binding to neuromelanin, can protect monkeys from MPTP neurotoxicity.

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.

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.

Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity.

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuropathological and clinical abnormalities in humans, monkeys, and mice that closely resemble idiopathic parkinsonism. N-Methyl-4-phenylpyridine (MPP+), a metabolite of MPTP formed by monoamine oxidase B, is accumulated into striatal and cerebral cortical synaptosomes by the dopamine and norepinephrine uptake systems, respectively, whereas MPTP itself is not accumulated. The potencies of drugs in inhibiting [3H]MPP+ or [3H]dopamine uptake into striatal synaptosomes are very similar, as are potencies in inhibiting [3H]MPP+ or [3H]norepinephrine uptake into cortical synaptosomes. The Km values for [3H]MPP+ uptake are 170 and 65 nM and the Vmax values are 2 and 0.1 nmol/g of tissue per min in rat striatum and cortex, respectively, similar to values for [3H]dopamine uptake, Autoradiography of accumulated [3H]MPP+ in slices of rat brain shows high densities in the caudate-putamen and nucleus accumbens. Furthermore, blockade of dopamine uptake by mazindol prevents MPTP-induced damage to nigrostriatal dopamine neurons, indicating that MPP+ concentration into dopamine neurons explains their selective destruction by MPTP.

Selective labeling of serotonin uptake sites in rat brain by [3H]citalopram contrasted to labeling of multiple sites by [3H]imipramine.

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes [3H]citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for [3H]citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of [3H]citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of [3H]citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of [3H]imipramine binding reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific [3H]imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of [3H]imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in [3H]citalopram and serotonin-sensitive [3H]imipramine binding with only a small effect on serotonin-insensitive [3H]imipramine binding. The dissociation rate of [3H]imipramine or [3H]citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive [3H]imipramine high affinity binding sites closely resembles that of [3H]citalopram binding.(ABSTRACT TRUNCATED AT 250 WORDS)