Sabine Kurz

Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone.

We tested the hypothesis that angiotensin II-induced hypertension is associated with an increase in vascular .O2- production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular .O2- production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/kg per d) produced a similar degree of hypertension, but did not increase vascular .O2- production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of .O2- activated by angiotensin II infusion is an NADH/NADPH-dependent, membrane-bound oxidase. Angiotensin II-, but not NE-, induced hypertension was associated with impaired relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. These relaxations were variably corrected by treatment of vessels with liposome-encapsulated superoxide dismutase. When Losartan was administered concomitantly with angiotensin II, vascular .O2- production and relaxations were normalized, demonstrating a role for the angiotensin type-1 receptor in these processes. We conclude that forms of hypertension associated with elevated circulating levels of angiotensin II may have unique vascular effects not shared by other forms of hypertension because they increase vascular smooth muscle .O2- production via NADH/NADPH oxidase activation.

Cigarette Smoking Potentiates Endothelial Dysfunction of Forearm Resistance Vessels in Patients With Hypercholesterolemia Role of Oxidized LDL

BACKGROUND Risk factors for atherosclerosis such as hypercholesterolemia and hypertension are associated with endothelial dysfunction of conduit and resistance vessels; however, the interaction of these risk factors and underlying mechanisms affecting endothelial function remain to be determined. The present study investigated the role of long-term smoking and hypercholesterolemia and their impact on endothelial function of peripheral resistance vessels in relation to plasma levels of autoantibodies against oxidized LDL, which has been implicated in the development of endothelial dysfunction and atherosclerosis. METHODS AND RESULTS The vascular responses to the endothelium-dependent agent acetylcholine (7.5, 15, 30, and 60 micrograms/min) and the endothelium-independent agent sodium nitroprusside (1,3, and 10 micrograms/min) were studied in normal control subjects (n = 10), patients with hypercholesterolemia (n = 15), long-term smokers (n = 15), and hypercholesterolemic patients who smoked (n = 15). Drugs were infused into the brachial artery, and forearm blood flow (FBF) was measured by venous occlusion plethysmography. The FBF responses to acetylcholine were significantly blunted in all three patient groups compared with normal control subjects (P < .05). The acetylcholine-induced increase in FBF was significantly attenuated in patients with hypercholesterolemia who smoked compared with hypercholesterolemic nonsmokers and normocholesterolemic smokers (P < .05 for both). The response to sodium nitroprusside was not statistically different in all four groups. Plasma levels of autoantibody titer against oxidized LDL were inversely related to acetylcholine-induced changes in FBF (r = -.53, P < .002) and were substantially increased in the group with both risk factors. CONCLUSIONS These results demonstrate that cigarette smoking and hypercholesterolemia synergistically impair endothelial function and that their combined presence is associated with increased plasma levels of autoantibodies against oxidized LDL. These observations raise the possibility that long-term smoking potentiates endothelial dysfunction in hypercholesterolemic patients by enhancing the oxidation of LDL.

Hydralazine prevents nitroglycerin tolerance by inhibiting activation of a membrane-bound NADH oxidase. A new action for an old drug.

Hydralazine has been shown to reduce mortality in patients with congestive heart failure when given concomitantly with isosorbide dinitrate. Recently, we demonstrated that nitrate tolerance is in part due to enhanced vascular superoxide .O2- production. We sought to determine mechanisms whereby hydralazine may prevent tolerance. Rabbits either received no treatment, nitroglycerin patches (1.5 micrograms/kg/min x 3 d), hydralazine alone (10 mg/kg/d in drinking water), or hydralazine and nitroglycerin. Aortic segments were studied in organ chambers and relative rates of vascular .O2- production were determined using lucigenin-enhanced chemiluminescence. Nitroglycerin treatment markedly inhibited relaxations to nitroglycerin (maximum relaxations in untreated: 92 +/- 1 vs. 64 +/- 3% in nitroglycerin-treated patients and increased vascular .O2- production by over two-fold (P < 0.05). Treatment with hydralazine in rabbits not receiving nitroglycerin significantly decreased .O2- production in intact rabbit aorta and increased sensitivity to nitroglycerin. When given concomitantly with nitroglycerin, hydralazine completely prevented the development of nitrate tolerance and normalized endogenous rates of vascular .O2- production. Studies of vessel homogenates demonstrated that the major source of .O2- was an NADH-dependent membrane-associated oxidase displaying activities of 67 +/- 12 vs. 28 +/- 2 nmol .O2-.min-1.mg protein-1 in nitroglycerin-treated vs. untreated aortic homogenates. In additional studies, we found that acute addition of hydralazine (10 microM) to nitroglycerin-tolerant vessels immediately inhibited .O2- production and NADH oxidase activity in vascular homogenates. The chemiluminescence signal was inhibited by a recombinant heparin-binding superoxide dismutase (HBSOD) demonstrating the specificity of this assay for .O2-. These observations suggest that a specific membrane-associated oxidase is activated by chronic nitroglycerin treatment, and the activity of this oxidase is inhibited by hydralazine, providing a mechanism whereby hydralazine may prevent tolerance. The ability of hydralazine to inhibit vascular .O2- anion production represents a novel mechanism of action for this drug.

Role of Endogenous Bradykinin in Human Coronary Vasomotor Control

BACKGROUND Bradykinin is a potent vasodilator that acts through B2 kinin receptors to stimulate the release of endothelium-derived nitric oxide, prostacyclin, and hyperpolarizing factor. In this study, we investigated the contribution of endogenous bradykinin to vasomotor control in the human coronary circulation. METHODS AND RESULTS The selective bradykinin B2 receptor antagonist HOE 140 was infused into the left main coronary artery (200 micrograms/min for 15 minutes) in 15 patients without significant coronary stenoses. Epicardial responses were evaluated by quantitative coronary blood flow with a Doppler flow-velocity wire. Flow-dependent dilation (n = 10; intracoronary papaverine) and acetylcholine responses (n = 5) were assessed before and after HOE 140. After HOE 140, there was a reduction in luminal area in the proximal (P < .001), mid (P < .001), and distal (P < .05) coronary arteries. HOE 140 led to an increase in coronary vascular resistance (P < .001) and a decrease in coronary blood flow (P < .001). After bradykinin B2 receptor blockade, there was a reduction in flow-dependent dilation (23.4 +/- 6.9% to 3.9 +/- 6.0%, P < .001), the extent of which correlated with the degree of basal vasoconstriction after HOE 140 in the same vessel segment (P < .05). Acetylcholine responses were unchanged after HOE 140. CONCLUSIONS The results of this study demonstrate for the first time a role for endogenous bradykinin in mediating normal vasomotor responses in resistance and epicardial coronary vessels under basal and flow-stimulated conditions in the human coronary circulation.

\E Role for Endothelin-1 in Angiotensin II– Mediated Hypertension

Experiments in cultured vascular smooth muscle cells have shown that angiotensin II (Ang II) stimulates expression of endothelin-1. We sought to examine role of endothelin-1 in the effects of Ang II in vivo. Ang II infusion in rats (0.7 mg/kg per day for 5 days) was associated with marked increases in vascular smooth muscle endothelin-1 levels, as assessed by immunostaining. Administration of the selective endothelin type A (ET(A)) receptor antagonist PD 155080 (50 mg/kg per day) abrogated the hypertensive response to a 5-day infusion of Ang II (0.7 mg/kg per day), as did losartan (25 mg/kg per day). ET(A) receptor blockade during Ang II-mediated hypertension was associated with marked elevations of plasma endothelin-1 levels. Ang II-mediated hypertension was associated with heightened vascular responsiveness to a variety of vasoconstrictor agents except endothelin-1. Blockade of ET(A) receptor invariably corrected this vasoconstrictor hyperresponsiveness. We conclude that some of the vascular effects of Ang II thought to be unique to this hormone are likely mediated by endothelin-1.

Evidence for a causal role of the renin-angiotensin system in nitrate tolerance

BACKGROUND We have previously shown that nitroglycerin (NTG) therapy increases vascular expression of endothelin 1 (ET-1) and stimulates vascular superoxide (O2.-) production via activation of NADH/NADPH oxidases. Both phenomena are stimulated by angiotensin II in vitro, and the renin-angiotensin system is activated during early nitrate therapy. We hypothesized that either angiotensin II or ET-1 may increase vascular O2.- production during nitrate therapy. METHODS AND RESULTS In New Zealand White rabbits, 3 days of treatment with NTG patches increased plasma renin activity for the entire treatment period. After 24 hours of NTG treatment, angiotensin II type 1 (AT1) receptor expression and vascular ACE activity were significantly decreased. At this time, constrictions to angiotensin I and II were depressed, but there was no loss of NTG vasodilator potency. Within 3 days of continuous NTG treatment, relaxations to NTG were markedly blunted. This was associated with an increase in AT1 receptor mRNA expression, a return of ACE activity back to baseline, and a marked increase in constrictions to angiotensin I and II despite continuously increased plasma renin activity. Tolerance was associated with a 2-fold increase in vascular O2.-, as estimated by lucigenin-enhanced chemiluminescence. Concomitant treatment with the AT1 receptor antagonist losartan (5 to 25 mg. kg-1. d-1) dose-dependently normalized vascular O2.- and prevented tolerance to NTG and cross-tolerance to endogenous nitric oxide released by acetylcholine. The nonselective ET-1 receptor blocker bosentan (100 mg. kg-1. d-1) had similar but less pronounced effects. CONCLUSIONS The positive effects of AT1 and ET-1 receptor blockade on tolerance and O2.- production imply a pathophysiological role for angiotensin II and to some extent for ET-1 in the development of nitrate tolerance.

New Insights Into Mechanisms UnderlyIng Nitrate tolerance

The hemodynamic and anti-ischemic efficacy of organic nitrates is rapidly blunted due to the development of nitrate tolerance. The mechanisms underlying this phenomenon remain poorly understood and likely involve several independent factors. More recent experimental observations suggest that tolerance may be the consequence of intrinsic abnormalities of the vasculature, including enhanced vascular superoxide and endothelin production. Superoxide anions degrade nitric oxide derived from nitroglycerin, whereas autocrine-produced endothelin within vascular smooth muscle sensitizes the vasculature to circulating neurohormones, such as catecholamines and angiotensin II, all of which may compromise the vasodilator potency of nitroglycerin. Interestingly, these vascular consequences of in vivo nitroglycerin treatment can be mimicked by incubating cultured endothelial and smooth muscle cells with angiotensin II. Further, nitrate tolerance and rebound following sudden cessation of prolonged nitroglycerin therapy can be prevented by concomitant treatment with high-dose angiotensin-converting enzyme inhibition or angiotensin-I receptor blockade. These data strongly suggest that increased circulating levels of angiotensin II, which are encountered during in vivo nitroglycerin treatment, initiate cellular events that ultimately attenuate the nitroglycerin vasodilator effects during prolonged treatment periods.

Effect of chronic angiotensin-converting enzyme inhibition on endothelial function in patients with chronic heart failure

Chronic heart failure is associated with neurohumoral activation and alterations of the peripheral circulation and skeletal muscle. Several mechanisms are involved in the impaired peripheral perfusion, including increased sympathetic tone and increased vascular stiffness. Recently, data suggest an important role of the endothelium for perfusion of skeletal muscle in heart failure. Endothelium-dependent dilation of resistance vessels is blunted in patients with severe chronic heart failure and may be involved in the impaired reactive hyperemia in these patients. In conductance vessels, flow-dependent dilation and the nitroglycerin-induced dilator response is attenuated in congestive heart failure as compared to normal subjects, indicating both endothelial dysfunction and a defect of smooth muscle relaxation. Recent data suggest that angiotensin-converting enzyme (ACE) inhibitors can improve endothelial function of resistance vessels, reduce serum level of the soluble endothelial (vascular cell) adhesion molecule (VCAM-1) and, in addition, improve peripheral vascular function by reducing or limiting the influence of cyclo-oxygenase-dependent vasoconstricting factor(s). It is conceivable that these beneficial effects of chronic ACE inhibition are due, in part, to blockade of bradykinin degradation by the ACE and the increased endothelial synthesis of prostaglandins and/or the release of nitric oxide by enhanced tissue levels of bradykinin.

Dissociation of coronary vascular tolerance and neurohormonal adjustments during long-term nitroglycerin therapy in patients with stable coronary artery disease

OBJECTIVES We sought to examine whether long-term nitroglycerin treatment causes tolerance in large coronary arteries and whether the loss of vascular effects parallels neurohormonal adjustments. BACKGROUND Nitroglycerin therapy is associated with increased plasma renin activity and aldosterone levels and a decrease in hematocrit. It is assumed that nitroglycerin tolerance results in part from these neurohormonal adjustments and intravascular volume expansion. METHODS Three groups were studied: group I (n = 10), no prior nitroglycerin therapy; and group II (n = 10) and group III (n = 8), 24- and 72-h long-term nitroglycerin infusion (0.5 micrograms/kg body weight per min), respectively. Coronary artery dimensions were assessed using quantitative angiography. Plasma renin activity, plasma aldosterone and vasopressin levels and hematocrit were monitored before and during nitroglycerin infusions. RESULTS In group I, increasing intravenous concentrations of nitroglycerin caused a dose-dependent increase of the midportion of the left anterior descending coronary artery (baseline diameter 2.13 +/- 0.07 mm [mean +/- SEM], maximally by 22 +/- 2%) and left circumflex coronary artery (baseline diameter 2.08 +/- 0.07) mm, maximally by 22 +/- 3%). An intracoronary nitroglycerin bolus (0.2 mg) caused no further significant increase in diameter, indicating maximal dilation. In group II (n = 10), the baseline large coronary artery diameter under ongoing nitroglycerin was significantly larger than that in group I (left anterior descending artery 2.61 +/- 0.08 mm, left circumflex artery 2.57 +/- 0.08 mm). Additional intravenous and intracoronary nitroglycerin challenges did not cause further dilation, indicating maximally dilated vessels. At the same time, plasma renin activity, plasma aldosterone and vasopressin levels were significantly increased, and hematocrit significantly decreased. In group III patients, the baseline diameter of the left anterior descending artery and the left circumflex artery did not differ from that in patients without nitroglycerin pretreatment, indicating a complete loss of nitroglycerin coronary vasodilative effects. These patients showed no significant increase in circulating neurohormonal levels but a significant decrease in hematocrit. CONCLUSIONS Within 24 h of continuous nitroglycerin treatment, the coronary arteries were maximally dilated despite neurohormonal adjustments and signs of intravascular volume expansion. Within 3 days of nitroglycerin infusion, tolerance developed in the absence of neurohormonal activation. The dissociation of neurohormonal adjustments and tolerance in large coronary arteries indicates that after long-term nitroglycerin treatment, true vascular tolerance, perhaps from an intracellular tolerance step, may have developed.

Neurohormonal inhibition and hemodynamic unloading during prolonged inhibition of ANF degradation in patients with severe chronic heart failure.

BackgroundThe purpose of this study was to investigate the therapeutic potential of prolonged inhibition of atrial natriuretic factor (ANF) degradation in patients with severe chronic heart failure. Methods and ResultsThe effects of repeated doses of the endopeptidase inhibitor candoxatrilat (150 mg i.v.) were examined over a 24-hour period in patients with severe chronic heart failure (New York Heart Association class III-IV). Plasma α-hANF(99–126) was elevated at baseline (235±59 pg/ml), increased 2.5-fold at 2 hours after the first dose, and remained significantly elevated throughout the 24-hour protocol. In contrast, pro-hANF(31–67) decreased from 3,151±616 to 2,072±362 pg/ml (p < 0.05). Cardiac index (CI) increased only transiently after the first dose of candoxatrilat (CI, 2.11±0.2 to 2.67±0.28 I/min/m2, p < 0.05). Sodium excretion increased sixfold (p < 0.05) 2 hours after the first dose of candoxatrilat and remained significantly elevated throughout the protocol. Degree of natriuresis and diuresis in response to candoxatrilat was closely related to baseline cardiac output. Glomerular filtration rate and volume excretion did not change significantly. Pulmonary capillary wedge pressure fell from 23±3 to 18±3 mm Hg (p < 0.05) and remained below baseline throughout the 24 hours. Arterial pressure, heart rate, and total peripheral resistance did not change significantly during the 24-hour period. Urinary cGMP excretion increased fivefold (p < 0.05), whereas urinary ANF immunoreactivity and plasma cGMP levels remained unchanged. Excretion of prostacyclin metabolite 6-keto-PGF-1α increased 3.3-fold (p < 0.05). Plasma norepinephrine and epinephrine levels decreased significantly after candoxatrilat and remained suppressed over the 24-hour period. There was also a transient reduction in plasma vasopressin, aldosterone levels, and plasma renin activity. Hematocrit, total protein content, and plasma albumin concentrations did not change, indicating that no fluid shift into the extravascular space had occurred. Conclusions1) The inhibition of ANF degradation causes sustained drop in left and right atrial pressures that appears to be mediated by an inhibition of neurohumoral activity; 2) concomitant inhibition of bradykinin breakdown (which in turn stimulates renal prostacyclin synthesis) contributes to natriuresis; 3) the close correlation between renal response and baseline cardiac index indicates that an inadequate renal perfusion secondary to low cardiac output diminishes the efficacy of this treatment modality. This spectrum of action would be advantagous for a first-line diuretic agent early in the course of disease rather than in patients with advanced chronic heart failure.