This work was funded by a Plaxgen Research grant awarded to SM (PLX-1008). We thank Palo Alto Medical Research Foundation Research Institute for collecting serum samples from atherosclerosis subjects under IRB approval.

1. Preparation of Fluorescent-labeled Cholesterol and Lipid-lowering Drugs
NOTE: Fluorescent-labeled cholesterol aggregates and statins were prepared as described in our previous article21. Please see Table of Materials for details of reagents, imaging flow cytometer, chemistry analyzers, and data analysis software used in this study.
<ol>
	<li>Solubilize fluorescent-labeled lyophilized cholesterol (1 mg, Ex/Em = 495 nm/507 nm) powder in 1 mL of 100% alcohol.</li>
	<li>Centrifuge the sample for 3 min at 2,040 x g and use the supernatants containing soluble fluorescence-labeled cholesterol aggregates in the plaque array assay.</li>
	<li>For the preparation of drugs, individually solubilize the powders (2 mg) of simvastatin, lovastatin, atorvastatin, ezetimibe, and omega-3 fatty acid in 1 ml of 100% alcohol.</li>
	<li>After centrifugation for 3 min at 2,040 x g, use the supernatants containing drugs in the assay.</li>
	<li>Similarly, solubilize individually the powders (2 mg) of fluvastatin, rosuvastatin, niacin, and fibrate in 1 mL of deionized water to make a stock solution of 2 mg/mL.</li>
	<li>After centrifugation for 3 min at 2,040 x g, use the supernatants containing drugs in the plaque array assay.</li>
</ol>
2. Plaque Array Assay for Examining In Vitro Cholesterol Particles Formation
<ol>
	<li>Use chemistry analyzer-1 (see Table of Materials) to set up the plaque array assay for cholesterol particles formation. Perform the assay in a round bottom, low protein binding 96-well plate using the reagents prepared in Section 1. Maintain the final reaction volume in each well at 200 &#181;L and perform all assays in triplicate.
	<ol>
		<li>First, load 194.5 &#181;L of phosphate buffered saline (PBS) to each well.</li>
		<li>To each well, add 2.5 &#181;L (5 &#181;g) of each lipid-lowering drug solution and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate to uniformly mix the drug into solution. No drug is added to the negative control samples.</li>
		<li>Finally, add 2 &#181;L (2 &#181;g) fluorescent-labeled cholesterol aggregate solution to each well and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
		<li>Incubate the plate on a lab shaker for 2 h set at 37 &#176;C and 200 rpm.</li>
		<li>After incubation, acquire the images of particles by imaging flow cytometry.</li>
	</ol>
	</li>
</ol>
3. Capturing Images of Cholesterol Particles Using Imaging Flow Cytometry
<ol>
	<li>Open the data acquisition template to load the correct instrument settings. Click &#34;File&#34;, then select &#34;Open Template&#8230;&#34; and select the template file.</li>
	<li>Click &#34;Flush, Lock, Load&#34; to prepare the instrument for sample loading.</li>
	<li>When the &#34;Load Sample&#34; dialogue box opens, click &#34;OK&#34; to load 50 &#181;L of the samples from each well prepared in Section 2 into the imaging flow cytometer.</li>
	<li>Click &#34;Run|Setup&#34; to view the particles in the imaging area in real time.</li>
	<li>When the particles in the imaging area are in good focus, click &#34;Run|Acquire&#34; to acquire images of each object simultaneously in a high-throughput manner for dark field (side scatter/SSC), bright field (BF), green fluorescence, and yellow fluorescence.</li>
	<li>Repeat steps 3.2 - 3.5 to acquire 5,000 - 10,000 particles from each sample.</li>
	<li>Analyze all raw image files using the image analysis software for determining object fluorescence intensity and morphological variations.
	<ol>
		<li>Click the &#34;Tools&#34; button at the top of the image analysis software, then select &#34;Batch Data Files&#34; from the drop-down menu, which will open the &#34;Batches&#34; window.</li>
		<li>In the &#34;Batches&#34; window, click the &#34;Add Batch&#34; button, which will open the &#34;Define a Batch&#34; window.</li>
		<li>In the &#34;Define a Batch&#34; window, click the &#34;Add Files&#34; button and select the raw image files, click the &#34;Template&#34; button and select the appropriate data analysis template file, then select &#34;OK&#34; and &#34;Submit Batches&#34; to begin processing and analysis of the raw image files.</li>
	</ol>
	</li>
</ol>
4. Plaque Array Assay Based Evaluation of Lipid-lowering Drugs Effect on Purified Cholesterol Particles
<ol>
	<li>As described in step 2.1, perform the cholesterol particles formation assay using purified VLDL, LDL, and HDL lipoproteins/particles. Perform the plaque array assay in a 96-well plate.
	<ol>
		<li>First, load all wells with PBS; use a 200 &#181;L final volume for each well.</li>
	</ol>
	</li>
	<li>For negative control, add 4 &#181;g VLDL, LDL, or HDL proteins/particles individually in each well without drugs and shake the plate for 30 s.
	<ol>
		<li>For examining the lipid-lowering drug effect, add 4 &#181;g VLDL, LDL, or HDL proteins/particles individually to each well and shake the plate for 30 s.</li>
		<li>For wells added with 4 &#181;g VLDL, LDL, or HDL, add 2.5 &#181;L (5 &#181;g) ezetimibe, lovastatin, simvastatin, or niacin solution and shake the plate for 30 s by placing it in chemistry analyzer-1.</li>
		<li>Finally, add 2 &#181;L (2 &#181;g) fluorescence-labeled cholesterol aggregate solution to all wells and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Incubate the plate for 2 h in a lab shaker set at 37 &#176;C and 200 rpm.</li>
	<li>Acquire the samples using imaging flow cytometry following steps 3.1 - 3.5.</li>
	<li>Analyze raw image files using image analysis software as described in step 3.7.</li>
	<li>In the analysis template, use the following gating scheme for identification of cholesterol particles subpopulations.
	<ol>
		<li>On a plot of green channel saturated pixel count (x-axis) and dark field channel saturated pixel count (y-axis), reject objects with one or more saturated pixels in either channel.</li>
		<li>Plot the objects/particles without saturated pixels using green channel intensity (x-axis) and SSC intensity (y-axis): objects fall into one of three regions (VLDL, LDL, HDL) based on their green channel and SSC intensities. 
		NOTE: These gates were created based on data generated from control experiments carried out using purified VLDL, LDL, and HDL particles/proteins without the drug.</li>
	</ol>
	</li>
	<li>To determine the lipid-lowering drug effect for each sample, calculate the total lipoprotein particles concentrations, as well as the percentage of each subpopulation (VLDL, LDL, HDL) as described in step 6.5.
	<ol>
		<li>Calculate the particle concentrations (particles/mL) using the following equation: 
		<img alt="Equation" src="/files/ftp_upload/56596/56596eq1.jpg" /> 
		NOTE: The VLDL, LDL, and HDL gating regions in the fluorescence dot-plot detect ~ 80% of their respective subpopulations, and the remaining ~ 20% of the particles overlap with other gating regions.</li>
	</ol>
	</li>
</ol>
5. Measurement of Lipid Concentrations in the Serum Samples
<ol>
	<li>For determining concentrations of LDL, HDL, cholesterol, and triglycerides by chemistry analyzer-2, use serum samples obtained from age-matched normal and dyslipidemia subjects.</li>
	<li>Define abnormal concentration of LDL, HDL, cholesterol, and triglycerides based on their concentration identified beyond the upper and lower reference values in serum samples.</li>
	<li>Follow the normal reference value recommended by the reagent manufacturer: cholesterol (4 - 200 mg/dL), HDL (3.0 - 65 mg/dL), LDL (4 - 100 mg/dL), and triglycerides (9 - 200 mg/dL).</li>
	<li>Identify serum samples that have an abnormal value as either below the lower reference value or above the higher reference value for cholesterol and triglycerides, and use them for screening in the presence and absence of lipid-lowering drugs.</li>
</ol>
6. Analyzing the Effect of Drugs on Cholesterol Particles Formation in Serum Samples
<ol>
	<li>Use the chemistry analyzer-1 to set up the plaque array assay for cholesterol particles formation in serum samples. Perform the assay in a round bottom, low protein binding 96-well plate. Use a final reaction volume of 200 &#181;L/well and perform all assays in triplicate.</li>
	<li>Prepare the plate by loading reagents in a stepwise manner.
	<ol>
		<li>Prepare the control wells.</li>
		<li>Load 193 &#181;L of PBS to each well.</li>
		<li>Add 2.5% (v/v) patient serum (only one serum sample per well) and shake the plate for 30 s by placing it on chemistry analyzer reaction plate.</li>
		<li>Add 2 &#181;L (2 &#181;g) of fluorescence-labeled cholesterol aggregate solution into each well and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Prepare the wells with drugs.
	<ol>
		<li>Load 191 &#181;L of PBS to each well.</li>
		<li>Add 2.5% (v/v) patient serum (only one serum sample per well) and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
		<li>Add 2 &#181;L ezetimibe, lovastatin, simvastatin or niacin solution (4 &#181;g) to all wells except the negative control wells. Add only one drug per well and shake the plate for 30 s by placing it on a chemistry analyzer-1 reaction plate.</li>
		<li>Add 2 &#181;L of fluorescence-labeled cholesterol aggregate solution (2 &#181;g) into each well and shake the plate for 30 s by placing it on chemistry analyzer-1 reaction plate.</li>
	</ol>
	</li>
	<li>Load drugs only after all wells (control and drug-treated) have been loaded with their respective serum samples, and add fluorescence-labeled cholesterol at the end of this step.</li>
	<li>Incubate the plate for 2 h in a lab shaker set at 37 &#176;C and 200 rpm.</li>
	<li>Acquire the samples by imaging flow cytometer following settings described in steps 3.1 - 3.6.</li>
	<li>Use the image analyzing software for batch processing all the image files using the same template as described in steps 3.7.
	<ol>
		<li>Perform the data analysis by drawing gates on the plot as described in steps 4.5 and 4.6.</li>
	</ol>
	</li>
	<li>To determine the lipid-lowering drug effect/response (low, moderate, and high) for each sample, calculate the total lipoprotein particle concentration, as well as the percentage of the total particles comprising of VLDL, LDL, and HDL subpopulations.</li>
	<li>Plot the VLDL, LDL, and HDL populations on a histogram of the object&#39;s bright field image, width subtracted from length (length - width), and gate into either globular or linear regions: objects with (length - width) &#8804;2&#160;&#181;m fall into the globular region for calculating the percentage of globular and linear shaped particles.</li>
	<li>Compare the percentage of globular and linear shaped LDL and HDL cholesterol particles identified for each serum sample incubated with and without each drug. Among triplicate values obtained for each serum sample, accept the percentage of globular and linear shaped LDL and HDL particles that fall within SD &#177;2 range for determination of the drug effect.</li>
</ol>

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

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,&#160;cellular, and plasma factors22,23. 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 samples21. 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 &#181;m2, 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.
Interestingly, screening of serum samples showed variations of drug effect on altering the profiles of VLDL, LDL, and HDL cholesterol particles formation, particularly their effect on the formations of linear shaped LDL and HDL particles. These drugs induced reduction on linear shaped 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 models24,25,26,27,28.
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 pathway29,30. 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-oxidative31. In addition, a number of studies have reported the effect of lipid-lowering therapy in increasing a low to moderate level of HDL in the plasma1,5,21. 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 &#181;g of fluorescence labeled cholesterol aggregates and 5 &#181;gof 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 &#181;2; (3) we observed a wide variations of drug response among serum samples incubated with the drugs (doses 300 ng to 5 &#181;g) 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.

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 events1,2,3,4,5. 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 blood6,7. 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 synthesis8. 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 intestine9. Fenofibrate, another lipid-lowering drug, substantially reduces plasma concentrations of triglycerides and also moderately decreases LDL cholesterol through the peroxisome proliferator-activated receptors pathway10. Besides, omega-3 fatty acid is reported to have the anti-atherosclerotic effect due to its ability to lower the plasma levels of LDL11.
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 aggregations12,13,14. 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 compositions15,16,17. Analytical methods such as ultra-centrifugation, NMR, and gradient gel electrophoresis are used to characterize LDL and HDL particles and their subfractions18,19. However, application of these methods is limited to studies aimed at determining the effect of drugs in modulating morphology and assembly of the lipid particles. The flow cytometer based plaque array is a functional biochemical assay developed for detection and visualization of serum derived lipid and amyloid plaque particles20. The advantages of in vitro imaging method described in this study enable identification of lipid-modulating drug effects in altering morphology and distribution of cholesterol particles in buffer and serum samples.

<table>
	<tbody>
		<tr>
			<td>TopFluor fluorescent cholesterol</td>
			<td>Avanti Polar lipids</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>simvastatin (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>lovastatin (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>rosuvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>atorvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>fluvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>ezetimibe (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Niacin</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>fibrate</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>omega-3 fatty acid</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>purified VLDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>purified LDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>purified HDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human age-matched serum</td>
			<td>Dx Biosamples</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human atherosclerosis serum</td>
			<td>Bioserve</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human normal serum</td>
			<td>Stanford Blood center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LDL measurement reagent pack</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDL measurement reagent pack</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Total cholesterol measurment</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtitre plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triglycerides measrument</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amnis Imaging Flow cytometer</td>
			<td>Amnis Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS image analysing software</td>
			<td>Amnis Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemistry Analyzer-1, ChemWel 2902</td>
			<td>Awarness Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemistry Analyzer-2, Intergra 400</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles at JoVE.com

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

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

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features 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 ...

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14.5K Views.  Plaxgen Inc. The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

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

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.