Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.


Introduction
Numerous clinical studies have demonstrated beneficial effects of the lipid-lowering drugs in reducing plasma levels of low-density lipoproteins (LDL) cholesterol and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol, which prevents both primary and secondary incidences of atherosclerosis-related adverse cardiovascular events 1,2,3,4,5 . Statins, a group of HMG-CoA reductase enzyme inhibitors, block endogenous cholesterol synthesis in the liver that in turn lead to lower circulating levels of LDL cholesterol in the blood 6,7 . Likewise, the lipid lowering effect of niacin is mediated by its direct and noncompetitive inhibition of hepatocyte diacylglycerol acyltransferase-2, a key liver enzyme involved in triglyceride synthesis 8 . Comparatively, ezetimibe reduces plasma level of LDL by limiting absorption of exogenous cholesterol by binding to Niemann-Pick C1-Like 1 (NPC1L1) protein located in the epithelial cells of the small intestine 9 . Fenofibrate, another lipid-lowering drug, substantially reduces plasma concentrations of triglycerides and also moderately decreases LDL cholesterol through the peroxisome proliferator-activated receptors pathway 10 . Besides, omega-3 fatty acid is reported to have the anti-atherosclerotic effect due to its ability to lower the plasma levels of LDL 11 .
The lipid-lowering drugs, in addition to their primary effect on lowering LDL cholesterol, have a number of beneficial pleiotropic effects including enhancing the HDL level, improving endothelial functions, reducing inflammation, and inhibiting platelet aggregations 12,13,14 . However, the underlying mechanism of these drugs in increasing HDL cholesterol particles and modifying their structural features are not fully understood. Since these medicines are widely prescribed to treat atherosclerosis-related cardiovascular diseases (CVDs), it is essential to further investigate their possible roles in determining morphological features and distribution of the lipid particles. The human plasma lipidome consists of approximately 600 distinct lipids and 22 different molecular types of cholesterols that are present in various sizes, shapes, densities, and compositions 15,16,17 . Analytical methods such as ultra-centrifugation, NMR, and gradient gel electrophoresis are used to characterize LDL and HDL particles and their subfractions

Plaque Array Assay Based Analysis of Lipid-lowering Drug Effects on Cholesterol Particles Formation:
For evaluating the effect of statins in modulating morphology of cholesterol particles, fluorescent-labeled cholesterol aggregates were individually incubated with lovastatin, simvastatin, atorvastatin, rosuvastatin, and fluvastatin in the buffer. All these samples were acquired using imaging flow cytometry to capture images of the cholesterol particles for morphology analysis as shown in Figure 1 and To further evaluate the effect of lipid-lowering drugs on particles formation, fluorescent-labeled cholesterol aggregates were individually incubated with ezetimibe, fibrate, niacin, and omega-3 fatty acid. As observed with the statins, these drugs induced the formation of cholesterol particles with heterogeneous sizes and shapes as displayed in Figure 4a-d. Among them, ezetimibe induced the formation of cholesterol particles exhibiting both globular and linear strand morphologies whereas fibrate, niacin, and omega-3 fatty acid induced the formation of only globularshaped cholesterol particles. Accordingly, the effect of drugs on the linear strand shaped cholesterol particles formation was in the order of 3% for ezetimibe, 0% for fibrate, 0% for niacin, and 0% for omega-3 fatty acid.
The morphological analysis revealed each globular or linear strand shaped cholesterol particle is composed of many smaller particles linked together. The sizes of fluorescent positive globular cholesterol particles identified are in the range of ~2 -30 µm 2 , whereas the sizes of the linear shaped particles are in the range of ~2 -60 µm 2 .

Analyzing the Effect of Lipid-Lowering Drugs on Purified VLDL and LDL Particles:
To further examine the cholesterol particles formation in the presence of lipoproteins, the fluorescent-labeled cholesterol aggregates were individually incubated with purified VLDL, LDL, and HDL proteins/particles. The results showed, compared to the incubation with LDL and HDL particles, that the cholesterol aggregates incubated with VLDL proteins caused the formation of a higher number of cholesterol particles, Figure  5. In addition, two major fractions of cholesterol particles were observed in VLDL populations suggesting their partial transformation into a LDL fraction during the incubation with the fluorescent-labeled cholesterol aggregates. The image analysis of particles indicated the presence of both globular (~97%) and linear shaped (~3%) particles among VLDL, LDL, and HDL populations. The size ranges of globular particles are ~2 -30 µm 2 , whereas the size ranges of linear particles are ~2 -60 µm 2 .
For examining the lipid-lowering drug effect on purified particles, the VLDL and LDL particles were individually incubated with ezetimibe, lovastatin, simvastatin, and niacin. As a result, compared to control experiments without the drug, the drug effect observed on VLDL particles formation was higher in ezetimibe, simvastatin, lovastatin, and niacin. The LDL particles incubated with the drugs showed a major single fraction and the drug-induced effect on particles formation was higher in ezetimibe, simvastatin, lovastatin, and niacin, Figure 6.

Variations of the Lipid-lowering Drug Effects in Altering the Distribution of Cholesterol Particles in Serum Samples:
The preceding experiments were performed in the buffer solution with purified lipoproteins for evaluating the drug effect. Hence, in the next step, the effectiveness of lipid-lowering drugs on cholesterol particles formation was examined using 50 serum samples collected from 25 subjects with dyslipidemia and 25 age-matched normal subjects. The drug response in each serum sample was measured based on changes in the profile of cholesterol particles formation in the presence and absence of drugs. In the plaque array assay, each serum sample was screened against ezetimibe, lovastatin, simvastatin, and niacin drugs. The results revealed disparity among these drugs in modulating the distribution of VLDL, LDL, and HDL particles in serum samples, particularly their effect on reducing LDL and increasing HDL cholesterol particles formation. Three representatives of dyslipidemia serum samples exhibiting unique responses to the lipid-lowering drugs are shown in Figure 7.

Identification of the Drug Effect on Modulating Morphology of Serum Derived LDL and HDL Cholesterol Particles:
The phenotype analysis of serum-derived cholesterol particles revealed the presence of both linear strands and globular shaped VLDL, LDL, and HDL subpopulations, thus confirming a similar morphology identified in the experiments performed both in the buffer and with purified lipoprotein particles as shown in Figure 8. However, the distribution of the globular and linear shaped cholesterol particles subpopulations widely varied among dyslipidemia and age-matched normal subjects. Notably, the control assays performed without the drugs showed differences in the distribution of linear strand shaped LDL cholesterol particles between dyslipidemia (mean of 2.0%) and age-matched normal (mean of 1.3%) serum samples. Similarly, an increased level of linear strand shaped HDL cholesterol particles was observed in dyslipidemia samples (mean of 18.3%) compared to the age-matched (mean of 11.1%) serum samples. In correlation, the assays performed in the presence of drugs in dyslipidemia serum samples showed a significant reduction in linear shaped HDL cholesterol particles formation for simvastatin (mean of 8.3%), ezetimibe (mean of 11.5%), lovastatin (mean of 11.7%), and no reduction for niacin (mean of 18.3%). In addition, a decrease in the formation of linear shaped LDL cholesterol particles was observed in dyslipidemia serum samples when incubated with the drugs displayed in Table 1.     Table 2: Screening of age-matched control serum samples in the plaque array assay show the differential effect of lipid-lowering drugs. Compared to controls without drug (columns 2, 3), serum samples incubated with ezetimibe (columns 4, 5), lovastatin (columns 6, 7), simvastatin (columns 8,9), and niacin (columns 10, 11) showed variations on inducing linear-shaped LDL and HDL cholesterol particles formation.

Discussion
In general, the distribution and functional properties of VLDL, LDL, and HDL cholesterol particles in the blood circulation are mainly determined by metabolic, genetic, epidemiological, cellular, and plasma factors 22,23 . In the present study, examining the effects of lipid-modifying drugs in the buffer revealed that highly lipophilic drugs such as ezetimibe, lovastatin, simvastatin, and atorvastatin induced a higher level complexity on the morphology of cholesterol particles compared to the lower level effect observed with highly hydrophilic rosuvastatin and fluvastatin drugs. These results are in good agreement with our previous study describing a non-enzymatic mechanism based effect of statins in modulating LDL and HDL cholesterol particles formation in the buffer and serum samples 21 . Accordingly, the results from present study revealed a non-enzymatic mechanism of action of ezetimibe, niacin, fibrate, and omega-3 fatty acid drugs that may play a direct role in modulating cholesterol particles formation.It is possible that the interactions between drugs and cholesterol aggregates leads to the assembly of large size cholesterol particles that are 2 -60 µm 2 , exhibiting globular and linear strand morphologies.
Besides, the results obtained using purified lipoprotein particles suggest interactions between cholesterol aggregates and plasma factors including VLDL, LDL, and HDL proteins that may alter the compositions and morphological properties of the cholesterol particles. The drug treatment results involving purified lipoprotein particles indicated a higher level drug effect on VLDL particles formation compared to their effect observed on LDL cholesterol particles formation. The lovastatin, simvastatin, and ezetimibe drugs were used as pro-drugs and their doses in the assays may be higher than the physiological concentrations.
LDL and HDL cholesterol particles formation in both dyslipidemia and age-matched normal serum samples. The drug effects observed on reducing linear shaped particles formation was higher in simvastatin, ezetimibe, lovastatin, and niacin. The identification of cholesterol particles with globular and linear strand morphologies in the normal and dyslipidemia serum samples suggests that particles with similar morphologies may form in in vivo conditions. Previous studies have identified the presence of disc and needle-like cholesterol crystals in the atherosclerotic plaques of human and ApoE -/and LDLR -/mice models 24,25,26,27,28 .
The HDL particles circulating in the blood exist as a heterogeneous mixture and the level of small and large size HDL particles along with functional activity are important factors to exert their cardio-protective effect via the reverse cholesterol transport pathway 29,30 . Recent studies have highlighted the importance of identifying HDL cholesterol particle subfractions for elucidating their role in multiple biological functions such as cholesterol efflux, anti-inflammation, anti-thrombotic, and anti-oxidative 31 . In addition, a number of studies have reported the effect of lipidlowering therapy in increasing a low to moderate level of HDL in the plasma 1,5,21 . Accordingly, the results from this study provide new insights on morphological features of cholesterol particles. Notably, the detection of a higher level of linear shaped HDL cholesterol particles in the serum samples of dyslipidemia subjects suggests that they may be the reliable biomarker for both diagnosis and evaluating effects of lipid-modifying drugs in patients. However, further investigation is required using large clinical samples to better understand the cholesterol particles with distinct morphologies and their association to CVD.
In the plaque array assay for examining the drug effect on assembly of cholesterol particles, we used 2 µg of fluorescence labeled cholesterol aggregates and 5 µgof each drug because: (1) drugs competitively bind to both fluorescence labeled cholesterol and endogenous lipids present in the serum samples; (2) from each sample, we acquired 5,000 to 10,000 cholesterol particles that are assembled into large sizes and shapes ranging from ~2 -60 µ 2 ; (3) we observed a wide variations of drug response among serum samples incubated with the drugs (doses 300 ng to 5 µg) and ~1 -5% of them incubated with high doses showed no detectable changes in the profile of cholesterol particles formation; and (4) the interaction between cholesterol aggregates and lipid-lowering drugs is mediated by a non-enzymatic process. Hence, the concentrations of the reagents used in the assay may be higher than their physiological level.
In conclusion, we have successfully demonstrated the advantages of an in vitro imaging method described in this study for determining the effect of a broad spectrum of lipid-lowering drugs on modulating morphology and composition of cholesterol particles. The approach of visualizing and quantifying the morphology of lipid particles by employing a constellation of image analysis algorithms may help both the diagnosis of atherosclerosis and to evaluate outcomes of lipid-lowering therapy in patients.

Disclosures
Dr. Madasamy received grant support from Plaxgen, Inc and has a competing financial interest. The other authors have no competing financial interests to disclose.