Martin Beauchamp

Modulation of Pro-inflammatory Gene Expression by Nuclear Lysophosphatidic Acid Receptor Type-1

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.

Cyclooxygenase-2 in Human and Experimental Ischemic Proliferative Retinopathy

Background—Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)–2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. Methods and Results—We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. Conclusion—These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP3 Receptors

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE2 in tissues and presence of perinuclear PGE2 receptors (EP). We presently studied mechanisms by which PGE2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE2 and selective EP3 receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP3 immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP3 receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear KCa channels were detected, and their functionality corroborated by NS1619-induced Ca2+ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca2+ chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as KCa channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-&kgr;B inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca2+ transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE2 through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope KCa channels, protein kinases, and NF-&kgr;B; the roles for nuclear EP3 receptors seem different from those on plasma membrane.

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.

Selective Neuromicrovascular Endothelial Cell Death by 8-Iso-Prostaglandin F2α Possible Role in Ischemic Brain Injury

Background and Purpose— Free radical-induced peroxidation is an important factor in the genesis of hypoxic-ischemic encephalopathy, including that of the preterm infant. Isoprostanes are major peroxidation products. Since microvascular dysfunction seems to contribute to ischemic encephalopathies, we studied the cytotoxicity of 8-iso-prostaglandin F2&agr; (PGF2&agr;) on cerebral microvascular cells. Methods— Microvascular endothelial, astroglial, and smooth muscle cells from newborn brain were cultured. The cytotoxicity of 8-iso-PGF2&agr; on these cells was determined by MTT assays and lactate dehydrogenase (LDH) release, propidium iodide incorporation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]). In addition, effects of intraventricular injections of 8-iso-PGF2&agr; and possible involvement of thromboxane in 8-iso-PGF2&agr;-induced cytotoxicity were determined. Results— 8-Iso-PGF2&agr; induced time- and concentration-dependent endothelial cell death (EC50=0.1 nmol/L) but exerted little effect on smooth muscle and astroglial cells; endothelial cell death seemed mostly of oncotic nature (propidium iodide incorporation and LDH release). Cell death was associated with increased endothelial thromboxane A2 (TXA2) formation and was prevented by TXA2 synthase inhibitors (CGS12970 and U63557A); TXA2 mimetics U46619 and I-BOP also caused endothelial cell death. Intraventricular injection of 8-iso-PGF2&agr; induced periventricular damage, which was attenuated by CGS12970 pretreatment. Conclusions— These data disclose a novel action of 8-iso-PGF2&agr; involving TXA2 in oxidant stress-induced cerebral microvascular injury and brain damage.

Understanding ischemic retinopathies: emerging concepts from oxygen-induced retinopathy

Ischemic retinopathies, such as retinopathy of prematurity and diabetic retinopathy are characterized by an initial microvascular degeneration, followed by an abnormal hypoxia-induced neovascularization. Oxygen-induced retinopathy (OIR) is a well-established in vivo model of ischemic retinopathies, which, although the triggering insult varies, all share a common end result of capillary loss. Understanding the mechanisms of normal retinal vascular development as well as the pathophysiological processes leading to the primary vascular loss is the key to develop treatments to prevent the sight-threatening neovascularization associated with human ischemic retinopathies. The importance of oxygen-dependant vascular endothelial growth factor in the pathophysiology of both phases of OIR has long been recognized. However, recent studies point out that OIR is a multifactorial disease, resulting from additive effects of an unbalanced expression of pro- and anti-angiogenic factors, interrelated with protective effects of nutritional factors and cytotoxic effects of oxidative and nitro-oxidative stress-dependant mediators. This review summarizes the most recent aspects of the research on OIR conducted in our laboratory and others, with a particular focus on the role of new mediators of nitro-oxidative stress, the trans-arachidonic acids, in microvascular degeneration, and on a novel pathway of metabolic signaling where hypoxia-driven succinate, via receptor GPR91, governs normal and pathological retinal angiogenesis.

Prolonged Hypercapnia-Evoked Cerebral Hyperemia via K+ Channel– and Prostaglandin E2–Dependent Endothelial Nitric Oxide Synthase Induction

Abstract— Mechanisms for secondary sustained increase in cerebral blood flow (CBF) during prolonged hypercapnia are unknown. We show that induction of endothelial NO synthase (eNOS) by an increase in prostaglandins (PGs) contributes to the secondary CBF increase during hypercapnic acidosis. Ventilation of pigs with 6% CO2 (Paco2≈65 mm Hg; pH ≈7.2) caused a ≈2.5-fold increase in CBF at 30 minutes, which declined to basal values at 3 hours and gradually rose again at 6 and 8 hours; the latter increase was associated with PG elevation, nitrite formation, eNOS mRNA expression, and in situ NO synthase (NOS) reactivity (NADPH-diaphorase staining). Subjecting free-floating brain sections to acidotic conditions increased eNOS expression, the time course of which was similar to that of CBF increase. Treatment of pigs with the cyclooxygenase inhibitor diclofenac or the NOS inhibitor N&ohgr;-nitro-l-arginine blunted the initial rise and prevented the secondary CBF increase during hypercapnic acidosis; neuronal NOS blockers 1-(2-trifluoromethylphenyl) imidazole and 3-bromo-7-nitroindazole were ineffective. Diclofenac abolished the hypercapnia-induced rise in cerebrovascular nitrite production, eNOS mRNA expression, and NADPH-diaphorase reactivity. Acidosis (pH ≈7.15, Pco2≈40 mm Hg; 6 hours) produced similar increases in prostaglandin E2 (PGE2) and eNOS mRNA levels in isolated brain microvessels and in NADPH-diaphorase reactivity of brain microvasculature; these changes were prevented by diclofenac, by the receptor-operated Ca2+ channel blocker SK&F96365, and by the KATP channel blocker glybenclamide. Acidosis increased Ca2+ transients in brain endothelial cells, which were blocked by glybenclamide and SK&F96365 but not by diclofenac. Increased PG-related eNOS mRNA and NO-dependent vasorelaxation to substance P was detected as well in rat brain exposed to 6 hours of hypercapnia. PGE2 was the only major prostanoid that modulated brain eNOS expression during acidosis. Thus, in prolonged hypercapnic acidosis, the secondary CBF rise is closely associated with induction of eNOS expression; this seems to be mediated by PGE2 generated by a KATP and Ca2+ channel–dependent process.

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.

Proangiogenic Effects of Protease-Activated Receptor 2 Are Tumor Necrosis Factor-α and Consecutively Tie2 Dependent

Objective—Angiogenesis is essential physiologically in growth and pathologically in tumor development, chronic inflammatory disorders, and proliferative retinopathies. Activation of protease-activated receptor 2 (PAR2) leads to a proangiogenic response, but its mechanisms have yet to be specifically described. Here, we investigated the mode of action of PAR2 in retinal angiogenesis. Methods and Results—PAR2-activating peptide, SLIGRL, increased retinal angiogenesis associated with an induction of vascular endothelial growth factor and angiopoetin-2 and most notably tie2 in the retina in vivo as well as in cultured neuroretinal endothelial cells. SLIGRL also induced release of the proinflammatory and angiogenic mediator tumor necrosis factor-α (TNF-α) via the MEK/extracellular signal-regulated kinase (ERK) (MEK/ERK) pathway in these endothelial cells. TNF-α, in turn, elicited tie2 expression by activating the MEK/ERK pathway. PAR2-evoked tie2 expression, endothelium proliferation (in vitro), and retinal neovascularization (in vivo) were abrogated by selective TNF-α blockers (neutralizing antibody infliximab and soluble TNF-α receptor-Fc fusion protein etanercept) as well as the MEK inhibitor PD98059. Conclusion—The proangiogenic properties of PAR2 are intertwined with its proinflammatory effects, such that in retinal vasculature, they depend on TNF-α and subsequent induction of tie2 via the MEK/ERK pathway.