Claude R. Benedict

Proinflammatory cytokine levels in patients with depressed left ventricular ejection fraction: a report from the Studies of Left Ventricular Dysfunction (SOLVD).

OBJECTIVES This study sought to assess proinflammatory cytokine levels in patients in the studies of left ventricular dysfunction trial (SOLVD) in relation to both their New York Heart Association functional classification and their neurohormonal status before randomization. BACKGROUND Elevated levels of tumor necrosis factor-alpha have been identified in 30% to 40% of patients with heart failure. However, it is unclear which subsets of patients with heart failure elaborate tumor necrosis factor-alpha. It is also unclear what the mechanism for the increased expression of proinflammatory cytokines is. METHODS Tumor necrosis factor-alpha and interleukin-6 levels were analyzed by enzymes-linked immunoassay using randomly selected plasma samples from patients in functional classes I to III who were enrolled in neurohormonal substudies of the SOLVD trial; age-matched healthy subjects served as the control group. RESULTS Plasma levels of tumor necrosis factor-alpha (p < 0.001) were elevated in patients in functional classes I to III ([mean +/- SD] 1.95 +/- 0.54, 2.63 +/- 0.48, 6.4 +/- 1.9 pg/ml, respectively) compared with age-matched control subjects (0.75 +/- 0.05 pg/ml) and were progressively elevated in relation to decreasing functional status of the patient. Plasma levels of interleukin-6 (p < 0.001) were elevated in patients in functional classes I to III (3.3 +/- 0.55, 6.2 +/- 1.1, 5.22 +/- 0.9 pg/ml, respectively) compared with age-matched control subjects (1.8 +/- 0.5 pg/ml and were progressively elevated in relation to decreasing functional status of the patient. Cox proportional-hazards analysis showed that there was a trend toward significance between plasma tumor necrosis factor-alpha (p < 0.07) and survival, whereas there was no significant relation for plasma interleukin-6 (p < 0.72). Except for atrial natriuretic factor, which correlated weakly (r = 0.23, p = 0.04) with circulating tumor necrosis factor-alpha levels, there was no significance correlation between neurohormonal and proinflammatory cytokine levels. CONCLUSIONS Circulating levels of proinflammatory cytokines increase in patients as their functional heart failure classification deteriorates. Moreover, activation of the neurohumoral axis is unlikely to completely explain the elaboration of proinflammatory cytokines in heart failure.

Prognostic Significance of Plasma Norepinephrine in Patients With Asymptomatic Left Ventricular Dysfunction

BACKGROUND Elevated plasma neurohormonal levels are associated with increased mortality rates in patients with symptomatic heart failure. A previous Studies of Left Ventricular Dysfunction (SOLVD) trial suggested that neurohumoral activation precedes the development of symptoms as demonstrated by increased neurohormonal levels in patients with asymptomatic left ventricular dysfunction. However, the significance of this early neurohumoral activation is unclear. The goals of this study were to determine the prognostic significance of the plasma concentrations of plasma norepinephrine (PNE) and atrial natriuretic peptide (ANP) and the renin activity (PRA) in patients with asymptomatic left ventricular dysfunction. METHODS AND RESULTS PNE and PRA were measured before randomization in 514 patients with left ventricular ejection fractions < or = 35% who did not require treatment for congestive heart failure and were enrolled in the SOLVD Prevention Trial. Plasma ANP levels were measured in a subset of 241 patients owing to study design. Using the Cox proportional hazards model that included left ventricular ejection fraction, New York Heart Association functional class, age, sex, treatment assignment to placebo or enalapril, and cause of heart failure, we examined whether these neurohormones predicted all-cause mortality, cardiovascular mortality, hospitalization for heart failure, development of heart failure, or development of ischemic events (myocardial infarction or unstable angina). PNE was the strongest predictor of clinical events in this patient population. PNE levels above the median of 393 pg/mL were associated with a relative risk of 2.59 (P = .002) for all-cause mortality, 2.55 (P = .003) for cardiovascular mortality, 2.55 (P = .005) for hospitalization for heart failure, 1.88 (P = .002) for development of heart failure, 1.92 (P = .001) for ischemic events, and 2.59 (P = .005) for myocardial infarction. PNE remained the most powerful predictor for all-cause mortality and ischemic events when the analysis included only the patients with histories of ischemic left ventricular dysfunction. The increases in other neurohormonal levels were not useful in predicting the subsequent development of clinical events. CONCLUSIONS Increased PNE levels in patients with asymptomatic left ventricular dysfunction appear to predict all-cause and cardiovascular mortalities and development of clinical events related to the onset of heart failure or acute ischemic syndromes. Thus, measurement of PNE may be a possible early marker for assessment of disease progression in patients with left ventricular dysfunction, and modulating the release or effect of PNE may lead to improved prognosis and/or a reduction in morbidity.

Effect of Serotonin, Thromboxane A2, and Specific Receptor Antagonists on Vascular Smooth Muscle Cell Proliferation

BACKGROUND Restenosis is a major complication that limits the long-term efficacy of coronary angioplasty. Migration and proliferation of activated medial smooth muscle cells (SMCs) is considered an important mechanism in this process. Because at sites of vascular injury, aggregating platelets release both serotonin (5-HT) and thromboxane A2 (TXA2), we examined whether 5-HT and TXA2 can induce SMC proliferation and whether there is synergistic interaction between these two mediators. METHODS AND RESULTS The mitogenic effects of 5-HT and TXA2 either alone or in combination was examined in serum-free medium on canine aortic SMCs by [3H]thymidine incorporation into DNA and by cell counting. 5-HT induced SMC proliferation at a concentration of 100 nmol/L, whereas the effect of TXA2 (U46619, a stable TXA2 mimetic) on inducing proliferation of SMCs was observed at a concentration of 100 nmol/L. When these two mediators were added together, there was a synergistic interaction on inducing SMC proliferation even at subthreshold concentrations. The mitogenic effect of 5-HT and its synergistic interaction with TXA2 on SMC proliferation was abolished by a 5-HT2 receptor antagonist, LY281067, without affecting the contribution of TXA2. Similarly, the TXA2 synthase inhibitor/receptor antagonist ridogrel abolished the mitogenic effect of TXA2 and the interaction between 5-HT and TXA2 without affecting the response to 5-HT. When LY281067 and ridogrel were used together, they abolished the mitogenic effects of 5-HT and TXA2. CONCLUSIONS At sites of vascular injury, platelet-induced SMC proliferation may also be modulated by nonpeptide growth mediators. A combination of a 5-HT2 receptor antagonist and TXA2 synthase inhibitor/receptor may be useful for attenuation of restenosis after angioplasty.

Local Effect of Serotonin Released during Coronary Angioplasty

BACKGROUND Serotonin is released after the aggregation of platelets, a phenomenon that may occur after coronary angioplasty. We sought to determine whether serotonin is released into the coronary circulation during coronary angioplasty and to assess whether serotonin can affect coronary-artery tone during angioplasty. METHODS Blood samples were drawn from the ascending aorta and the coronary sinus of eight patients scheduled to undergo angioplasty of the left anterior descending or circumflex coronary artery. Samples were obtained before angioplasty and after each balloon dilation. The dimensions of arterial segments distal to the site of dilation were measured angiographically before angioplasty and 5 and 15 minutes after the last dilation in these eight patients and in seven similar patients; the latter group was treated with ketanserin, a serotonin2-receptor antagonist, before angioplasty. RESULTS Before the eight patients underwent angioplasty, their mean (+/- SE) plasma serotonin level in the aorta was 2.5 +/- 0.7 ng per milliliter and that in the coronary sinus was 2.3 +/- 0.6 ng per milliliter (P = 0.34). The serotonin level in plasma from the coronary sinus rose significantly, to 31.5 +/- 13.5, 17.6 +/- 5.3, and 29.1 +/- 8.1 ng per milliliter after the first, second, and third dilations, respectively (P = 0.014 for the comparison with preoperative levels). In contrast, the serotonin level in plasma from the ascending aorta did not change. The cross-sectional area of the coronary artery was significantly reduced 5 and 15 minutes after the last dilation (from a preoperative value of 3.7 +/- 0.5 mm2 to 2.7 +/- 0.4 mm2 15 minutes after the last dilation; P = 0.011). This vasoconstriction was significantly blunted in the seven patients who received ketanserin (from 3.7 +/- 0.5 mm2 before angioplasty to 3.9 +/- 0.4 mm2 after 15 minutes) (P = 0.017 for comparison with the eight patients who did not receive ketanserin). CONCLUSIONS Serotonin is released into the coronary circulation during angioplasty, and this vasoactive substance may contribute to the occurrence of vasoconstriction distal to the dilated site. The vasoconstriction is attenuated by ketanserin, a serotonin2-receptor antagonist.

Comparative neurohormonal responses in patients with preserved and impaired left ventricular ejection fraction: Results of the studies of left ventricular dysfunctions (SOLVD) registry

Abstract Objectives . The aim of this study was to determine the differences in neurohumoral responses between patients with pulmonary congestion with and without impaired left ventricular ejection fraction. Background . Previous studies have established the presence of neurohumoral activation in patients with congestive heart failure. It is not known whether the activation of these neurohumoral mechanisms is related to the impairment in systolic contractility. Methods . The 898 patients recruited into the Studies of Left Ventricular Dysfunction (SOLVD) Registry substudy were examined to identify those patients with pulmonary congestion on chest X-ray film who had either unpaired ( 45%, group II) left ventricular ejection fraction. Plasma norepinephrine, plasma renin activity, arginine vasopressin and atrial natriuretic peptide levels were measured in these two groups of patients and compared with values in matched control subjects, Results . Distribution of the New York Heart Association symptom classification was the same in the two groups of patients. Compared with control subjects, patients in group II with pulmonary congestion and preserved ejection fraction had no activation of the neurohumoral mechanisms, except for a small but statistically significant increase in arginine vasopressin and plasma renin activity. Compared with patients in group II, those in group I with pulmonary congestion and unpaired ejection fraction had significant increases in plasma norepinephrine (p Conclusions . The results point to the importance of the decrease in left ventricular ejection fraction as one of the mechanisms for activation of neurohormones in patients with heart failure.

New Variant of Human Tissue Plasminogen Activator (TPA) With Enhanced Efficacy and Lower Incidence of Bleeding Compared With Recombinant Human TPA

BACKGROUND The thrombolytic properties of a new variant of tissue plasminogen activator (TPA) (T103N, N117Q, KHRR 296-299 AAAA, or TNK-TPA) with longer plasma half-life, greater fibrin specificity, and increased resistance to inhibition by plasminogen activator inhibitor (PAI-1) were investigated in a rabbit thrombosed carotid artery model. METHODS AND RESULTS After 60 minutes of arterial occlusion, TPA (1.5, 3.0, 6.0, or 9.0 mg/kg as a front-loaded IV infusion for 90 minutes; n = 22) or TNK-TPA (0.38, 0.75, or 1.5 mg/kg as IV bolus; n = 16) was administered. Blood flow through the artery was monitored for an additional 120 minutes. Bleeding was assessed by weighing the amount of blood absorbed in a gauze pad placed in a subcutaneous muscular incision. Recanalization rates and duration of recanalization were dose dependent. The doses that produced > 80% recanalization rates with the longest duration of recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg for TNK-TPA. At these doses, time to reperfusion (mean +/- SEM) was significantly faster (11 +/- 2 versus 23 +/- 7 minutes) and duration of recanalization longer (77 +/- 9 versus 51 +/- 18 minutes) for TNK-TPA compared with TPA (P < .025). Weights of the residual thrombi of the TPA group were greater than those of the TNK-TPA group (P = .004). Concentrations of fibrinogen, plasminogen, and alpha 2-antiplasmin at 120 minutes were significantly higher for TNK-TPA-treated animals compared with TPA-treated animals (P < .001). ANOVA of the blood loss data determined that there were significant differences between thrombolytic agents but not between doses. After correction for saline controls, total blood loss for pooled doses of TPA and TNK-TPA was 82 +/- 6 mg and 40 +/- 4 mg, respectively (P < .01). CONCLUSIONS From these data, we conclude that TNK-TPA, given as a bolus, produces faster and more complete recanalization of occluded arteries in a rabbit experimental model compared with TPA, without increasing systemic plasmin generation or peripheral bleeding. In addition, we observed that TNK-TPA, unlike TPA, did not potentiate collagen-induced aggregation of platelets obtained from human plasma. This lack of effect on platelet aggregation by TNK-TPA potentially could be associated with a decreased risk of reocclusion after successful thrombolysis.

Correlation of plasma serotonin changes with platelet aggregation in an in vivo dog model of spontaneous occlusive coronary thrombus formation.

The role of platelets in contributing to occlusive coronary artery thrombus formation remains unresolved. A large number of studies have utilized in vitro techniques to study platelet aggregation. This report describes a model of spontaneous in vivo thrombus formation which involves application of current in the left circumflex coronary artery of the dog. Changes in mean coronary blood flow velocity (50% above control) are used to predict the point at which current can be discontinued without interrupting the ongoing process of thrombus formation. Thrombus formation proceeds to total vessel occlusion within 62 ± 18 minutes after discontinuation of current. Coronary sinus plasma serotonin concentrations are used as an in vivo index of platelet aggregation during thrombus formation. Plasma serotonin levels increased only slightly above baseline levels during initial thrombus formation. Coronary sinus serotonin levels rose markedly after cessation of current, reaching a peak just prior to total vessel occlusion. The marked increase in serotonin concentration observed in the latter stages of thrombus formation strongly suggests that platelet aggregation is a significant factor in the evolution of an occlusive coronary thrombus.

Progression of left ventricular dysfunction secondary to coronary artery disease, sustained neurohormonal activation and effects of ibopamine therapy during long-term therapy with angiotensin-converting enzyme inhibitor☆

Left ventricular function and neurohormonal status in patients with heart failure remaining symptomatic during therapy with angiotensin-converting enzyme inhibitors were assessed, and the effects of dopaminergic receptor stimulation in this setting were determined. Neurohormonal and left ventricular function (radionuclide angiography) data were obtained in 19 patients with symptomatic ischemic heart failure. Measurements were repeated after 4 to 6 weeks of therapy with the dopamine agonist ibopamine (100 mg, 3 times/day) or placebo administered in a double-blind, randomized, parallel group design. At baseline, despite therapy with enalapril, the angiotensin II levels (mean 39.4 pg/ml; p < 0.01 vs controls) were significantly increased, as were plasma norepinephrine (497 +/- 240 pg/ml; p < 0.01 vs controls), endothelin-1, atrial natriuretic peptide and arginine vasopressin. Moreover, in comparison with pretreatment values, left ventricular ejection fraction had decreased substantially (-9.1%) in patients with plasma norepinephrine > or = 600 pg/ml, but not in those with lower values of norepinephrine. With ibopamine, plasma norepinephrine decreased from 516 +/- 241 to 391 +/- 208 pg/ml (n = 8; p < 0.025 vs placebo), whereas it increased with placebo. In conclusion, the neurohormonal control provided by an angiotensin-converting enzyme inhibitor is reduced in a large subset of patients during prolonged therapy; ibopamine appears to be a potentially useful drug to improve neurohormonal control in this setting.

Mitogenic effect of serotonin on vascular endothelial cells.

BACKGROUND Recent studies indicate that serotonin (5-HT) has a growth-promoting effect on several different cell types, including smooth muscle cells. After percutaneous transluminal coronary angioplasty, there is damage and denudation of vascular endothelial cells, which promotes platelet aggregation at the site of injury. Aggregating platelets release 5-HT; thus, a high concentration of the amine may be present at sites of endothelial damage, which may act as a mitogen to endothelial cells. METHODS AND RESULTS The mitogenic effect of 5-HT was examined on canine and bovine aortic endothelial cells by (1) assessing the increase in [3H]thymidine incorporation into DNA and (2) assessing the increase in the absolute number of cells after stimulation with 5-HT. 5-HT at an added concentration of 200 to 1000 mumol/L in the media induced a significant increase in [3H]thymidine incorporation into endothelial cells and an increase in cell number. This effect was not observed with fibroblasts. As the concentrations of added 5-HT were decreased, the endothelial cells had to be stimulated with 5-HT for longer periods to induce the same degree of cellular proliferation. The precursors and metabolic breakdown products of 5-HT were inactive. The 5-HT-induced endothelial proliferation was reversed by 5-HT2 receptor antagonists and pertussis toxin. These data suggest that the mitogenic effect of 5-HT on endothelial cells is mediated by the 5-HT2 receptor, which is coupled to a G protein. CONCLUSIONS 5-HT is a mitogen for endothelial cells at concentrations likely to be present at sites of vascular injury. This effect is probably mediated via the 5-HT2 receptor. The growth-promoting effects of 5-HT on endothelial cells may facilitate the healing of intima after vascular damage.

Platelet Microparticles Promote Platelet Interaction With Subendothelial Matrix in a Glycoprotein IIb/IIIa–Dependent Mechanism

BACKGROUND Platelets, on activation, release vesicular particles called platelet microparticles. Despite their procoagulant activity, their functional role in platelet-vessel wall interactions is not known. METHODS AND RESULTS We examined the binding of microparticles to vessel wall components in vitro and in vivo. Microparticles bound to fibrinogen-, fibronectin-, and collagen-coated surfaces. Compared with activated platelets, we observed minimal binding of microparticles to vitronectin and von Willebrand factor. The glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors abciximab and eptifibatide (Integrilin) inhibited the binding to fibrinogen and fibronectin but had minimal effect on binding to collagen. Furthermore, monoclonal antibodies to GP Ib or anionic phospholipid-binding proteins (beta2-glycoprotein I or annexin V) had no effect in these interactions. Microparticles did not bind to monolayers of resting or stimulated human umbilical vein endothelial cells (HUVECs), even in the presence of fibrinogen or von Willebrand factor. However, under similar conditions, microparticles bound to extracellular matrix produced by cultured HUVECs. Abciximab inhibited this interaction by approximately 50%. In a rabbit model of arterial endothelial injury, the infusion of 51Cr-labeled microparticles resulted in a 3- to 5-fold increase of microparticle adhesion to the injured site compared with the uninjured site (P<0.05%). Furthermore, activated platelets bound to surface-immobilized microparticles in a GP IIb/IIIa-dependent mechanism. This binding increased in the presence of fibrinogen by approximately 30%. CONCLUSIONS Platelet microparticles bind to subendothelial matrix in vitro and in vivo and can act as a substrate for further platelet binding. This interaction may play a significant role in platelet adhesion to the site of endothelial injury.