Tommy A. Brock

Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization.

Cultured endothelial cells release a potent vasoconstrictor peptide, endothelin. Cumulative addition of synthetic endothelin to isolated rabbit aortic rings elicited a concentration-dependent increase in contractile tension which was endothelium-independent. In cultured rabbit vascular smooth muscle cells loaded with the fluorescent dye fura 2, endothelin induced a concentration-dependent increase in [Ca2+]i over the range of 0.01 to 100 nM. Moreover, in the absence of extracellular Ca2+, endothelin could still induce an increase in [Ca2+]i. In addition, endothelin stimulated 45Ca2+ efflux from preloaded vascular smooth muscle cells in the presence and absence of extracellular Ca2+, as well as stimulating 45Ca2+ influx in a concentration-dependent manner. Measurement of inositol phosphates in [3H]-myoinositol-labelled vascular vascular trisphosphate. Unlabelled endothelin inhibited (125I)-endothelin binding to cultured rabbit vascular smooth muscle cells in a concentration-dependent manner. Binding was not inhibited by other vasoactive hormones or calcium channel ligands, suggesting cell surface receptors specific for endothelin. We conclude that one of the initial membrane events in the action of endothelin is to induce phospholipase C-stimulated PIP2 hydrolysis and that this signalling mechanism is initiated by endothelin/receptor interaction at the plasma membrane.

JAM2 interacts with alpha4beta1. Facilitation by JAM3.

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn2+-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and α4β1 in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an α4-specific inhibitor, to attenuate the Mn2+-enhanced component. Intriguingly, the engagement of α4β1 by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/α4β1 interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and α4β1counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on α4β1 interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin.

Bradycardia-Induced Coronary Angiogenesis Is Dependent on Vascular Endothelial Growth Factor

A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm(3)) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased >2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.

Na+-Ca2+ exchange in cultured vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) contract as intracellular free calcium ([Ca2+]1) rises. While Na+-Ca2+ exchange has been proposed to contribute to transmembrane Ca2+ flux, its role in cultured VSMC is unknown. Accordingly, we have investigated the role of Na+-Ca2+ exchange in unidirectional and net transmembrane Ca2+ fluxes in cultured rat aortic VSMC under basal conditions and following agonist-mediated stimulation. Transmembrane Ca2+ uptake was significantly increased in response to a low external Na+ concentration ([Na+]0) compared with 140 mM [Na+]0. Na+-dependent Ca2+uptake in response to low [Na+]0 was further increased by intracellular Na+ loading by preincubation of the VSMC with 1 mM ouabain. Under steady-state conditions, Ca2+ content varied inversely with [Na+]0, increasing from 1.0 nmol Ca2+/mg protein at 140 mM [Na+]0 to 4.0 nmol Ca2+/mg protein at 20 mM [Na+]0. Increasing [K+]0 to 55 mM also enhanced Na+-dependent Ca2+ influx. Augmentation of Ca2+ uptake with K+ depolarization was not significantly inhibited by the calcium channel antagonist verapamil. Transmembrane Ca2+ efflux was increased in response to 130 mM [Na+]0 compared with zero [Na+]0 (iso-osmotic substitution with choline+), and was further stimulated by the vasoconstrictor angiotensin II, which is known to elevate [Ca2+]1. These changes in [Ca2+]1 were studied directly using fura-2 fluorescence measurements. Elevated [Ca2+]1 levels returned to baseline more rapidly in the presence of normal (130 mM) [Na+]0 compared with zero [Na+]0 (iso-osmotic substitution with choline+). These findings suggest that a bidirectional Na+-Ca2+ exchange mechanism is present in cultured rat aortic VSMC. Na+-Ca2+ exchange appears to play a part in Ca2+ homeostasis, particularly under conditions of altered intracellular Na+ or increased [Ca2+]1 following agonist stimulation.

STRIDE 1: Effects of the selective ETA receptor antagonist, sitaxsentan sodium, in a patient population with pulmonary arterial hypertension that meets traditional inclusion criteria of previous pulmonary arterial hypertension trials

Sitaxsentan (SITAX; Thelin®, Encysive Corporation, Bellaire, TX, U.S.A.) is a highly selective oral endothelin-A receptor antagonist. STRIDE-1, a 12-week randomized, doubleblind, placebo-controlled trial of sitaxsentan for pulmonary arterial hypertension showed significant benefit in 6-minute walk distance, functional class and hemodynamics. Pulmonary arterial hypertension clinical trials traditionally limit enrolment to class III/IV patients with idiopathic pulmonary arterial hypertension or pulmonary arterial hypertension related to connective tissue disease, who have a baseline 6-minute walk distance of < 450 m. In contrast, STRIDE-1 included milder cases: class II patients, no baseline 6- minute walk cut-off, and congenital heart disease patients. We now present the STRIDE-1 subset who would have qualified under traditional inclusion criteria. The results were: change for placebo (mean ± SE) vs change for sitaxsentan (mean ± SE) vs treatment effect (mean), all statistically significant: 6-minute walk (m): -26 ± 13, 39 ± 10, 65; mean right atrial pressure (mmHg): 2.1 ± 0.8, -1.2 ± 0.5, -3.3; mean pulmonary arterial pressure (mmHg): 0.4 ± 1.5, -4.7 ± 1.5, -5.1; cardiac index (L/min per m2): -0.09 ± 0.09, 0.38 ± 0.06, 0.47; pulmonary vascular resistance (dyne·s·cm-5): 85 ± 60, -274 ± 47, -359. A 45% improvement in functional class was seen in sitaxsentan-treated patients (P = 0.0005). Thus, in the STRIDE-1 subpopulation that met enrolment criteria of previous pulmonary arterial hypertension trials, improvement in efficacy parameters with sitaxsentan therapy was even greater than seen in the entire STRIDE-1 population.

KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells.

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.

Protein kinase C-mediated intracellular alkalinization in rat and rabbit aortic smooth muscle cells

The influence of protein kinase C (C-kinase) activation on intracellular pH (pHi) of cultured rat (RASM) and rabbit (RBASM) aortic smooth muscle cells was studied by employing a pH-sensitive fluorescent-dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The known C-kinase activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) and mezerine as well as the agonist angiotensin II each caused an intracellular alkalinization of approximately 0.1-0.15 pH units in RASM and RBASM cells grown in serum-free conditions. TPA-induced alkalinization was sensitive to the Na+/H+ exchange blockers amiloride and 5-N-ethylisopropyl-amiloride (EIPA). These results suggest that protein kinase C activation leads to intracellular alkalinization in vascular smooth muscle cells and the increase in pHi might play an important role in receptor-coupled arterial contraction.

Therapeutic potential of endothelin receptor antagonists and nitric oxide donors in pulmonary hypertension.

Pulmonary hypertension can occur idiopathically as a primary disorder of the pulmonary circulation or more commonly, it can exist as a haemodynamic manifestation of a wide variety of pulmonary and cardiovascular diseases, including acute lung injury, chronic obstructive lung disease, congenital heart disease, mitral stenosis, chronic left-sided congestive heart failure and connective tissue diseases such as scleroderma. Pulmonary hypertension is associated with changes in vascular tone as well as vascular structure, with the relative contribution of each dependent upon the aetiology of the increased pulmonary vascular resistance. Most currently available treatments utilise anticoagulants as well as vasodilator drugs that only attenuate the vasoconstrictive component of the disease. The latter category includes oral calcium channel blockers, iv. and aerosolised prostacyclin analogues and inhaled nitric oxide but all three classes of vasodilators have disadvantages and limitations. Treatment with vasodilators is often ineffective in patients with longstanding pulmonary hypertension in which structural changes contribute significantly to the pulmonary hypertension, blood flow obstruction and right heart failure. In view of the immense clinical need, new therapies are being developed by pharmaceutical companies to treat pulmonary hypertension. This update will focus on the current development status of endothelin receptor antagonists and nitric oxide donors for the treatment of pulmonary hypertension.

Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.

Subtype-specific increase in G-protein α-subunit mRNA by interleukin 1β

The guanine nucleotide regulatory proteins (G‐proteins) which are substrates for ADP‐ribosylation by pertussis toxin (αi−1, αi−2, αi−3 and α0) transduce a variety of hormonal signals. Endothelial cells express mRNA for three αi subtypes although the level of αi−1 mRNA is very low. Interleukin 1β (IL1β), a pleiotropic inflammatory mediator which stimulates a complex series of responses in human endothelial cells leading to increased coagulation and platelet adhesion, increases expression of one subtype of αi (αi−2) mRNA in human endothelial cells as determined by Northern blot analysis without affecting the level of mRNA for other α‐subunits. These studies show that mRNA levels for αi subtypes are independently regulated, suggesting that there may be subtype specificity in the cells requirements for the Gi class of signal‐transducing proteins.