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Variation in Coronary Atherosclerosis Severity Related to a Distinct LDL (Low-Density Lipoprotein) Profile

Supplemental Digital Content is available in the text.


Follow-up time on atherogenic diet
After baseline imaging (T1), the invasive imaging was repeated at 9 (T2) and 10-13 months (T3) after the start of the atherogenic diet. The timing of T3 was determined based on the size of the coronary plaques at T2. Because of the exponential growth pattern of the plaques and to prevent death due to a major cardiovascular event, pigs that displayed significant disease at T2 (n=2) were sacrificed within 1 month (T3 at 10 months). The other animals (n=8) were planned to be kept in experiment up to 12 months. Of these pigs, 1 pig died one day after the invasive imaging procedure at T2 due to an unknown, but acute cause of death during feeding. Another pig had to be sacrificed between T2 and T3 due to suspected thrombosis in one leg.
To compare the plaque types found in the proximal versus the distal regions of the coronary arteries, the first half of the excised artery (up to 14 (LAD), 9 (LCX) and 16 (RCA) 3mm-blocks) were regarded as proximal and the other half distal. These numbers were determined based on a median split of the total number of blocks of the individual arteries. Furthermore, a 3mm region was marked as 'around a side branch' when a side branch was present in at least one of the slices derived from that respective block.
Positive staining for lipids and macrophages was quantified by using user-specified standard Hue-Saturation-Brightness values. Necrotic core was delineated manually as a lipidrich area, scarce in nuclei and fibrous tissue with an overlaying fibrous cap 1 . Quantification of the average plaque area was based on all Oil-red-O, CD68 and Miller-stained sections and lipid, macrophage and necrotic core content were presented as an average percentage of the total plaque area. The presence of intraplaque haemorrhage was assessed on both HE and MSB stained sections as bright pink (HE) and red (MSB) stained areas. Neovascularisation in the plaque area could be detected using CD31 staining. The presence of calcifications was assessed on the Oil-red-O staining and calcifications were subdivided in micro (spotty appearance, <10µm/<5% of plaque area 2 ) or macrocalcifications.

Imaging analysis
Before analysis, IVUS pullbacks were ECG-triggered by selecting the frame that was recorded 6 frames before the R-peak by in-house developed software. Hereby differences in lumen size were removed that were induced by movement of the catheter or because of cardiac contraction. The triggered IVUS pullbacks were analysed each frame by semi-automatic delineation (with manual correction) of the vessel wall and lumen contours. Based on these contours, total vessel area (VA), lumen area (LA) and plaque area (PA=VA-LA) (mm 2 ) were quantified. Plaque burden (PB) was calculated as PA/VA*100%. For final analysis, data were averaged over 3mm in longitudinal direction to reduce the influence of manual drawing errors and to reduce statistical dependence amongst the data points. The plaque size was also assessed by classifying the maximal intima-media thickness (IMT) per 3mm-segment into 4 grades (<0.5mm, 0.5-0.7mm, 0.7-1.0mm and >1.0mm) according to the method of Chatzizisis et al. 3 . The percentage of the segments occupied by the respective grade was quantified per artery and averaged over all arteries.
For the OCT analysis, frames with a poor flush were excluded. Lumen contours were automatically segmented and plaque type and component angles were indicated manually. Fibrous plaques were defined as homogeneous, signal-rich intimal thickening with an IMT>0.5mm. Lipid-rich plaques were defined as displaying an inhomogeneous, fading signal combined with an absent 3-layered structure. Lipid-pools were identified when a signal-low region with a diffuse border was present with an overlying signal-rich layer: the fibrous cap. Minimal, maximal and average fibrous cap thickness were determined automatically by the QCU-CMS software. Plaque classification was based on the most severe classification in that respective frame: fibrous, lipid-rich or fibrous-cap atheroma (FCA). The latter classification was given when a lipid-pool was present in the plaque.

Plasma analysis pigs
At the start of every imaging procedure, blood samples were drawn from the carotid sheath into EDTA and clotting tubes. Blood tubes were spun at 1460 g for 10 minutes (Thermo scientific Heraeus centrifuge 3 S-R) to isolate the plasma and serum, which was stored at -80°C. Standard plasma analysis was performed on fresh plasma by the internal clinical chemistry department to determine leucocyte count and levels of total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL).

Patient plasma collection
Blood was collected in EDTA tubes from three homozygous FH (hoFH) patients who were under treatment at the Erasmus MC, The Netherlands. EDTA-plasma was isolated from blood by centrifugation (3000 rpm, 10 min, 4 °C) and stored at -80 °C until analysis. The diagnosis of hoFH was based on an LDL plasma level >13 mmol/L 4 and a genetic analysis.
Human pool plasma, used as a reference, was a mixture of plasma of approximately 100 non-FH patients that was residual material remaining after diagnostic analyses from the department of clinical chemistry. No informed consent was needed for the use of this residual material.

Lipoprotein profiling
Density-gradient ultracentrifugation A detailed lipoprotein profile was obtained using 1 mL of EDTA plasma or serum by densitygradient ultracentrifugation (DGUC) as previously reported by Versmissen et al. [5][6][7][8] . In short, potassium bromide (KBr) (0.35 g/mL plasma) was added to plasma to obtain a density of 1.26 g/mL. 1 mL of this mix was placed in an ultracentrifuge tube (331372, Beckmann Coulter) and 1.9 mL of 1.21, 1.10, 1.063, 1.04, and 1.02 g/mL KBr in physiological salt was layered on top, followed by 1 mL of water. Samples were centrifuged at 207,000 g for 18 h at 4 °C using a SW41 rotor in an Optima XPN-80 Beckman ultracentrifuge (Beckman Instruments, Indianapolis, IN, USA). Thereafter, DGUC-fractions were collected starting from the bottom of the tube.

Size exclusion chromatography (fast protein liquid chromatography)
A fast protein liquid chromatography (FPLC) profile was obtained from 200 µl plasma. Plasma was added to a Tricorn Superose200 10-300 GL column and a Tricorn Superdex 6 10-300 GL column with a flow rate of 0.5 ml/min. FPLC-fractions of 0.5 mL were collected.

Cholesterol and triglyceride content
Cholesterol and triglyceride content was determined in the fractions obtained by DGUC and FPLC using standard laboratory methods as previously described [7][8][9] .
An autosampler (Shimadzu, Kyoto, Japan) injected 5 µL lipid extracts into a Shimadzu HPLC system (Shimadzu) equipped with a Kinetex C8 column (50 x 2.1 mm, 2.6 µm, Phenomenex, Maarssen, the Netherlands) at 30 °C using a gradient, starting from 95% mobile phase A (H2O/MeOH (50/50, v/v) containing 1.5 mM ammonium formate and 0.1% formic acid) for 2 minutes and increased to 93% mobile phase B (100% MeOH containing 1 mM ammonium formate and 0.1% formic acid) at 5.5 minutes. After 10 minutes, the column was flushed with 99% mobile phase B for 2 minutes before a 2-minute re-equilibration. The flow rate was set at 0.25 ml/min and total run time was 14 minutes. The effluent was directed to a Sciex Qtrap 5500 quadruple mass spectrometer (AB Sciex Inc., Thornhill, Ontario, Canada) and analyzed in positive ion mode following electrospray ionization using multiple reaction monitoring (MRM). Detailed HPLC MS/MS settings are given in Supplemental table XII. Nine-point calibration curves were constructed by plotting the analyte to the internal standard peak area ratios versus the corresponding analyte concentration for Cer   Tables   Supplemental table I: General pig characteristics   Advanced-diseased pigs  Mildly-diseased pigs  p-value   T1  T2  T3  T1  T2  T3  AD