Julie Nigro

Regulation of glycosaminoglycan structure and atherogenesis

Cardiovascular disease is the major cause of premature death in modern society, and its impact is increasing due to rising rates of obesity and type 2diabetes. Clinical studies based on targeting metabolic abnormalities and biomarkers demonstrate significant benefits, but always an element of disease remains which is resistant to treatment. Recent evidence has strongly implicated an early interaction of atherogenic lipoproteins with vascular matrix proteoglycans as the initiating step in atherogenesis. Expert commentary has pointed to the need for vascular directed therapies to provide reductions in the residual disease component. We propose that the regulation of synthesis and thus structure of glycosaminoglycans on proteoglycans provides a potential pathway to this reduction. We review existing evidence that the vascular synthesis of glycosaminoglycan chains can be regulated in a manner which reduces lipoprotein binding and the potential application of this strategy to attenuation of the current cardiovascular disease pandemic.

Differential effects of gemfibrozil on migration, proliferation and proteoglycan production in human vascular smooth muscle cells.

The aim of this study was to determine, if gemfibrozil has anti-atherogenic actions on human vascular smooth muscle cells (SMCs) and whether these actions are affected by high glucose concentrations, which mimic the hyperglycemia of diabetes. Proliferation of SMCs treated with gemfibrozil was estimated by cell counting (Coulter Counter) and [3H]thymidine incorporation, migration in a scrape-wound assay, proteoglycan (PG) biosynthesis and glycosaminoglycan (GAG) synthesis on xyloside by [35S]sulfate labeling and sizing by sodium dodecyl sulphide-polyacrylamide gel electrophoresis (SDS-PAGE). Gemfibrozil (100 micromol/l) did not affect migration in low or high glucose media. Gemfibrozil caused concentration-dependent inhibition of proliferation in low glucose media (24% inhibition at 100 micromol/l, P<0.01) and inhibited the re-initiation of DNA synthesis by 33.3% (100 micromol/l, P<0.05) in low glucose and 31.4% (100 micromol/l, P<0.001) in high glucose conditions. In low and high glucose media, gemfibrozil (100 micromol/l) reduced total PG production in the presence of TGF-beta 1, which was associated with a decrease in the apparent size of PGs. Gemfibrozil and another PPAR-alpha ligand, WY-14643, significantly inhibited basal and TGF-beta1 stimulated GAG synthesis. We conclude that some SMCs properties associated with atherogenesis are favorably affected by gemfibrozil. Hence, direct vascular actions of gemfibrozil observed in this study may contribute to the reduction in cardiovascular disease observed in clinical studies with gemfibrozil.

Fenofibrate modifies human vascular smooth muscle proteoglycans and reduces lipoprotein binding

Aims/hypothesisVascular 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.MethodsProteoglycans synthesised by human VSMCs treated with fenofibrate (30xa0µmol/l) were assessed for binding to human LDL using a gel mobility shift assay, metabolically labelled with [35S]-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.ResultsProteoglycans synthesised in the presence of fenofibrate showed an increase in the half-maximum saturation concentration of LDL from 36.8±12.4xa0µg/ml to 77.7±17xa0µg/ml under basal conditions, from 24.9±4.6xa0µg/ml to 39.1±6.1xa0µg/ml in the presence of TGF-β1, and from 9.5±4.4xa0µg/ml to 31.1±3.4xa0µg/ml in the presence of platelet-derived growth factor/insulin. Fenofibrate treatment in the presence of TGF-β1 inhibited the incorporation of [35S]-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/interpretationFenofibrate 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.

Anti-proliferative activity of oral anti-hyperglycemic agents on human vascular smooth muscle cells: thiazolidinediones (glitazones) have enhanced activity under high glucose conditions

BackgroundInhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [3H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [3H]-thymidine incorporation.MethodsVSMC proliferation was investigated by [3H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [3H]-thymidine uptake. Cell cycle events were analyzed by FACS.ResultsVSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [3H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [3H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [3H]-thymidine incorporation or cell numbers.ConclusionTZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.

High glucose potentiates mitogenic responses of cultured ovine coronary smooth muscle cells to platelet derived growth factor and transforming growth factor-β1

Macrovascular complications in diabetes are associated with exaggerated growth responses of vascular smooth muscle cells. We studied the effect of high glucose media on the growth responses of vascular smooth muscle cells from the left anterior descending (LAD) coronary artery of young sheep. Experiments were conducted in DMEM containing 5.5 or 25 mmol/l glucose and mitogenic responses assessed by 3H-thymidine incorporation. In the absence of growth factors there was a slight and variable response to high glucose but the maximum response to platelet derived growth factor-bb (PDGF-bb) (100 ng/ml) was increased more than 2-fold. Transforming growth factor-beta1 (1 ng/ml) caused a 100% increase of the PDGF-bb response in both normal and high glucose media. The acute stimulatory effect of high glucose was not affected by pre-incubation of the cells for 24 h in the high glucose medium. The mitogenic response occurring in the presence of PDGF-bb and high glucose was totally inhibited by the tyrosine kinase inhibitors (imatinib and genistein) and could not be mimicked by increasing diacylglycerol in low glucose media with the diacylglycerol kinase inhibitor, R59949. In conclusion, high glucose, per se, only very weakly stimulates smooth muscle cell growth but it interacts positively to potentiate the responses to the vascular derived growth factors PDGF and TGF-beta1. The effect of high glucose is transduced via receptor tyrosine kinases and may not involve diacylglycerol that is subject to diacylglycerol kinase catabolism. The data provide explanations for the accelerated vascular smooth muscle cell proliferation in diabetes.

Phosphorylated Troglitazone Activates PPARγ and Inhibits Vascular Smooth Muscle Cell Proliferation and Proteoglycan Synthesis

Phosphorylation of α-tocopherol produces an entity with enhanced antiatherogenic properties. Troglitazone, an α-tocopherol derivative of a 2,4-thiazolidinedione nucleus, is an antidiabetic agent that shows fatal idiosyncratic hepatotoxicity, a property not shared by later agents. We investigated the effects of phosphorylation of troglitazone (to yield “phosphoglitazone”) on the biochemical pharmacologic properties of troglitazone. We investigated its ability to act as a PPARγ agonist and to inhibit 2 atherogenic properties of vascular smooth muscle cells (vSMC)-proliferation and proteoglycan synthesis. PPARγ activity was assessed in a transfection assay. Proliferation was assessed by [3H]-thymidine incorporation and cell counting and proteoglycan synthesis by [35S]-sulfate incorporation using human vSMCs stimulated with platelet-derived growth factor (PDGF; 50 ng/mL) and transforming growth factor (TGF)-β (2 ng/mL). Phosphoglitazone was a full agonist for PPARγ with a potency and efficacy similar to troglitazone. Phosphoglitazone also inhibited cell proliferation and proteoglycan synthesis with potency similar to troglitazone. We conclude that phosphorylation retains the pharmacologic activity of troglitazone while decreasing its lipophilicity and therefore potentially its toxicity. A phosphorylated derivative of a 2,4-thiazolidinedione warrants further investigation as a potential new therapeutic agent for the treatment of insulin resistance and Type 2 diabetes.

Anti-Atherogenic Role of Peroxisome Proliferator-Activated Receptor Ligands

Two families of peroxisome proliferating-activated receptor (PPAR) ligands are utilized clinically to address abnormalities of blood-borne cardiovascular risk factors. PPAR-α ligands, the fibrates, lower plasma triglycerides and increase high-density lipoprotein (HDL)-cholesterol leading to positive outcomes in clinical trials such as the Veterans Affairs HDL Intervention Trial. PPAR-γ ligands, the recently introduced family of thiazolidinediones, act as insulin sensitizers to alleviate the hyperglycemia of insulin resistant states such as Type 2 diabetes. The primary aim of treating the cardiovascular risk factors is to reduce the burden of cardiovascular disease, mostly atherosclerotic vascular disease. Considerable evidence is emerging that PPAR ligands can exert anti-atherogenic activity. Although the agents acting on their target nuclear receptors are primarily regulators of gene transcription it may be that some actions are independent of gene transcription and represent direct inhibition of atherogenic signalling pathways in the vasculature. The direct actions including inhibition of vascular smooth muscle cell proliferation and inhibition of glycosaminoglycan elongation on proteoglycans, the latter leading to reduced low-density lipoprotein (LDL) binding, provide in vitro examples of antiatherogenic actions. PPAR ligands reduce atherosclerosis in animal models such as the ApoE-null mouse and PPAR-γ attenuate in-stent restenosis in patients with diabetes. The actions of these agents have lead to suggestions that they may be useful in vascular therapy even in the absence of the underlying risk factor abnormality. However, they may also represent tools and provide insights into areas where agents directly targeting atherosclerotic mechanisms may be developed to provide combined therapy with agents targeting traditional risk factors.

Androgens: New Effects on Proteoglycan Biosynthesis and Its Consequences for Atherosclerosis

The pro- and antiatherogenic role of sex hormones is very controversial. These hormones have metabolic actions, but may also display actions directly on cells of the vasculature involved in atherosclerosis. We recently examined the effect of the male sex hormones, the androgens, on proteoglycan biosynthesis and LDL binding and found that androgens have a growth factor-like effect to increase the size of proteoglycans and enhance LDL binding. These studies demonstrate, at least in this in vitro model, that androgens can be proatherogenic.