Sickle Cell Anemia Is Associated With Reduced Nitric Oxide Bioactivity in Peripheral Conduit and Resistance Vessels

Transcription

1 American Journal of Hematology 74: (2003) Sickle Cell Anemia Is Associated With Reduced Nitric Oxide Bioactivity in Peripheral Conduit and Resistance Vessels Robert T. Eberhardt, 1,3 * Lillian McMahon, 1,3 Stephen J. Duffy, 4 Martin H. Steinberg, 1,3 Susan P. Perrine, 1,3 Joseph Loscalzo, 1,2 Jay D. Coffman, 1 and Joseph A. Vita 1,2 1 Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 2 Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 3 Boston Comprehensive Sickle Cell Center, Boston, Massachusetts 4 Heart Centre, The Alfred Hospital and Baker Heart Research Institute, Melbourne, Victoria, Australia The purpose of the study was to examine endothelium-dependent and -independent vasodilation of conduit and resistance vessels in sickle cell anemia (HbSS). We measured the effects of intra-arterial infusion of methacholine, sodium nitroprusside, and N G -monomethyl-l-arginine (L-NMMA) on forearm blood flow using venous occlusion plethysmography in eight patients with HbSS and 11 HbAA controls. We assessed vasodilation of the conduit brachial artery using ultrasound in 17 patients with HbSS and 41 nonanemic controls. Forearm blood flow response to methacholine was similar in HbSS and HbAA, while the response to sodium nitroprusside was greater in those with HbSS. Nitric oxide synthase inhibition with L-NMMA attenuated methacholine-induced forearm blood flow to a lesser extent in HbSS compared to HbAA, suggesting diminished nitric oxide (NO) contribution to endothelium-dependent vasodilation. Flow-mediated and nitroglycerin-induced dilation were impaired in HbSS compared to controls and were not affected by the antioxidant vitamin C or the precursor substrate for NO synthesis L-arginine. NO bioactivity is reduced but differs in peripheral conduit and resistance vessels in HbSS. Resistance vessels have preserved response to exogenous NO donors but have diminished contribution of NO to endothelium-dependent vasodilation. Conduit vessels demonstrate impaired vasodilation to endogenous and exogenous NO. Am. J. Hematol. 74: , ª 2003 Wiley-Liss, Inc. Key words: vascular function; nitric oxide; endothelium; sickle cell anemia; L-arginine; vitamin C INTRODUCTION Sickle cell anemia (HbSS) is characterized by recurrent vaso-occlusive manifestations including painful crisis, acute chest syndrome, and stroke [1]. Vasoocclusion has traditionally been ascribed to plugging of the microcirculation by deformed sickle erythrocytes generated by deoxygenation-induced polymerization of mutated hemoglobin. However, evidence suggests that sickle erythrocytes may also induce changes in the vascular wall that contribute to the occlusive process [2]. The endothelium plays a central role in the regulation of vascular homeostasis by releasing a number of factors, including nitric oxide (NO) [3]. Loss of the biological activity of NO ª 2003 Wiley-Liss, Inc. Contract grant sponsor: NIH; Contract grant number: HL (Sickle Research Scholar Program to RTE); Contract grant sponsor: National Health and Medical Research Council of Australia Career Development Award; Contract grant number: (to SJD); Contract grant sponsor: the National Institutes of Health; Contract grant numbers: HL55993 (Specialized Center of Research in Ischemic Heart Disease), PO1HL60886, HL *Correspondence to: Robert T. Eberhardt, MD, Boston University Medical Center, 88 East Newton Street, Boston, MA robert.eberhardt@bmc.org Received for publication 21 March 2003; Accepted 15 June 2003 Published online in Wiley InterScience ( DOI: /ajh.10387

2 NO Bioactivity in Sickle Cell Anemia 105 could predispose to vaso-occlusion by promoting erythrocyte adhesion, increasing the propensity toward spasm, and impairing the regional regulation of blood flow. Several lines of evidence suggest that there is vascular dysfunction and impaired NO bioactivity in HbSS. In experimental models, despite increased NO production and perhaps NOS (NO synthase) expression, the response to endothelium-dependent vasodilators is impaired in both micro- and macrocirculations [4 6]. In humans with HbSS, abnormalities of endothelium-dependent vasodilation in conduit vessels has been reported; however, uncertainty remains regarding resistance vessel response to NO-mediated vasodilators [7 9]. Despite these recent findings, vascular function and NO bioactivity in peripheral conduit and resistance vessels in HbSS remain incompletely defined. The objective of the present study was to characterize further endothelium-dependent and -independent vasodilation in peripheral conduit and resistance vessels in HbSS with a view towards defining NO bioactivity. The hypothesis was that HbSS is associated with endothelial dysfunction and reduced NO bioactivity. We also sought to determine if oxidant mechanisms or inadequate substrate availability might play a role in impaired NO bioactivity. SUBJECTS AND METHODS Study Population Adults with HbSS and racially matched, healthy controls were considered eligible. HbSS was defined by hemoglobin electrophoresis with all subjects having the HbSF phenotype. Control subjects having brachial ultrasound evaluation were not anemic (Hg > 13 g/dl in male and >11 g/dl in females), while control subjects undergoing intra-arterial evaluation had HbA only on electrophoresis (HbAA). We excluded subjects with a history of vaso-occlusive crisis within 1 week of study. Other exclusion criteria included hypertension (blood pressure 150/90 mmhg), diabetes mellitus (fasting glucose >125 mg/dl), hypercholesterolemia (LDL cholesterol >160 mg/dl), tobacco use within the past month, and known atherosclerosis. Racial determination was made by self-report. The Boston Medical Center Institutional Review Board approved the study, and all subjects provided informed consent. Forearm Blood Flow Assessment Subjects were studied after fasting overnight in a quiet, dimly lit, laboratory at 23 C. A 20-g polyethylene catheter (Arrow International, Reading, PA) was inserted in the brachial artery and vehicle (5% dextrose in water) was infused at 0.6 ml/min using an infusion pump (Harvard Apparatus, Dover, MA). Forearm blood flow measurements with cuff exclusion of the hand circulation were performed by venous occlusion plethysmography [10]. After vehicle infusion for at least 30 min, vasoactive drugs were infused as follows: 1) sodium nitroprusside (0.3, 1.0, 3.0, and 10 mg/min; Elkins-Sinn, Cherry Hill, NJ); 2) methacholine (0.3, 1.0, 3.0, and 10 mg/min; Roche Laboratories, Nutley, NJ); 3) N G -monomethyl- L-arginine (L-NMMA; Clinalpha, Switzerland) at 8 mmol/min for 10 min; and 4) repeat methacholine infusions while coinfusing L-NMMA. Each dose was infused for 4 min and forearm blood flow was measured during the last 2 min of each infusion. Vehicle infusions of at least 30 min were made between agonists to reestablish baseline conditions. Brachial Artery Ultrasound Assessment Endothelium-dependent, flow-mediated, and endothelium-independent nitroglycerin-induced dilation of the conduit brachial artery was determined using vascular ultrasound [11,12]. Briefly, Doppler flow signals and two-dimensional ultrasound images were digitized using an R-wave trigger. Images (10 per time point) were recorded at baseline and 1 min after induction of reactive hyperemia by 5-min cuff occlusion of the upper arm. After a 10-min rest period, images were recorded before and 4 min after administration of sublingual nitroglycerin (0.4 mg). Nitroglycerin was omitted in subjects with a history of migraine headache or with a systolic blood pressure <100 mmhg. Images were analyzed by personnel blinded to hemoglobin status using commercially available software (Brachial Analyzer, Medical Imaging Applications, Iowa City, IA). Vitamin C and L-Arginine Effect Using a cross-over design study, subjects received vitamin C (2 g orally), L-arginine (0.1 g/kg orally), and placebo in random order on separate days at least 2 days apart. Endothelium-dependent and -independent dilation of the brachial artery was assessed at baseline and 3 hr following each treatment. Biochemical Analyses Fasting serum glucose, total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol were determined using a Hitachi model 917 automated analyzer (Hitachi Instruments, Indianapolis, IN). LDL cholesterol was calculated with the Friedewald formula.

3 106 Eberhardt et al. Statistical Analysis Results are expressed as mean ± SD, except in the figures (mean ± SE). Group differences in clinical characteristics and ultrasound parameters were compared using the unpaired t-test, w 2 test or Fisher s exact test, as appropriate. Adjusted forearm blood flow was obtained by subtracting baseline forearm blood flow from the flow response to each agonist. Group differences in forearm blood flow responses were examined using two-way (dose and sickle status) repeated measure analysis of variance (RM-ANOVA). The effect of L-NMMA on the methacholine response was made according to sickle status using RM-ANOVA. To determine if HbSS was an independent predictor of flow-mediated dilation, analysis of covariance (ANCOVA) was performed with adjustment for differences in clinical characteristics. The effect of vitamin C and L-arginine on flow-mediated dilation was made by two-way RM-ANOVA. Statistical significance was defined as P < RESULTS Baseline Characteristics The study population included 18 subjects with HbSS and 52 controls. Baseline clinical and laboratory characteristics are displayed in Table I. As shown, the groups were comparable in age, gender, and racial distribution (primarily black). Subjects with HbSS had lower body mass index, diastolic and mean blood pressure, lipid parameters (except triglycerides), and serum creatinine. As expected, both hemoglobin and hematocrit were lower in subjects with HbSS. Resistance Vessel Function Forearm blood flow assessment was performed in eight subjects with HbSS and 11 HbAA controls. Of those with HbSS, there were three males and five females with a hemoglobin level of 8.2 ± 0.8 g/dl, a fetal hemoglobin of 6.2 ± 2.1%, and of whom three were on hydroxyurea. Baseline forearm blood flow was three-fold greater and vascular resistance nearly four-fold lower in patients with HbSS compared to HbAA (7.9 ± 3.0 vs. 2.6 ± 1.1 ml/min/dl, P ¼ 0.001; 11.5 ± 5.0 vs ± 13.7U, P ¼ 0.001). L-NMMA reduced baseline forearm blood flow to a greater extent in patients with HbSS than in HbAA, both the absolute flow decrease (Fig. 1A) and relative decrease expressed as percent flow inhibition (Fig. 1B). Methacholine produced a dose-dependent increase in forearm blood flow in both groups (Fig. 2A). After adjustment for differences in baseline flow by subtracting baseline flow from observed flow, forearm blood flow was 18.8 ± 7.4 and 22.0 ± 9.7 ml/min/ dl at peak methacholine dose in HbAA and HbSS, respectively (P ¼ 0.23 by RM-ANOVA). Similarly, sodium nitroprusside produced dose-dependent increases in forearm blood flow in both groups, but a greater response was seen in subjects with HbSS (P ¼ by RM-ANOVA) (Fig. 2B). After adjustment for differences in baseline flow, forearm blood flow at peak sodium nitroprusside was greater in subjects with HbSS compared with HbAA (20.9 ± 10.3 vs.12.8 ± 3.3 ml/min/dl; P < 0.001). Similar results were seen when dilator responses were expressed as vascular resistance (data not shown). The contribution of NO to methacholine-induced flow was determined by comparing flow with and without coinfusion of the nitric oxide synthase (NOS) TABLE I. Baseline Characteristics Sickle Control P-value Number of subjects Age (years) 29.2 ± ± Gender: M/F 9/9 25/ Race: black/non-black 16/2 47/ Body mass index (kg/m 2 ) 21.6 ± ± Systolic blood pressure (mmhg) 118 ± ± Diastolic blood pressure (mmhg) 62 ± ± 9 <0.001 Mean blood pressure (mmhg) 81 ± ± Heart rate (beat/min) 73 ± ± Total cholesterol (mg/dl) 118 ± ± 28 <0.001 LDL cholesterol (mg/dl) 53 ± ± 27 <0.001 HDL cholesterol (mg/dl) 41 ± ± Triglycerides (mg/dl) 118 ± ± Glucose (mg/dl) 91 ± ± Creatinine (mg/dl) 0.39 ± ± 0.21 <0.001 Hemoglobin (g/dl) 8.4 ± ± 1.3 <0.001 Hematocrit (%) 24.6 ± ± 3.9 <0.001

4 NO Bioactivity in Sickle Cell Anemia 107 Fig. 1. Effect of L-NMMA on baseline forearm blood flow in sickle cell anemia (HbSS). (A) Forearm blood flow before and during infusion of L-NMMA in patients with HbSS (n = 8) and HbAA controls (n = 11). &, HbSS;, HbAA control. (B) Percent of forearm blood flow inhibition with L-NMMA in HbSS and HbAA. inhibitor L-NMMA. L-NMMA attenuated the vasodilator response to methacholine in HbAA and HbSS subjects (compared with methacholine alone; P ¼ and P ¼ 0.01, respectively) (Fig. 3).However,theeffect of L-NMMA on the methacholine-induced flow response differed according to sickle cell status (P ¼ 0.004) with less attenuation in those with HbSS. The increase in forearm blood flow to peak methacholine was reduced by only 7 ± 8% (or 1.5 ± 1.4 ml/min/ dl) in HbSS compared with 72 ± 21% (or 6.8 ± 8.4 ml/ min/dl) in HbAA (P ¼ and P ¼ 0.02). In these studies, L-NMMA had no effect on systemic blood pressure in either group. No apparent gender effect was seen in microvascular function expressed as percent change in flow to methacholine and sodium nitroprusside. Conduit Vessel Function Brachial ultrasound studies were performed in 17 subjects with HbSS and 41 nonanemic controls. Of Fig. 2. Forearm blood flow in response to methacholine (A) and sodium nitroprusside (B) in sickle cell anemia (HbSS). Forearm blood flow (minus baseline flow) during infusion of methacholine and sodium nitroprusside in patients with HbSS (n = 8) and HbAA controls (n = 11 for MCh, n = 10 for SNP). &, HbSS;, HbAA control. Fig. 3. Effect of L-NMMA on forearm blood flow response to methacholine in controls (A) and sickle cell anemia (HbSS) (B). Forearm blood flow (minus baseline flow) during infusion of methacholine prior to and during co-infusion of L-NMMA (8 mmol/min) in HbAA control subjects (n = 11) and subjects with HbSS (n = 8). D, no L-NMMA;, L-NMMA.

5 108 Eberhardt et al. those with HbSS, there were eight males and nine females with a hemoglobin level of 8.4 ± 1.1 g/dl, a fetal hemoglobin of 7.9 ± 7.1%, and of whom four were on hydroxyurea. The brachial ultrasound results are shown in Table II. Resting and peak hyperemic flows were higher in the subjects with HbSS compared to controls, but the relative changes were equivalent in the two groups. As shown in Figure 4A, relative flow-mediated dilation was lower in the HbSS subjects compared to the control subjects (8.9 ± 4.2% vs ± 7.2%; P ¼ 0.012). Similarly, absolute change in vessel diameter in response to hyperemic flow was lower in HbSS, as shown in Table II. As shown in Figure 4B, there was an impairment in nitroglycerin-mediated dilation in the HbSS subjects compared to controls expressed as percent dilation controls (17.6 ± 6.8% vs ± 10.7%; P ¼ 0.041), but not expressed as an absolute change in vessel diameter, as shown in Table II. Most of the observed group differences in clinical characteristics would be expected to be associated with higher, rather than lower, flow-mediated dilation in the subjects with HbSS and, thus, are unlikely to account for the observed findings. Furthermore, after adjusting for body mass index, diastolic blood pressure, total cholesterol, triglycerides, creatinine, and hemoglobin by ANCOVA, sickle cell status continued to be associated with lower flow-mediated dilation (P ¼ 0.012). Similarly, after adjusting for baseline brachial artery diameter by ANCOVA, sickle status continued to be associated with lower flow-mediated dilation (P ¼ 0.024). There was no observed effect of hydroxyurea status or fetal hemoglobin level on flowmediated or nitroglycerin-mediated dilation in those with HbSS (data not shown). Although gender was predictive of flow-mediated dilation in both groups, there was no interaction of gender and sickle status (data not shown). The effect of acute administration of vitamin C and L-arginine on brachial artery function was assessed in 11 subjects with HbSS. This subgroup of patients consisted of four males and seven females with a hemoglobin level of 8.6 ± 1.3 g/dl, a fetal hemoglobin of 12.4 ± 9.3%, and of whom five were on hydroxyurea. The baseline flow-mediated dilation was 10.4 ± 3.3% and nitroglycerin-mediated dilation was 18.9 ± 7.0. As shown in Figure 5, there was no significant change in flow-mediated dilation expressed as absolute change with either treatment. Similarly, baseline diameter, reactive hyperemia, and nitroglycerin-mediated dilation were not affected by either treatment (data not shown). Fig. 4. Flow-mediated dilation (A) and nitroglycerin-induced dilation (B) of the brachial artery in sickle cell anemia (HbSS). The percent change in the brachial artery diameter above baseline in response to hyperemic flow (A) and sublingual nitroglycerin (B) in patients with HbSS (n = 17) and controls (n = 41). TABLE II. Ultrasonography Data Sickle Control P-value Baseline diameter (mm) 3.74 ± ± Flow-mediated diameter (mm) 4.08 ± ± Absolute D flow-mediated diameter (mm) 0.33 ± ± Baseline flow (ml/min) 179 ± ± Hyperemic flow (ml/min) 1121 ± ± 317 <0.001 Percent flow increase (%) 579 ± ± Pre-NTG diameter (mm) 3.85 ± ± Post-NTG diameter (mm) 4.51 ± ± Absolute NTG-induced dilation (mm) 0.67 ± ±

6 NO Bioactivity in Sickle Cell Anemia 109 Fig. 5. Effect of vitamin C and L-arginine on flow-mediated dilation in sickle cell anemia (HbSS). The absolute change in flow-mediated dilation above baseline response with vitamin C (2 gms), L-arginine (0.1 g/kg) and placebo in HbSS (n = 11). No significant effect. DISCUSSION The present study demonstrates divergent effects of HbSS on NO-dependent vasodilation according to vessel type (conduit vs. resistance vessels) and specific vasodilator stimuli (methacholine, flow, and NO-donor). Resting forearm blood flow was greater in patients with HbSS and was inhibited to a greater extent by L- NMMA, suggesting increased basal NO production in resistance vessels. Although the forearm blood flow response to methacholine was preserved, the degree to which the methacholine response was inhibited by L- NMMA was much less in HbSS patients, suggesting an impairment in the bioavailability of endothelium-derived NO under stimulated conditions in resistance vessels. The forearm blood flow response to sodium nitroprusside was actually enhanced in HbSS patients, arguing against an impaired ability to respond to NO donors. These results also suggest that non-no vasodilators contribute to the muscarinic response to a greater extent in HbSS subjects compared to HbAA, resulting in an overall equivalent vasodilator response to methacholine. In peripheral conduit vessels, we found a reduction in flow-mediated dilation in patients with HbSS compared to age, gender, and race-matched control subjects. There were no significant group differences in baseline brachial artery diameter, or extent of reactive hyperemia, suggesting that the observed findings were not attributable to differences in vessel size, or the stimulus for vasodilation. The response to nitroglycerin was also impaired, suggesting that there is a generalized impairment in NO-mediated vasodilation in conduit vessels in HbSS. The impairment in flowmediated dilation and nitroglycerin-mediated dilation were not affected by acute administration of either vitamin C or L-arginine, suggesting that oxidative stress and substrate deficiency do not explain the observed impairment in conduit vessel function. Hemodynamic consequences of anemia including increased cardiac output and blood flow stimulate NO production and reduce peripheral vascular resistance [13]. In HbSS, additional effects of increased shear stress and tissue hypoxemia may further stimulate NOS expression and NO production. Transgenic murine models of sickle cell disease have shown greater basal NO production with a greater rise in blood pressure with NOS inhibition, although results regarding NOS expression are controversial [5,6]. In humans with HbSS, the contribution of NO to increased basal flow and reduced peripheral resistance is unclear [7]. Our findings of greater resting forearm blood flow with greater absolute and relative flow decreases with NOS inhibition supports increased basal NO production contributes to the regulation of basal tone and tissue perfusion in HbSS in the steady state. Despite enhanced basal NO production, several basic investigations support a disturbance in vascular function and stimulated NO bioactivity in sickle cell disease. Transgenic murine models of HbSS have shown diminished responses to endogenous and exogenous NO-mediated dilators [4 6]. However, in humans with HbSS, there is controversy regarding microvascular dysfunction of the resistance vessel of the forearm. Belhassen et al. [7] found preserved responses to endogenous and exogenous NO vasodilators, while Reiter et al. [8] suggested diminished NO bioavailability in HbSS. Our findings differ from previous studies regarding responses to cholinergic stimulation and nitrovasodilators in HbSS. We found that methacholine-induced forearm blood flow response was preserved in HbSS, while others have demonstrated a heightened response to acetylcholine [7,9]. The reasons for these apparently discrepant findings are not immediately clear, but could relate to differences in the study populations. Of note, the peak acetylcholine response in the prior studies was unusually low in the control subjects. Our response to an exogenous nitrovasodilator also differs from those previously reported [7,9]. Belhassen et al. [7] reported

7 110 Eberhardt et al. equivalent responses to nitroprusside in HbSS and HbAA subjects, while Reiter et al. [8] found diminished responses to nitroprusside that was dependent on cellfree hemoglobin (or other forms of redox active iron). In contrast, we found a heightened response to this exogenous NO donor in the resistance vessels. In addition, Gladwin et al. [9] found a significant effect of gender on NO bioavailability in HbSS, which we did not observe, with men having diminished response. Another finding of this study was the preserved response to methacholine with a diminished portion of that response attributable to NO. A similar finding was observed in men with HbSS with L-NMMA having no effect on acetylcholine-induced flow [9]. One might consider the possibility that anemia itself may cause the observed abnormalities. However, anemia due to other causes, including aplastic anemia and iron deficiency, is not associated with an impairment of endothelium-derived NO, as the response to methacholine is preserved, with a comparable reduction in peak flow with L-NMMA [13]. Evidence of enhanced non-no-dependent responses to methacholine has been reported in patients with congestive heart failure [14]. A possible explanation for preserved endothelium-dependent dilation but diminished NO contribution is an upregulation of other pathways stimulated by methacholine, such as prostacyclin, endothelium-derived hyperpolarizing factor, or hemeoxygenase-1 [15]. In contrast to resistance vessels, information regarding conduit vessel function in HbSS is more limited. Belhassen et al. [7] found a defect in shear stressmediated vasodilation in patients with HbSS with diminished brachial artery flow-mediated dilation, despite similar increases in flow. Based on resting flow, brachial artery diameter, and measured viscosity, calculated wall shear stress was higher in the HbSS patients, and these investigators concluded that the equivalent baseline brachial diameter reflects impairment of endothelium-mediated regulation of vessel size in response to chronic shear stress [7]. The present study is consistent with that prior study in several respects, including the findings of higher resting flow, equivalent baseline diameter, and impaired brachial artery flow-mediated dilation in HbSS patients. The present findings also include an impaired response to nitroglycerin, which was not previously evaluated. The impaired response to a non-endothelium-dependent NO donor raises the possibility that there is a generalized loss of the bioavailability of NO or impairment of the function of vascular smooth muscle. Overall, the present study and previous human and experimental studies suggest that there is impairment in vascular function with diminished NO bioactivity in HbSS when endothelial cells are stimulated to produce NO. We considered potential mechanisms for this finding. Several prior studies suggest that HbSS is associated with increased oxidative stress [5,16,17]. This concept is further supported by the observation that NO-mediated dilation may be improved by pretreatment with antioxidant enzymes in models of sickle cell disease [4]. Reactive oxygen species, such as superoxide anion, are known to react with and decrease the biological activity of NO and alteration in the cellular redox status. Ascorbic acid is a potent antioxidant and is known to favorably affect cellular redox status and the activity of endothelial NO synthase [18]. In human subjects with coronary artery disease, a 2-g dose has been shown to reverse impaired brachial artery flow-mediated dilation [11]. The lack of an effect of ascorbic acid in the present study argues against the importance of specific forms of oxidative stress that would be reversed by ascorbic acid when administered in this manner. We also investigated the possibility that an absolute or relative impairment of L-arginine availability might explain impaired flow-mediated dilation in HbSS. Inadequate L-arginine bioavailability may lead to decreased endothelial production of NO production. A prior study suggested that serum L-arginine levels are reduced in patients with HbSS (40 50 mmol/l) compared with controls (80 90 mmol/l) [19]. However, these levels of L-arginine far exceed the K m for enos (5 mmol/l), making the relevance of these findings uncertain [20]. We evaluated the effect of L-arginine on brachial artery reactivity using a dose (0.1 g/kg) previously shown to acutely increase L-arginine levels (uptonearly145mmol/l in 2 hr) in patients with HbSS [21]. Our findings do not support inadequate precursor substrate bioavailability; however, the effect of longterm treatment was not tested and arginase levels were not measured, which may also limit L-arginine levels. There are a number of additional limitations of the present study that warrant discussion. Higher resting blood flow was seen in HbSS patients compared with control subjects. Although we attempted to adjust for this difference by subtracting baseline flow, the ultimate impact on the measurements of vascular function is unknown. The difference observed in baseline blood flow makes evaluating the response to vasodilators problematic, as the same absolute increase in flow would appear depressed if expressed as percent flow increase. Furthermore, since the study examined conduit and resistance vessels in the forearm, it remains unclear whether the present findings may be extended to other vascular beds. Finally, the study is cross-sectional in nature and it remains possible that unmeasured differences in the population may have confounded the results. Regarding the clinical relevance of the findings, it seems likely that impaired NO bioactivity may con-

8 NO Bioactivity in Sickle Cell Anemia 111 tribute to the vaso-occlusive complications of HbSS. In the resistance vessels, NO production appears to be increased at baseline but has diminished augmentation with further stimulation. A further limitation of NO bioavailability from either heightened destruction or diminished production may predispose to vaso-occlusion. The finding of impaired response of conduit vessels to NO may be particularly important in the carotid circulation contributing to the reversible cerebral vasculopathy and propensity for stroke observed in children with HbSS [22]. Consistent with this idea is the finding that continued NO production is important for maintaining cerebral blood flow during experimental stroke in the presence of sickle erythrocytes [23]. Another potential therapeutic implication in treating the pain associated with vaso-occlusive crisis relates to the possibility of a compensatory response with upregulation of non- NO-mediated, endothelium-dependent vasodilators. If vasodilator prostaglandins are key determinants of blood flow and its regulation, then the inhibition of cyclooxygenase with nonsteroidal antiinflammatory agents for their analgesic properties may have a detrimental role and exacerbate the vaso-occlusive process. Further investigations are required to explore the relevance of these findings. In conclusion, the present study demonstrates divergent NO bioactivity in peripheral conduit and resistance vessels in HbSS in the steady state. There is a generalized defect in NO responses in peripheral conduit vessels in humans with HbSS that effects flowmediated dilation and nitroglycerin-induced dilation. In resistance vessels, basal NO production is increased, but the availability of NO is decreased upon methacholine stimulation. The resistance response to exogenous NO is enhanced. It seems likely that such abnormalities in vascular function in various vascular beds might contribute to vaso-occlusive manifestations of the disease. Further studies are required to determine whether these insights will lead to new approaches for therapy of the vascular complications of this disease. ACKNOWLEDGMENTS We thank Lynn Rescorl and Catherine Denson for technical work. REFERENCES 1. Steinberg MH. Management of sickle cell disease. N Engl J Med 1999;340: Hebbel RP. Adhesive interactions of sickle erythrocytes with endothelium. J Clin Invest 1997;100(11 Suppl):S83 S Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329: Aslan M, Ryan TM, Adler B, et al. Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease. Proc Natl Acad Sci USA 2001;98: Nath KA, Shah V, Haggard JJ, et al. Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease. Am J Physiol Regul Integr Comp Physiol 2000;279:R1949 R Kaul DK, Liu XD, Fabry ME, Nagel RL. Impaired nitric oxidemediated vasodilation in transgenic sickle mouse. Am J Physiol Heart Circ Physiol 2000;278:H1799 H Belhassen L, Pelle G, Sediame S, et al. Endothelial dysfunction in patients with sickle cell disease is related to selective impairment of shear stress-mediated vasodilation. Blood 2001;97: Reiter CD, Wang X, Tanus-Santos JE, et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002;8: Gladwin MT, Schechter AN, Ognibene FP, et al. Divergent nitric oxide bioavailability in men and women with sickle cell disease. Circulation 2003;107: Sherman DL, Keaney JF Jr, Biegelsen ES, Duffy SJ, Coffman JD, Vita JA. Pharmacological concentrations of ascorbic acid are required for the beneficial effect on endothelial vasomotor function in hypertension. Hypertension 2000;35: Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF Jr, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation 1996;93: Gokce N, Keaney JF Jr, Frei B, et al. Long-term ascorbic acid administration reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation 1999;99: Anand IS, Chandrashekhar Y, Wander GS, Chawla LS. Endothelium-derived relaxing factor is important in mediating the high output state in chronic severe anemia. J Am Coll Cardiol 1995;25: Katz SD, Krum H. Acetylcholine-mediated vasodilation in the forearm circulation of patients with heart failure: indirect evidence for the role of endothelium-derived hyperpolarizing factor. Am J Cardiol 2001;87: Platt JL, Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med 1998;4: Hebbel RP, Eaton JW, Balasingam M, Steinberg MH. Spontaneous oxygen radical generation by sickle erythrocytes. J Clin Invest 1982;70: Klings ES, Christman BW, McClung J, et al. Increased F2 isoprostanes in the acute chest syndrome of sickle cell disease as a marker of oxidative stress. Am J Respir Crit Care Med 2001; 164: Huang A, Vita JA, Venema RC, Keaney JF xjr. Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem 2000;275: Morris CR, Kuypers FA, Larkin S, Vichinsky EP, Styles LA. Patterns of arginine and nitric oxide in patients with sickle cell disease with vaso-occlusive crisis and acute chest syndrome. J Ped Hem Onc 2000;22: Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 1997;100: Morris CR, Kuypers FA, Larkin S, et al. Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease. Br J Haem 2000;111: Adams RJ, McKie VC, Hsu L, et al. Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography. N Engl J Med 1998;339: French JA, Kenny D, Scott JP, et al. Mechanisms of stroke in sickle cell disease: sickle erythrocytes decrease cerebral blood flow in rats after nitric oxide synthase inhibition. Blood 1997;89: