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Sobenin et al. Desialylated LDL in diabetes

(desialylated) LDL] using affinity chromatography on the whole set of experiments was performed in the
Ricinus communis agglutinin (RCA )-agarose (Sigma primary culture obtained from one autopsy sample.
120
Chemical Co., St. Louis, MO) as described elsewhere. The results were reproduced twice in primary cultures
[21]
Briefly, approximately 2 mg of LDL (by protein) was obtained from two other autopsy samples and, hence,
applied on the column containing 2 mL of the affinity gel considered reliable. The autopsy material was taken
equilibrated with PBS. Non-bound (sialylated) LDL was from men aged 52-62 years who had died suddenly in
washed from the gel with 30 volumes of PBS containing an accident. The cells were suspended in Medium 199
0.5 mol/L NaCl, and bound (desialylated) LDL was containing standard additives: 2 mmol/L L-glutamine,
eluted with 5 volumes of PBS containing 50 mmol/L 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL
galactose (Sigma Chemical Co., St. Louis, MO). LDL Fungizone, and 10% fetal calf serum (FCS). Cells were
fractions were adjusted with solid NaBr to a density of seeded onto 24-well tissue culture plates at a density
1.065 g/mL and concentrated by ultracentrifugation. For of 3 × 10 cells/cm of growth area, and cultured at
4
2
cell culture experiments, aliquots of LDL preparations 37 ºC in a humidified CO -incubator (95% air and 5%
2
were dialyzed against PBS and sterilized by filtration CO ). The medium was changed every other day. On
2
(pore size, 0.45 μm). The larger portions of LDL fractions the 7th day in culture, the total, non-bound, and bound
were subdivided in a density gradient as described LDL fractions (50 μg/mL) were added in quadruplicate
elsewhere. [22,23] Briefly, total, non-bound, and bound in Medium 199 containing 10% human lipoprotein-
LDL were dialyzed against 2000 volumes of NaCl/ deficient serum (LDS). Control cells were incubated in
NaBr solution with a density of 1.050 g/mL overnight Medium 199 containing 10% LDS without LDL addition.
at 4 ºC. The gradients were formed into 14 × 95-mm After 24 h of incubation, cells were rinsed thoroughly
tubes by successively layering 2.6 mL of NaCl/KBr with PBS, and cellular lipids were extracted thrice with
solutions having densities of 1.1289, 1.0637, 1.0500 n-hexane/isopropanol (3:2, v/v) according to Hara and
(containing the LDL sample), 1.0398, and 1.0282 g/mL. Radin’s protocol. [24] Total cholesterol in the extracts
The gradients were centrifuged for 42 h in a Beckman was determined enzymatically. [25] Cellular protein was
L-80 ultracentrifuge (Beckman Instruments Inc., Palo measured according to Lowry et al. [26]
Alto, CA) using Type SW40Ti swinging bucket rotor
operated at 36,000 g at 10 ºC. After centrifugation, the Electrophoresis
gradients were fractionated by upward displacement The aliquots of LDL samples (20 μg LDL by protein)
with KBr solution (density, 1.300 g/mL) at a speed of were run on agarose gel films (Ciba Corning Diagnostics
0.5 mL/min (Isco Density Gradient Fractionator, Model GmbH, Giessen, Germany) according to Lipid
640, Isco, Lincoln, NE). The absorbance of the effluent Research Clinics Manual of Laboratory Operations. [27]
was monitored with an Isco UA-5 absorbance detector The same LDL sample from healthy donors was used
(Isco, Lincoln, NE) at 280 nm, and 0.4 mL fractions were as the reference sample throughout all experiments,
collected. Equal salt gradients without LDL were run and and its mobility was assumed as 1.00. Nondenaturing
fractionated simultaneously, and the densities of these polyacrylamide gradient gel electrophoresis was
fractions were measured to reconstruct the density run on continuous 2-16% gradient gels as described
profile of the gradient (DMA 45 Calculating Digital elsewhere. [28] After gel staining with Coomassie Brilliant
Density Meter, Anton Paar, Graz, Austria). Blue R-250, LDL-migration distance was measured
by scanning densitometry at 560 nm. LDL particle
Cell culture studies size was calculated according to the calibration curve
Subendothelial cells were isolated from uninvolved reconstructed from migration distances of standards
human aortic intima by dispersion with 0.15% with known diameters using Exponential Curve Fit
collagenase. Subendothelial cells represented a utility in the Sigmaplot 12.0 program package.
[7]
mix of typical smooth muscle cells (about 30-40%),
atypical (pericyte-like or stellate) smooth-muscle Other analyses
cells (about 40-45%), resident macrophages (3-8%), LDL sialic acid content was measured according
and very small number of cells that migrated from to Warren’s modified method. [29] LDL protein was
circulation (monocytes and lymphocytes). Because measured according to Lowry et al. [26]
atherosclerosis develops only in the subendothelial
intima, this type of primary culture is adequate for Statistical methods
reproducing in vitro the major traits of atherogenesis Results are reported as mean and standard deviation
at the cellular level (e.g. lipid deposition, proliferation, (SD). Significance of differences was evaluated using
fibrosis, and inflammation), although this model has ANOVA and Mann-Whitney tests (IBM SPSS 20.0
certain limitations with the respect to inter-experiment statistical program package), and P values < 0.05 were
standardization. These limitations were avoided when considered statistically significant.
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