Janet J. Maguire

Orphan‐receptor ligand human urotensin II: receptor localization in human tissues and comparison of vasoconstrictor responses with endothelin‐1

We have determined the distribution of receptors for human urotensin‐II (U‐II) in human and rat CNS and peripheral tissues. In rat, [125I]‐U‐II binding density was highest in the abducens nucleus of brainstem (139.6±14 amol mm−2). Moderate levels were detected in dorsal horn of spinal cord and lower levels in aorta (22.5±6 amol mm−2). In human tissues density was highest in skeletal muscle and cerebral cortex (∼30 amol mm−2), with lower levels (<15 amol mm−2) in kidney cortex and left ventricle. Little binding was identified in atria, conducting system of the heart and lung parenchyma. Receptor density was less in human coronary artery smooth muscle (14.6±3 amol mm−2, n=10) than rat aorta with no significant difference between normal and atherosclerotic vessels. In human skeletal muscle [125I]‐U‐II bound to a single receptor population with KD=0.24±0.17 nM and Bmax=1.97±1.1 fmol mg−1 protein (n=4). U‐II contracted human coronary, mammary and radial arteries, saphenous and umbilical veins with sub‐nanomolar EC50 values. U‐II was 50 times more potent in arteries and <10 times more potent in veins than endothelin‐1 (ET‐1). The maximum response to U‐II (∼20% of control KCl) was significantly less than to ET‐1 (∼80% KCl). In contrast, in rat aorta, U‐II and ET‐1 were equipotent with similar maximum responses. This is the first report of high affinity receptors for [125I]‐U‐II in human CNS and peripheral tissues. This peptide produces potent, low efficacy, vasoconstriction in human arteries and veins. These data suggest a potential role for U‐II in human physiology.

Chronic Apoptosis of Vascular Smooth Muscle Cells Accelerates Atherosclerosis and Promotes Calcification and Medial Degeneration

Vascular smooth muscle cell (VSMC) accumulation is implicated in plaque development. In contrast, VSMC apoptosis is implicated in plaque rupture, coagulation, vessel remodeling, medial atrophy, aneurysm formation, and calcification. Although VSMC apoptosis accompanies multiple pathologies, there is little proof of direct causality, particularly with the low levels of VSMC apoptosis seen in vivo. Using a mouse model of inducible VSMC–specific apoptosis, we demonstrate that low-level VSMC apoptosis during either atherogenesis or within established plaques of apolipoprotein (Apo)E−/− mice accelerates plaque growth by two-fold, associated with features of plaque vulnerability including a thin fibrous cap and expanded necrotic core. Chronic VSMC apoptosis induced development of calcified plaques in younger animals and promoted calcification within established plaques. In addition, VSMC apoptosis induced medial expansion, associated with increased elastic lamina breaks, and abnormal matrix deposition reminiscent of cystic medial necrosis in humans. VSMC apoptosis prevented outward remodeling associated with atherosclerosis resulting in marked vessel stenosis. We conclude that VSMC apoptosis is sufficient to accelerate atherosclerosis, promote plaque calcification and medial degeneration, prevent expansive remodeling, and promote stenosis in atherosclerosis.

[125I]-(Pyr1)Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man

We have determined the binding characteristics of [125I]‐(Pyr1)Apelin‐13, a putative ligand for the APJ orphan receptor in human cardiovascular and rat tissue and investigated the functional properties of (Pyr1)Apelin‐13 in human saphenous vein. The binding of [125I]‐(Pyr1)Apelin‐13 to sections of human heart tissue was time dependent and rapid at 23°C. Data were fitted to a single site model with an association rate constant (kobs) of 0.115 min−1. [125I]‐(Pyr1)Apelin‐13 also dissociated from a single site with a dissociation rate constant of 0.0105 min−1. In saturation binding experiments [125I]‐(Pyr1)Apelin‐13 bound to human left ventricle with a KD value of 0.35±0.08 nM, Bmax of 4.3±0.9 fmol mg−1 protein with a Hill slope of 0.97±0.04 and to the right atria with a KD of 0.33±0.09 nM, Bmax of 3.1±0.6 fmol mg−1 protein and a Hill slope of 0.93±0.05. [125I]‐(Pyr1)Apelin‐13 binding sites were localized using autoradiography to human cardiovascular tissue, including coronary artery, aorta and saphenous vein grafts. In rat tissue a high density of receptors were localized to the molecular layer of the rat cerebellum, rat lung, rat heart and low levels in the rat kidney cortex. (Pyr1)Apelin‐13 potently contracted human saphenous vein with a pD2 value of 8.4±0.2 (n=8). The maximum response elicited by the peptide was 22.6±6% of 100 mM KCl. We provide the first evidence of APJ receptor expression, relative densities and functional properties of (Pyr1)Apelin‐13 in human cardiovascular tissue.

[Pyr1]Apelin-13 Identified as the Predominant Apelin Isoform in the Human Heart: Vasoactive Mechanisms and Inotropic Action in Disease

Apelin receptors, present on vascular smooth muscle cells, endothelium, and cardiomyocytes, are activated by the family of apelin peptides to elicit cardiovascular effects in experimental animals, but functional activity in humans has not been studied in detail. We detected low levels of apelin immunoreactivity in plasma of volunteers consistent with an autocrine/paracrine action and detected apelin immunoreactivity in the supernatant from human cultured endothelial cells. We found that [Pyr1]apelin-13 was the predominant isoform in cardiac tissue from patients with coronary artery disease. We tested the hypothesis that apelins have vascular and cardiac actions in human tissues in vitro and compared responses to [Pyr1]apelin-13, apelin-13, and apelin-36. In endothelium-intact mammary artery, all 3 of the apelins induced concentration-dependent vasodilatation with comparable potency (EC50: 0.6 to 1.6 nM; maximum response: 40% to 50%). Vasodilatation was abolished after endothelial removal or preincubation with indomethacin but was unaffected by preincubation with NG-nitro-l-arginine methyl ester, indicating involvement of prostanoids but not NO in dilatation by apelins in this patient group. Apelins were potent constrictors of endothelium-denuded saphenous vein (EC50: 0.6 to 1.6 nM; maximum response: 17% to 26%) and mammary artery ([Pyr1]apelin-13; EC50: 0.2 nM; maximum response: 29%). In paced atrial strips, all 3 of the peptides increased the force of contraction with subnanomolar potencies (EC50: 40 to 125 pM). For the first time, we demonstrate that the 3 principal forms of apelin have comparable potency and efficacy in human cardiovascular tissues. Apelins are potent endothelium-dependent vasodilators acting via a prostanoid-dependent mechanism; however, removal of the endothelium revealed direct vasoconstrictor actions in both the artery and vein. Furthermore, in human cardiac tissue, the apelin peptides are among the most potent endogenous positive inotropic agents yet reported.

ETA receptor-mediated constrictor responses to endothelin peptides in human blood vessels in vitro

1 We have characterized the constrictor endothelin receptors present in human isolated blood vessels using ETA and ETB selective agonists and antagonists. 2 Monophasic dose‐response curves were obtained for ET‐1 with EC50 values of 6.8 nM in coronary artery, 3.9 nM in internal mammary artery, 17.4 nM in pulmonary artery, 14.5 nM in aorta and 3.2 nM in saphenous vein. In coronary artery, ET‐2 was equipotent with ET‐1 with an EC50 value of 5.7 nM. The non‐selective peptide, sarafotoxin 6b, was 2–3 times less potent than ET‐1 but the maximum responses to these two were comparable. 3 In each vessel ET‐3 was much less active than ET‐1. No response was obtained to ET‐3 in aorta and pulmonary artery or in up to 50% of coronary artery, mammary artery and saphenous vein preparations. In those preparations that did respond, dose‐response curves were incomplete at 300 nM. Variable contractions were also obtained with the ETB‐selective agonist, sarafotoxin 6c (S6c). Where responses were detected, although S6c was more potent than ET‐1 (EC50 values of 0.6‐1.2 nM), the maximum response produced was always less than 20% of that to ET‐1. 4 The synthetic ETB agonists, BQ3020 and [l,3,11,15Ala]‐ET‐l, were without effect in any of the five blood vessels at concentrations up to 3 μM. 5 ET‐1‐induced vasoconstriction was blocked by the ETA‐selective antagonists, BQ123 and FR139317. Schild‐derived pA2 values were 7.0, 7.4 and 6.9 for BQ123 and 7.6, 7.9 and 7.3 for FR139317 in coronary artery, mammary artery and saphenous vein, respectively, consistent with antagonism of ETA receptors. Slopes of the Schild regressions were not significantly different from one. Comparable values of pA2 were estimated for 3 μM BQ123 in aorta (7.4 ±0.5) and pulmonary artery (6.9) from the Gaddum‐Schild equation. 6 In conclusion we have shown that in human isolated blood vessels, ET‐1 is more potent than ET‐3 suggesting the presence of vasoconstrictor ETA receptors. This is supported by the lack of effect of the ETB agonists, BQ3020 and [1,3,11,15Ala]‐ET‐l and the ability of the ETA antagonists, BQ123 and FR 139317 to block ET‐1 responses. Some preparations did contract in response to low concentrations of the ETB‐selective sarafotoxin 6c but responses were variable and the maximum was always much less than that to ET‐1 in the same preparations. Therefore although constrictor ETB receptors were present on the smooth muscle of human blood vessels, vasoconstriction elicited by the endothelin peptides in vitro is via ETA receptor activation.

Human endothelin receptors characterized using reverse transcriptase-polymerase chain reaction, in situ hybridization, and subtype-selective ligands BQ123 and BQ3020 : evidence for expression of ETB receptors in human vascular smooth muscle

Our aim was to characterize and determine the function of endothelin (ET) receptor subtypes in human vascular tissue. Reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers detected the presence of mRNA encoding both ETA and ETB receptors in the media from aorta and pulmonary and coronary arteries. In situ hybridization confirmed the presence of mRNA for both subtypes in the media of coronary arteries. Saturation binding assays using 125I-ET-1 found a single population of high-affinity ET receptors (n = three patients, +/- SEM) in aorta (Kd = 0.507 +/- 0.020 nM; Bmax = 9 +/- 4 fmol/mg protein) and pulmonary (Kd = 0.845 +/- 0.245 nM; Bmax = 15 +/- 10 fmol/mg protein) and coronary arteries (Kd = 0.141 +/- 0.020 nM; Bmax = 71 +/- 21 fmol/mg protein). Using media from coronary arteries, the ETA-selective ligand BQ123 (cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp]) and the ETB-selective ligand BQ3020 (Ala11,15-Ac-ET-1[6-21]) both produced biphasic competition binding curves against 125I-ET-1, confirming the presence of high- and low-affinity sites corresponding to the two subtypes: BQ123 (KdETA = 0.85 +/- 0.03 nM; KdETB = 7.58 +/- 2.27 microM; ETA/ETB, 87%:13%) and BQ3020 (KdETA = 0.22 +/- 0.04 microM; KdETB = 0.77 +/- 0.34 nM; ETA/ETB, 62%:38%). BQ123 (0.1 microM) caused a significant parallel rightward shift of ET-1-induced vasoconstriction of coronary arteries in vitro, but BQ3020 and Ala1,3,11,15-ET-1 failed to show any agonist activity when tested at concentrations of < or = 3 microM in three vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Is urotensin-II the new endothelin?

Urotensin‐II (U‐II), a peptide isolated from the urophysis of teleost fish 35 years ago, is the endogenous ligand of the mammalian orphan receptor GPR14/SENR. Recently, human homologues of both the receptor (UT‐II) and the peptide (hU‐II) have been discovered. Following de‐orphanization, hU‐II was declared the ‘new endothelin’ as initial studies suggested similarities between the peptides, and in isolated arteries of cynomolgus monkey U‐II was a more potent constrictor than endothelin‐1 (ET‐1), with equal efficacy. However, effects of U‐II in vascular tissue from other mammalian species are variable and although potent, U‐II exhibits a lesser maximal response than ET‐1. In contrast, in humans U‐II has emerged as a ubiquitious constrictor of both arteries and veins in vitro and elicits a reduction in blood flow in the forearm and skin microcirculation in vivo. In addition to direct vasoconstrictor activity on smooth muscle receptors, endothelium‐dependent U‐II‐mediated vasodilatation has also been observed. Non‐vascular, peripheral actions of U‐II include potent inotropy and airway smooth muscle constriction and U‐II and its receptor are present throughout rat brain implying a possible neurotransmitter or neuromodulatory role in the central nervous system. U‐II is proposed to contribute to human diseases including atherosclerosis, cardiac hypertrophy, pulmonary hypertension and tumour growth. The development of selective receptor antagonists should help to clarify the relative importance of hU‐II as a multifunctional peptide in mammalian systems and its role in disease. What is clear is that U‐II is emerging as a new and potentially important mammalian transmitter.

Intracellular Interleukin-1 Receptor 2 Binding Prevents Cleavage and Activity of Interleukin-1α, Controlling Necrosis-Induced Sterile Inflammation

Summary Necrosis can induce profound inflammation or be clinically silent. However, the mechanisms underlying such tissue specificity are unknown. Interleukin-1α (IL-1α) is a key danger signal released upon necrosis that exerts effects on both innate and adaptive immunity and is considered to be constitutively active. In contrast, we have shown that necrosis-induced IL-1α activity is tightly controlled in a cell type-specific manner. Most cell types examined expressed a cytosolic IL-1 receptor 2 (IL-1R2) whose binding to pro-IL-1α inhibited its cytokine activity. In cell types exhibiting a silent necrotic phenotype, IL-1R2 remained associated with pro-IL-1α. Cell types possessing inflammatory necrotic phenotypes either lacked IL-1R2 or had activated caspase-1 before necrosis, which degraded and dissociated IL-1R2 from pro-IL-1α. Full IL-1α activity required cleavage by calpain after necrosis, which increased its affinity for IL-1 receptor 1. Thus, we report a cell type-dependent process that fundamentally governs IL-1α activity postnecrosis and the mechanism allowing conditional release of this blockade.

Chemokine receptor CCR5: from AIDS to atherosclerosis

There is increasing recognition of an important contribution of chemokines and their receptors in the pathology of atherosclerosis and related cardiovascular disease. The chemokine receptor CCR5 was initially known for its role as a co‐receptor for HIV infection of macrophages and is the target of the recently approved CCR5 antagonist maraviroc. However, evidence is now emerging supporting a role for CCR5 and its ligands CCL3 (MIP‐1α), CCL4 (MIP‐1β) and CCL5 (RANTES) in the initiation and progression of atherosclerosis. Specifically, the CCR5 deletion polymorphism CCR5delta32, which confers resistance to HIV infection, has been associated with a reduced risk of cardiovascular disease and both CCR5 antagonism and gene deletion reduce atherosclerosis in mouse models of the disease. Antagonism of CCL5 has also been shown to reduce atherosclerotic burden in these animal models. Crucially, CCR5 and its ligands CCL3, CCL4 and CCL5 have been identified in human and mouse vasculature and have been detected in human atherosclerotic plaque. Not unexpectedly, CC chemokines have also been linked to saphenous vein graft disease, which shares similarity to native vessel atherosclerosis. Distinct roles for chemokine–receptor systems in atherogenesis have been proposed, with CCR5 likely to be critical in recruitment of monocytes to developing plaques. With an increased burden of cardiovascular disease observed in HIV‐infected individuals, the potential cardiovascular‐protective effects of drugs that target the CCR5 receptor warrant greater attention. The availability of clinically validated antagonists such as maraviroc currently provides an advantage for targeting of CCR5 over other chemokine receptors.

Cellular distribution of immunoreactive urotensin-II in human tissues with evidence of increased expression in atherosclerosis and a greater constrictor response of small compared to large coronary arteries

We detected urotensin-II-like immunoreactivity in the endothelium of normal human blood vessels from heart, kidney, placenta, adrenal, thyroid and umbilical cord. Immunoreactivity was also detected in endocardial endothelial and kidney epithelial cells. In atherosclerotic coronary artery, immunoreactivity localized to regions of macrophage infiltration. Urotensin-II constricted human atherosclerotic epicardial coronary arteries with pD2=10.58 +/- 0.46 (mean +/- S.E.M.) and Emax=11.4 +/- 4.2% KCl and small coronary arteries with pD2=9.25 +/- 0.38 and Emax=77 +/- 16% KCl. Small coronary arteries clearly exhibited a greater maximum response to urotensin-II than epicardial vessels. This enhanced responsiveness may be of importance in heart failure, where circulating concentrations of U-II are increased, or in atherosclerosis where focally up-regulated urotensin-II production may act down stream to produce significant vasospasm, compromising blood flow to the myocardium. We conclude that urotensin-II is a locally released vasoactive mediator that may be an important regulator of blood flow particularly to the myocardium and may have a specific role in human atherosclerosis.