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Emerging Treatments and Technologies ORIGINAL ARTICLE

Anti-Inflammatory and Effects of Pravastatin in Patients With Type 2 Diabetes

1,2 4 DIRKJE W. SOMMEIJER, MD ANTON P. VAN ZANTEN, PHD of cardiovascular disease in type 2 diabe- 2 1 MELVIN R. MACGILLAVRY, MD, PHD PIETER H. REITSMA, PHD tes focus not only on optimal regulation of 3 1,2,5 JOOST C.M. MEIJERS, PHD HUGO TEN CATE, MD, PHD hyperglycemia but also on treatment of other cardiovascular risk factors (3,4). A subgroup analysis of several large randomized clinical trials (5,6) shows OBJECTIVE — Type 2 diabetes is associated with increased plasma concentrations of coagula- that the relative risk for cardiovascular tion and inflammation markers. Different studies have shown that treatment with hydroxy- complications in type 2 diabetic patients methylglutaryl-CoA reductase inhibitors () is associated with and anti- can be reduced by 25% using aggressive inflammatory effects in addition to a cholesterol-lowering effect. Our objective was to evaluate the treatment of dyslipidemia with hy- effect of pravastatin (40 mg/day) on and inflammation markers in type 2 diabetic patients. droxymethylglutaryl-CoA reductase in- RESEARCH DESIGN AND METHODS — This was an open, randomized, crossover hibitors, also known as statins. Treatment study designed with an 8-week intervention period. The study group was comprised of 50 with statins may be beneficial not only patients with type 2 diabetes (median HbA1c 7.1%) and serum total cholesterol of 5–10 mmol/l. because of these agents’ lipid-lowering ac- We evaluated plasma levels of fibrinogen, F1 ϩ 2, D-dimer, soluble (sTF), von tion, but also because of their effect on Willebrand Factor antigen (vWFag), and C-reactive protein (CRP) in samples drawn after inflammation, endothelial function, ad- fasting on day 1 and after 8 and 16 weeks. hesion of leukocytes to endothelium, and formation (7). Although statins RESULTS — Significant reductions of total cholesterol (Ϫ22%; P Ͻ 0.001), LDL cholesterol Ϫ Ͻ Ϫ Ͻ have proven to be effective in the preven- ( 32%; P 0.001), and triglycerides ( 10%; P 0.05) were achieved after 8 weeks of tion of cardiovascular disease in type 2 treatment with pravastatin. In addition, significant reductions of plasma levels of F1 ϩ 2 (Ϫ4.4%; P Ͻ 0.05), vWFag (Ϫ5.3%; P Ͻ 0.05), and sTF (Ϫ3.4%; P Ͻ 0.05) were observed after diabetes, little is known about these so- treatment with pravastatin. Furthermore, plasma levels of CRP were also significantly reduced called pleiotropic effects in patients with (Ϫ13%; P Ͻ 0.05). Levels of fibrinogen and D-dimer did not decrease after treatment with type 2 diabetes. pravastatin. Our objective was to determine if treatment with pravastatin has potential CONCLUSIONS — The results indicated that pravastatin reduces levels of coagulation and antithrombotic and anti-inflammatory ef- inflammation markers in type 2 diabetic patients. These antithrombotic and anti-inflammatory fects in patients with well-controlled type effects of treatment with statins could play a role in reducing cardiovascular complications in 2 diabetes. Therefore, we evaluated the type 2 diabetic patients. effect of pravastatin on coagulation and inflammation markers in patients with Diabetes Care 27:468–473, 2004 type 2 diabetes and serum total choles- terol of 5–10 mmol/l.

ype 2 diabetes is a leading cause of terolemia, hypertension, obesity, and RESEARCH DESIGN AND vascular morbidity and death. It is increased markers of coagulation (1) and METHODS — In this crossover trial, T often complicated by other cardio- inflammation (2). It has therefore been 50 type 2 diabetic patients were studied to vascular risk factors such as hypercholes- recommended that therapeutic prevention evaluate the effect of pravastatin on ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● plasma coagulation and inflammation From the 1Laboratory for Experimental Internal Medicine, Academic Medical Center, University of Amster- markers. A crossover design was chosen 2 dam, Amsterdam, the Netherlands; the Department of Internal Medicine, Slotervaart Hospital, Amsterdam, to allow treatment comparisons in one the Netherlands; the 3Department of Vascular Medicine, Academic Medical Center, University of Amster- dam, Amsterdam, the Netherlands; the 4Department of Clinical Chemistry, Slotervaart Hospital, Amster- subject rather than between subjects and dam, the Netherlands; and the 5Department of Internal Medicine and Cardiovascular Research Institute because the sample size needed for detec- Maastricht, Academic Hospital and University of Maastricht, Maastricht, the Netherlands. tion of treatment effects is smaller. Pa- Address correspondence and reprint requests to Dirkje W. Sommeijer, MD, Laboratory of Experimental tients were recruited from the outpatient Internal Medicine, G2–108, Meibergdreef 9, 1105 AZ Amsterdam, Netherlands. E-mail: d.w.sommeijer clinic of the Slotervaart Hospital (Amster- @amc.uva.nl. Received for publication 28 January 2003 and accepted in revised form 23 October 2003. dam, the Netherlands). Men and women Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine phos- ages 18–80 years who were diagnosed phokinase; ELISA, enzyme-linked immunosorbent assay; hs-CRP, high-sensitivity C-reactive protein; IL, with type 2 diabetes for at least 1 year and interleukin; sTF, soluble tissue factor; TNF-␣, tumor factor-␣; vWFag, presented with serum cholesterol levels of antigen. A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion 5.0–10.0 mmol/l were eligible for the factors for many substances. study. Patients with acute medical condi- © 2004 by the American Diabetes Association. tions; surgery during the previous 3

468 DIABETES CARE, VOLUME 27, NUMBER 2, FEBRUARY 2004 Sommeijer and Associates months; deep venous or pul- ity, defined as the lowest measurable con- 50 patients are presented in Table 1. The monary embolism during the previous 3 centration that can by distinguished from median age of the patients was 59 years. months; significant renal, hepatic, meta- zero with 95% confidence, was 0.2 mg/ Patients were overweight, with a median bolic, or thyroid disease; abuse; or ml. Measurements of the prothrombin BMI of 29 kg/m2. Patients’ diabetes was ϩ known familial hypercholesterolemia fragment F1 2 (Dade Behring, Mar- well controlled, with the median HbA1c were excluded. Included patients were burg, Germany), the von Willebrand fac- being 7.1%. In all, 62% of the patients not concurrently receiving other lipid- tor antigen (vWFag) (antibodies from were treated with insulin alone or in com- lowering, antithrombotic, or hormonal Dako, Glostrup, Denmark), and soluble bination with oral antidiabetic agents, treatment, but were allowed to use an ace- tissue factor (sTF) (American Diagnos- whereas the other 38% were treated with tylsalicylic acid. Patients maintained their tica, Greenwich, CT) were performed by oral alone. In addition, 42% regular diet during the study period. enzyme-linked immunosorbent assay of the patients used medication for hyper- An open-label, randomized, cross- (ELISA). D-Dimers were measured with tension and 26% used acetylsalicylic acid. over design was used. One-half of the an automated quantitative latex particle subjects (group A) began with pravastatin immunoassay (BioMe´rieux, Durham, Serum lipids and safety parameters (Selectin; Bristol-Myers Squibb, Woer- NC). Interleukin (IL)-12-p70, IL-1␤, The effects of are shown in Table den, the Netherlands; 40 mg/day), and IL-6, IL-10, and IL-8 were measured by 2. Data are expressed as medians with the the other half (group B) began with no cytometric bead array analysis (Becton 25th and 75th percentile. Statistically sig- treatment. Patients visited the outpatient Dickinson Biosciences, San Diego, CA). nificant reductions of total cholesterol clinic on day 1, after the first period of 8 Tumor necrosis factor-␣ (TNF-␣) and (Ϫ1.4 mmol/l [Ϫ1.9 to Ϫ1.0]), LDL cho- weeks, at which time pravastatin or no IL-6 were measured with a high- lesterol (Ϫ1.3 mmol/l [Ϫ1.74 to treatment was crossed over for another sensitivity ELISA (Quantikine HS human Ϫ0.95]), and triglycerides (Ϫ0.19 8-week period, and after 16 weeks at the TNF-␣ and IL-6 ELISA; R&D Systems Eu- mmol/l [Ϫ0.55 to 0.08]) were achieved end of the study. At each visit, blood sam- rope, Abingdon, Oxon, U.K.). after treatment with pravastatin, indicat- ples were taken and patients’ blood pres- ing satisfactory compliance with the study sure was measured. The active treatment Statistics medication. HDL levels did not change and its possible effects on the measured Results are presented as medians with during treatment. Pravastatin did variables were presupposed to be washed 25th and 75th percentiles. After testing not influence glycemic control: HbA1c out after 8 weeks. Laboratory outcomes at for normality, Student’s paired t test or and glucose levels remained unchanged day 1 and at 8 and 16 weeks were com- Wilcoxon’s signed-rank test was used to during treatment. Treatment with prava- pared, with each patient being his or her compare values after a treatment or no- did not significantly change safety own control. All patients gave their in- treatment period. The main outcome data parameters CK, ALT, and AST. formed consent, and the study was ap- were tested for carryover effect by com- proved by the institutional Ethical Review paring treatment effects between the two Coagulation and inflammation Board of the Slotervaart Hospital, Amster- patients groups (⌬ group A vs. ⌬ group B) markers dam. (8a). No carryover effect was determined. The effects of therapy on the principal Thus, we pooled data from patients after study outcome markers are summarized Blood sampling and laboratory the treatment period, irrespective of in Table 2. A statistically significant re- methods whether they started out or ended with duction in plasma levels of hs-CRP Blood samples were obtained by standard the treatment period, and compared (Ϫ0.52 mg/dl [Ϫ1.34 to 0.27]) was venepuncture between 9:00 and 11:00 those with pooled data from the no- achieved by pravastatin treatment. The A.M., after a 12-h fast. Total, HDL, and treatment period. Spearman’s rank corre- prothrombin activation marker F1 ϩ 2 LDL cholesterol; triglycerides; and fibrin- lation coefficient analysis was used to was slightly, but significantly, lower ogen were determined using standard examine associations between measured (Ϫ0.04 nmol/l [Ϫ0.2 to 0.04]) after active laboratory procedures within 1 h after parameters. A two-tailed P Յ 0.05 was treatment. The selected markers of endo- sampling. HDL cholesterol was deter- considered to indicate statistical thelial dysfunction, vWFag (Ϫ7% [Ϫ12 mined using a direct assay. Safety param- significance. to 3]) and sTF (Ϫ4 pg/ml [Ϫ45 to 4.5]), eters included creatinine, alanine were also significantly reduced after prav- aminotransferase (ALT), aspartate amino- RESULTS — A total of 56 patients astatin treatment. Despite the overall re- transferase (AST), and creatine phos- were randomized to begin the study with duction in vWFag levels, the median phokinase (CK) were measured with an 8-week period of either pravastatin concentration of vWFag was increased af- standard techniques. Glycemic control therapy or no treatment. Of those 56, 50 ter treatment compared with before treat- was monitored by evaluating fasting glu- completed the study; 4 patients stopped ment (138% ([103 to 175] vs. 131% [114 cose, measured with standard techniques, during the treatment period because of to 162]) because of the extreme nonnor- and HbA1c, determined by high- side effects attributed to the medication mal distribution (Fig. 1). performance liquid chromatography, as (skin and gastrointestinal complaints), 1 Treatment did not significantly lower described elsewhere (8). Levels of high- patient stopped during the treatment pe- the levels of fibrinogen, D-dimer, IL-8, or sensitivity C-reactive protein (hs-CRP) riod because he had a myocardial infarc- TNF-␣. In 20% of the patients, IL-12- were determined with a near infrared par- tion, and 1 patient stopped because lung p70, IL-1␤, IL-6, and IL-10 levels were ticle immunoassay rate methodology carcinoma was detected. detected with the cytometric bead array (Beckman, Brea, CA). Analytical sensitiv- Baseline clinical characteristics of the analysis. In this group of patients, no

DIABETES CARE, VOLUME 27, NUMBER 2, FEBRUARY 2004 469 Pleiotropic effects of pravastatin in type 2 diabetes

Table 1—Patient characteristics

Type 2 diabetic patients n 50 Age (years) 59 (54–64) Sex ratio (male/female) 25/25 BMI (kg/m2) 28.9 (26.8–33.1) Diabetes duration (years) 6.0 (3.0–10.3)

HbA1c (%) 6.9 (6.4–7.7) Insulin treatment 31 ACE inhibitor 11 A2-antagonist 5 Acetylsalicylic acid 13 Current smoker 12 (Figure 1—The reduction of vWFag was Ϫ5.3% after pravastatin treatment (ۙ) despite in- Plasma glucose (mmol/l) 9.7 (8.2–12.2 creased median concentration of vWFag (no treatment 131% [114–162] vs. pravastatin 138% Total cholesterol (mmol/l) 6.3 (5.7–6.9) [103–175]), the latter possibly because of the extreme non-normal distribution of vWFag. NT, LDL cholesterol (mmol/l) 4.0 (3.6–4.6) after 8 weeks of no treatment; PRAV, after 8 weeks of pravastatin treatment. HDL cholesterol (mmol/l) 1.2 (1.0–1.5) Triglycerides (mmol/l) 1.7 (1.4–2.8) changes were measured after treatment ated with fibrin formation and proteolytic Data are median (25th–75th percentile). with pravastatin (data not shown). We re- cleavage (D-dimer). No correlation be- peated the measurements of IL-6 using a tween a change in F1 ϩ 2 and changes in high-sensitivity ELISA (R&D Systems Eu- fibrinogen was observed. No correlations rope) with a detection limit at ϳ0.05 pg/ were found between reductions of hs- eficial effects of statins are attributable not ml. IL-6 was still detectable in only 35% CRP, F1 ϩ 2, sTF, and vWFag and only to their lipid-lowering action, but of patients. In the patients with detectable changes in total, LDL, or HDL cholesterol, also to the “pleiotropic” actions of statins. IL-6 levels, no changes were observed be- or triglycerides. A weak correlation was Current knowledge of such pleiotro- fore and after pravastatin treatment (2.9 observed between the degree of change of pic effects is largely derived from in vitro [1.8–3.2] vs. 2.6 pg/ml [1.9–4.5]; P ϭ vWFag and the change of hs-CRP (r ϭ experiments and studies in patients with 0.3). 0.312; P ϭ 0.031). No other correlations hypercholesterolemia. To specifically de- To identify possible mechanisms for were found between the observed reduc- termine the effects of pravastatin on in- the decrease of CRP, F1 ϩ 2, sTF, and tions in hs-CRP, F1 ϩ 2, sTF, and vWFag. flammation, coagulation, and endothelial vWFag after pravastatin treatment, corre- activation markers in type 2 diabetic pa- lations with changes in other parameters CONCLUSIONS — Statins comprise tients, we performed the present study. were assessed. A statistically significant a group of agents that are increasingly Our data demonstrated that 2 months of correlation was observed between the prescribed to counteract treatment with pravastatin reduced the change in F1 ϩ 2 and the degree of and related cardiovascular complications. levels of CRP, F1 ϩ 2, sTF, and vWFag. change of D-dimer (r ϭ 0.534; P Ͻ Statins also show marked clinical efficacy These biological alterations may have 0.0001), a finding that fits the notion that in individuals with type 2 diabetes. Sev- clinical significance, as type 2 diabetes is generation (F1 ϩ 2) is associ- eral lines of evidence suggest that the ben- associated with increased inflammation

Table 2—Effect of pravastatin on lipids and coagulation and inflammation markers

⌬ (after treatment Ϫ No treatment After pravastatin P after no treatment) Total cholesterol (mmol/l) 6.3 (5.6–7.0) 4.9 (4.1–5.4) Ͻ0.001* Ϫ1.4 (Ϫ1.9 to Ϫ1.0) LDL cholesterol (mmol/l) 4.0 (3.6–4.6) 2.7 (2.4–3.0) Ͻ0.001* Ϫ1.3 (Ϫ1.74 to Ϫ0.95) HDL cholesterol (mmol/l) 1.2 (1.0–1.5) 1.2 (1.0–1.4) 0.699* 0.03 (Ϫ0.11 to 0.11) Hs-CRP (mg/dl) 4.0 (2.0–6.2) 3.3 (1.3–4.7) 0.019† Ϫ0.52 (Ϫ1.34 to 0.27) TNF-␣ (pg/ml) 2.6 (2.1–3.8) 2.7 (2.2–3.9) 0.967† 0.0015 (Ϫ0.98 to 1.5) IL-8 (pg/ml) 6.2 (3.0–11.7) 5.7 (3.0–10.3) 0.956† 0(Ϫ2.7 to 3.9) (g/l) 3.2 (2.9–3.9) 3.3 (2.8–3.8) 0.231† 0(Ϫ1.0 to 3.0) D-Dimer (␮g/ml) 0.26 (0.19–0.39) 0.27 (0.19–0.45) 0.104† Ϫ0.02 (Ϫ0.09 to 0.05) F1 ϩ 2 (nmol/l) 0.92 (0.67–1.29) 0.91 (0.63–1.18) 0.007† Ϫ0.04 (Ϫ0.2 to 0.04) vWFag (%) 131 (114–162) 138 (103–175) 0.027† Ϫ7(Ϫ12 to 3) sTF (pg/ml) 119 (87–158) 104 (64–146) 0.044* Ϫ4(Ϫ45 to 4.5) Data are median (25–75% quartile). *Normally distributed variable; †not a normally distributed variable.

470 DIABETES CARE, VOLUME 27, NUMBER 2, FEBRUARY 2004 Sommeijer and Associates and coagulation activity and impaired en- did not diminish thrombin production in statin (26). To our knowledge, our study dothelial function. these patients. The mechanism by which is the first to show that pravastatin re- CRP is a marker of inflammation; its statins might influence -mediated duces levels of vWFag and sTF in diabetic plasma concentration levels correlate thrombin production remains unknown. patients. with the severity and extent of the athero- In contrast to F1 ϩ 2, we observed no Fibrinogen has been claimed as an in- sclerotic process in the arterial wall and is significant reduction in D-dimer levels af- dependent cardiovascular risk factor, and consistently associated with prognosis in ter pravastatin treatment. On the basis of increased levels of fibrinogen have been ischemic disease. Several studies our study, we were not able to unravel the observed in patients with various athero- have shown that treatment with statins pathophysiological mechanism behind sclerotic diseases. One study in patients lowers CRP (9–11) in hypercholester- this observation. with poorly controlled diabetes showed a olemic individuals and that patients with Theoretically, a lowered cellular sTF decrease of fibrinogen after treatment higher levels of CRP have greater benefit production or exposure may also be re- with pravastatin (27). In the present from treatment with statins than patients sponsible for reduced thrombin produc- study, no change in fibrinogen was found with lower concentrations (12). In our tion. At this stage, it remains uncertain after 2 months’ treatment. This finding is study population of type 2 diabetic pa- whether a reduced level of sTF, as ob- in line with the majority of studies in hy- tients, we observed a significant reduction served in our study, may translate into percholesterolemic patients, where no re- in plasma levels of CRP after treatment lower thrombin production, because the duction in fibrinogen levels after statin with pravastatin. This observation con- role of sTF as a stimulus of coagulation treatment have been observed. firms previous observations (13,14) and has not been established. The small de- Some of the effects of pravastatin on suggests that statins have anti- crease in sTF should probably be inter- thrombin formation and endothelial inflammatory properties in type 2 dia- preted as diminished proteolytic cleavage function may be induced by an anti- betic patients also. from injured endothelial cells, which inflammatory action of this class of The anticoagulant potential of prava- would support the concept of stabiliza- agents. Statins are able to block the syn- statin was assessed by measuring two rel- tion of endothelial cell function by statins thesis of important isoprenoid intermedi- evant markers, F1 ϩ 2 and D-dimer. The (21). ates, which serve as lipid attachments for F1 ϩ 2 peptide fragment is released when The level of circulating vWFag is an- a variety of intracellular signaling mole- prothrombin is converted into thrombin, other marker that is considered to reflect cules. To identify a mechanism for the ob- with concentrations of F1 ϩ 2 in plasma endothelial injury. The vWFag is a glyco- served changes in inflammation, reflecting the amount of in vivoϪ protein stored in endothelial cells and se- coagulation, and endothelial function af- generated thrombin. Several studies have creted into the circulation. It increases in ter pravastatin treatment, correlations shown that statins reduce circulating lev- parallel with the degree of endothelial cell with a reduction of lipids were assessed. els of F1 ϩ 2 (15,16) and F1 ϩ 2 in sam- damage. In our patients, the basal levels of The fact that no significant correlation ples from time wounds in vWFag were quite high (median 131%), a was found between reductions in CRP, patients with hypercholesterolemia finding that might reflect the longer term F1–2, vWFag, and sTF levels and the re- (17,18). Aoki et al. (19) showed that in- vascular perturbation inflicted by type 2 duction of lipids supports the concept creased platelet-dependent thrombin diabetes (median duration of type 2 dia- that effects other than the lipid-lowering generation in hypercholesterolemic pa- betes in these patients was 6 years). The action play a role in these changes. In ad- tients normalizes after pravastatin treat- observed decrease of vWFag during treat- dition, the change of vWFag after prava- ment, whereas Szczeklik et al. (17) found ment, confirming observations on statins statin treatment was associated with the that simvastatin inhibits thrombin forma- in patients with hypercholesterolemia degree of change of CRP, suggesting that tion in bleeding time blood. had (16,22,23), could be explained by an en- the endothelial cellϪimproving effect was no further effect on thrombin formation. dothelial improvingϪeffect of pravasta- attributable to an anti-inflammatory and Likewise, Dangas et al. showed that prav- tin. Data on endothelial cellϪimproving not a cholesterol-lowering effect of prav- astatin (18) reduced ex vivo thrombus effects of statins in type 2 diabetic patients astatin. We speculated that the regulation formation, whereas the reduction was at- are scarce and conflicting. Endothelial of vWFag and sTF were related, with both tenuated in patients on aspirin. The re- cellϪmediated vasoreactivity improves in being markers of endothelial cell dysfunc- duction in thrombin or thrombus diabetic rats after treatment with prava- tion. However, we did not find an associ- formation in these studies may have been statin or cerivastatin (24,25). In a study ation between changes of both secondary to an antiplatelet effect of st- with diabetic patients, vasoreactivity im- parameters. This finding might be ex- atins (20). Our study extends the above proved as soon as after 3 days of treatment plained by a different pattern of cleavage, findings to type 2 diabetic patients, in with cerivastatin. In addition, the plasma secretion, or elimination from the whom we observed that pravastatin low- level of soluble vascular adhesion mole- circulation. ered levels of F1 ϩ 2. A possible explana- cule-1, a plasma marker for endothelial In conclusion, our data demonstrate tion for this reduction of in vivo thrombin dysfunction, was decreased in these pa- that treatment with pravastatin for 2 formation in these diabetic patients with tients after 3 months of treatment (13). months induces anti-inflammatory, anti- mild hypercholesterolemia is that like in However, another study reported the ab- thrombotic, and endothelial-improving the previous described studies with hy- sence of any effect on nitric oxide– actions in patients with type 2 diabetes percholesterolemic patients, platelet- dependent vasoreactivity in type 2 and mild hypercholesterolemia. These mediated thrombin formation is reduced diabetic patients after 4 weeks of aggres- findings provide an additional biological by statin treatment. In contrast, aspirin sive lipid-lowering treatment with atorva- basis for the presumed importance of

DIABETES CARE, VOLUME 27, NUMBER 2, FEBRUARY 2004 471 Pleiotropic effects of pravastatin in type 2 diabetes pleiotropic effects of statin treatment in and their reduction with pravastatin in dia- on thrombogenic and inflammatory pa- patients with type 2 diabetes and cardio- betic and glucose-intolerant myocardial in- rameters in patients with hypercholester- vascular disease. farction survivors with average cholesterol olemia. Thromb Haemost 85:47–51, 2001 levels: subgroup analyses in the Cholesterol 17. Szczeklik A, Musial J, Undas A, Gajewski and Recurrent Events (CARE) Trial. Circu- P, Gora P, Swadzba J, Jankowski M: Inhi- lation 98:2513–2519, 1998 Acknowledgments— H.T.C. is a Clinical Es- bition of thrombin generation by simva- 7. Palinski W: New evidence for beneficial tablished Investigator of the Netherlands statin and lack of additive effects of effects of statins unrelated to lipid lower- Heart Foundation. The study costs were de- aspirin in patients with marked hyper- ing. 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