Supplementary methods
Western blot analysis. Extracellular matrix extracts were obtained and western blotted as
previously described (4,6) .
Native Low-Density-Lipoprotein (LDL) preparation, oxidation and glycation. LDL isolation,
oxidation and LDL glycation were performed as previously described (16). Pooled human LDL
was obtained from healthy, normolipidaemic volunteers. The purity of LDL particles was tested
by agarose gel electrophoresis, which showed a single band, i.e. lack of contamination from
other lipoprotein subclasses. To obtain LDL oxidation, native LDL (1 mg protein/ml) was
incubated with CuSO4 (20 µmol/l final concentration) in 1 mol/l PBS pH 7.4 for 17 h at 37°C.
Oxidation was stopped by adding EDTA (200 µmol/l final concentration). The quality of
oxidation was tested by the increase in relative mobility on agarose gel, indicating an enhanced
negative charge of oxidised LDL. Non-enzymatic glycation of LDL was induced by incubating
native LDL with 0.3 mmol/l EDTA at 37°C for 6 weeks in the dark, in the presence of 25 mmol/l
glucose, preventing oxidation by argon gas, and removing unbound sugars by repeated and
extensive dialysis. Control LDL incubated in the absence of glucose showed no change in
mobility. Before use, LDL solutions were filtered twice to remove Cu2+ and/or EDTA. LDL
particle concentration was adjusted to a concentration of 50 µg protein/ml by measuring total
proteins with the bicinchoninic acid method (Sigma, St Louis, MO, USA).
Cell culture
Coronary Artery Smooth Muscle Cells (CASMC, Lonza) were cultured as described (4). Cells
were treated for 24 hours respectively with glucose (20 mM, HG), mannitol (20 mM, Man),
insulin (10-7M, INS), 100 g/ml native LDL, 100 g/ml oxidized LDL (oxLDL), 100 g/ml
Glycated-LDL (glyLDL) (11), 10 M 22-R-hydroxycholesterol (RH, Sigma-Aldrich), 10 M
22-S-hydroxycholesterol (SH, Sigma-Aldrich), 5 M T0901317 (T09, Sigma-Aldrich), 3 M
GW3965 (GW, Sigma-Aldrich), and 50 M Sirtinol (Calbiochem). Proteins and total RNA were
extracted and TIMP3 levels and ADAM17 activity were assayed. Knockdown of SirT1 in
CASMC was performed trasfecting 1 g of SIRT1 siRNA (sc-40986, Santa Cruz Biotechnology)
and 0.8 g of control siRNA-A (sc-40986, Santa Cruz Biotechnology) by Dharmafect1
(Dharmacon) according to manufacturer’s instructions.
Human THP-1 monocytic cells were cultured in RPMI-1640 medium as described (26). HUVEC
cells culture was previously described (27). For high glucose culture, cells were treated with 20
mM glucose (HG) for 24 hours.
Supplementary references
26)
Narkunaraja Shanmugam, Irene T. Gaw Gonzalo, and Rama Natarajan Molecular
Mechanisms of High Glucose-Induced Cyclooxygenase-2 Expression in Monocytes Diabetes
53: 795-802
27)
Federici M, Pandolfi A, De Filippis EA, Pellegrini G, Menghini R, Lauro D, Cardellini
M, Romano M, Sesti G, Lauro R, Consoli A. G972R IRS-1 variant impairs insulin regulation
of endothelial nitric oxide synthase in cultured human endothelial cells. Circulation. 109:399405, 2004