Thiazolidinediones reduce the LDL binding affinity of non-human primate vascular cell proteoglycans

Abstract

Aims/hypothesis. Retention of atherogenic lipoproteins in the artery wall by proteoglycans is a key step in the development of atherosclerosis. Thiazolidinediones have been shown to reduce atherosclerosis in mouse models. The aim of this study was to determine whether thiazolidinediones modify vascular proteoglycan synthesis in a way that decreases LDL binding. Methods. Primate aortic smooth muscle cells were exposed to troglitazone or rosiglitazone, or no stimulus at all for a 24-hour steady-state labelling period. Sulphate incorporation, size and LDL binding affinity of proteoglycans were determined. Proteoglycans secreted by cells in the presence or absence of troglitazone were separated into large and small classes by size exclusion chromatography, and LDL binding affinity was determined. Results. Proteoglycans synthesised by cells exposed to troglitazone or rosiglitazone were smaller, with decreased sulphate incorporation and decreased LDL binding affinity. However, troglitazone had a greater effect than rosiglitazone. Troglitazone reduced the LDL binding affinities of both the large and small proteoglycans compared with control. The binding differences persisted when glycosaminoglycan chains released from proteoglycans were incubated with LDL, indicating that troglitazone affects the glycosaminoglycan synthetic machinery of these cells. Conclusions/interpretation. Thiazolidinediones decrease the LDL binding affinity of the proteoglycans synthesised by primate aortic smooth muscle cells. This could, in part, account for the reduced atherosclerosis observed in animal models.

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans

“…Radiolabel incorporation into proteoglycans was quantified using the CPC precipitation assay [38]. The sizes of the proteoglycans and GAG chains (cleaved chemically from the proteoglycan core proteins via a β-elimination reaction [39]) were analysed by gradient SDS-PAGE [38]. GAG chain lengths also were analysed by size exclusion chromatography as described previously [40] and the data standardised by calculation of K av values.…”

Serum Amyloid A, but Not C-Reactive Protein, Stimulates Vascular Proteoglycan Synthesis in a Pro-Atherogenic Manner

“…Wight, Seattle, WA) were maintained in commercially available Dulbecco's modified Eagle's medium with 5.6 mmol/L glucose as previously described. 27,28 Cells were grown to confluence then made quiescent by reducing the serum concentration to 0.1% for 48 hours. Quiescent cells were stimulated with SAA (1 to 100 mg/L) or CRP (1 to 100 mg/L) for 0 to 72 hours.…”

“…29,32 The hydrodynamic size of glycosaminoglycans was determined by molecular sieve chromatography using Sepharose CL-6B columns. 27,28 To estimate the size of proteoglycans, equal counts (20,000 dpm) of purified 35 S0 4 -labeled proteoglycans, were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis 4 to 12% gradient gels with a 3.5% stacking gel, as previously described. 29,32 Dried gels were exposed to Fuji Imaging Plates for 48 to 72 hours and then visualized using FLA-5000 (Fujifilm, Tokyo, Japan) and analyzed using Multi Gauge software (Fujifilm).…”

“…27,28,34 The amount of proteoglycan bound by LDL in each lane is expressed as the proportion of radioactivity retained at the lane origin relative to the total radioactivity per lane.…”

Proteoglycans can mediate renal lipoprotein retention

“…Cells were metabolically labelled with 3.7 MBq/ml [ 35 S]sulphate or 1.85 MBq/ml [ 35 S]methionine, then secreted proteoglycans were collected as previously described [3]. Proteoglycans secreted by mesangial cells were incubated with native human LDL for 1 h under physiological conditions of pH, temperature and ionic strength, and subjected to gel mobility shift assays [3]. Analyses of the binding kinetics (using GraphPad Prism Software) demonstrated that mesangial proteoglycans have high binding affinity for LDL (K d =14±5 μg/ml LDL; mean±SEM, n=3; Fig.…”