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

The overall goal of this study is to evaluate the effect of lipid-lowering drugs in modulating morphological features of cholesterol particles using a plaque array method, and to identify potential biomarkers for diagnosis of atherosclerosis and determining drug effects. The role of lipid-lowering drugs in modulating cholesterol particle formation is poorly understood. Here we demonstrate that lipid-lowering drugs play a direct role in altering profiles and morphological features of cholesterol particle formation.

The main advantage of the plaque array based in vitro imaging method over other particle detection methods is that it enables visualization of cholesterol particles for evaluating the effect of lipid-lowering drugs in serum. Jason Lundry, laboratory technician for Plaxgen will demonstrate how we set up the plaque array assay using a chemistry analyzer. Raymond Kong, senior scientist at Millipore Sigma, will demonstrate how we do the sample processing using imaging flow cytometry.

Finally, David Liu, laboratory technician for Plaxgen will demonstrate data analysis for cholesterol particle images. Use a chemistry analyzer-1 to set up the plaque array assay for cholesterol particle formation. Throughout the assay, maintain the final reaction volume in each well at 200 microliters, and perform all assays in triplicate.

First, load 194 point five microliters of phosphate buffered saline, or PDS to each well of a round bottom, low protein binding 96-well plate. To each well, add two point five microliters of each lipid lowering drug solution, no drug is added to the negative control samples. Then, shake the plate for 30 seconds, by placing it on the chemistry analyzer-1 reaction plate to uniformly mix the drug into solution.

Finally, add two microliters of fluorescent-labeled cholesterol aggregate solution to each well. Shake the plate for 30 seconds by placing it on chemistry analyzer-1 reaction plate as before. Then, incubate the plate on a lab shaker for two hours set at 37 degrees Celsius and 200 rpm.

After incubation, acquire the images of particles by imaging flow cytometry. Open the data acquisition template to load the correct instrument settings. Click File, then select Load Template and select the template file.

Click Load, to prepare the instrument for sample loading. When the Load Sample dialog box opens, click OK to load 50 microliters of the samples into the imaging flow cytometer. Wait until the particles can be seen in the imaging area in real time.

When the particles in the imaging area are in good focus, click Acquire to acquire images of each object simultaneously in a high-throughput manner for dark field, bright field, green fluorescence, and yellow fluorescence. At the end of the acquisition, click the Return button to return sample. Repeat these steps to acquire 5, 000 to 10, 000 particles from each sample.

Analyze all raw image files using the image analysis software for determining object fluorescence intensity, and morphological variations, as described in the text protocol. Prepare a round-bottom low protein binding 96 well plate by loading reagents in a stepwise manner, using a final reaction volume of 200 microliters per well. First, prepare the control wells.

Load 193 microliters of PBS to each well. Add 2.5%of patient serum, loading only one serum sample per well. Then, shake the plate for 30 seconds by placing it on chemistry analyzer reaction plate.

Next, add two microliters of fluorescence-labeled cholesterol aggregate solution into each well. Again, shake the plate for 30 seconds by placing it on the chemistry analyzer-1 reaction plate. To prepare the wells with drugs, load 191 microliters of PBS to each well.

Then, add 2.5%of each patient serum per well, and shake the plate for 30 seconds, by placing it on a chemistry analyzer-1 reaction plate. Add two microliters of ezetimibe, lovastatin, simvastatin or niacin solution to all wells except the negative control wells. Adding only one drug per well.

Proceed to shake the plate for 30 seconds as before. Next, add two microliters of fluorescence labeled cholesterol aggregate solution into each well before shaking the plate for 30 seconds. Then, incubate the plate for two hours in a lab shaker, set at 37 degrees Celsius, and 200 rpm.

Following incubation, acquire the samples by imaging flow cytometry following the same settings as before. Use the image analyzing software for batch processing all the image files as described in the text protocol. Shown here are representative results showing identification of globular, and linear strand shaped cholesterol particles, induced by statins, by a non-enzymatic mechanism.

Confirmation of two distinct morphologies of cholesterol particle formation, induced by other lipid lowering drugs is shown. Including ezetimibe, niacin, fibrate, and omega-3 fatty acid. Shown here are dot plots, where the X-axis displays a spectrum of cholesterol particles detected in the green fluorescence channel.

And the Y-axis displays side scatter, gating shows regions of very low density lipo-protein, low-density lipoprotein, and high-density lipoprotein particles, detected in the fluorescence dot plots. These results demonstrate how the lipid-lowering effect alters the profiles of purified VLDL and LDL cholesterol particles, in the presence of no drug, ezemide, lovastatin, simvastatin and niacin. Shown here is a screen of three different serum samples for the identification of a differential effect of various lipid lowering drugs, that modulate profiles of VLDL, LDL and HDL cholesterol particle formation.

These profiles indicate there is also a variation in the response levels among the three different serum samples. Screening of serum sample one for globular and linear-shaped cholesterol particles formed without the drug and in the presence of various lipid lowering drugs, confirms two distinct morphologies of serum derived cholesterol particles, displaying both globular and linear strand shapes. So we have successfully demonstrated an in-vitro imaging approach for identification of lipid-lowering drug effect, that modulate relationship to cholesterol particle formation and their clinical significance.

Our preliminary results are promising, and we are in the process of further validating this plaque array assay using large scale clinical samples. Your standard lipid panel measures LDL and HDL cholesterol levels in serum. Our plaque array assay, hopes to improve the accuracy of atherosclerosis risk and predicts the lipid lowering drug response using serum.