Stephanie T. de Dios

Inhibitory Activity of Clinical Thiazolidinedione Peroxisome Proliferator Activating Receptor-γ Ligands Toward Internal Mammary Artery, Radial Artery, and Saphenous Vein Smooth Muscle Cell Proliferation

Background—The proliferation of vascular smooth muscle cells (VSMCs) is a known response to arterial injury that is an important part of the process of restenosis and atherosclerosis. People with diabetes have an increased risk of cardiovascular disease resulting from accelerated coronary atherosclerosis. The newest drugs for Type 2 diabetes are thiazolidinediones, which are insulin-sensitizing peroxisome proliferator activating receptor-&ggr; (PPAR&ggr;) ligands. We investigated the antiproliferative effects of troglitazone, rosiglitazone, and pioglitazone on VSMCs derived from the three vascular beds used for coronary artery by-pass grafting: the internal mammary and radial artery and saphenous veins. Methods and Results—The three vessels yielded proliferating cells of slightly differing morphology. Inhibition of cell proliferation was assessed by cell counting and cell cycle studies by Western blotting for phosphorylated retinoblastoma protein. All three thiazolidinediones showed inhibitory potency toward cell proliferation with a potency troglitazone>rosiglitazone≈pioglitazone, and this potency profile was maintained toward the growth factor and insulin-stimulated phosphorylation of the retinoblastoma protein, which controls cell cycle progression. Conclusion—The inhibitory potency of clinical thiazolidinediones toward different vascular sources is dependent on the individual thiazolidinedione and very little on the vascular source.

Troglitazone Stimulates Repair of the Endothelium and Inhibits Neointimal Formation in Denuded Rat Aorta

Objective—Vascular endothelium is emerging as a therapeutic target for atherosclerotic macrovascular disease in diabetes using oral hypoglycemic agents with pleiotropic actions. We have addressed whether the thiazolidinedione troglitazone has effects on the endothelial cell response to injury in rat aorta and its interaction with the growth response of underlying vascular smooth muscle. Methods and Results—Repair of rat aorta after balloon catheter injury in troglitazone-treated (400 mg/kg per day by mouth) rats showed early acceleration of reendothelialization and late reduction in neointima formation. Complementary in vitro studies showed that troglitazone dose-dependently inhibited migration and proliferation of cultured macrovascular endothelial and vascular smooth muscle cells in low-glucose (5 mmol/L) and high-glucose (25 mmol/L) media. However, in endothelial cells, the inhibitory response at low (<3 &mgr;mol/L) troglitazone concentrations resulted from direct inhibition of proliferation, whereas inhibition at higher (10 &mgr;mol/L) concentrations was secondary to apoptosis and necrosis. Additional studies indicated a concentration-specific activity of troglitazone to protect endothelial cells from apoptosis. Conclusions—Troglitazone had effects consistent with maintenance of vascular integrity and protection against mechanisms of atherosclerosis and restenosis, which may arise from a concentration-specific effect to reduce high rates of apoptosis occurring in cultured cells and repairing vessels.

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.

The effect of PPAR ligands to modulate glucose metabolism alters the incorporation of metabolic precursors into proteoglycans synthesized by human vascular smooth muscle cells

Abstract PPAR ligands are important effectors of energy metabolism and can modify proteoglycan synthesis by vascular smooth muscle cells (VSMCs). Describing the cell biology of these important clinical agents is important for understanding their full clinical potential, including toxicity. Troglitazone (10 μM) and fenofibrate (30 μM) treatment of VSMCs reduces (35S)-sulphate incorporation into proteoglycans due to a reduction of glycosaminoglycan (GAG) chain length. Conversely, under physiological glucose conditions (5.5 mM), the same treatment increases (3H)-glucosamine incorporation into GAGs. This apparent paradox is the consequence of an increase in the intracellular (3H)-galactosamine specific activity from 48.2 ± 3.2 μCi/ μmol to 90.7 ± 11.0 μCi/ μmol (P < 0.001) and 57.1 ± 2.6 μCi/ μmol (P < 0.05) when VSMCs were treated with troglitazone and fenofibrate, respectively. The increased specific activity observed with troglitazone (10 μM) treatment correlates with a two-fold increase in glucose consumption, while fenofibrate (50 μM) treatment showed a modest (14.6%) increase in glucose consumption. We conclude that the sole use of glucosamine precursors to assess GAG biosynthesis results in misleading conclusions when assessing the effect of PPAR ligands on VSMC proteoglycan biosynthesis.

Oxidative stress and endothelial dysfunction

Oxidative stress is a hallmark of all cardiovascular risk states (e.g. hypertension, diabetes, hypercholesterolemia, cigarette smoking) and a major underlying cause of endothelial dysfunction, vascular inflammation and blood vessel pathology. Under physiological conditions, cells of the vessel wall produce reactive oxygen species (ROS) such as superoxide (O2•–) and hydrogen peroxide (H2O2) in a deliberate and tightly regulated manner for use as second messengers in redox signalling pathways. However, in vascular pathophysiology, the production of ROS in vascular cells is elevated such that these molecules escape detoxification by cellular antioxidant pathways. When present at higher concentrations, ROS may undergo direct chemical interactions with other biomolecules. Of particular importance are the reactions between O2•– and nitric oxide (NO), which give rise to peroxynitrite (ONOO–), and the iron-catalysed Haber-Weiss reaction between O2 and H2O2, which gives rise to hydroxyl radicals (OH•).Peroxynitrite and OH• are extremely powerful oxidising species and, along with O2•– and H2O2, cause endothelial dysfunction through direct oxidative damage to cellular macromolecules, impairment of the NO signalling pathway, and activation of pro-inflammatory signalling cascades. Recent evidence suggests that the elevated ROS production in vascular pathophysiology is the result of a complex feed-forward mechanism whereby a primary source of ROS (NADPH oxidases) leads to dysfunction of endothelial nitric oxide synthase, xanthine oxidase and the mitochondrial electron transport chain, so that these enzymes become secondary sources of ROS and major contributors to vascular oxidative stress.