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.

Lipocalin-2 derived from adipose tissue mediates aldosterone-induced renal injury.

Lipocalin-2 is not only a sensitive biomarker, but it also contributes to the pathogenesis of renal injuries. The present study demonstrates that adipose tissue-derived lipocalin-2 plays a critical role in causing both chronic and acute renal injuries. Four-week treatment with aldosterone and high salt after uninephrectomy (ANS) significantly increased both circulating and urinary lipocalin-2, and it induced glomerular and tubular injuries in kidneys of WT mice. Despite increased renal expression of lcn2 and urinary excretion of lipocalin-2, mice with selective deletion of lcn2 alleles in adipose tissue (Adipo-LKO) are protected from ANS- or aldosterone-induced renal injuries. By contrast, selective deletion of lcn2 alleles in kidney did not prevent aldosterone- or ANS-induced renal injuries. Transplantation of fat pads from WT donors increased the sensitivity of mice with complete deletion of Lcn2 alleles (LKO) to aldosterone-induced renal injuries. Aldosterone promoted the urinary excretion of a human lipocalin-2 variant, R81E, in turn causing renal injuries in LKO mice. Chronic treatment with R81E triggered significant renal injuries in LKO, resembling those observed in WT mice following ANS challenge. Taken in conjunction, the present results demonstrate that lipocalin-2 derived from adipose tissue causes acute and chronic renal injuries, largely independent of local lcn2 expression in kidney.

Endothelium-Dependent Hyperpolarization and Endothelial Dysfunction.

The endothelium controls vascular tone not only by releasing various vasoactive substances but also by another pathway associated with the hyperpolarization of both endothelial and vascular smooth muscle cells and is termed endothelium-dependent hyperpolarization (EDH). These responses involve an increase in the endothelial intracellular Ca concentration by the activation of transient receptor potential channels (predominantly TRPV4) followed by the opening of Ca-activated K channels of small and intermediate conductance (SKCa and IKCa). These channels show a distinct subcellular distribution. SKCa are widely distributed over the plasma membrane but segregates at sites of homocellular endothelial junctions, whereas IKCa are preferentially expressed in the myoendothelial projections. Following KCa activation, smooth muscle hyperpolarization is evoked by electrical coupling through myoendothelial gap junctions and/or by the potassium efflux that subsequently activates smooth muscle Kir2.1 and/or Na/K-ATPase. Alteration of the EDH contributes to the endothelial dysfunctions observed in various pathologies or conversely compensates for the loss in NO bioavailability. A better characterization of EDH should allow determining whether new druggable targets can be identified for the treatment of cardiovascular diseases.

Vascular dysfunctions in the isolated aorta of double-transgenic hypertensive mice developing aortic aneurysm.

Angiotensin-II and oxidative stress are involved in the genesis of aortic aneurysms, a phenomenon exacerbated by endothelial nitric oxide synthase (eNOS) deletion or uncoupling. The purpose of this work was to study the endothelial function in wild-type C57BL/6 (BL) and transgenic mice expressing the h-angiotensinogen and h-renin genes (AR) subjected to either a control, or a high-salt diet plus a treatment with a NO-synthase inhibitor, N-ω-nitro-L-arginine-methyl-ester (L-NAME; BLSL and ARSL). BLSL showed a moderate increase in blood pressure, while ARSL became severely hypertensive. Seventy-five percent of ARSL developed aortic aneurysms, characterized by major histo-morphological changes and associated with an increase in NADP(H) oxidase-2 (NOX2) expression. Contractile responses (KCl, norepinephrine, U-46619) were similar in the four groups of mice, and relaxations were not affected in BLSL and AR. However, in ARSL, endothelium-dependent relaxations (acetylcholine, UK-14304) were significantly reduced, and this dysfunction was similar in aortae without or with aneurysms. The endothelial impairment was unaffected by catalase, superoxide-dismutase mimetic, radical scavengers, cyclooxygenase inhibition, or TP-receptor blockade and could not be attributed to sGC oxidation. Thus, ARSL is a severe hypertension model developing aortic aneurysm. A vascular dysfunction, involving both endothelial (reduced role of NO) and smooth muscle cells, precedes aneurysms formation and, paradoxically, does not appear to involve oxidative stress.

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.

Deamidated lipocalin-2 induces endothelial dysfunction and hypertension in dietary obese mice.

BACKGROUND: Lipocalin-2 is a proinflammatory adipokine upregulated in obese humans and animals. A pathogenic role of lipocalin-2 in hypertension has been suggested. Mice lacking lipocalin-2 are protected from dietary obesity-induced cardiovascular dysfunctions. Administration of lipocalin-2 causes abnormal vasodilator responses in mice on a high-fat diet (HFD). METHODS AND RESULTS: Wild-type and lipocalin-2 knockout mice were fed with standard chow or HFD. Immunoassays were performed for evaluating the circulating and tissue contents of lipocalin-2. The relaxation and contraction of arteries were studied using a wire myograph. Blood pressure was monitored with implantable radio telemetry. Dietary obesity promoted the accumulation of lipocalin-2 protein in blood and arteries. Deficiency of this adipokine protected mice from dietary obesity-induced elevation of blood pressure. Mass spectrometry analysis revealed that human and murine lipocalin-2 were modified by polyamination. Polyaminated lipocalin-2 was rapidly cleared from the circulation. Adipose tissue was a major site for lipocalin-2 deamidation. The circulating levels and the arterial accumulation of deamidated lipocalin-2 were significantly enhanced by treatment with linoleic acid (18:2n-6), which bound to lipocalin-2 with high affinity and prevented its interactions with matrix metalloproteinase 9 (MMP9). Combined administration of linoleic acid with lipocalin-2 caused vascular inflammation and endothelial dysfunction and raised the blood pressure of mice receiving standard chow. A human lipocalin-2 mutant with cysteine 87 replaced by alanine (C87A) contained less polyamines and exhibited a reduced capacity to form heterodimeric complexes with MMP9. After treatment, C87A remained in the circulation for a prolonged period of time and evoked endothelial dysfunction in the absence of linoleic acid. CONCLUSIONS: Polyamination facilitates the clearance of lipocalin-2, whereas the accumulation of deamidated lipocalin-2 in arteries causes vascular inflammation, endothelial dysfunction, and hypertension.

Mechanisms of the vasorelaxing effects of CORM-3, a water-soluble carbon monoxide-releasing molecule: interactions with eNOS.

The purpose of the present work was to elucidate the mechanisms underlying the endothelium-dependent and endothelium-independent components of the vascular relaxation induced by a water-soluble and ruthenium-based carbon monoxide (CO)-releasing agent, tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). Changes in isometric tension and cyclic guanosine monophosphate (cGMP) production were measured in isolated aortic rings from normotensive Wistar-Kyoto rats. Nitric oxide (NO) generation was assessed in cultured human umbilical vein endothelial cells (HUVEC) by electron spin resonance. In rat aortic rings, CORM-3, but not the inactivated compound, iCORM, induced relaxations. In rings with but not in those without endothelium relaxations were partially inhibited by L-nitro-arginine (L-NA), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ), or hydroxocobalamin, inhibitors of NO-synthase, soluble guanylyl cyclase, and scavenger of NO, respectively. In rings with and without endothelium, deoxyhemoglobin abolished the relaxations. A combination of potassium channel blockers (barium, glibenclamide, and iberiotoxin) blunted the relaxation in rings without endothelium. CORM-3 produced an endothelium-dependent generation of cGMP that was inhibited by L-NA. CORM-3, but not iCORM, inhibited the endothelium-dependent relaxation to acetylcholine without affecting the response to sodium nitroprusside. In HUVEC, CORM-3 produced a concentration-dependent release of NO. Therefore, CORM-3-induced relaxations involve the soluble guanylyl cyclase-independent activation of smooth muscle potassium channels. Additionally, CO can produce concomitantly activation and inhibition of NO synthase, the former being responsible for the endothelium- and cGMP-dependent effect of CORM-3, the latter for the inhibition of acetylcholine-induced endothelium-dependent relaxations.

KCa 3.1 channels maintain endothelium-dependent vasodilatation in isolated perfused kidneys of spontaneously hypertensive rats after chronic inhibition of NOS.

BACKGROUND AND PURPOSE: The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury. EXPERIMENTAL APPROACH: Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar-Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N(ω) -nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg(-1) ·day(-1) ). KEY RESULTS: Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N(w) -nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca(2+) -activated K(+) channels of intermediate conductance (K(Ca) 3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca(2+) -activated K(+) channels of small conductance (K(Ca) 2.3), was ineffective. Dilatation induced by an opener of K(Ca) 3.1/K(Ca) 2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K(Ca) 3.1, but not K(Ca) 2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR). CONCLUSIONS AND IMPLICATIONS: In SHR kidneys, an EDHF-mediated response, involving activation of K(Ca) 3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.

Nitric oxide: orchestrator of endothelium-dependent responses.

The present review first summarizes the complex chain of events, in endothelial and vascular smooth muscle cells, that leads to endothelium-dependent relaxations (vasodilatations) due to the generation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) and how therapeutic interventions may improve the bioavailability of NO and thus prevent/cure endothelial dysfunction. Then, the role of other endothelium-derived mediators (endothelium-derived hyperpolarizing (EDHF) and contracting (EDCF) factors, endothelin-1) and signals (myoendothelial coupling) is summarized also, with special emphasis on their interaction(s) with the NO pathway, which make the latter not only a major mediator but also a key regulator of endothelium-dependent responses.

Endothelium-mediated control of vascular tone: COX-1 and COX-2 products.

Endothelium-dependent contractions contribute to endothelial dysfunction in various animal models of aging, diabetes and cardiovascular diseases. In the spontaneously hypertensive rat, the archetypal model for endothelium-dependent contractions, the production of the endothelium-derived contractile factors (EDCF) involves an increase in endothelial intracellular calcium concentration, the production of reactive oxygen species, the predominant activation of cyclooxygenase-1 (COX-1) and to a lesser extent that of COX-2, the diffusion of EDCF towards the smooth muscle cells and the subsequent stimulation of their thromboxane A2-endoperoxide TP receptors. Endothelium-dependent contractions are also observed in various models of hypertension, aging and diabetes. They generally also involve the generation of COX-1- and/or COX-2-derived products and the activation of smooth muscle TP receptors. Depending on the model, thromboxane A(2), PGH(2), PGF(2α), PGE(2) and paradoxically PGI(2) can all act as EDCFs. In human, the production of COX-derived EDCF is a characteristic of the aging and diseased blood vessels, with essential hypertension causing an earlier onset and an acceleration of this endothelial dysfunction. As it has been observed in animal models, COX-1, COX-2 or both isoforms can contribute to these endothelial dysfunctions. Since in most cases, the activation of TP receptors is the common downstream effector, selective antagonists of this receptor should curtail endothelial dysfunction and be of therapeutic interest in the treatment of cardiovascular disorders.

TP receptors and oxidative stress hand in hand from endothelial dysfunction to atherosclerosis.

Thromboxane A(2) and the activation of TP receptors that it causes play an important role in platelet aggregation and therefore in thrombosis. However, TP receptors are also involved in the pathologies of the vascular wall including impaired endothelium-dependent vasodilation, increased oxidant generation, and increased expression of adhesion molecules. The beneficial effects of TP antagonists on the vascular wall attenuate these features of vascular disease. They are not shared by aspirin. In fact, TP antagonists are active in patients treated with aspirin, indicating that their potential beneficial effects are mediated by mechanisms different from the antithrombotic actions of aspirin. Our studies have demonstrated the vascular benefits of TP antagonists in experimental animals, particularly in models of diabetes mellitus, in which elevated levels of eicosanoids play a role not only in vascular pathologies but also in those of the kidney and other tissues. They suggest that TP blockade protects against fundamental and widespread tissular dysfunction associated with metabolic disease including hyperlipidemia and hyperglycemia. TP receptor antagonists represent a promising avenue for the prevention of vascular disease in part because of these pleiotropic actions that extend beyond their antithrombotic properties.

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.

Endothelium-derived vasoactive factors and hypertension: possible roles in pathogenesis and as treatment targets.

Endothelial cells regulate vascular tone by releasing various contracting and relaxing factors including nitric oxide (NO), arachidonic acid metabolites (derived from cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases), reactive oxygen species, and vasoactive peptides. Additionally, another pathway associated with the hyperpolarization of the underlying smooth muscle cells plays a predominant role in resistance arteries. Endothelial dysfunction is a multifaceted disorder, which has been associated with hypertension of diverse etiologies, involving not only alterations of the L-arginine NO-synthase-soluble guanylyl cyclase pathway but also reduced endothelium-dependent hyperpolarizations and enhanced production of contracting factors, particularly vasoconstrictor prostanoids. This brief review highlights these different endothelial pathways as potential drug targets for novel treatments in hypertension and the associated endothelial dysfunction and end-organ damage.

Endothelium-derived hyperpolarising factors and associated pathways: a synopsis.

The term endothelium-derived hyperpolarising factor (EDHF) was introduced in 1987 to describe the hypothetical factor responsible for myocyte hyperpolarisations not associated with nitric oxide (EDRF) or prostacyclin. Two broad categories of EDHF response exist. The classical EDHF pathway is blocked by apamin plus TRAM-34 but not by apamin plus iberiotoxin and is associated with endothelial cell hyperpolarisation. This follows an increase in intracellular [Ca(2+)] and the opening of endothelial SK(Ca) and IK(Ca) channels preferentially located in caveolae and in endothelial cell projections through the internal elastic lamina, respectively. In some vessels, endothelial hyperpolarisations are transmitted to myocytes through myoendothelial gap junctions without involving any EDHF. In others, the K(+) that effluxes through SK(Ca) activates myocytic and endothelial Ba(2+)-sensitive K(IR) channels leading to myocyte hyperpolarisation. K(+) effluxing through IK(Ca) activates ouabain-sensitive Na(+)/K(+)-ATPases generating further myocyte hyperpolarisation. For the classical pathway, the hyperpolarising "factor" involved is the K(+) that effluxes through endothelial K(Ca) channels. During vessel contraction, K(+) efflux through activated myocyte BK(Ca) channels generates intravascular K(+) clouds. These compromise activation of Na(+)/K(+)-ATPases and K(IR) channels by endothelium-derived K(+) and increase the importance of gap junctional electrical coupling in myocyte hyperpolarisations. The second category of EDHF pathway does not require endothelial hyperpolarisation. It involves the endothelial release of factors that include NO, HNO, H(2)O(2) and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BK(Ca) and/or K(ATP), which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.