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(Circulation. 1997;95:76-82.)
© 1997 American Heart Association, Inc.

Articles

Single LDL Apheresis Improves Endothelium-Dependent Vasodilatation in Hypercholesterolemic Humans

Osamu Tamai, MD; Hidehiro Matsuoka, MD, PhD; Hiroyuki Itabe, PhD; Yoshifumi Wada, MD; Keisuke Kohno, MD; Tsutomu Imaizumi, MD, PhD

the Department of Internal Medicine III (O.T., H.M., Y.W., K.K., T.I.), Kurume University School of Medicine, Fukuoka, Japan, and the Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan (H.I.).

	Abstract

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Background Although long-term lipid-lowering therapy improves endothelium-dependent vasodilatation in humans, it remains unknown whether the short-term removal of LDL per se ameliorates endothelial dysfunction.

Methods and Results To examine the effects of a single session of LDL apheresis on endothelial function in patients with hypercholesterolemia, we measured forearm blood flow (FBF) by strain-gauge plethysmography before and after single LDL apheresis while infusing acetylcholine (ACh; 4 to 24 µg/min) and sodium nitroprusside (SNP; 0.2 to 1.2 µg/min). The single session of LDL apheresis reduced total LDL (from 142.2±15.0 to 32.6±5.0 mg/mL, P<.0005) and oxidized LDL (from 111.6±22.8 to 30.0±5.4 ng/mL, P<.005). Although ACh and SNP increased FBF dose-dependently before and after LDL apheresis, the endothelium-dependent vasodilatation responses to ACh were significantly augmented (P<.01) after the single session of LDL apheresis without changes in the endothelium-independent vasodilatation responses to SNP. The plasma levels of total and oxidized LDL correlated with the degree of ACh-induced vasodilatation. Furthermore, the local production of nitrate/nitrite, metabolites of NO, during ACh infusion was significantly (P<.05) augmented by LDL apheresis, and there was a significant correlation between the degree of ACh-induced vasodilatation and the production in nitrate/nitrite (r=.99, P<.0005).

Conclusions We demonstrated that even a single session of LDL apheresis with the reduction of total LDL and oxidized LDL improved endothelial function. Our results suggest that total LDL and/or oxidized LDL may directly impair endothelial function in the human forearm vessel.

Key Words: acetylcholine • arteriosclerosis • blood flow • endothelium-derived factors • lipoproteins

	Introduction

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Impaired endothelium-dependent vasodilatation has been demonstrated in patients with atherosclerosis^{1 2 3} and in conditions predisposing to the development of atherosclerosis, such as hypercholesterolemia, even before structural vascular changes are established.^{4 5 6 7 8 9} Several lines of evidence showed that the impaired endothelium-dependent vasodilatation is restored by long-term lipid-lowering and/or antioxidant therapies,^{10 11 12} with regression of the atherosclerotic lesions.¹³ Precise mechanisms of amelioration in endothelium-dependent vasodilatation by lipid-lowering therapy remain to be elucidated.

LDL alters vasomotion by inhibiting endothelium-dependent vasodilatation.^{14 15 16} It has been suggested that oxidized LDL plays a role in the development of atherosclerosis.² Several lines of evidence demonstrated that oxidized LDL decreases endothelium-dependent vasodilatation in ex vivo experiments.^{17 18 19 20} Oxidized LDL may impair the signal transduction between endothelial cell surface receptors and NO production,^{21 22} inhibit NO synthase activity,²³ and inactivate NO released from endothelial cells.²⁴

LDL apheresis is currently used for the treatment of familial hypercholesterolemia.²⁵ Repeated LDL apheresis produces an increase of blood flow to the skeletal muscle²⁶ and induces the regression of coronary artery disease.^{27 28} It has been proposed that the former effect is due to an improvement of rheological properties of blood by apheresis,^{26 29} although the role of endothelium remains unclear.

Accordingly, we hypothesized that the short-term removal of LDL may restore endothelium-dependent vasodilatation in patients with hypercholesterolemia. To test this hypothesis, we measured total LDL, oxidized LDL, and forearm blood flow responses to acetylcholine and sodium nitroprusside before and after a single session of LDL apheresis. In addition, we evaluated changes in local NO metabolites by single LDL apheresis during the infusion of acetylcholine.

	Methods

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Subjects
Seven patients with hypercholesterolemia (6 men, 1 woman) were enrolled in our study. Mean age was 57.6±3.9 years (range, 44 to 70 years). Hypercholesterolemia was defined as LDL cholesterol level greater than the 75th percentile adjusted for age and sex. The lipid profiles of the patients are shown in Table 1. Two of 7 patients had xanthoma, and 5 had thickness of the Achilles tendon. Three patients had a well-documented history of chronically elevated blood pressure (140/90 mm Hg). Six patients were smokers. The female patient was postmenopausal. No patients had diabetes mellitus. Five patients had stable angina pectoris with coronary stenosis verified by angiography. Two patients had intermittent claudication, with femoral artery stenosis verified by angiography. None had stroke, large-vessel aneurysm, renal failure, or congestive heart failure.

View this table: [in this window] [in a new window]   Table 1. Blood Measurements Before and After Single Apheresis

General Procedures
The protocol was explained, and informed written consent was obtained from each subject. The study was approved by Ethical Committee for Human Study in our institution. Although all patients had been treated with lipid-lowering agents, antiplatelet agents, calcium antagonists, and nitrates, all medications were discontinued 12 hours before the study. The study was done with subjects in a supine position and in an air-conditioned room at a temperature of 25°C to 26°C. Under local anesthesia with 1% mepibacaine, the left brachial artery was cannulated with a 20-gauge catheter (Angiocath; Becton Dickinson Vascular Access) for drug infusion. The catheter was connected by a three-way stopcock to a pressure transducer (UK4006SD; Trabenol Laboratories, Inc) and to a carrier amplifier (AP-601G, Nihon Kohden) sequentially for direct measurement of arterial pressure. The arterial line was kept open by infusion of heparinized saline (0.1 mL/min) when no drug was being administered. In all subjects, a vein in the antecubital region of the ipsilateral arm was cannulated to obtain blood samples.

Measurements of Forearm Blood Flow
Forearm blood flow was measured by a mercury-in-Silastic strain-gauge plethysmograph as previously described.³⁰ The strain gauge (Whitney Straingauge, University of Iowa) was placed 5 cm below the antecubital crease. Forearm blood flow (mL·min^-1·100 mL forearm^-1) was calculated from the rate of increase in forearm volume while venous return from the forearm was prevented by a cuff inflated to 40 mm Hg with a rapid cuff inflator (model E-10, Hokanson) on the upper arm. Circulation to the hand was arrested by a cuff inflated to a suprasystolic pressure around the wrist. The wrist cuff was inflated before the determination of forearm blood flow and remained inflated throughout the measurements. An average of four flow measurements made at 15-second intervals, calculated by an author who did not know the order of the results, was used for later analysis. Intraobserver variation was 1.9±1.8%.

Forearm Vascular Responses to Drugs
After the placement of the cannula and the strain-gauge plethysmograph, 15 minutes was allowed for subjects to become accustomed to the study conditions before the experiments were begun. To evaluate the effectiveness of LDL apheresis on forearm vascular endothelial function, we examined forearm vasodilator responses to intra-arterial infusions of acetylcholine (4, 8, 16, and 24 µg/min) and sodium nitroprusside (0.2, 0.4, 0.8, and 1.2 µg/min) for 2 minutes at each dose just before and immediately after LDL apheresis. The sequence of these two drugs was alternated. Forearm blood flow reached the steady state by 1 minute after infusion of acetylcholine and sodium nitroprusside was begun.

LDL Apheresis
For LDL apheresis, the MA-01 system (Kaneka Chemical) equipped with a Sulflux (FS-05 or FS-08, Kaneka Chemical) plasma separator was used. A catheter (HP602-18G, Medikit) was inserted into the femoral vein. Blood flow from the femoral vein was in the range of 100 to 130 mL/min, with the plasma flow rate at 30 to 40 mL/min. Heparin was given as an intravenous bolus (2000 IU) at the beginning of the procedure and then continuously infused at the rate of 1000 IU/h. LDL was removed selectively from plasma by perfusion of plasma through dextran sulfate cellulose beads (Liposorber LA-15, Kaneka Chemical) packed in columns. Four to 5 L plasma was generally processed in each 2-hour procedure.

Chemical Analysis
Oxidized LDL
Blood samples were collected in a tube containing EDTA, and plasma was stored in a refrigerator at 4°C. Measurement of plasma oxidized LDL was performed by a sandwich ELISA method as previously described.^{31 32} Briefly, LDL fractions were obtained from the samples by sequential ultracentrifugation. Diluted LDL fractions (5 µg/well) were added to the microtiter wells precoated with 0.5 µg of an anti–oxidized LDL monoclonal antibody, FOH1a/DLH3. After extensive washing, the remaining oxidized LDL was detected with a sheep anti-human apolipoprotein B antibody and an alkaline phosphatase–conjugated anti-sheep IgG antibody. In each ELISA plate, various concentrations of standard oxidized LDL, which was prepared by incubation of LDL with 5 µmol/L CuSO₄ at 37°C for 3 hours, were run simultaneously to determine a standard curve.

Nitrate/Nitrite
Plasma NO_x concentrations were measured by a colorimetric assay based on the Griess reaction.³³

Cyclic GMP
The plasma cGMP level was determined in duplicate with a radioimmunoassay kit (Yamasa) as previously described.³⁴

Endothelin
Plasma concentrations of endothelin-1 were determined by a radioimmunoassay (¹²⁵I-endothelin-1, Amersham International).

Local Production of NO_x and cGMP
Local productions of NO_x and cGMP were determined at rest and during the maximal dose of acetylcholine by multiplying the concentration of the venous effluent by the forearm blood flow. These were determined before and after apheresis.

Statistical Analysis
All values are expressed as mean±SEM, and P<.05 was considered to be statistically significant. The values were analyzed by one- or two-way ANOVA for repeated measures. Linear regression analyses were performed between plasma LDL, oxidized LDL, and the ratio of maximal to baseline forearm blood flow or the ratio of maximal to baseline NO_x production.

	Results

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Effects of Single LDL Apheresis
Total cholesterol and LDL cholesterol levels decreased by 60.5±4.1% (P<.0005) and 76.5±3.0% (P<.0005), respectively, by LDL apheresis (Tables 1 and 2). Similarly, oxidized LDL level decreased significantly, by 67.3±8.9% (P<.005). HDL cholesterol, hematocrit, plasma osmolarity, and the serum albumin level were not altered by LDL apheresis. Systemic blood pressure, heart rate, and baseline forearm blood flow were not altered by LDL apheresis.

Forearm Vascular Responses to Drugs
Representative recordings during the infusion of acetylcholine and sodium nitroprusside are shown in Fig 1. Direct intra-arterial infusion of acetylcholine and sodium nitroprusside increased forearm blood flow (P<.01) dose-dependently without changes in blood pressure before and after LDL apheresis (Table 2, Fig 2). After LDL apheresis, vasodilatory responses to infusions of acetylcholine were significantly augmented (P<.01), with no changes in responses to sodium nitroprusside (Table 2, Fig 2).

View larger version (77K): [in this window] [in a new window]   Figure 1. A, Representative recordings of forearm blood flow (FBF) during intra-arterial infusion of acetylcholine (ACh) before and after single LDL apheresis. ACh caused dose-dependent increases in FBF. Although baseline FBF was not altered, vasodilatory responses to ACh were significantly augmented by single LDL apheresis. B, Representative recordings of FBF during intra-arterial infusion of sodium nitroprusside (SNP) before and after single LDL apheresis. SNP caused dose-dependent increases in FBF. Neither baseline FBF nor vasodilatory response to SNP was altered by single LDL apheresis.

View this table: [in this window] [in a new window]   Table 2. Responses of Mean Arterial Pressure, Heart Rate, and Forearm Blood Flow to Acetylcholine and Sodium Nitroprusside

View larger version (15K): [in this window] [in a new window]   Figure 2. A, Responses of forearm blood flow (FBF) to intra-arterially infused acetylcholine (ACh) before () and after () single LDL apheresis. ACh caused dose-dependent increases in FBF. Increases in FBF to ACh were augmented after single LDL apheresis (P<.01, ANOVA) (n=7). B, Responses of FBF to intra-arterially infused sodium nitroprusside (SNP) before () and after () single LDL apheresis. Increases in FBF to SNP were similar before and after single LDL apheresis (n=7).

Endothelium-Derived Vasoactive Substances
Production of NO_x before and after LDL apheresis was significantly increased (P<.05) by intra-arterial infusion of acetylcholine (24 µg/min) (Table 3). LDL apheresis significantly augmented production of NO_x in response to acetylcholine (P<.05). The production of cGMP was increased by infusion of acetylcholine only after apheresis but not before apheresis. Plasma immunoreactive endothelin was significantly increased by LDL apheresis, from 1.7±0.2 to 2.8±0.4 pg/mL (P<.001).

View this table: [in this window] [in a new window]   Table 3. Local Productions at Baseline and During Acetylcholine Infusion Before and After LDL Apheresis

Relationship Among LDL, Oxidized LDL, Acetylcholine-Induced Vasodilatation, and Local NO_x Production
There were significant correlations between the ratio of the maximal to baseline blood flow and the levels of total LDL (r=-.62, P<.05; Fig 3A) and oxidized LDL (r=-.67, P<.01; Fig 3B). There were significant correlations between the ratio of the maximal to baseline production of NO_x and the plasma level of total LDL (r=-.57, P<.05; Fig 4A) and oxidized LDL (r=-.60, P<.05; Fig 4B), whereas there was no correlation between the ratio of the maximal to baseline production of cGMP and total LDL or oxidized LDL.

View larger version (19K): [in this window] [in a new window]   Figure 3. A, Relation between ratio of maximal blood flow induced by acetylcholine to baseline forearm blood flow (FBF) and level of plasma total LDL before () and after () single LDL apheresis (n=7). B, Relation between ratio of maximal blood flow induced by acetylcholine to baseline FBF and level of plasma oxidized LDL before () and after () single LDL apheresis (n=7).

View larger version (20K): [in this window] [in a new window]   Figure 4. A, Relation between ratio of maximal NOx production induced by acetylcholine to baseline NOx production and level of plasma total LDL before () and after () single LDL apheresis (n=7). B, Relation between ratio of maximal NOx production induced by acetylcholine to baseline NOx production and level of plasma oxidized LDL before () and after () single LDL apheresis (n=7).

Consequently, there was a strong correlation between the ratio of the maximal forearm blood flow induced by acetylcholine to baseline forearm blood flow and the ratio of maximal NO_x production induced by acetylcholine to baseline NO_x production (r=.99, P<.0005; Fig 5).

View larger version (17K): [in this window] [in a new window]   Figure 5. Relation between ratio of maximal NOx production induced by acetylcholine to baseline NOx production and ratio of maximal forearm blood flow induced by acetylcholine to baseline forearm blood flow before () and after () single LDL apheresis (n=7).

	Discussion

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The major findings of this study were that (1) a single session of LDL apheresis augmented vasodilator responses to acetylcholine but not to sodium nitroprusside in patients with hypercholesterolemia; (2) NO_x production in response to acetylcholine was significantly potentiated by a single session of LDL apheresis; and (3) plasma levels of oxidized LDL correlated significantly with the degree of acetylcholine-induced vasodilatation and with NO_x production. Our results suggest that the short-term removal of oxidized LDL ameliorates endothelium-dependent vasodilatation in patients with hypercholesterolemia, possibly via the augmented production of NO.

Effect of LDL Apheresis on Endothelial Function
LDL apheresis, a potent therapeutic modality that removes cholesterol within several hours, has been reported to increase the skeletal muscle blood flow when repeated for 3 weeks.²⁶ In the present study, we first demonstrated that a single session of LDL apheresis improved acetylcholine-induced endothelium-dependent vasodilatation associated with the reduction in plasma LDL cholesterol. This observation supports the concept that hypercholesterolemia causes endothelial dysfunction and indicates that short-term correction of hypercholesterolemia could restore endothelial function.

Although the precise mechanisms of amelioration of endothelium-dependent vasodilatation by lipid-lowering therapy remain to be elucidated, several possibilities have been suggested: (1) improvement of endothelium-derived relaxing factor (EDRF) production, (2) decrease in the inactivating factors of EDRF, (3) decrease in the endothelium-derived contracting factors, (4) regression of the diffusional barrier for EDRF, (5) improvement of the rheological property of blood, and (6) improvement of smooth muscle responsiveness to EDRF.

The reduced endothelial NO bioactivity has been demonstrated in hypercholesterolemic animals.^{35 36 37} Thus, the single LDL apheresis might have ameliorated endothelium-dependent vasodilatation by enhancing NO production in this study. To determine this possibility, we measured NO_x in the venous effluents as an index of local NO production, because NO is rapidly oxidized to NO_x in vivo.³⁸ We demonstrated that acetylcholine-induced vasodilatation and the production of NO_x in response to acetylcholine were significantly augmented by LDL apheresis. Furthermore, in response to acetylcholine, the improvement of vasodilator responses significantly correlated with the increase in the production of NO_x, suggesting that the improvement of endothelium-dependent vasodilatation may be caused by the augmented NO production after single LDL apheresis. Because NO decreases intracellular calcium via cGMP, a second messenger of NO-dependent mechanisms,³⁸ we evaluated cGMP levels in the venous effluent. Production of cGMP was increased by the infusion of acetylcholine only after apheresis but not before apheresis. There was no significant correlation between the magnitude of forearm vasodilatation and the acetylcholine-induced production of cGMP. However, this finding is not surprising, because cGMP is the intracellular second messenger, which may not be shed into blood in response to acetylcholine.

A possible mechanism underlying the impaired NO formation in our hypercholesterolemic patients may be increased oxidative stress. It has been demonstrated that hypercholesterolemia or LDL increases vascular production of superoxide anion,^{39 40} which can inactivate NO rapidly.⁴¹ Dietary correction of hypercholesterolemia,⁴² long-term antioxidant therapy,^{43 44} and polyethylene glycol superoxide dismutase⁴⁵ not only improve endothelium-dependent vasodilatation but also normalize endothelial superoxide anion production. It has been reported in vitro that oxidatively modified LDL per se plays a role in endothelial dysfunction² and that oxidized LDL but not native LDL decreases endothelium-dependent vasodilatation in ex vivo experiments.^{17 20} Oxidized LDL may also impair the function of signal transduction pathways that link endothelial cell surface receptors with NO production^{21 22} and inhibit NO synthase activity.²³ In our present study, the single session of LDL apheresis decreased total and oxidized LDL and improved endothelium-dependent vasodilatation. There was a significant correlation between the plasma level of total or oxidized LDL and NO_x production or the degree of forearm vasodilatation. Furthermore, the amelioration of endothelium-dependent vasodilatation and the augmented NO production by LDL apheresis significantly correlated with the amount of reduced oxidized LDL (Fig 4). Thus, it is likely that short-term removal of oxidized LDL by apheresis may have potentiated the production of endothelium-derived NO and that oxidized LDL may diminish the production or release of NO in hypercholesterolemic humans. However, the role of nonoxidized LDL could not be excluded, since native LDL also has been reported to inhibit endothelial function.^{14 16 18} Indeed, there were similar correlations between total LDL, the improvement of endothelial function, and NO_x production (Figs 3 and 4).

In hyperlipidemic patients, elevated plasma levels of endothelin, a potent endothelium-derived vasoconstricting factor, have been reported.⁴⁶ Recently, Lerman et al⁴⁷ demonstrated that endothelin immunoreactivity is enhanced in the coronary and systemic circulation in humans with endothelial dysfunction. Thus, it is possible that increased endothelin instead of decreased EDRF may contribute to the impaired endothelium-dependent vasodilatation in hypercholesterolemic patients.⁴⁷ In the present study, we examined the plasma concentrations of endothelin before and after the single session of LDL apheresis. Interestingly, the plasma endothelin increased after the apheresis despite the amelioration of endothelium-dependent vasodilatation. Thus, our results indicate that improved endothelium-dependent vasodilatation after apheresis was not due to decreased endothelin production.

Because of its short half-life, NO may not be biologically effective on smooth muscle cells in the presence of lipid deposition and intimal hyperplasia in the vascular wall.³ Recent lines of evidence suggest that long-term LDL apheresis may decrease lipid deposition on the arterial wall^{27 28} and may improve the biological efficacy of NO. In the present study, this possibility was not likely, because only a single session of LDL apheresis improved endothelium-dependent vasodilatation. Rubba and colleagues²⁶ demonstrated that repeated sessions of LDL apheresis decreased blood viscosity, possibly because of changes in the deformational property of erythrocytes. However, the contribution of the rheological property to improved endothelium-dependent vasodilatation was unlikely in our study, because there were no changes in basal blood flow and hematocrit, although we did not examine the rheological parameters. Finally, the improved smooth muscle responsiveness after LDL apheresis to NO was unlikely, because the vasodilator responsiveness to sodium nitroprusside was unaltered by LDL apheresis.

Study Limitations
For ethical reasons, we could not justify performing extracorporeal erythrocyte filtration in hypercholesterolemic patients or LDL apheresis in healthy control subjects. It has been suggested that long-term heparin treatment augments endothelium-dependent relaxation in the aorta from hypercholesterolemic animals in ex vivo.⁴⁸ Thus, it is possible that the heparin used in this study may have improved endothelium-dependent vasodilatation. However, since the dosage of heparin (4000 IU) used in apheresis was very small compared with that used in the ex vivo experiment,⁴⁸ the possibility that extracorporeal erythrocyte filtration with the small dose of heparin without LDL removal could augment endothelium-dependent vasodilatation is less likely but still exists.

Clinical Implications
Recent clinical trials demonstrated that lipid-lowering therapy markedly reduced cardiovascular events associated with a modest regression of atherosclerotic stenosis.^{11 13 27 28} Of note was that the lipid-lowering therapy decreased ischemic symptoms within a few months of treatment, in which time the regression of atherosclerosis is unlikely to occur. Furthermore, LDL apheresis is known to improve the ischemic symptoms more rapidly than other pharmacological or nonpharmacological therapies.⁴⁹ Thus, it is possible that marked reductions in clinical events and symptoms by lipid-lowering therapies may result from an immediate improvement of the NO production with or without the regression of atherosclerotic lesions.

In summary, we demonstrate that a single session of LDL apheresis augmented vasodilator responses to acetylcholine, with the increased production of NO. The plasma level of total or oxidized LDL correlated significantly with the magnitude of acetylcholine-induced vasodilatation and with the acetylcholine-induced NO_x production. Our results may suggest in patients with hypercholesterolemia that the short-term removal of oxidized LDL ameliorates endothelium-dependent vasodilatation, possibly via the augmented production of NO.

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (635-08770534) from the Ministry of Education, Science, Sports, and Culture, Tokyo. The authors are grateful to Drs Nobuhiko Koga and Hidemi Nishida for comments and Yasuko Adachi for technical assistance.

	Footnotes

 
Reprint requests to Hidehiro Matsuoka, MD, PhD, Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830, Japan.

Received May 14, 1996; revision received September 23, 1996; accepted September 28, 1996.

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