Isabelle Lahaie

A novel mechanism for vasoconstrictor action of 8-isoprostaglandin F2α on retinal vessels

Using a video-imaging technique, we characterized the effects of 8-isoprostaglandin F2α(8-iso-PGF2α) on retinal vasculature from piglets. 8-Iso-PGF2α potently contracted (EC50 = 5.9 ± 0.5 nM) retinal vessels. These effects were completely antagonized by the cyclooxygenase inhibitor indomethacin, the thromboxane synthase blocker CGS-12970, the thromboxane receptor antagonist L-670596, and the putative inhibitor of the non-voltage-dependent receptor-operated Ca2+ pathway SKF-96365; constrictor effects of 8-iso-PGF2α were also partly attenuated by the ETA-receptor blocker BQ-123 and an inhibitor of endothelin-converting enzyme, phosphoramidon, but was negligibly affected by the L-type voltage-gated Ca2+ channel blocker nifedipine. Correspondingly, 8-iso-PGF2αelicited endothelin release from retinal preparations, which was markedly reduced by SKF-96365. 8-Iso-PGF2α also increased thromboxane production in the retina and cultured endothelial cells, but not on retinovascular smooth muscle cells; these effects of 8-iso-PGF2α were blocked by indomethacin, CGS-12970, SKF-96365, and EGTA, but not by nifedipine. 8-Iso-PGF2α also increased Ca2+ transients in retinal endothelial cells, which were inhibited by SKF-96365 and EGTA, but not by nifedipine, whereas in smooth muscle cells U-46619, but not 8-iso-PGF2α, stimulated a rise in Ca2+ transients. Finally, H2O2+ FeCl2 (in vitro) and anoxia followed by reoxygenation (in vivo) stimulated formation of 8-iso-PGF2α in the retina. In conclusion, 8-iso-PGF2α-induced retinal vasoconstriction is mediated by cyclooxygenase-generated formation of thromboxane and, to a lesser extent, by endothelin after Ca2+ entry into cells, possibly through receptor-operated channels. Retinal vasoconstriction to 8-isoprostanes might play a role in the genesis of ischemic retinopathies.

Altered Vascular Function in Fetal Programming of Hypertension

Background and Purpose— Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension. Methods— Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 &mgr;m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and KCa channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining. Results— Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156±2 and 155±1 mm Hg, respectively) than that of control offspring (137±1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A2 mimetic and dilation to carba-prostaglandin I2 did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of KCa (another important mediator of NO action) and relaxation to the KCa opener NS1619 were unchanged by antenatal diet. Conclusions— Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.

Role of brain and peripheral angiotensin II in hypertension and altered arterial baroreflex programmed during fetal life in rat.

Intrauterine programming of hypertension is associated with evidence of increased renin-angiotensin system (RAS) activity. The current study was undertaken to investigate whether arterial baroreflex and blood pressure variability are altered in a model of in utero programming of hypertension secondary to isocaloric protein deprivation and whether activation of the RAS plays a role in this alteration. Pregnant Wistar rats were fed a normal-protein (18%) or low-protein (9%) diet during gestation, which had no effect on litter size, birth weight, or pup survival. Mean arterial blood pressure (MABP; 126 ± 3 mm Hg 9%versus 108 ± 4 mm Hg 18%; p < 0.05) and blood pressure variability were significantly greater in the adult offspring of the 9% protein–fed mothers. Arterial baroreflex control of heart rate, generated by graded i.v. infusion of phenylephrine and nitroprusside, was significantly shifted toward higher pressure; i.v. angiotensin-converting enzyme inhibitor normalized MABP and shifted the arterial baroreflex curve of the 9% offspring toward lower pressure without affecting the 18% offspring. For examining whether brain RAS is also involved in programming of hypertension, angiotensin-converting enzyme inhibitor and losartan (specific AT1 receptor antagonist) were administered intracerebroventricularly; both significantly reduced MABP of the 9% but not the 18% offspring. Autoradiographic receptor binding studies demonstrated an increase in brain AT1 expression in the subfornical organ and the vascular organ of the lamina terminalis in the 9% offspring. These data demonstrate a major tonic role of brain and peripheral RAS on hypertension associated with antenatal nutrient deprivation.

Development of a Novel Noncompetitive Antagonist of IL-1 Receptor

IL-1 is a major proinflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein subunits. Currently available strategies to counter pathological IL-1 signaling rely on a recombinant IL-1 receptor antagonist, which directly competes with IL-1 for its binding site. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1R accessory protein, which are putative interactive sites with the IL-1RI subunit. In this study, we substantiate the merits of one of these peptides, rytvela (we termed “101.10”), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC50 ≈ 1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed noncompetitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. Findings describe the discovery of a potent and specific small (peptide) antagonist of IL-1RI, with properties in line with an allosteric negative modulator.

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

Interleukin-1 and Ischemic Brain Injury in the Newborn: Development of a Small Molecule Inhibitor of IL-1 Receptor

Inflammation participates in the genesis and progression of hypoxic-ischemic brain injury. Interleukin (IL)-1 is a major pro-inflammatory cytokine, which plays a dominant role in hypoxic-ischemic (and postinfectious) brain damage. Abundant evidence reveals the principal involvement of IL-1 over other pro-inflammatory cytokines. IL-1 interacts with the IL-1 receptor I (IL-1RI). The natural IL-1 receptor antagonist (IL-1ra) is a large 17.5-kDa peptide that competes with IL-1 for its binding site on IL-1RI. Recombinant IL-1ra (Kineret) is effective in human inflammatory conditions. However, a number of drawbacks of IL-1ra limit its broader use; these include injection site reactions [70%], broad immunosuppression, and high costs. We hereby report the characterization of a small (peptide) IL-1RI antagonist we developed, namely rytvela (termed 101.10), and its efficacy in models of (gut) inflammation and of newborn hypoxic-ischemic brain injury. Experiments reveal that 101.10 is selective for the IL-1RI and inhibits to a variable extent different effects induced by IL-1. 101.10 is effective in vivo (on systemic as well as oral administration) in established models of inflammation involving IL-1, notably in inflammatory bowel disease, and is superior to dexamethasone. In a rat pup model of hypoxic-ischemic brain injury (Rice-Vannucci model), where IL-1 and IL-1R expression is increased, 101.10 preserved microvascular density, parenchymal integrity, and brain mass. In conclusion, we hereby describe for the first time the discovery of a stable, potent, and effective specific IL-1RI small (peptide) antagonist, namely 101.10 (rytvela), which exhibits allosteric modulatory properties, and is effective in vivo in models of inflammation (known to involve IL-1) and in particular in hypoxic-ischemic newborn brain injury. 101.10 (and small alike compounds) may be suitable alternatives to IL-1ra.

Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.

Cyclooxygenase- (COX) 2 is the main intrinsic source of prostaglandins (PG) in the ductus arteriosus (DA) of the newborn (NB) but not of the fetus. |[bull]| 109

Non-selective COX inhibitors are the mainstay for treatment of patent DA. COX-1 is the principal source of PG in the systemic circulation of the NB; but inhibition of COX-1 compromises gut and renal function. Selective COX-2 blockers would be effective in closing NB DA provided this enzyme is the principal source of PG in this tissue and intrinsic DA PG contribute more than circulating PG (from COX-1) to ductal tone. We studied the ontogeny of COX-1 and 2 expression in DA, and evaluated its functional significance. DA of NB( 90%, <10% and ≈50% of PG synthesis respectively in the NB DA, pulmonary artery and aorta, arose from COX-2. In contrast to NB, DA of fetal (95% gestation) pig expressed simply COX-1, and was confirmed in sheep and human fetus. Functional significance of predominant COX-2 expression in NB DA was evaluated by measuring contraction of isolated pig DA (bath[O2]=5%) to COX-2 blocker DuP697 (10 μM); DuP697 elicited DA contraction not augmented by non-selective COX blocker ibuprofen (0.1 mM). Finally, administration of DuP697 (5 mg/kg iv, which reduces COX-2 activity but not circulating PGE2 levels) to NB pig (<2½ h old) and fetal sheep (90% gestation) did not affect DA patency (echocardiogram), whereas indomethacin decreased plasma PGE2 levels and closed the DA. Data indicate that the DA of the NB, in contrast to that of the fetus, expresses abundantly more COX-2 than COX-1; but the circulating PGs, mostly from COX-1, exert the major control on DA patency in vivo. Thus COX-2 inhibitors are not an alternative to non-selective COX inhibitors to treat patent DA. On the other hand, because COX-2 is induced in inflamed tissue as well as in fetal membranes during labor, blockers of COX-2 to women during gestation (eg. early tocolysis) may be preferable to non-selective COX inhibitors which may undesirably close fetal DA.

MODE OF ACTION OF ISOPROSTANES ON RETINAL VESSELS: Novel intermediate mediators of oxidation exerting greater vasoconstriction in newborn (NB) than adult (A). ♦ 325

MODE OF ACTION OF ISOPROSTANES ON RETINAL VESSELS: Novel intermediate mediators of oxidation exerting greater vasoconstriction in newborn (NB) than adult (A). ♦ 325