Atorvastatin improves blood rheology in patients with familial hypercholesterolemia (FH) on long-term LDL apheresis treatment

Atherosclerosis 159 (2001) 513– 519 www.elsevier.com/locate/atherosclerosis

Atorvastatin improves blood rheology in patients with familial hypercholesterolemia (FH) on long-term LDL apheresis treatment S. Banyai a,*, M. Banyai a, J. Falger b, M. Jansen b, E. Alt a, K. Derfler b, R. Koppensteiner a a

Di6ision of Angiology, Department of Medicine, Uni6ersity Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland b Di6ision of Nephrology and Dialysis, Uni6ersity Hospital Vienna, Waehringer Guertel 18 -20, A-1090 Vienna, Austria Received 3 August 2000; received in revised form 5 February 2001; accepted 9 April 2001

Abstract To determine the effect of atorvastatin on blood rheology in patients with familial hypercholesterolemia (FH) on regular LDL apheresis, we prospectively studied the rheological variables fibrinogen, plasma viscosity, red cell aggregation, whole blood viscosity, hematocrit and platelet aggregation in 12 patients (two homozygous, ten heterozygous) before and during treatment with atorvastatin. Baseline values of red cell aggregation and whole blood viscosity were increased in FH patients on regular LDL apheresis compared with healthy controls (P B0.05), whereas fibrinogen, plasma viscosity and hematocrit were similar in the two groups. Treatment with atorvastatin reduced red cell aggregation (PB 0.01), whole blood viscosity (PB 0.01), plasma viscosity (PB 0.01) and platelet aggregation (P B0.05), but caused a slight increase in plasma fibrinogen (by 5%; P B 0.01). Our findings suggest that atorvastatin improves blood rheology in patients with FH on regular LDL-apheresis. This improvement in blood flow properties may contribute to the well-known beneficial effects of atorvastatin on cardiovascular risk in patients with severe hyperlipidemia and atherosclerotic vascular disease. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Familial hypercholesterolemia; LDL apheresis; Atorvastatin; Blood viscosity; Red cell aggregation; Plasma viscosity; Fibrinogen

1. Introduction Patients with familial hypercholesterolemia (FH) have a high risk of cardiovascular morbidity and mortality due to the early development of atherosclerosis [1]. Prothrombotic effects associated with hyperlipoproteinemia such as platelet activation, increased whole blood and plasma viscosity and increased plasma fibrinogen levels might further enhance the risk of acute vascular events [2– 4]. Plasma fibrinogen, which is a major determinant of blood rheology, is an established cardiovascular risk factor and is associated with the initiation and progression of atherosclerosis [5,6]. In patients with hyperlipoproteinemia, the risk can be reduced by aggressive lipid-lowering therapy [7]. LDL immunapheresis is an excellent therapeutic option in * Corresponding author. Tel.: + 41-62-8367120; fax: + 41-1-2552671. E-mail address: [email protected] (S. Banyai).

patients with FH: long-term LDL apheresis treatment is known to reduce plasma LDL-cholesterol (LDL-C) by 50–60% [8]. Atorvastatin, the most powerful member of the HMG-CoA reductase inhibitor class, has also proved effective in heterozygous FH. Used as a single agent, it was shown to reduce LDL-cholesterol by \ 50% [9]. A combination of regular LDL apheresis and atorvastatin treatment in patients with FH resulted in a more effective reduction in LDL-C levels (up to 80% of baseline values) than LDL apheresis alone [10 –12]. Thus, the combination of LDL apheresis and atorvastatin is the most powerful lipid-lowering strategy available today. Positive effects of regular LDL apheresis on blood rheology are well known [11,13]. Clinical improvement in patients with symptomatic atherosclerotic disease during LDL apheresis has been attributed to improved blood flow properties, resulting in better microcirculatory blood flow (i.e. a beneficial effect on myocardial perfusion) [14,15].

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Several statins have been shown to improve blood rheology in addition to lowering cholesterol [16– 18]. However, data concerning the effects of atorvastatin on blood rheology in FH patients on regular LDL apheresis are not available so far. This is of major interest as reports about the effects of atorvastatin on plasma fibrinogen are controversial [9,19– 22], a marked increase of plasma fibrinogen levels up to 44% found in uncontrolled trials might have unfavourable effects on the rheological behaviour of blood [9,19]. Therefore, the aim of the present study was to investigate the effect of adjunctive treatment with atorvastatin on several determinants of blood rheology in patients with FH on regular LDL apheresis.

2. Patients and methods

2.1. Patients Twelve patients with FH (ten severe heterozygous, two homozygous) were studied. Patients had been on regular LDL apheresis treatment at weekly intervals for several years, and continued the treatment during the study. LDL apheresis had been initiated in the individual patient after dietary restrictions recommended by the American Heart Association and conventional pharmacological interventions had failed to sufficiently reduce hypercholesterolemia. All patients had been adhering to dietary restrictions in accordance with the American Heart Association Step II guidelines [23,24]. Demographic data and characteristics of the patients are summarised in Table 1. All patients were examined for mutations in LDLreceptor in the exons 1– 18, exon location, nucleotide changes, and codon changes were determined by denaturing gradient gel electrophoresis and Southern blot analysis as recently reported [25]. For eight out of ten patients with heterozygous hypercholesterolemia, the locations of the mutations have been already identified. Mutations are located in exon 4.2 Table 1 Baseline characteristics in 12 patients with familial hypercholesterolemia (2 homozygous, 10 heterozygous) maintained on long-term LDL apheresis treatment Age

489 13 years range 24–70

Male/female Body mass Index (BMI)

10/12 239 1.9 kg/m2 range 19–25 549 18, 24–84 5670 9 480 ml, range 5500–5730

Months of apheresis treatment at study entry Mean desorbed plasma volume per apheresis treatment

(N= 1), in exon 7 (N= 1), in exon 10 (N =2), in exon 13 (N= 1) and in exon 14 (N= 3). In the remaining two patients, examinations could not disclose the definite gene defect at this time. For both patients with homozygous disease investigation exhibited the basal genetic defect of hyperlipoproteinemia (exon 4.2, N= 1; exons 8 and 14, N = 1). Tendon xanthomas were present in both homozygous patients. All patients had established atherosclerotic vascular disease (coronary heart disease, 12; cerebrovascular disease, 4; peripheral arterial occlusive disease, 5). Arterial hypertension was present in five patients and three patients were active smokers. Co-medication consisted of conventional antianginal therapy, acetylsalicylic acid, b-blockers and ACE inhibitors, and was not altered during the study period. Patients with significant hepatic, renal or endocrine disease, alcohol consumption \ 210 g/w, uncontrolled hypertension, risk of conception and a body mass index \ 32 kg/m2 were excluded from the study.

2.2. Study design All patients were on weekly LDL apheresis treatment; this treatment was continued during the study period. Earlier lipid-lowering drugs that had been inadequately effective were withdrawn for a run-in period of 4 weeks. Subsequently, atorvastatin (initial dose, 20 mg per day) was administered. The dose was increased in a stepwise fashion every two weeks (40 and 60 mg, up to a maximal dose of 80 mg per day) to achieve target LDL-C levels below 5 100 mg/dl (2.59 mmol/l) as recommended by the NCEP Expert Panel [24]. Only two patients achieved a sufficient reduction in LDL-C levels to values below 100 mg/dl by administration of a maximal dose of 60 mg per day. In the remaining ten patients, target LDL-C values were not achieved despite a daily dose of 80 mg of atorvastatin. Plasma lipids — total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triglycerides (TG) and lipoprotein(a) (Lp(a)) — and several determinants of blood rheology-fibrinogen (Fgen), plasma viscosity (PV), red cell aggregation (RCA), whole blood viscosity (WBV), hematocrit (Hc) and platelet aggregation (PA) — were monitored at the end of the run-in period to obtain baseline values and after 8 weeks (at the maximal dose) of treatment with atorvastatin, while regular LDL-apheresis was continued. Twelve age- and sex-matched healthy subjects served as controls for the baseline values. The study has been approved by the local official Ethics Committee. For ethical reasons, no patient control group was constituted. All patients gave informed consent to participate in the study.

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2.3. Methods

2.4. LDL immunoadsorption apheresis

Blood samples were obtained from the antecubital vein by venipuncture without veno-occlusion immediately before LDL apheresis treatment was started. Whole blood 6iscosity was measured with the Contraves Low Shear 30 viscometer at 37 °C at native hematocrit and at three different shear rates (high: 94.5 s − 1; medium: 2.37 s − 1 and low: 0.695 s − 1) [26]. Plasma 6iscosity was determined at 25 °C (with the Coulter– Harkness viscometer) [27]. For red blood cell aggregation studies, the Myrenne aggregometer (Myrenne, GmBH, Aachen, Germany) was used. Aggregation was determined at stasis (after shearing at 600 s − 1) and at low shear (3 s − 1) at room temperature, based on the analysis of transmitted light in a cone plate chamber. The results were expressed as an aggregation index [28]. Fibrinogen (Fgen, mg/dl) was determined according to the method of Clauss [29]. For in vitro platelet aggregation studies, venous blood was collected through siliconised syringes and anticoagulated with 1 part in 10 of 3.8% sodium citrate. Platelets were analysed within 30 min of blood collection. Platelet aggregation studies were done with freshly prepared platelet-rich plasma (PRP) obtained by low-speed centrifugation (10 min, 100× g, ambient temperature). The remaining sample was recentrifuged (15 min, 3000 × g, 23 °C) to obtain platelet-poor plasma (PPP). Platelets in PRP were counted and the latter was diluted with PPP to achieve a concentration of 250× 109 platelets per l. For induction of platelet aggregation, ADP (2 mM), epinephrine (1 mM) and collagen (5 mg ml − 1) (Dade Cluster Platelet Aggregation Reagents, Dade International Inc. Miami, USA) were used. Platelet aggregation was registered photometrically as a change in light transmission over time (Aggrecorder Daichii II, Japan). For quantification of the aggregation response, the amplitude (Amax: the maximal amplitude of light transmission during recording) was used. Blood cell counts were performed with an automatic electronic counter (SYSMEX). Total cholesterol and triglycerides were measured enzymatically using a commercial kit (Boehringer Mannheim, Mannheim, Germany). Lipoprotein lipids were measured according to the Lipid Research Clinic methods with slight modifications as recently described [30]. Very low density lipoproteins were removed by ultracentrifugation (d B1.006 g/ml), LDLs were separated from an infranatant (d B1.063 g/ml) by heparin and polyanion precipitation using manganese chloride, and high-density lipoprotein (HDL)-cholesterol was determined from the supernatant. Lp(a) was determined quantitatively using an enzyme immunoassay (Innotest Lp(a); Innogenetics, Belgium).

For LDL immunoadsorption, blood was drawn from an antecubital vein via a 17-gauge needle at a flow rate of 50–80 ml/min. The Autopheresis-C therapeutic plasma system (TPS, Baxter, Deerfield, IL) was used for primary plasma separation [31]. The functional separation unit of the device is the plasmacell-C, a rotating cylindrical membrane housed in a plastic casing. The plasmacell-C is capable of fast and highly efficient plasma separation using a small membrane surface area (70 cm2) with a blood processing volume of only 7 ml. Standard sodium heparin at a rate of 1000 U/h and acid citrate dextrose (ACD- formula A, Baxter, Munich, Germany) were added for anticoagulation. The volume of ACD-A to whole blood flow was maintained at a ratio of 1:20 (5%). LDL immunapheresis was performed in an automated double-needle, continuous-flow operation in which the TPS is connected with an adsorption desorption automate (Medicap, Du¨ sseldorf, Germany). Two columns, each containing 150 ml Sepharose 4B gel coupled with polyclonal sheep apolipoprotein B-100 antibodies (LDL-Therasorb; Therasorb, Munich-Unterschleissheim, Germany), were used for lipoprotein removal [30]. In each adsorption cycle, approximately 100 ml of plasma were loaded on one column (plasma flow rate, 25–35 ml/min), while the other column was regenerated. A total of 5–7 cycles were performed at each immunoadsorption session with a duration of 3.59 0.4 h. Columns were regenerated by elution of apo B-containing lipoproteins with glycine buffer at pH 2.8 and a subsequent rinse with phosphate-buffered saline (PBS) and physiological saline. Two columns were assigned to each patient; the columns were reused and stored under sterile conditions.

2.5. Statistical analysis Values are presented as mean9 S.D. The Mann – Whitney test was used to compare baseline values of patients with healthy controls. Rheological variables and plasma lipids at baseline and at the individual maximal dose of atorvastatin were analysed by Wilcoxon’s signed rank test. Coefficients of correlation were calculated according to Pearson. P-values less than 0.05 were considered significant.

3. Results

3.1. Lipid and lipoprotein le6els Lipid and lipoprotein levels at baseline and after adjunctive atorvastatin treatment are shown in Table 2.

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Table 2 Lipid and lipoprotein levels in patients with FH on long-term LDLapheresis at baseline and after treatment with atorvastatin (N= 12)

TC HDL-C LDL-C Lp(a) TG

Baseline values

Values at the maximal atorvastatin dose

P-values

340.7 971 42.6 912 239.6 9 73 38.5 938.2 24.9a 186.1 955 178.2a

201.3 9 22 45.8 911 140.49 28 37.4 9 35.5 23.5a

0.002 0.002 0.002 n.s.

151.6 945 141.6a

0.002

TC, total cholesterol (mg/dl); HDL-C, HDL-cholesterol (mg/dl); LDL-C, LDL-cholesterol (mg/dl); Lp(a), Lipoprotein(a)(mg/dl);TG, triglycerides (mg/dl), n.s., not significant. a Geometric means.

In all patients, atorvastatin induced a marked decrease in LDL-C, TC and TG compared with baseline. An increase was seen in HDL-C, while Lp(a) remained unchanged (Table 2, Fig. 1). Only two patients achieved a sufficient reduction in LDL-C levels to values below 100 mg/dl by administration of a maximal dose of 60 mg per day. In the remaining ten patients, target LDL-C values were not achieved despite a daily dose of 80 mg of atorvastatin. Atorvastatin was well tolerated by all patients; no adverse events were observed.

3.2. Rheological 6ariables At baseline, whole blood viscosity (at all shear rates) and red cell aggregation were increased in patients with FH compared with healthy controls, whereas plasma fibrinogen, plasma viscosity and hematocrit did not differ between the groups (Table 3). Treatment with atorvastatin at the maximal dose resulted in a decrease in whole blood viscosity (at all shear rates), red cell aggregation and plasma viscosity (PB0.01). Platelet aggregation was also reduced (PB 0.05), whereas plasma fibrinogen was increased by 5% (PB 0.01) (Table 4, Fig. 1). Correlations were found between plasma fibrinogen and plasma viscosity at baseline (r= 0.81, PB0.01) and at the maximal dose of atorvastatin (r=0.66, PB 0.05).

4. Discussion The present study was designed to investigate the effect of concomitant atorvastatin therapy on blood rheology in patients with homozygous or severe heterozygous FH on regular LDL apheresis. In these patients, atorvastatin therapy resulted in an improvement in several rheological variables.

Fig. 1. Mean percent change from baseline in rheological variables and lipids and lipoproteins produced by atorvastatin (maximal dose) in patients with FH on regular LDL apheresis (Red cell aggregation: S, at stasis; L, at low shear; Whole blood viscosity: H, high; M, medium, L, low shear).

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Table 3 Rheological variables in patients with FH on long-term LDL-apheresis and in healthy controls (N= 12)

Fgen PV RCA-S RCA-L WBV high WBV medium WBV low Hc

Healthy controls

Patients with FH (Baseline values)

P-values

2.37 9 0.18 1.61 9 0.02 6.70 9 1.5 32.5 9 8.7 5.64 9 0.18 19.569 4 31.9 9 7 42.5 9 3

2.55 90.48 1.59 90.09 8.1 9 1.0 40.19 3.8 5.919 0.55 22.89 1.1 37.3 92.4 40.79 1.5

n.s. n.s. 0.08 0.03 0.02 0.02 0.03 n.s.

Fgen, fibrinogen (g/l); PV, plasma viscosity (mPas, milli-Pascal s), RCA-S (red cell aggregation at stasis, arbitrary units); RCA-L, red cell aggregation at low shear (3 s−1; arbitrary units); WBV, whole blood viscosity (mPas; at high [94.5 s−1], medium [2.37 s−1], low [0.695 s−1] shear rate); Hc, hematocrit (%). n.s., not significant.

At baseline, we found an increase in red cell aggregation (RCA) and whole blood viscosity (WBV) in FH patients compared with healthy controls. RCA is mainly determined by hematocrit and by bridging macromolecules such as fibrinogen, but also by high molecular weight lipids like LDL and VLDL [32– 35]. In our study, baseline fibrinogen and hematocrit were similar in patients and in controls. Therefore, changes in lipids and lipoproteins might be responsible for the increase in RCA in the patients. An increase in RCA in FH patients has been reported earlier and has been also attributed to plasma lipids [33– 35]. Further, red cell aggregability might be enhanced by an increase in the erythrocyte membrane cholesterol found in FH patients [34]. WBV, which was also increased at baseline, depends on hematocrit, red cell rigidity, plasma viscosity and red cell aggregation [36]. Changes in the red cell membrane lipids might reduce red cell flexibility and thus contribute to an increase in WBV [34,37,38]. Hyperfibrinogenemia has been reported in untreated patients with FH [33]. In our study, fibrinogen levels were not increased at baseline, as patients had been receiving long-term regular LDL apheresis treatment, which is known to reduce plasma fibrinogen levels by 15–20% [13]. Prolonged treatment with atorvastatin had significantly reduced RCA, WBV and plasma viscosity (PV). The decrease in these rheological variables is most likely attributable to the parallel reduction in plasma lipids, as the major determinants of blood rheology, hematocrit and plasma fibrinogen remained unaltered or even slightly increased during follow-up. The effect of plasma lipids and lipid lowering treatment on blood rheology has been investigated before [16,18,33,35,39,40]. Several studies showed a reduction in plasma viscosity by treatment with statins [16,18,33,35,39]. A decrease in the cholesterol/phospholipid ratio of the red cell membrane in FH patients treated with statins might further contribute to reduce WBV by improving red cell deformability [35]. We

suggest that the decrease in WBV in our patients being on treatment with atorvastatin might result from the decrease in RCA and plasma viscosity, but also from an increase in red cell flexibility. Reports concerning the effects of various statins on plasma fibrinogen levels are controversial. With lovastatin an increase by 19–24%, but also a reduction by 10% has been reported [40,41]. Conflicting results have been published for pravastatin [33,42] and for atorvastatin [19–22,39,43]. In uncontrolled trials, treatment with atorvastatin was associated with an increase in plasma fibrinogen levels up to 44% [9,19]. The clinical relevance of this finding is still a matter of debate. As fibrinogen is a major determinant of plasma viscosity, a marked increase might have adverse rheological consequences and deteriorate blood flow in the microcirculation. Persistent hyperfibrinogenemia might increase the risk of vascular events at long-term and counterbalance the risk reduction achieved by aggressive lipid-lowering treatment [5,6,44–46]. In our patients, atorvastatin therapy increased plasma fibrinogen levels by no more than 5.5%. The fact that several other rheological variables (RCA, WBV, PV) improved during follow-up indicates that the potential adverse rheological effect of this small increase in plasma fibrinogen is entirely counterbalanced by the favourable effects of atorvastatin on other determinants of blood rheology. All patients were on long-term antiplatelet therapy with acetylsalicylic acid at a dose of 100 mg per day for secondary prevention of their atherosclerotic vascular disease. Treatment with atorvastatin resulted in a further reduction in platelet aggregation by various inducers in in-vitro aggregation tests. This effect of atorvastatin on platelets might be beneficial in vivo in terms of reducing the risk of atherothrombosis in these high-risk patients. The extent of the hypocholesterolemic response to atorvastatin in our patients is consistent with earlier reports [9,47]. LDL-C was reduced by 41% and total cholesterol by 40%. In only two of the 12 patients was

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Table 4 Rheological variables in patients with FH on long-term LDL-apheresis at baseline and after treatment with atorvastatin (N= 12)

Fgen PV RCA-S RCA-L WBV high WBV medium WBV low Hc PA-ADP PA-E PA-C

Baseline values

Values at the maximal atorvastatin dose

P-values

2.559 0.48 1.599 0.09 8.19 1.0 40.19 3.8 5.91 9 0.55 22.89 1.1 37.39 2.4 40.79 1.5 65.59 13 56.09 15 65.19 15

2.69 90.53 1.49 9 0.07 6.69 90.6 35.4 94.3 5.51 90.2 18.8 90.75 32.4 9 2.8 40.8 9 1.6 53.9 9 21 39.0 9 15 51.0 9 23

0.009 0.002 0.002 0.002 0.018 0.002 0.003 n.s. 0.006 0.003 0.034

Fgen, fibrinogen (g/l); PV, plasma viscosity (mPas, milli-Pascal seconds), RCA-S (red cell aggregation at stasis, arbitrary units); RCA-L, red cell aggregation at low shear (3 s−1; arbitrary units); WBV, whole blood viscosity (mPas; at high [94.5 s−1], medium [2.37 s−1], low [0.695] shear rate); Hc, hematocrit (%), PA-ADP, platelet aggregation ADP-induced; PA-E, epinephrine-induced; PA-C, collagen-induced (% maximal amplitude). n.s., not significant

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