[PPAR gamma: a novel pharmacological target against retinal and choroidal neovascularization]. / Le PPAR gamma: une nouvelle cible pharmacologique contre la néovascularisation rétinienne et choroïdienne.

PPARg (peroxisome proliferator-activated receptor gamma) is a nuclear receptor that regulates the transcription of numerous genes involved in the differentiation, proliferation and apoptosis of various cell types. It was initially discovered in adipocytes as a differentiation agent, then was characterized in vascular endothelium and recently in choroidal and retinal endothelial cells. Agonists that bind to PPARgamma and stimulate its transcriptional activity are endogenous lipids such as lysophosphatidic acid and 15-d-PGJ2 as well as the synthetic pharmacological compounds, thiazolidinediones, used for treating type 2 diabetes. These ligands prevent choroidal and retinal neovascularization in several experimental animal models, notably through the inhibition of vascular endothelial growth factor (VEGF) receptor expression. Because of the high affinity and the low molecular weight of agonists, suitable for good bioavailability, PPARgamma could potentially be a novel pharmacological target of angiostatic agents, particularly useful to treat age-related macular degeneration and diabetic retinopathy.

Leukotriene B4 photoaffinity probes: design, synthesis and evaluation of new arylazide-1,3-disubstituted cyclohexanes.

The synthesis and the binding affinities of new leukotriene B4 receptor photoaffinity probes, where a 1,3-disubstituted cyclohexane ring replaces the conjugated delta6,7 and delta8,9 double bonds of the natural eicosanoid, are described. One enantiomeric compound, 4b alpha, is specifically cross-linked upon photolysis to the recombinant leukotriene B4 receptor from human origin (h-BLTR) solubilized in a micellar medium. This probe appears as a good candidate for identifying the ligand binding site of this receptor.

Synthesis and structure-activity relationships of new 1, 3-disubstituted cyclohexanes as structurally rigid leukotriene B(4) receptor antagonists.

A series of 1-hydroxy-3-¿3-hydroxy-7-phenyl-1-hepten-1-yl cyclohexane acetic acid derivatives was designed based on postulated active conformation of leukotriene B(4) (LTB(4)) and evaluated as human cell surface LTB(4) receptor (BLTR) antagonists. Binding was determined through ¿(3)HLTB(4) displacement from human neutrophils and receptor antagonistic assays by in vitro measurements of inhibition of leukocyte chemotaxis induced by LTB(4). On the basis of these assays, a structure-affinity relationship was investigated. Optimization of the acid chain length and omega-substitution of a phenyl group on the lipophilic tail were shown to be critical for binding activity. These modifications led to the discovery of compounds with submicromolar potency and selective BLTR antagonism. The most potent compound 3balpha (IC(50) = 250 nM) was found to significantly inhibit oedema formation in a topical model of phorbolester-induced inflammation. Substantial improvement of in vitro potency was achieved by modification of the carboxylic acid function leading to the identification of the N,N-dimethylamide series. Compound 5balpha, free of agonist activity, displayed higher potency in receptor binding with an IC(50) of 40 nM. These results support the hypothesis that the spatial relationship between the carboxylic acid and allylic hydroxyl functions is crucial for high binding affinity with BLTR.

Free radicals in retinal ischemia.

1. Reactive oxygen species (ROS) can be generated in biological tissues, including the retina, in particular under or after ischemia. They can provoke cell necrosis by reacting with cell components or they can trigger programmed cell death by activating specific targets. 2. Experiments based on electroretinography and electron spin resonance spin trapping analysis show that ROS are produced in the rabbit retina during ischemic episodes themselves as well as reperfusion. ROS are also generated as a consequence of ischemia by overstimulation of glutamate ionotropic receptors and calcium-dependent activation of enzymes such as phospholipase A2 and nitric oxide synthase. 3. The targets of ROS that can be responsible for functional damage of the retina are numerous: Na+-K+-ATPase inhibition leads to ionic imbalance and electroretinogram alteration; inhibition of glutamate transporter contributes to excitotoxicity. In addition, ROS can be deleterious by inducing protein synthesis (e.g., apoptotic proteins, vascular endothelial growth factor/vascular permeability factor). 4. In this short review, we consider the various mechanisms of ROS generation in retinal ischemia and the different effects of ROS so as to suggest possible effects of neuroprotective agents.

Free radicals and glutamate uptake in the retina.

1. Glutamate (Glu) uptake in neurons and astrocytes is essential to prevent the persistence of excitotoxic levels of Glu in the synaptic cleft. 2. We investigated the effect of oxidative stress, which is also involved in ischemia-reperfusion, on the Glu transporter in isolated rat retinal cells. 3. Hydrogen peroxide (H2O2 3-300 microM) decreases the Na+-dependent Glu uptake. This effect is not related to a free radical production and is partly reversed by reducing agents, suggesting a transporter modulation by a redox-related event.

Free radicals in rabbit retina under ocular hyperpressure and functional consequences.

Pharmacological experiments have suggested that ocular ischemia, induced by high intraocular pressure in the rabbit, provokes an oxidative stress responsible for functional alteration of the retina. However, the nature of the oxidant chemical species and their mode of generation were not elucidated. The aim of the present studies was to characterize the oxygen-derived free radicals produced during and/or after the hyperpressure period. The technique used was based on electron spin resonance spin trapping analysis of the signals obtained in microdialysates of the retina perfused with the nitrone 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). The oxidative stress was also evaluated under ischemia and reperfusion periods by measuring the level of ascorbate in the retina via electron spin resonance detection of the ascorbyl free radical-dimethyl sulfoxide (AFR-DMSO) complex. Electroretinograms were recorded to determine the functional consequences of high intraocular pressure and free radical generation. Our results show that superoxide dismutase-inhibitable DEPMPO/hydroxyl radical adducts were generated during the high intraocular pressure period and that the oxidative stress was not increased at reperfusion as assessed by spin trapping and AFR-DMSO measurements. Functional protection provided by free radical scavengers (superoxide dismutase+catalase, TEMPO nitroxide+catalase and dimethylthiourea) against high intraocular pressure-induced electroretinogram alteration confirmed these observations. In conclusion, these experiments demonstrate for the first time by direct measurement that oxygen-derived free radicals are produced by the retina during acute ischemia. This generation could be the explanation for electroretinogram alteration.

Synthesis and biochemical/pharmacological profile of the novel leukotriene B4 receptor antagonist sodium (1S*, 3S*)-1-hydroxy-3-[(3 R*S*,E)-3-hydroxy-7-phenyl-1-hepten-1-yl]-1-cyclohexane acetate.

A novel series of leukotriene B4 (LTB4) antagonists is reported. These compounds present a cyclohexane ring in their chemical structure, which mimics the three conjugated double bonds of LTB4. The biochemical/pharmacological profile of the leader compound, PH-163 (sodium (1S*,3S*)-1-hydroxy-3-[(3R*S,E)-3-hydroxy-7-phenyl-1-hepten-1-yl]- 1 -cyclohexane acetate, CAS 163251-41-0) is described. This compound competes with [3H]LTB4 binding to its receptor in human neutrophils and guinea pig lung membranes with IC50's of 0.8 mumol/l and 0.2 mumol/l, respectively, i.e. relative binding affinities of 1% as compared to LTB4. PH-163 does not elicit any agonist activity, but inhibits leucocyte chemotaxis induced by LTB4 (pKB = 6.57) and lung parenchymal strip contraction (IC50 = 0.1 mumol/l). In conclusion, PH-163 or derivatives could be useful in the treatment of inflammatory diseases where LTB4 seems to be involved.

Differential effect of ischemia/reperfusion on pigmented and albino rabbit retina.

The purpose of the study was to compare the retinal sensitivity of pigmented and albino rabbits to ischemia/reperfusion-induced electroretinogram (ERG) alterations and optic nerve morphological changes. High intraocular pressure (HIOP) was induced by applying a suction-cup on the eye and a depression with an ophthalmodynamometer. HIOP was maintained for lengths of time (30-75 min). Flash ERGs were recorded in dark-adapted animals for ischemia and 2 h reperfusion periods. Two weeks later, histological examination of the retinas and optic nerves was done. Albino rabbits submitted to 45 min HIOP failed to recover b-wave ERG amplitude after 2 h reperfusion, whereas pigmented animals presented a total ERG recovery even if ischemia was maintained as long as 75 min. Intravenous treatment of albino animals with Lazaroid U74389G led to significant ERG recovery at reperfusion. Histological studies show that pigmented rabbit optic nerves suffered less damage than the albino ones. These results emphasize the role of the pigmentary status of the animals in the retinal sensitivity to ischemia. Neuroprotection afforded by the antioxidant U74389G suggests that ocular pigments could also protect the retinal functional integrity through a free radical scavenging activity.

Calcium-dependent free radical generation in cultured retinal neurons injured by kainate.

Cultured rat retinal neurons exposed to kainate produced free radicals, as demonstrated by electron spin resonance (ESR) spin trapping using the nitrone 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the generation of DMPO hydroxyl adduct (DMPO-OH). This DMPO-OH production was abolished by EGTA, nitro-arginine and oxypurinol, suggesting that it was dependent on Ca2+ influx and subsequent activation of nitric oxide synthase and xanthine oxidase. Moreover, kainate induced a receptor-mediated Ca2+ influx and neuronal injury assessed by lactate dehydrogenase release. Neuroprotection afforded by nitro-arginine and oxypurinol shows that calcium-dependent free radical production plays a major role in kainate retinal toxicity.

Insulin-like growth factor-1 (IGF-1) specifically binds to bovine lens epithelial cells and increases the number of fibronectin receptor sites.

The purpose of the study was to determine the quantitative characteristics of IGF-1 binding sites in lens cells and to investigate its ability to modulate cell growth, in particular by inducing integrin expression. Studies were carried out in bovine lens epithelial cells in culture. IGF-1 receptor binding parameters were measured from saturation experiments with 125I-IGF-1. Scatchard plot indicates one class of high affinity sites (Kd = 2.5 +/- 1.5 nM). In addition, we showed that this growth factor was synthesized and released by lens cells. The characteristics of the receptor sites are in accordance with the effects of the growth factor (e.g. stimulation of DNA synthesis) in the range of nM concentrations. Moreover, by incubating cells with IGF-1 (12 nM) for 40 hr we demonstrated that the expression of integrin, the fibronectin receptor, was activated (N = 885 +/- 169 x 10(3) sites/cell vs N = 453 +/- 105 x 10(3) sites/cell in control cultures) without modification of its affinity (Kd congruent to 16 x 10(-8) M). These new data emphasize the role of IGF-1 in the regulation of migration, proliferation and differentiation of mature lens cells.

Protection by prostaglandins from glutamate toxicity in cortical neurons.

The growing evidence that glutamate may be an important agent mediating ischemic damage to neurons, led us to investigate the possible protective effects of pharmacological agents against glutamate in a model system of cortical neurons. In this study we examined, in particular, the cytoprotective effect of prostaglandins. Experiments were carried out in vitro by using rat cortical neurons in culture for 10 days. They were incubated for 3h with glutamate (10 microM) in the presence or absence of various pharmacological agents including prostaglandins (PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-Keto-PGF1 alpha, carba-TXA2, carba-PGI2 and PGF2 alpha-methylester). Increase in lacticodehydrogenase (LDH) release into the culture medium has been measured as an index of cell injury. When neurons were incubated with glutamate they released LDH due to NMDA-receptor activation since D-L-2-amino-5-phosphonovaleric acid, a specific receptor antagonist, protected the cells. The protective activity of oxypurinol, amflutizole, superoxide dismutase, NG nitro-L-arginine and quinacrine, also suggests that xanthine oxidase activation, the generation of superoxide radical, and nitrix oxide, as well as phospholipase A2 stimulation are responsible for neuron injury (i.e. LDH release). All the tested prostaglandins, except PGF2 alpha-methylester, afforded significant protection at concentrations between 0.1 and 10 microM. The order of potency of the prostanoids was: PGF2 alpha = PGE2 > Carba-TXA2 > PGE1 > PGD2 > PGI2 = Carba-PGI2 > 6-Keto-PGF1 alpha. Additional experiments showed that prostaglandins did not compete for the NMDA binding site and that they did not inhibit free radical-related membrane damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cell adhesion to lens capsule by LCM 1910, an RGD-derived peptide.

Opacification of the posterior lens capsule, (secondary cataract), is one of the major complications of extracapsular cataract extraction. The lens epithelial cells remaining after surgery migrate and proliferate along posterior capsule, and give rise to structures such as pearls and cells with contractile properties, which considerably hamper vision. One pharmacological approach aimed at limiting this phenomenon would be to stop this cell migration, thus inhibiting their proliferation. It has been shown that cells adhere and migrate on their support via adhesion molecules such as integrins. Generally, the tripeptide sequence Arg-Gly-Asp (RGD) is the recognition motif for these receptors. In this study, cell adhesion inhibition in the presence of RGD peptides and derivatives was measured on extracellular matrix and lens capsule. One of these compounds, the [N alpha-acetyl-NG(H+)-arginyl]-glycyl-[C beta (H)-C alpha -benzyl]-aspartamid- HCl] (LCM 1910), significantly inhibited cell migration at millimolar concentrations, and could be of interest in prevention of secondary cataract.

[New perspectives in the pharmacological treatment of glaucoma]. / Nouvelles perspectives dans le traitement pharmacologique du glaucome.

Open angle glaucoma is an optic neuropathy, the etiology of which is still unknown and which has not yet satisfactory therapy. Intraocular hypertension due to a decrease in trabecular out flow facility, is a risk factor. The side effects caused by ocular hypotensive treatments justify the search for better-tolerated drugs. A survey of the new approaches is reported: carbonic anhydrase inhibition, renin-angiotensin system inhibition, glucocorticoid antagonism, the use of prostaglandins etc. are evoked, as well as other more speculative ways: antioxidant treatments. Glaucoma which is characterized by ganglion cell degeneration probably related to a vascular defect, deserves to be studied from this point of view. Recent data on vascular and haemorheological abnormalities and the possible involvement of excitotoxic neurotransmitters in pathological process open a novel way for really innovative pharmacological research.

Prostacyclin (PGI2) protects rat cortical neurons in culture against hypoxia/reoxygenation and glutamate-induced injury.

Arachidonic acid and its metabolites are released in brain extracellular fluids as a result of ischemia and may participate in either damaging or protecting neural tissues. This study investigates the neuroprotective effect of prostacyclin (PGI2) on hypoxia (5 h)/reoxygenation (3 h) and on the excitotoxic neurotransmitter, glutamate (10 microM), in rat cortical neuron cultures. At microM concentrations, PGI2 inhibits lactate dehydrogenase release, a cell-injury marker. These results, showing a direct cytoprotective effect of PGI2 on brain cells, reinforce its beneficial properties on vessels and circulating cells in cerebral ischemia.

Superoxide and nitric oxide cooperation in hypoxia/reoxygenation-induced neuron injury.

Oxygen-derived free radicals are implicated in hypoxia- and reoxygenation-related brain injury. In addition, excitatory amino acid neurotransmitters seem to be involved in this neurotoxicity and could act through the L-arginine/nitric oxide (NO) synthase pathway. In the present study we have used rat forebrain neurons in culture submitted to hypoxia/reoxygenation to investigate the relative role of free radicals, glutamate, and nitric oxide in hypoxic neuronal injury. Hypoxia (5 h) followed by reoxygenation (0-24 h) induced cell damage assessed by lacticodehydrogenase release into culture medium. Superoxide dismutase (SOD, 500 U/mL), D-L-2-amino-5-phosphonovaleric acid (100 microM), a glutamate receptor antagonist, and NG-nitro-L-arginine (100 microM), an NO synthase inhibitor, protected the neurons. The effect of NG-nitro-L-arginine was reversed by adding L-arginine (10 mM) in the culture medium, and hemoglobin, which scavenges NO, also afforded protection. Hypoxia (5 h) provoked glutamate release from neurons, and this effect was inhibited by SOD. Exogenous glutamate (1-100 microM) induced lacticodehydrogenase release, and this effect was inhibited by glutamate antagonism, NO synthase inhibition, or superoxide radical scavenging. These data are consistent with the following sequence of events in hypoxia-related neurotoxicity: free radical formation, glutamate release, and activation of NO synthase leading to superoxide and NO cooperative toxicity.

Antioxidant activity of plasma from subjects with and without senile cataract.

The relationship between overall plasma antioxidant activity and senile cataract was examined in 148 subjects aged 37-90 years. Antioxidant activity of plasma from 52 control and 96 patients with senile cataract was determined by a method which measures the capacity of plasma to inhibit auto-oxidation of bovine brain homogenate. In contrast to previous studies which demonstrated a relationship between decreased levels of antioxidant components in blood and the occurrence of lens opacities, the present study does not show any significant difference between subjects with and without senile cataract.

Oxygen free radicals adversely affect the regulation of vascular tone by nitric oxide in the rabbit retina under high intraocular pressure.

Flash electroretinograms (ERG) were recorded in the rabbit eye submitted to high intraocular pressure (HIOP) induced by the suction-cup method. When intraocular pressure rose to 100 mmHg, ERG was suppressed but rapidly recovered even under HIOP when animals were pre-treated either intravitreously with a nitric oxide donor, sodium nitroprusside, or intravenously with free radical scavengers, superoxide dismutase (SOD)+catalase. In contrast, injection of a nitric oxide synthesis inhibitor, nitro-L-arginine, into the vitreous cavity inhibited the protective effect of SOD+catalase during HIOP-induced ERG extinction. These results suggest that nitric oxide could play a role in the regulation of ocular vessel tone and that severe ischemia can impede this effect through oxygen-derived free radical generation.