Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens.

The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma-derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12-O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha-phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become differentiated to organize into nongrowing tubes.

Extension of life-span with superoxide dismutase/catalase mimetics.

We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems of Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wild-type worms increased their mean life-span by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their life-span (a 67 percent increase). It appears that oxidative stress is a major determinant of life-span and that it can be counteracted by pharmacological intervention.

Blood-brain barrier penetration and in vivo activity of an NGF conjugate.

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.

Mitigating effect of EUK-207 on radiation-induced cognitive impairments.

The brain could be exposed to irradiation as part of a nuclear accident, radiological terrorism (dirty bomb scenario) or a medical radiological procedure. In the context of accidents or terrorism, there is considerable interest in compounds that can mitigate radiation-induced injury when treatment is initiated a day or more after the radiation exposure. As it will be challenging to determine the radiation exposure an individual has received within a relatively short time frame, it is also critical that the mitigating agent does not negatively affect individuals, including emergency workers, who might be treated, but who were not exposed. Alterations in hippocampus-dependent cognition often characterize radiation-induced cognitive injury. The catalytic ROS scavenger EUK-207 is a member of the class of metal-containing salen manganese (Mn) complexes that suppress oxidative stress, including in the mitochondria, and have been shown to mitigate radiation dermatitis, promote wound healing in irradiated skin, and mitigate vascular injuries in irradiated lungs. As the effects of EUK-207 against radiation injury in the brain are not known, we assessed the effects of EUK-207 on sham-irradiated animals and the ability of EUK-207 to mitigate radiation-induced cognitive injury. The day following irradiation or sham-irradiation, the mice started to receive EUK-207 and were cognitively tested 3 months following exposure. Mice irradiated at a dose of 15Gy showed cognitive impairments in the water maze probe trial. EUK-207 mitigated these impairments while not affecting cognitive performance of sham-irradiated mice in the water maze probe trial. Thus, EUK-207 has attractive properties and should be considered an ideal candidate to mitigate radiation-induced cognitive injury.

Using superoxide dismutase/catalase mimetics to manipulate the redox environment of neural precursor cells.

Past work has shown that neural precursor cells are predisposed to redox sensitive changes, and that oxidative stress plays a critical role in the acute and persistent changes that occur within the irradiated CNS. Irradiation leads to a marked rise in reactive oxygen species (ROS) that correlates with oxidative endpoints in vivo and reductions in neurogenesis. To better understand the impact of oxidative stress on neural precursor cells, and to determine if radiation-induced oxidative damage and precursor cell loss after irradiation could be reduced, a series of antioxidant compounds (EUK-134, EUK-163, EUK-172, EUK-189) were tested, three of which possess both superoxide dismutase (SOD) and catalase activities and one (EUK-163) whose only significant activity is SOD. Our results show that these SOD/catalase mimetics apparently increase the oxidation of a ROS-sensitive fluorescent indicator dye, particularly after short (12 h) treatments, but that longer treatments (24 h) decrease oxidation attributable to radiation-induced ROS. Similarly, other studies found that cells incubated with CuZnSOD showed some increase in intracellular ROS levels. Subsequent data suggested that the dye-oxidising capabilities of the EUK compounds were linked to differences in their catalase activity and, most likely, their ability to catalyse peroxidative pathways. In unirradiated mice, the EUK-134 analogue induced some decrease of proliferating precursor cells and immature neurons 48 h after radiation, an effect that may be attributable to cytotoxicity and/or inhibition of precursor proliferation. In irradiated mice, a single injection of EUK-134 was not found to be an effective radioprotector at acute times (48 h). The present results support continued development of our in vitro model as a tool for predicting certain in vivo responses, and suggest that in some biological systems the capability to scavenge superoxide but produce excess H(2)O(2), as is known for CuZnSOD, may be potentially deleterious. Our results also show that the ability of catalase mimetics, like true catalases, to catalyse peroxidase reactions can complicate the interpretation of data obtained with certain fluorescent ROS-indicator dyes.

Lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase-catalase mimetics.

Superoxide is produced as a result of normal energy metabolism within the mitochondria and is scavenged by the mitochondrial form of superoxide dismutase (sod2). Mice with inactivated SOD2 (sod2 nullizygous mice) die prematurely, exhibiting several metabolic and mitochondrial defects and severe tissue pathologies, including a lethal spongiform neurodegenerative disorder (Li et al., 1995; Melov et al., 1998, 1999). We show that treatment of sod2 nullizygous mice with synthetic superoxide dismutase (SOD)-catalase mimetics extends their lifespan by threefold, rescues the spongiform encephalopathy, and attenuates mitochondrial defects. This class of antioxidant compounds has been shown previously to extend lifespan in the nematode Caenorhabditis elegans (Melov et al., 2000). These new findings in mice suggest novel therapeutic approaches to neurodegenerative diseases associated with oxidative stress such as Friedreich ataxia, spongiform encephalopathies, and Alzheimer's and Parkinson's diseases, in which chronic oxidative damage to the brain has been implicated.

Inhibition of phosphatidylinositol kinase in vascular smooth muscle membranes by adenosine and related compounds.

Adenosine and 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of phosphatidylinositol in membranes prepared from aortic smooth muscle. The nucleosides did not affect the breakdown of phosphatidylinositol-4-phosphate. Under certain conditions, the membrane-bound phosphatidylinositol kinase phosphorylated exogenous phosphatidylinositol. The nucleosides inhibited the enzyme competitively with respect to magnesium-ATP and non-competitively with respect to phosphatidylinositol. Adenosine analogs modified in the ribose moiety were inhibitors with potencies comparable to that of adenosine, whereas adenine nucleotides and purine-modified adenosine analogs were much weaker inhibitors. Density gradient fractionation studies showed that phosphatidylinositol kinase is primarily associated with the sarcoplasmic reticulum. Vascular smooth muscle contraction is associated with increased phosphatidylinositol turnover. Inhibition of phosphatidylinositol kinase by intracellular adenosine may, therefore, be a factor involved in regulating vasodilation.

Role of oxidant stress and iron delocalization in acidosis-induced intestinal epithelial hyperpermeability.

Using Caco-2BBe monolayers as a model of the intestinal epithelium, we tested the hypothesis that reactive oxygen metabolites contribute to lactic acid-induced hyperpermeability. Compared to monolayers incubated at normal pH (i.e., 7.4) monolayers incubated in medium titrated to extracellular pH (pHo) 5.0 with 10 mM lactic acid demonstrated increased permeability to both fluorescein sulfonic acid (FS) and fluorescein isothiocyanate-dextran (average molecular mass = 4000 Da; FD4). Lactic acid-induced hyperpermeability to both FS and FD4 was reduced by adding either 30 microM EUK-8, a superoxide dismutase/catalase mimetic, or catalase (10(4) U/mL). Incubation of monolayers with lactic acid increased cellular malondialdehyde content, a measure of lipid peroxidation. EUK-8 (30 microM) completely abrogated this effect. Incubation with ferrous sulfate (100 microM) exacerbated both lactic acid-induced hyperpermeability to FS and lactic acid-induced lipid peroxidation. Iron chelation with 1 mM diethylene triamine pentaacetic acid (DTPA)-trisodium calcium salt attenuated lactic acid-induced hyperpermeability, whereas iron-loaded DTPA (1 mM) was not protective. Treatment with DTPA-trisodium calcium salt also ameliorated lactic acid-induced lipid peroxidation. Incubation with lactic acid (pHo 5.0) for 16 h increased the cellular content of low molecular weight iron species. Incubation with lactic acid (pHo 5.0) for 24 h significantly increased the percentage of oxidized protein-bound thiols in Caco-2BBe cells. We conclude that lactic acidosis induces hyperpermeability in Caco-2BBe monolayers, in part, via an iron-dependent increase in reactive oxygen metabolite-mediated damage.

Role of oxidant stress in the adult respiratory distress syndrome: evaluation of a novel antioxidant strategy in a porcine model of endotoxin-induced acute lung injury.

Reactive oxygen metabolites (ROMs) are thought to play a key role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Accordingly, the use of ROM scavengers, such as N-acetyl-cysteine or dimethylthiourea, as therapeutic adjuncts to prevent oxidant-mediated damage to the lung have been evaluated extensively in animal models of ARDS. Results with this approach have been quite variable among studies. Another strategy that has been examined in animal models of ARDS is the administration of various enzymes, particularly superoxide dismutase (SOD) or catalase (CAT), in an effort to promote the conversion of ROMs to inactive metabolites. In theory, this strategy should be more effective than the use of ROM scavengers since a single molecule of a catalytically active molecule can neutralize a large number of molecules of a reactive species, whereas most scavengers act in a stoichiometric fashion to neutralize radicals on a mole-for-mole basis. This notion is supported by studies showing that prophylactic treatment with CAT provides impressive protection against acute lung injury induced in experimental animals by the administration of lipopolysaccharide (LPS). Results with SOD have been more variable. Recently, we have utilized a porcine model of LPS-induced ARDS to investigate the therapeutic potential of EUK-8, a novel, synthetic, low molecular salen-manganese complex that exhibits both SOD-like and CAT-like activities in vitro. Using both pre- and post-treatment designs, we have documented that treatment with EUK-8 significantly attenuates many of the features of LPS-induced acute lung injury, including arterial hypoxemia, pulmonary hypertension, decreased dynamic pulmonary compliance, and pulmonary edema. These findings support the view that salen-manganese complexes warrant further evaluation as therapeutic agents for treatment or prevention of sepsis-related ARDS in humans.

Prevention of 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide dismutase and catalase mimetic, in cultured dopaminergic neurons.

Oxidative stress has been implicated in the selective degeneration of dopaminergic (DAergic) neurons in Parkinson's disease (PD). In this study, we tested the efficacy of EUK-134, a superoxide dismutase (SOD) and catalase mimetic, on the nitration of tyrosine hydroxylase (TH), a marker of oxidative stress, and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP(+)) and 6-hydroxydopamine (6-OHDA) in primary DAergic neuron cultures. Exposure of cultures to 10 microM MPP(+) reduced dopamine (DA) uptake and the number of tyrosine hydroxylase immunoreactive (THir) neurons to 56 and 52% of control, while exposure to 30 microM 6-OHDA reduced DA uptake and the number of THir neurons to 58 and 59% of control, respectively. Pretreatment of cultures with 0.5 microM EUK-134 completely protected DAergic neurons against MPP(+)- and 6-OHDA-induced neurotoxicity. Exposure of primary neuron cultures to either MPP(+) or 6-OHDA produced nitration of tyrosine residues in TH. Pretreatment of cultures with 0.5 microM EUK-134 completely prevented MPP(+)- or 6-OHDA-induced nitration of tyrosine residues in TH. Taken together, these results support the idea that reactive oxygen species (ROS) are critically involved in MPP(+)- and 6-OHDA-induced neurotoxicity and suggest a potential therapeutic role for synthetic catalytic scavengers of ROS, such as EUK-134, in the treatment of PD.

The tight-junction-specific protein ZO-1 is a component of the human and rat blood-brain barriers.

Continuous tight junctions between vascular endothelial cells, the principal anatomical basis for the blood-brain barrier, have been investigated functionally and morphologically but their molecular components have not been defined. This communication reports that the protein ZO-1, a specific constituent of epithelial tight junctions, is found in human and rat brain vasculature. ZO-1-positive immunocytochemical staining forms a tightly banded pattern outlining individual endothelial cells in blood vessels of the human cerebral cortex. Rat brain exhibits a similar staining of blood vessels as well as ZO-1-positive staining around individual epithelial cells of the choroid plexus. The antiserum used for immunocytochemistry recognizes a protein of about 200 kDa in rat brain microvessels by Western blot. These findings indicate that ZO-1 is located at the interendothelial junctions of brain vasculature, implicating its importance as a component of the blood-brain barrier.

Interaction of basic fibroblast growth factor with bovine growth plate chondrocytes.

The basic fibroblast growth factor (bFGF) family of peptides influences a wide range of cellular actions. To better understand the possible role of bFGF in the growth plate, we have characterized the interaction of this growth factor with isolated bovine growth plate chondrocytes. Basic FGF interacts with two classes of binding sites on these cells. One is consistent with high-affinity bFGF receptors and the other with low-affinity heparin-like binding sites on the chondrocyte surface. Radiolabeled bFGF binding studies revealed approximately 4 x 10(6) binding sites per cell, with a Kd of approximately 42 nM. Graded concentrations of heparin or NaCl competed with [125I]-labeled bFGF in a dose-dependent fashion, reducing [125I]-labeled bFGF binding by 75 and 97%, respectively. The data suggest the presence of a high-capacity, low-affinity class of binding sites with the properties of a heparin-like moiety. Affinity cross-linking of [125I]-labeled bFGF to chondrocytes labeled two principal species with apparent molecular masses of 135 and 160 kDa. Labeled bFGF was specifically displaced from both species by subnanomolar concentrations of unlabeled bFGF. These high-affinity, low-capacity binding sites are characteristic of classical bFGF receptors. Binding of [125I]-labeled bFGF to these sites was also influenced by heparin, consistent with coregulation of binding to the two classes of binding sites. The data suggest that bFGF participates in the regulation of skeletal growth at the growth plate and that this regulation may involve bFGF interaction with at least two distinct classes of binding sites.

Oxidative neuropathology and putative chemical entities for Alzheimer's disease: neuroprotective effects of salen-manganese catalytic anti-oxidants.

Considerable evidence exists that the brains of individuals with Alzheimer's disease are subject to elevated levels of oxidative stress, particularly in regions exhibiting pathological damage. A major contributor to this oxidative stress appears to be the inflammatory process. Activation of rodent microglial cells by LPS or beta-amyloid peptide results in a marked up-regulation of inducible nitric oxide synthase (iNOS) and corresponding nitric oxide (NO) production. Elevated levels of iNOS are also observed in the brains of Alzheimer patients. The reaction of NO with superoxide leads to the generation of the highly reactive and damaging peroxynitrite free radical species. Peroxynitrite appears to play a key role in the generation of an oxidative stress in the Alzheimer brain as evidenced by widespread nitrotyrosine immunoreactivity. We have employed SIN-1 as a peroxynitrite generating system in cell cultures in order to characterize the effects of this free radical on neurons. SIN-1 treatment of primary rat hippocampal neurons in culture results in neurotoxicity by a necrosis mechanism according to electron microscopic criteria. One approach to limiting peroxynitrite mediated damage is to limit superoxide production. An approach we have evaluated is treatment with salen manganese compounds, a class of catalytic antioxidant compounds which behave as superoxide dismutase (SOD)/catalase mimetics to detoxify superoxide. A number of such salen manganese compounds, including EUK-8 and EUK-134, can markedly protect primary rat cortical neurons from hydrogen peroxide mediated oxidative stress. Such salen manganese compounds can similarly afford marked neuroprotection to an oxidative stress imposed by SIN-1, potentially attributable at least in part to their inherent SOD activity. The salen manganese SOD/catalase mimetics represent a promising class of catalytic antioxidant for attenuating oxidative stress.

Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblast growth factor.

A study was performed in order to investigate the possible functional roles of insulin-like growth-factor I (IGFI) and basic fibroblast growth factor (bFGF) in the regulation of mitotic and metabolic activity of growth-plate chondrocytes. Chondrocytes from the distal radial growth plates of calves and the costal physeal cartilage of rats were exposed to these factors, individually and in combination, in primary monolayer culture, to assess their effects. The data showed that bFGF had both a greater potency and a greater efficacy as a mitogen for bovine growth-plate chondrocytes than did IGF-I. The maximum incorporation of 3H-thymidine by bFGF was 8.3 times that in serum-free (control) cultures; the maximum stimulation of incorporation by IGF-I was 2.5 times that in the control medium. In contrast, IGF-I stimulated a maximum incorporation of 35S-sulfate into glycosaminoglycans that was 2.6 times that in the IGF-I serum-free control cultures, while bFGF had no effect or was mildly inhibitory. When used together, these two factors acted synergistically. Incorporation of 3H-thymidine was more than two times greater than the sum of the effects of the growth factors when used alone and 20.5 times greater than that of the growth factor-free control cultures. Physeal chondrocytes from six-day-old rats were mitotically more responsive to bFGF than to IGF-I, but they were more responsive to IGF-I when they had been derived from twenty-eight-day-old rats. Interaction between bFGF and factors in the serum enhanced the mitotic activity of the rat chondrocytes, but bFGF did not interact with IGF-I under the same experimental conditions. In the presence of bFGF, there was a reduction in the stimulation by IGF-I of incorporation of 35S-sulfate and a decrease in the percentage of chondrocytes containing alkaline phosphatase. These growth factors also influenced cellular morphology in culture. In the presence of IGF-I or serum, the rat chondrocytes manifested the polygonal morphology typical of chondrocytes in culture, while bFGF promoted a more elongated spindle shape. Removal of bFGF and replacement by IGF-I restored the polygonal morphology, indicating that this transition is reversible.

Mitigation of radiation-induced lung injury with EUK-207 and genistein: effects in adolescent rats.

Exposure of civilian populations to radiation due to accident, war or terrorist act is an increasing concern. The lung is one of the more radiosensitive organs that may be affected in people receiving partial-body irradiation and radiation injury in lung is thought to be associated with the development of a prolonged inflammatory response. Here we examined how effectively damage to the lung can be mitigated by administration of drugs initiated at different times after radiation exposure and examined response in adolescent animals for comparison with the young adult animals that we had studied previously. We studied the mitigation efficacy of the isoflavone genistein (50 mg/kg) and the salen-Mn superoxide dismutase-catalase mimetic EUK-207 (8 mg/kg), both of which have been reported to scavenge reactive oxygen species and reduce activity of the NFkB pathway. The drugs were given by subcutaneous injection to 6- to 7-week-old Fisher rats daily starting either immediately or 2 weeks after irradiation with 12 Gy to the whole thorax. The treatment was stopped at 28 weeks post irradiation and the animals were assessed for levels of inflammatory cytokines, activated macrophages, oxidative damage and fibrosis at 48 weeks post irradiation. We demonstrated that both genistein and EUK-207 delayed and suppressed the increased breathing rate associated with pneumonitis. These agents also reduced levels of oxidative damage (50-100%), levels of TGF-ß1 expression (75-100%), activated macrophages (20-60%) and fibrosis (60-80%). The adolescent rats developed pneumonitis earlier following irradiation of the lung than did the adult rats leading to greater severe morbidity requiring euthanasia (∼37% in adolescents vs. ∼10% in young adults) but the extent of the mitigation of the damage was similar or slightly greater.

Mitigation of lung injury after accidental exposure to radiation.

There is a serious need to develop effective mitigators against accidental radiation exposures. In radiation accidents, many people may receive nonuniform whole-body or partial-body irradiation. The lung is one of the more radiosensitive organs, demonstrating pneumonitis and fibrosis that are believed to develop at least partially because of radiation-induced chronic inflammation. Here we addressed the crucial questions of how damage to the lung can be mitigated and whether the response is affected by irradiation to the rest of the body. We examined the widely used dietary supplement genistein given at two dietary levels (750 or 3750 mg/kg) to Fischer rats irradiated with 12 Gy to the lung or 8 Gy to the lung + 4 Gy to the whole body excluding the head and tail (whole torso). We found that genistein had promising mitigating effects on oxidative damage, pneumonitis and fibrosis even at late times (36 weeks) when drug treatment was initiated 1 week after irradiation and stopped at 28 weeks postirradiation. The higher dose of genistein showed no greater beneficial effect. Combined lung and whole-torso irradiation caused more lung-related severe morbidity resulting in euthanasia of the animals than lung irradiation alone.

Adenosine and 5'-chloro-5'-deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle.

Smooth muscle from calf aorta is homogenized and centrifuged. The insoluble material is subjected to sucrose density gradient centrifugation. When the heaviest fraction so obtained is incubated with radioactive ATP, two components incorporate most of the acid-insoluble radioactivity. One is a phosphoprotein with a molecular weight of 21,000. It has been identified as myosin light chain by its molecular weight, isoelectric point, and precipitation by antibody to calf aorta myosin. Its phosphorylation is strongly inhibited by EGTA, in agreement with published reports that myosin light chain kinase of smooth muscle is Ca2+ dependent. The other product is of low molecular weight, is extracted into acidic chloroform-methanol, and has been identified as phosphatidylinositol 4-phosphate. Adenosine and 5'-chloro-5'-deoxyadenosine, which are vasodilators, inhibit the phosphorylation of both substrates. Phosphorylation of phosphatidylinositol is inhibited at lower concentrations of the nucleosides than is the phosphorylation of myosin light chain. The inhibitory effects of the two nucleosides are not associated with changes in the concentration of cyclic AMP. The precise function of phosphatidylinositol phosphorylation in smooth muscle is not known, but correlations between smooth muscle contraction and increased turnover of phosphatidylinositol and its mono- and diphosphates have been reported. Myosin light chain is phosphorylated under conditions which favor smooth muscle contraction. We conclude that the inhibitory effects of adenosine described here are consistent with their physiological action as vasodilators.

Neutralizing antibodies inhibit the binding of basic fibroblast growth factor to its receptor but not to heparin.

Polyclonal antibodies were prepared against recombinant basic fibroblast growth factor (bFGF) that reacted only with bFGF but not acidic FGF. These antibodies were able to inhibit various biological activities of bFGF such as the ability of bFGF to stimulate DNA synthesis in 3T3 cells, proliferation and migration of bovine capillary endothelial cells (BCEC), and neurite extension in pheochromocytoma (PC12) cells. The anti-bFGF antibodies also inhibited the mitogenic activity of subendothelial cell extracellular matrix for BCEC, demonstrating that the growth factor component in extracellular matrix required for supporting BCEC proliferation was bFGF. Anti-bFGF antibodies inhibited the cross-linking of bFGF to its high affinity receptor on BCEC cells. However, these antibodies did not inhibit the binding of bFGF to heparin-Sepharose or to the low affinity receptors of BCEC which have been demonstrated to be heparin-like molecules. These results suggest that bFGF has distinct domains for binding to high affinity cellular receptors and for binding to heparin.

EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology.

The present study tested the effects of EUK-134, a synthetic superoxide dismutase/catalase mimetic, on several indices of oxidative stress and neuropathology produced in the rat limbic system as a result of seizure activity elicited by systemic kainic acid (KA) administration. Pretreatment of rats with EUK-134 did not modify the latency for or duration of KA-induced seizure activity. It did produce a highly significant reduction in increased protein nitration, activator protein-1- and NF-kappaB-binding activity, and spectrin proteolysis as well as in neuronal damage resulting from seizure activity in limbic structures. These results support the hypothesis that kainate-induced excitotoxicity is caused, at least in part, by the action of reactive oxygen species. Furthermore, they suggest that synthetic superoxide dismutase/catalase mimetics such as EUK-134 might be used to prevent excitotoxic neuronal injury.