Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo

Abstract

The 'response to retention' hypothesis of atherogenesis proposes that proteoglycans bind and retain low-density lipoproteins (LDL) in the vessel wall. Platelet-derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF-mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated (35)S-SO(4) incorporation into proteoglycans by 31% (P < 0.01) and inhibited PDGF-mediated size increases in both chemically cleaved and xyloside associated glycosaminoglycan (GAG) chains by 19%, P < 0.05 and 27%, P < 0.05, respectively. Imatinib decreased PDGF stimulation of the 6:4 position sulphation ratio of disaccharides. The half maximal saturation value for LDL binding for proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5-fold higher than for PDGF treatment alone. In high fat fed ApoE(-/-) mice, imatinib reduced total lipid staining area by approximately 31% (P < 0.05). Carotid artery lipid accumulation in imatinib treated mice was also reduced. Furthermore, we demonstrate that imatinib inhibits phosphorylation of tyrosine 857, the autophosphorylation site of the PDGF receptor, in vSMCs. Thus imatinib inhibits GAG synthesis on vascular proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease.

Fig 1

Imatinib dose dependently decreases radiolabel incorporation into proteoglycans and increases electrophoretic mobility in the presence of PDGF. Human vSMC were treated with imatinib (A, B 0.01–10 μM, C, D 1 μM) and PDGF (50 ng/ml) in the presence of (A) Sulfur-35 Na₂SO₄ (1.85 MBq/ml) or (C) D-glucosamine-HCl, [6–3H] (0.37 MBq/ml) or (D) Tran³⁵S-label (1.85 MBq/ml). Media containing secreted proteoglycans was blotted onto chromatography paper and precipitated via cetylpyridinium chloride, followed by measurement of radiolabel incorporation. Results are the mean ± S.E.M. of normalized data from three experiments in triplicate, **P < 0.01 using a one-way ANOVA. For the dose–response curve shown in (A) IC₅₀ value 0.5 μM r² 0.95. (B) Secreted proteoglycans were isolated using ion exchange chromatography (DEAE Sephacel) and concentrated by ethanol precipitation. Proteoglycans were separated on a 4−13% acrylamide gradient gel. Gel was performed twice from two separate experiments, with a representative shown.

Fig 2

Imatinib treatment of human vSMCs decreases the size of chemically cleaved and xyloside initiated GAG chains. (A) Human vSMC were treated with imatinib (1 μmol/l) and PDGF (50 ng/ml) for 12 hrs then Sulfur-35 Na₂SO₄ (1.85 MBq/ml) was added for a further 16 hrs. Secreted proteoglycans were isolated and concentrated. GAG chains were cleaved from proteoglycan core proteins via a β-elimination reaction and were separated by SDS-PAGE on 4–20% acrylamide gradient gels. (C) Human vSMC were treated with imatinib (1 μmol/l) and xyloside (0.5 mmol/l) under basal conditions and in the presence of PDGF (50 ng/ml) for 4 hrs prior to the addition of Sulfur-35 Na₂SO₄ (50 μCi/ml) for a further 24 hrs. Secreted proteoglycans and xyloside initiated GAG chains were isolated and concentrated and separated by SDS-PAGE on 4–13% acrylamide gradient gels. In each case gels were performed three times from three separate experiments, with a representative shown. Size analysis of the B cleaved GAG chains and D xyloside associated GAG chains were performed by size exclusion chromatography (sepharose CL-6B). Vertical lines indicate the calculated K_av of GAG chains of proteoglycans from control cells. Three measurements were performed from three separate experiments in each case with a representative shown, **P < 0.01 using a paired Student’s t-test.

Fig 3

Imatinib treatment of human vSMC decreases the GAG chain 6:4 position sulphation ratio and results in decreased binding to LDL. (A) Human vSMC were treated with imatinib (1 μmol/l) under basal conditions and in the presence of PDGF (50 ng/ml) for 24 hrs. Secreted proteoglycans were isolated, concentrated, dialysed into sterile water and vacuum concentrated. Proteoglycan core proteins were digested (Proteinase K), GAG chains precipitated (ethanol) and digested with chondroitinase (ABC). Disaccharides were then fluorotagged with AMAC and separated (20% acrylamide gel). Images were captured using a FACE imager and the 6:4 position sulphation ratios of monosulphated disaccharides calculated. Normalized data from three separate experiments is shown as mean ± S.E.M., **P < 0.01 versus control, ##P < 0.01 versus PDGF using a one-way ANOVA. Human vSMC were treated with imatinib (1 μmol/l) under B basal conditions and C in the presence of PDGF (50 ng/ml) in the presence of ³⁵S-met/cys (1.85 MBq/ml). Core protein radiolabelled proteoglycans were isolated and equal counts combined with increasing concentrations of LDL. Flat bed agarose gels were utilized for separation and calculation of bound and free proteoglycans was performed with image analysis software. Three separate experiments were performed, with a representative gel shown and results shown as mean ± S.E.M., ***P < 0.001 using a two-way interaction ANOVA comparing the two data sets. Half maximal saturation binding values: Control 0.031 mg/ml, Imatinib 0.193 mg/ml, PDGF 0.028 mg/ml, PDGF + Imatinib 0.123 mg/ml.

Fig 4

Imatinib decreases lipid area in ApoE^−/– mice and blocks PDGFR autophosphorylation. (A) Eight-week-old ApoE^−/– mice (n= 15 per group) were fed a high fat diet and treated for 12 weeks with imatinib (at 10, 20 or 40 mg/kg body weight, respectively) by daily gavage. The proportion of aortic surface area stained with Sudan-IV for lipid was analysed en face by image analysis. Results are shown as mean ± S.E.M., *P < 0.05 using a multi-factor one-way ANOVA. (B) Representative examples of aortic arch of ApoE^−/– mice fed a high fat diet and treated for 12 weeks with imatinib (at 10, 20 or 40 mg/kg body weight, respectively) by daily gavage. Aortic surface was stained with Sudan-IV to identify lipid-rich plaques. (C) Effect of imatinib on tyrosine phosphorylation on the autophosphorylation site (Tyr857) of the PDGF receptor. Western blot of human vSMC from imatinib-treated cells (Imat lanes 3 and 5) in the absence (lane 3) or presence of PDGF (50 ng/ml) for 20 min. (lane 5). Samples were lysed and subjected to 10% SDS-PAGE. Phospho-PDGFR-β was visualized using anti-phosphotyrosine 857 antibody and anti-goat-HRP conjugated antibody followed by ECL detection. Membranes were reprobed with anti-smooth muscle α-actin and antimouse-HRP conjugated antibody.