Evacetrapib reduces preβ-1 HDL in patients with atherosclerotic cardiovascular disease or diabetes
Introduction
Epidemiological studies have shown high-density lipoprotein cholesterol (HDL-C) levels to be independently and inversely correlated with cardiovascular disease (CVD) [1]. This relationship is thought to be largely mediated by the ability of HDL to transport excess cholesterol from peripheral tissues back to the liver for excretion, a process known as reverse cholesterol transport [2]. Potent cholesteryl ester transfer protein (CETP) inhibitors, like torcetrapib, evacetrapib and anacetrapib, significantly increase HDL-C and decrease LDL-C levels, as monotherapy and in combination with statins [[3], [4], [5], [6]]. However, except anacetrapib [7], these lipoprotein changes have not resulted in reduction of major cardiovascular events (MACE) [[8], [9], [10]]. It has been hypothesized that small increase in blood pressure and hsCRP, as well as qualitative changes in HDL subclasses observed with CETP inhibitors, could partly offset the beneficial effect of LDL-C reduction [11].
It is well known that HDL particles are very heterogeneous [12], and many HDL subclasses have been described depending on the analytical technique used for separation. One of these subclasses, preβ1-HDL, plays a critical role in reverse cholesterol transport but it is particularly challenging to quantify. Two-dimensional nondenaturing linear gel electrophoresis followed by apoA-I immunoblotting and image analysis were first utilized to identify preβ1-HDL, preβ2-HDL, and α-HDL [13]. Subsequently, a further separation of lipid-free apoA-I from preβ1-HDL using the same technique was described [14]. However, other investigators have not been able to distinguish lipid-free monomolecular apoA-I from preβ1-HDL using an in-house sandwich enzyme immunoassay [15,16]. A modified two-dimensional gel system has also been used to define 12 subfractions of HDL, including preβ1-HDL and preβ2-HDL [17], however, there was no clear evidence to show that the method can distinquish preβ1-HDL and lipid free apoA-I [17]. We have recently established a native polyacrylamide gel electrophoresis (PAGE) system with lipid pre-staining for quantification of preβ1-HDL and other HDL subclasses, as well as LDL, in human serum [18]. In the present study, we utilized this method to evaluate the effect of evacetrapib on HDL subclasses in patients with atherosclerotic cardiovascular disease (ASCVD) or diabetes, on background of atorvastatin 40 mg daily, enrolled in the ACCENTUATE trial [19].
Section snippets
Study design
Patient disposition and characteristics, as well as baseline and post-treatment laboratory value for the ACCENTUATE trial (ClinicalTrials.gov NCT02227784), have previously been reported [19]. Samples were available for 30 patients on atorvastatin 40 mg daily and 70 patients on atorvastatin 40 mg plus evacetrapib 130 mg daily, at baseline and after 90 days of treatment. Eli Lilly and Company provided Xian-Cheng Jiang's laboratory at SUNY Downstate Medical Center with the samples and some
Results
As shown in Fig. 1A, our tube native PAGE system can separate HDL (HDL total) into four fractions, preβ1-HDL, large HDL (HDL1), medium HDL (HDL2), and small HDL (HDL3). The percentage of each HDL subclass in total HDL is preβ1-HDL (about 5%), HDL1 (about 10%), HDL2 (about 50%), and HDL3 (about 35%) (Fig. 1B). HDL1, HDL2, and HDL3 were confirmed by their densities, as previously described [18]. Preβ1-HDL was also confirmed by the treatment with 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB), an
Discussion
The novel finding of the present study is the reduction of preβ-1 HDL particles with evacetrapib treatment in patients with ASCVD or diabetes on atorvastatin 40 mg per day, when a recently developed native PAGE system was used to quantify this particular HDL species. Moreover, we found that preβ-1 HDL and medium HDL are negatively interrelated. As previously shown by other authors, here we also observe a dramatic increase of large HDL [[21], [22], [23], [24]]. Finally, our native PAGE system
Trial registration
ClinicalTrials.gov NCT02227784
Conflicts of interest
The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.
Author contributions
Y.C. and J.D. designed the study and performed most of experiments, and they contributed equally to this work; X.Z. and X.C. did some experiments and statistical analysis; L.W., H.C., and J.G. involved in designing the study and edited the paper; X.C.J. designed the study and wrote the manuscript.
Acknowledgements
We would like to thank Dr. Bingsheng Yin (Southern Medical University, China) for his effort in the establishment of the native polyacrylamide gel electrophoresis system. We would also like to thank Dr. Akihiro Inazu (Kanazawa University, Japan) for providing human CETP mutant serums. We would like to thank Eli Lilly & Company for providing the samples from the ACCENTUATE trial. Submission of this manuscript for publication has followed the terms of Material Transfer Agreement (between Lilly
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Both Yunqin Chen and Jibin Dong contributed equally as first authors.