S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

Preserved regulation of renal perfusion pressure by small and intermediate conductance KCa channels in hypertensive mice with or without renal failure.

The purpose of this study was to assess, in the murine kidney, the mechanisms underlying the endothelium-dependent control of vascular tone and whether or not, in a severe model of hypertension and renal failure, KCa channels contribute to its regulation. Wild-type (BL) and double-transgenic female mice expressing human angiotensinogen and renin (AR) genes received either control or a high-salt diet associated to a nitric oxide (NO) synthase inhibitor treatment (BLSL and ARSL). Changes in renal perfusion pressure (RPP) were measured in isolated perfused kidneys. BLSL and AR were moderately hypertensive without kidney disease while ARSL developed severe hypertension and renal failure. In the four groups, methacholine induced biphasic endothelium-dependent responses, a transient decrease in RPP followed by a cyclooxygenase-dependent increase in RPP. In the presence or not of indomethacin, the vasodilatations were poorly sensitive to NO synthase inhibition. However, in the presence of cyclooxygenase and NO synthase inhibitors, apamin, and/or TRAM-34, blockers of KCa2.3 and KCa3.1, respectively, abolished the decrease in RPP in response to either methacholine or the two activators of KCa2.3/KCa3.1, NS309, and SKA-31. Thus, KCa2/3 channels play a major role in the regulation of murine kidney perfusion and this mechanism is maintained in hypertension, even when severe and associated with kidney damage.

Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure.

Adenosine A1 receptors (A1R) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for A1R-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the A1R agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the A1R biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca2+ influx relative to NECA and the cAMP pathway at the A1R, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the A1R. In contrast to VCP746, which displays more 'adenosine-like' signaling at the A2BR, neladenoson was a highly selective A1R agonist, with biased, weak agonism at the A2BR. Together these results show that unwanted hemodynamic effects of A1R agonists can be avoided by compounds biased away from Ca2+ influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that A1R-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.

Soluble Siglec-5 associates to PSGL-1 and displays anti-inflammatory activity.

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general.

Role of NADPH oxidase-mediated superoxide production in the regulation of E-selectin expression by endothelial cells subjected to anoxia/reoxygenation.

OBJECTIVE: Anoxia followed by reoxygenation (A/R) increases endothelial cell superoxide (O2-) generation which is implicated in E-selectin overexpression. The mechanisms which govern these processes are not fully understood and therefore the goal of our study was to determine the functional importance of NADPH oxidase in the regulation of E-selectin expression in human umbilical veins endothelial cells (HUVECs) submitted to A/R. METHODS: O2- production was estimated using lucigenin chemiluminescence and formazan accumulation. NADPH oxidase expression in HUVECs was studied by RT-PCR and Western blot and E-selectin by Northern blot analysis. NFkappaB activation was assessed by electrophoretic mobility shift assay. RESULTS: A/R caused an increased O2- production which was inhibited by the superoxide dismutase mimetic M40403 (50 micromol/l), the protein kinase C inhibitor chelerythrine (10 micromol/l), the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 micromol/l) and the NADPH oxidase assembly blocker apocynin (600 micromol/l). At the end of the anoxic period, the mRNA expression and the protein p47phox was increased as compared to normoxic HUVECs. NFkappaB activation of anoxic HUVECs was maximal after 1 h of reoxygenation and returned to basal normoxic levels after 2 h of reoxygenation. Apocynin reduced the NFkappaB activation at 1 h of reoxygenation. E-selectin mRNA expression was increased after 3 h of reoxygenation of anoxic HUVECs and the SOD mimetic M40403 as well as apocynin prevented this overexpression. CONCLUSIONS: Activated NADPH oxidase is a critical enzyme in E-selectin overexpression after A/R of HUVECs. Moreover, A/R increased expression of membranous and cytosolic NADPH oxidase subunits as well as the protein p47phox. Strategies aimed at preventing endothelial NADPH oxidase activation and/or activity may be useful in controlling leukocyte adhesion during ischemia/reperfusion.

Effect of NADPH oxidase inhibition on E-selectin expression induced by concomitant anoxia/reoxygenation and TNF-alpha.

The aim of this study was to quantify the expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vascular endothelial cells (HUVECs) exposed to anoxia/reoxygenation (A/R) in the presence or absence of an inflammatory context (0.1 IU/ml tumor necrosis factor-alpha [TNF-alpha]) and to investigate the effects of two different NADPH inhibitors, apocynin and diphenyleneiodonium (DPI), on the expression of the endothelial cell adhesion molecules. Confluent HUVECs were exposed to anoxia for 3 hours (100% N2), followed by a reoxygenation period of 4 hours. TNF-alpha at 0.1 IU/ml was added to the medium either under normoxic conditions for 7 hours (TNF-alpha) or just before the start of anoxia (A/R + TNF-alpha). Levels of E-selectin, VCAM-1, and ICAM-1 were quantified using specific monoclonal antibodies revealed by an alkaline phosphatase-labeled goat F(ab)'2 fragment against mouse IgG antibody and the fluorescent substrate Attophos. Adhesion experiments were also performed using calcein-labeled U937 leukocytes. HUVECs submitted to A/R overexpressed E-selectin but not VCAM-1 or ICAM-1, whereas TNF-alpha at 0.1 IU/ ml increased the expression of all three adhesion molecules. In endothelial cells subjected to A/R in the presence of TNF-alpha, a synergistic increase of E-selectin expression and a synergistic adhesion of U937 cells was noted. The NADPH oxidase inhibitors apocynin and DPI both decreased significantly the U937 adhesion and the E-selectin overexpression on HUVECs submitted to A/R, TNF-alpha, or A/R + TNF-alpha. These results suggest that E-selectin expression is implicated in the leukocyte adhesion to HUVECs caused by A/R in the presence or absence of an inflammatory context. NADPH oxidase appears to participate in the E-selectin overexpression on HUVECs subjected either to A/R and/or TNF-alpha, suggesting a major role of this enzyme in the ischemia/reperfusion syndrome.

The anti-aggregating effect of BAY 41-2272, a stimulator of soluble guanylyl cyclase, requires the presence of nitric oxide.

BACKGROUND AND PURPOSE: The purpose of the present study was to determine whether a stimulator of soluble guanylyl cyclase, BAY 41-2272, inhibits platelet aggregation and to clarify its interaction with nitric oxide (NO). EXPERIMENTAL APPROACH: Blood was collected from anaesthetized Wistar Kyoto rats. The aggregation of washed platelets was measured and the production of cAMP and cGMP was determined. KEY RESULTS: In adenosine 5'-diphosphate (ADP)-induced platelet aggregation, the anti-aggregating effects of BAY 41-2272, nitroglycerin, sodium nitroprusside and DEA-NONOate were associated with increased levels of cGMP while that of beraprost, a prostacyclin analogue, was correlated with an increase in cAMP. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) prevented the effects of BAY 41-2272 and that of nitroglycerin and sodium nitroprusside, but only inhibited the increase in cGMP produced by of DEA-NONOate. Hydroxocobalamin, an NO scavenger, inhibited the effects of the three NO donors and BAY 41-2272 but did not affect those of beraprost. ADP-induced aggregation and the effects of BAY 41-2272 were not affected by L-nitroarginine. A positive interaction was observed between BAY 41-2272 and the three NO donors. BAY 41-2272 potentiated also the anti-aggregating effects of beraprost, and again this potentiation was inhibited by hydroxocobalamin. CONCLUSIONS AND IMPLICATIONS: Inhibition of platelet aggregation by BAY 41-2272 requires the reduced form of soluble guanylyl cyclase and the presence of NO. The positive interaction observed between BAY 41-2272 and various NO donors is qualitatively similar whatever the mechanism involved in NO release. Furthermore, a potent synergism is observed between BAY 41-2272 and a prostacyclin analogue, but only in the presence of NO.

Anti-aggregating effect of BAY 58-2667, an activator of soluble guanylyl cyclase.

The purpose of the present study was to determine whether an activator of soluble guanylyl cyclase (sGC), BAY 58-2667, inhibits platelet aggregation and to clarify its mechanism of action. Blood was collected from anesthetized WKY rats. The aggregation of washed platelet was measured and the production of cAMP and cGMP was determined. BAY 58-2667 produced a partial inhibition of the ADP- and collagen-induced platelet aggregation, but did not significantly affect thrombin-induced aggregation. In ADP-induced platelet aggregation, the inhibitory effects of BAY 58-2667 were associated with an increased level of both cGMP and cAMP while that of the prostacyclin analogue, beraprost, was correlated only with an increase in cAMP. The inhibitor of sGC, ODQ, enhanced the effects of BAY 58-2667. The presence of L-nitroarginine, an inhibitor of NO-synthase, hydroxocobalamin, a scavenger of NO, or that of three different NO-donors did not affect the anti-aggregating effect of BAY 58-2667. However, the anti-aggregating effects of beraprost were potentiated by BAY 58-2667. Therefore, the platelet inhibitory effects of BAY 58-2667 are associated with the generation of cGMP and a secondary increase in cAMP, both being totally NO-independent. When the sGC is oxidized, BAY 58-2667 becomes a relevant anti-aggregating agent, which synergizes with the cAMP-dependent pathway.

Aging and prostacyclin responses in aorta and platelets from WKY and SHR rats.

In spontaneously hypertensive rat (SHR) aorta, prostacyclin is an endothelium-derived contracting factor contributing to the endothelial dysfunction. This study was designed to determine whether the impairment of the prostacyclin response is influenced by aging and whether such a dysfunction is observed in platelets. Isometric tension was measured in aortic rings, and aggregation was studied in platelet-rich plasma taken from 3-, 6-, and 15-mo-old Wistar-Kyoto rats (WKY) and SHR. In aorta from 3- and 6-mo-old WKY, prostacyclin and beraprost [prostacyclin receptor (IP) agonists] produced relaxations that were enhanced by Triplion (thromboxane-prostanoid receptor antagonist). In 15-mo-old WKY, the relaxations to beraprost were maintained, but not those to prostacyclin. In SHR aorta, prostacyclin or beraprost produced no or minor relaxations, which, in younger SHR, were enhanced by Triplion. In both strains, the relaxations were inhibited by CAY-10441 (IP receptor antagonist). The relaxations to forskolin and isoproterenol were reduced with aging. When compared with those of WKY, the relaxations to isoproterenol were reduced in 3- but not in 6- or 15-mo-old SHR, whereas those to forskolin were consistently diminished at any given age. Whatever the age, prostacyclin and beraprost produced CAY-10441-sensitive inhibitions of ADP-induced platelet aggregation. Both agonists were more potent in SHR than in WKY. Therefore, in platelets from WKY and SHR, the IP receptor-dependent antiaggregant response is functional and maintained during aging. In aorta from WKY those responses are reduced by aging and, in SHR, are already compromised at 3 mo. This dysfunction of the IP receptor is only partially explained by a general dysfunction of the adenylate cyclase pathway.

Altered TP receptor function in isolated, perfused kidneys of nondiabetic and diabetic ApoE-deficient mice.

Early manifestations of kidney disease occur in atherosclerosis and activation of TP (thromboxane A(2)) receptors is implicated in atherosclerotic, diabetes, and renal diseases. The purpose of the present study was to analyze, in isolated, perfused mouse kidneys, the participation of TP receptors in renal vasoconstrictions and vasodilatations. In kidneys, taken from wild-type C57BL6, apolipoprotein E-deficient (ApoE-KO) and diabetic ApoE-KO mice, changes in perfusion pressure were recorded. Constrictions to TP receptor ligands U 46619, arachidonic acid, PGH(2), and 8-iso-PGF(2alpha), but not those to angiotensin II, endothelin, or norepinephrine, were inhibited by the selective TP receptor antagonist Triplion (S 18886; 10 nM). Acetylcholine and prostacyclin evoked biphasic responses during methoxamine constrictions; the constrictor part was blocked by Triplion. In ApoE-KO mouse kidneys, compared with C57BL6, a specific decrease in norepinephrine response and no modification in dilator responses were observed. In diabetic ApoE-KO mouse kidneys, constrictions to U 46619 and those to 8-iso-PGF(2alpha) were significantly and selectively augmented, without modification in the expression of the TP receptor, and again without any significant change in vasodilator activity. Thus TP receptors are functional, and their activation is not involved in norepinephrine, endothelin, and angiotensin II vasoconstrictions but is implicated in the unusual vasoconstrictions to acetylcholine and prostacyclin. Increased responsiveness of TP receptors occurs in diabetic ApoE-KO mouse kidneys. Thus early changes in TP receptor-mediated vasoconstrictor activity may participate in the development of kidney disease in atherosclerosis and diabetes.

In SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors.

In mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), acetylcholine and the calcium ionophore A-23187 release endothelium-derived contracting factors (EDCFs), cyclooxygenase derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine are most likely prostacyclin and prostaglandin (PG)H(2), whereas those released by A-23187 remain to be identified. Isometric tension and the release of PGs were measured in rings of isolated aortas of WKY and SHR. A-23187 evoked the endothelium-dependent release of prostacyclin, thromboxane A(2), PGF(2alpha), PGE(2), and possibly PGH(2) (PGI(2) >> thromboxane A(2) = PGF(2alpha) = PGE(2)). In SHR aortas, the release of prostacyclin and thromboxane A(2) was significantly larger in response to A-23187 than to acetylcholine. In response to the calcium ionophore, the release of thromboxane A(2) was significantly larger in aortas of SHR than in those of WKY. In both strains of rat, the inhibition of cyclooxygenase-1 prevented the release of PGs and the occurrence of endothelium-dependent contractions. Dazoxiben, the thromboxane synthase inhibitor, abolished the A-23187-dependent production of thromboxane A(2) and inhibited by approximately one-half the endothelium-dependent contractions. U-51605, an inhibitor of PGI synthase, reduced the release of prostacyclin elicited by A-23187 but induced a parallel increase in the production of PGE(2) and PGF(2alpha), suggestive of a PGH(2) spillover, which was associated with the enhancement of the endothelium-dependent contractions. These results indicate that in the aorta of SHR and WKY, the endothelium-dependent contractions elicited by A-23187 involve the release of thromboxane A(2) and prostacyclin with a most likely concomitant contribution of PGH(2).

Long-term incubation with IL-4 and IL-10 oppositely modifies procoagulant activity of monocytes and modulates the surface expression of tissue factor and tissue factor pathway inhibitor.

Monocytes can be induced to express both tissue factor (TF) and its inhibitor, TF pathway inhibitor-1 (TFPI-1). A short incubation (<6 h) with interleukin (IL)-4 and IL-10, two potent deactivators of monocyte functions, has been shown to modulate the synthesis and expression of TF by monocytes activated by lipopolysaccharide, but the consequences of longer incubations (up to 96 h) on both TF and TFPI-1 are unknown. The results of this study showed that adherent monocytes in culture spontaneously expressed TF and TFPI and that prolonged incubation with IL-10 induced a time- and dose-dependent decrease of monocyte TF synthesis, and an accumulation of TF/TFPI-1 complexes at the moncyte surface, suggesting a decreased clearance of these complexes. In contrast, IL-4 induced a time- and dose-dependent increase in TF synthesis, which remained intracytoplasmic, as shown by confocal microscopy. Surprisingly, TF:antigen (Ag) was decreased at the monocyte surface, but the procoagulant activity (PCA) of IL-4-treated monocytes was increased, as a result of more pronounced decrease of TFPI-1:Ag expression than that of TF. In conclusion, prolonged incubation with IL-4 and IL-10 oppositely modified PCA of cultured monocytes, and altered TF and TFPI trafficking and clearance. These data explain the respective deleterious or benefit effects of IL-4 or IL-10 in atherothrombosis.