Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding

Abstract

Aims/hypothesis. Vascular disease in type 2 diabetes is associated with an up-regulation of atherogenic growth factors, which stimulate matrix synthesis including proteoglycans. We have examined the direct actions of fenofibrate on human vascular smooth muscle cells (VSMCs) and have specifically investigated proteoglycan synthesis and binding to LDL. Methods. Proteoglycans synthesised by human VSMCs treated with fenofibrate (30 µmol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [ 35 S]-sulphate and quantitated by cetylpyridinium chloride. They were then assessed for electrophoretic mobility by SDS-PAGE, for size by gel filtration, for sulphation pattern by fluorophore-assisted carbohydrate electrophoresis, and for glycosaminoglycan (GAG) composition by enzyme digestion. Results. Proteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8±12.4 µg/ml to 77.7±17 µg/ml under basal conditions, from 24.9±4.6 µg/ml to 39.1±6.1 µg/ml in the presence of TGF-β1, and from 9.5±4.4 µg/ml to 31.1±3.4 µg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-β1 inhibited the incorporation of [ 35 S]-sulphate into secreted and cell-associated proteoglycans synthesised by human VSMCs by 59.2% (p<0.01) and 39.8% (p<0.01) respectively. The changes in sulphate incorporation following treatment with fenofibrate were associated with a concentration-related increase in the electrophoretic mobility due to a reduction in GAG length. There was no change in the sulphation pattern; however, there was an alteration in the disaccharide composition of the GAGs. Conclusions/interpretation. Fenofibrate modifies the structure of vascular proteoglycans by reducing the length of the GAG chains and GAG composition, resulting in reduced binding to human LDL, a mechanism which may lead to a reduction of atherosclerosis and cardiovascular disease in people with diabetes treated with fenofibrate.

Smad and p38 MAP Kinase-mediated Signaling of Proteoglycan Synthesis in Vascular Smooth Muscle

“…Increasing concentrations of LDL (0 -0.5 mg/ml) purified from human blood (22) were incubated with equal counts (1250 cpm) of proteoglycans for 1 h at 37°C. Samples were run on flat-bed agarose gels (0.7%) as described previously (23).…”

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

“…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.…”

“…Bound and free proteoglycans were separated on flat bed agarose gels as described previously. [40] Dried agarose gels were exposed to phosphorscreens and calculation of bound proteoglycans was performed using MacBas software (v1, Fuji Photo Film Co., Japan).…”

Aortic Smooth Muscle Cells from ApoE(-/-) Mice Secrete Biglycan with Hyperelongated Glycosaminoglycan Chains

“…Furthermore, isolated (chemically cleaved) GAG chains from biglycan from growth factor treated cells as well as the small free GAG chains that arise in cells supplemented with exogenous β-D-xyloside also show enhanced binding to LDL compared to the respective entities from untreated cells [26]. Treatment of cells with many mechanistic antagonists and cardiovascular drugs blocks this response [27][28][29] and furthermore, treatment of ApoE -/-mice with antagonists such as imatinib blocks PDGF-stimulated GAG hyperelongation in vitro and blocks lipid deposition in the vessel wall of mice ex vivo and in vivo [30,31]. Other drugs such as the angiotensin receptor blocker, telmisartan, reduce biglycan expression and lipid deposition in ApoE -/-mice [32].…”