Changes in lipoprotein lipase and endothelial lipase mass in familial hypercholesterolemia during three-drug lipid-lowering combination therapy

This study was performed to compare the effects of three different lipid-lowering therapies (statins, ezetimibe, and colestimide) on lipoprotein lipase and endothelial lipase masses in pre-heparin plasma (pre-heparin LPL and EL mass, respectively) from patients with familial hypercholesterolemia (FH). FH is usually treated by coadministration of these three drugs. The pre-heparin LPL and EL masses were measured in fresh frozen plasma drawn and stored at various time points during coadministration of the three drugs from patients with heterozygous FH harboring a single mutation in the LDL receptor (n = 16, mean age 63 years). The patients were randomly divided into two groups based on the timing when ezetimibe was added. Plasma LPL mass concentration was significantly reduced by rosuvastatin at 20 mg/day (median = 87.4 [IQR: 71.4–124.7] to 67.5 [IQR: 62.1–114.3] ng/ml, P < 0.05). In contrast, ezetimibe at 10 mg/day as well as colestimide at 3.62 g/day did not alter its level substantially (median = 67.5 [IQR: 62.1–114.3] to 70.2 [IQR: 58.3–106.2], and to 74.9 [IQR: 55.6–101.3] ng/ml, respectively) in the group starting with rosuvastatin followed by the addition of ezetimibe and colestimide. On the other hand, the magnitude in LPL mass reduction was lower in the group starting with ezetimibe at 10 mg/day before reaching the maximum dose of 20 mg/day of rosuvastatin. Plasma EL mass concentration was significantly increased by rosuvastatin at 20 mg/day (median = 278.8 [IQR: 186.7–288.7] to 297.0 [IQR: 266.2–300.2] ng/ml, P < 0.05), whereas other drugs did not significantly alter its level. The effects on changes of LPL and EL mass differed depending on the lipid-lowering therapy, which may impact the prevention of atherosclerosis differently.


Background
Lipoprotein lipase (LPL) and endothelial lipase (EL) hydrolyze triglyceride (TG) in circulating chylomicrons and very low-density lipoprotein (VLDL) on the surface of endothelial cells [1]. It has been demonstrated that a reduced concentration of plasma LPL mass is associated with an increased risk of coronary artery disease [2,3].
Several studies have demonstrated that plasma LPL mass concentration could be altered by drug manipulations, such as fibrate, insulin sensitizer, and statins in patients with diabetes, potentially affecting the progression of their atherosclerosis [4][5][6]. In addition, recent genetic studies have suggested that genetic modulators of LPL mediate cardiovascular risk. For example, loss-of-function mutations in apolipoprotein C3 (APOC3), which is an inhibitor of LPL, decrease the risk of coronary artery disease (CAD) [7], while loss-of-function mutations in apolipoprotein A5 (APOA5), which is an activator of LPL, increases the risk of CAD [8]. In contrast, large-scale clinical trials assessing the efficacy of HDL cholesterol-raising therapies have failed [9,10] based on the neutral impact of HDL cholesterol concentration on CAD estimated by Mendelian randomization studies [11,12]. Accordingly, targeting plasma LPL (and EL) concentrations, rather than plasma HDL cholesterol concentration, could be much more reasonable in terms of preventive cardiology. However, no study has yet assessed the changes in plasma lipase mass concentration during lipid-lowering therapy in patients with familial hypercholesterolemia (FH), which usually requires the coadministration of different types of such drugs [13]. Therefore, we compared the effects of three different lipid-lowering therapies (statins, ezetimibe, and colestimide) on LPL and EL mass in pre-heparin plasma (pre-heparin LPL and EL mass, respectively) in patients with FH.

Study subjects
The study population consisted of patients with heterozygous FH who participated in a previous clinical trial conducted as a prospective open randomized study to investigate the efficacy and safety of coadministration of rosuvastatin (20 mg/day), ezetimibe (10 mg/day), and granulated colestimide (3.62 g/day) at the maximum doses permitted in Japan. All 17 subjects were heterozygous with a confirmed LDL receptor gene mutation and fulfilled our clinical diagnostic criteria for heterozygous FH: patients with primary hyper-LDL cholesterolemia (>160 mg/dl) with tendon xanthoma or those with firstdegree relatives with previously diagnosed heterozygous FH showing primary hyper-LDL cholesterolemia (>160 mg/dl). Exclusion criteria of the present study were FH patients with a homozygous gene mutation, patients under LDL apheresis therapy or any immunomodulatory medication, patients with fasting serum triglyceride levels >500 mg/dl, patients with hepatic disease, or patients within 12 weeks after the onset of an acute myocardial infarction or stroke. Details of this study have been described elsewhere [14], and 16 of 17 patients whose fresh frozen plasma was available were included in this study. The subjects were divided into two groups using a sealed envelope-based method according to the timing when ezetimibe was added at 10 mg/day. All participants were started on a 4week treatment with rosuvastatin at 5 mg/day followed by another 4-week treatment of rosuvastatin at 10 mg/day. The dose of rosuvastatin in group 1 was increased to 20 mg with an 8-week follow-up, whereas group 2 received ezetimibe at 10 mg/day with an 8-week follow-up (phase 1). After phase 1, group 1 received ezetimibe at 10 mg/day added to rosuvastatin for 8 weeks, whereas in group 2 the doses of rosuvastatin were increased to 20 mg with an 8-week follow-up (phase 2). In phase 3, groups 1 and 2 were given 3.62 g of granulated colestimide (twice a day before meals, once in the morning and once in the evening) added to the phase 2 treatment regimen ( Fig. 1).

Determination of LPL and EL mass concentrations
Fasting blood samples were drawn and stored at −80°C during the study period. Pre-heparin LPL concentrations were determined with a highly sensitive and specific ELISA kit (Code No.27184; IBL, Fujioka, Japan) [15,16]. Fig. 1 Study design. The present study was conducted as a prospective open randomized study to investigate the efficacy and safety of coadministration of rosuvastatin at 20 mg/day, ezetimibe at 10 mg/day, and granulated colestimide at 3.62 g/day. Any lipid-lowering agents had been washed out ≥ 4 weeks before entry into the study. Study subjects were divided into two groups by an envelope-based method to elucidate the secondary end point of the present study: rosuvastatin at 20 mg/day (group 1) versus rosuvastatin at 10 mg/day coadministered with ezetimibe at 10 mg/day (group 2). The white, blue, pink, and purple arrows indicate baseline, second, third, and last blood sampling, respectively EL mass concentrations were determined using our ELISA system (Code No. 27182; IBL) [17].

Biochemical analysis
Fasting blood samples were drawn for assays. Plasma concentrations of total cholesterol, TG, and HDL cholesterol were determined enzymatically as described previously [18]. Remnant-like particles (RLP) were determined as described previously [19].

Statistical analysis
Categorical variables are expressed as percentages. Continuous variables with a normal distribution are shown as means (± SD), and those with a skewed distribution are shown as medians (interquartile [IQR]). The changes in each lipase mass concentration were compared by analysis of variance (ANOVA). All statistical analyses were conducted using R. In all analyses, P < 0.05 was assumed to indicate statistical significance.

Ethical considerations
This study was approved by the Ethics Committees of Kanazawa University (Number: 1883-1) and Kanazawa Medical University (Number: 272). All procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent for genetic analyses was obtained from subjects with FH for inclusion in the study.

Baseline characteristics of study subjects
Sixteen Japanese subjects with heterozygous FH were enrolled in the present study. Baseline characteristics and concomitant drug therapies are listed in Table 1 Fig. 2a). Similar trends were observed in group 2, in which the statistical significance of the reduction during phase 1 was diminished using combination therapy consisting of rosuvastatin at 10 mg/day and ezetimibe at 10 mg/day (median = 79.3 [IQR: 58.8-85.0] to 77.5 [IQR: 60.0-84.6] ng/ml, P = NS, Fig. 3a). On the other hand, the significant reduction in LPL mass concentration achieved by adding colestimide at 3.62 g/day was replicated in group 2 (median = 81.9 [IQR: 47.9-87.0] to 75.1 [IQR: 43.3-85.5] ng/ml, P < 0.05, Fig. 3a).

Changes in EL mass during coadministration of three drugs
In group 1, the EL mass concentration was significantly increased by rosuvastatin at 20 Fig. 2b). In group 2, combination therapy with rosuvastatin at 10 mg/day and ezetimibe at 10 mg/day also increased its level significantly (median = 250.6 [IQR: 175.6-351.8] to 300.3 [IQR: 238.0-375.0] ng/ml, P < 0.05, Fig. 3b). On the other hand, there were no significant changes in EL mass concentration when ezetimibe was added at 10 mg/day and colestimide at 3.62 g/day (median = 300. 3 Fig. 3b).

Association between plasma lipids and lipase mass
We investigated the associations between changes in LPL/EL and those in LDL cholesterol, TG, or RLP cholesterol during each phase. There were no significant associations between the changes in LPL mass concentrations and those in lipids (Additional file 1: Figures S1-S6). Significant associations between the changes in EL mass concentrations and TG/RLP cholesterol were observed during phase 2 in group 2 (Additional file 1: Figures S7-S12).

Discussion
We measured the pre-heparin LPL and EL mass at each time point during coadministration of three drugs (statin, ezetimibe, and colestimide) in patients with heterozygous FH harboring a single mutation in the LDL receptor to compare the effects of these drugs on the plasma lipase mass concentration. Our results indicate that (1) statin and colestimide significantly reduced plasma LPL mass, but ezetimibe did not alter its level; (2) statin significantly increased plasma EL mass, but ezetimibe and colestimide did not change its level; (3) there was no clear association between changes in lipase mass and changes in plasma lipid levels.
Recent Mendelian randomization trials have suggested that plasma TG is one of the causal factors for the development of coronary artery disease, rather than merely a marker [7,20]. In addition, plasma TG level is one of the residual risks in this statin era [21]. Accordingly, reassessment of TG-rich lipoprotein metabolism seems to be a reasonable strategy to combat such residual risk. In this regard, genetic studies have indicated that the LPL and APOC3 pathway is strongly associated with plasma TG as well as coronary artery disease, gathering a great deal of attention as a novel therapeutic target. Increasing plasma LPL concentration as well as reducing plasma APOC3, rather than increasing plasma HDL cholesterol, could be beneficial targets.
The results of one study that investigated the effect of statins on changes in LPL mass concentration in patients with diabetes showed results that were contrary to ours [6]. This difference may have been because we used the a b Fig. 2 Lipase mass concentration during coadministration of three drugs in group 1. a LPL mass concentration. b EL mass concentration. White indicates the baseline. Blue indicates the second blood sampling when rosuvastatin was administered at 20 mg/day. Pink indicates the third blood sampling when rosuvastatin at 20 mg/day and ezetimibe at 10 mg/day were coadministered. Purple indicates the last blood sampling when rosuvastatin at 20 mg/day, ezetimibe at 10 mg/day, and colestimide at 3.62 g/day were coadministered a b Fig. 3 Lipase mass concentration during coadministration of three drugs in group 2. a LPL mass concentration. b EL mass concentration. White indicates the baseline. Blue indicates the second blood sampling when rosuvastatin at 10 mg/day and ezetimibe at 10 mg/day were coadministered. Pink indicates the third blood sampling when rosuvastatin at 20 mg/day and ezetimibe at 10 mg/day were coadministered.
Purple indicates the last blood sampling when rosuvastatin at 20 mg/day, ezetimibe at 10 mg/day, and colestimide at 3.62 g/day were coadministered maximum dose of rosuvastatin in patients with FH whose TG-rich lipoprotein levels were shown to be impaired. Our study has several limitations. First, the size of the study population was small because of the rarity of the disease (diagnosed genetic heterozygous FH). However, we observed similar tendencies between the two groups divided according to the timing of the initiation of ezetimibe. Second, this study did not have a crossover design, which could potentially lead to biased assessment of the differences among drugs. Third, we measured pre-heparin lipase mass levels instead of post-heparin levels, which could potentially affect the results, especially LPL mass levels.

Conclusion
In conclusion, the effects on changes of LPL and EL mass were different depending on the lipid-lowering therapy, which may have different impacts on the preventive effects of the therapy on atherosclerosis. Authors' contributions HT participated in the collection and analysis of data and writing of the manuscript. MK, KM, AN, AI, KN, HM, and MY participated in data collection and analysis. JK participated in conception of the study, supervision, data analysis and manuscript editing. All authors read and approved the final version of the manuscript.