Simon K. Jackson

Expression and Function of PPARγ in Rat and Human Vascular Smooth Muscle Cells

Background—Peroxisome proliferator–activated receptor-γ (PPARγ) is activated by fatty acids, eicosanoids, and insulin-sensitizing thiazolidinediones (TZDs). The TZD troglitazone (TRO) inhibits vascular smooth muscle cell (VSMC) proliferation and migration in vitro and in postinjury intimal hyperplasia. Methods and Results—Rat and human VSMCs express mRNA and nuclear receptors for PPARγ1. Three PPARγ ligands, the TZDs TRO and rosiglitazone and the prostanoid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), all inhibited VSMC proliferation and migration. PPARγ is upregulated in rat neointima at 7 days and 14 days after balloon injury and is also present in early human atheroma and precursor lesions. Conclusions—Pharmacological activation of PPARγ expressed in VSMCs inhibits their proliferation and migration, potentially limiting restenosis and atherosclerosis. These receptors are upregulated during vascular injury.

Ciprofibrate therapy improves endothelial function and reduces postprandial lipemia and oxidative stress in type 2 diabetes mellitus.

BACKGROUND Exaggerated postprandial lipemia (PPL) is a factor in atherogenesis, involving endothelial dysfunction and enhanced oxidative stress. We examined the effect of ciprofibrate therapy on these parameters in type 2 diabetes mellitus. METHODS AND RESULTS Twenty patients entered a 3-month, double-blind, placebo-controlled study. Each subject was studied fasting and after a fatty meal, at baseline, and after 3 months of treatment. Glucose and lipid profiles were measured over an 8-hour postprandial period. Endothelial function (flow-mediated endothelium-dependent vasodilatation [FMD]) and oxidative stress (electron paramagnetic resonance spectroscopy) were measured after fasting and 4 hours postprandially. At baseline, both groups exhibited similar PPL and deterioration in endothelial function. After ciprofibrate, fasting and postprandial FMD values were significantly higher (from 3.8+/-1. 8% and 1.8+/-1.3% to 4.8+/-1.1% and 3.4+/-1.1%; P<0.05). This was mirrored by a fall in fasting and postprandial triglycerides (3. 1+/-2.1 and 6.6+/-4.1 mmol/L to 1.5+/-0.8 and 2.8+/-1.3 mmol/L, P<0. 05). Fasting and postprandial HDL cholesterol was also elevated (0. 9+/-0.1 and 0.8+/-0.1 mmol/L and 1.2+/-0.2 and 1.2+/-0.1 mmol/L, P<0. 05). There were no changes in total or LDL cholesterol. Fasting and postprandial triglyceride enrichment of all lipoproteins was attenuated, with cholesterol depletion of VLDL and enrichment of HDL. There were similar postprandial increases in oxidative stress in both groups at baseline, which was significantly attenuated by ciprofibrate (0.3+/-0.6 versus 1.5+/-1.1 U, P<0.05). CONCLUSIONS This study demonstrates that fibrate therapy improves fasting and postprandial endothelial function in type 2 diabetes. Attenuation of PPL and the associated oxidative stress, with increased HDL cholesterol levels, may be important.

The relationships between post-prandial lipaemia, endothelial function and oxidative stress in healthy individuals and patients with type 2 diabetes

Post-prandial lipaemia (PPL) is a factor in atherogenesis and results in reversible endothelial dysfunction in healthy individuals. Oxidative stress and triglyceride (TG)-rich lipoproteins have been implicated. Type 2 diabetes (NIDDM) results in exaggerated PPL. We attempted to delineate the mechanisms of PPL induced, endothelial dysfunction (EF) and oxidative stress in 12 NIDDM and 12 matched healthy subjects. Subjects underwent a fat tolerance test, with endothelial function assessed by flow-mediated vasodilatation and oxidative stress measured by venous lipid-derived free radicals ex vivo and lipid peroxidation products over the postprandial phase. Fasting TG, post-prandial hypertriglyceridaemia and the TG enrichment of all lipoproteins was significantly greater in NIDDM. Post-prandial endothelial function inversely correlated with fasting HDL-C (r=-0.84, P=0.001) in both the control and NIDDM groups. The deterioration in EF in the NIDDM group also correlated with TG enrichment of VLDL and LDL. PPL in both groups also resulted in increased oxidative stress. The increment in free radicals correlated with TG enrichment of VLDL in both groups and was, therefore, greater in NIDDM. Thus, PPL -- with the production of TG-enrichment of VLDL -- results in endothelial dysfunction by an oxidative stress mechanism in both groups. The magnitude is greater in NIDDM. Fasting HDL-C appears to contribute to the protection of the endothelium against this phenomenon. Hence, exaggerated PPL associated with reduced HDL-C may be important in the pathogenesis of vascular disease, particularly in NIDDM.

Electron spin resonance spectroscopic detection of oxygen-centred radicals in human serum following exhaustive exercise

Abstract Free radicals or oxidants are continuously produced in the body as a consequence of normal energy metabolism. The concentration of free radicals, together with lipid peroxidation, increases in some tissues as a physiological response to exercise – they have also been implicated in a variety of pathologies. The biochemical measurement of free radicals has relied in the main on the indirect assay of oxidative stress by-products. This study presents the first use of electron spin resonance (ESR) spectroscopy in conjunction with the spin-trapping technique, to measure directly the production of radical species in the venous blood of healthy human volunteers pre- and post-exhaustive aerobic exercise. Evidence is also presented of increased lipid peroxidation and total antioxidant capacity post-exercise.

Comparison of cross-linked polyethylene materials for orthopaedic applications.

Cross-linked polyethylenes are being marketed by orthopaedic manufacturers to address the problem of osteolysis caused by polyethylene particulate wear debris. Wear testing of these cross-linked polyethylenes in hip simulators has shown dramatic reduction in wear rate compared with standard ultrahigh molecular weight polyethylene, either gamma irradiated in air or nitrogen - or ethylene oxide-sterilized. However, this reduction in wear rate is not without cost. The cross-linking processes can result in materials with lower mechanical properties than standard ultrahigh molecular weight polyethylene. To evaluate the effect of the various cross-linking processes on physical and mechanical properties of ultrahigh molecular weight polyethylene, commercially available cross-linked polyethylenes from six orthopaedic manufacturers were tested. This study was the culmination of collaboration with these manufacturers, who provided cross-linked polyethylene for this study, wear characteristics of the material they provided, and review of the physical and mechanical properties measure for their polyethylene. Cross-linked materials were evaluated as received and after an accelerated aging protocol. Free radical identity and concentration, oxidation, crystallinity, melt temperature, ultimate tensile strength, elongation at break, tensile stress at yield, and toughness are reported for each material. By comparing these physical and mechanical properties, surgeons can evaluate the trade-off that results from developing materials with substantially lower wear rates.

Neutrophil superoxide anion--generating capacity, endothelial function and oxidative stress in chronic heart failure:effects of short- and long-term vitamin C therapy

OBJECTIVES First, we sought to study the effects of short- and long-term vitamin C therapy on oxidative stress and endothelial dysfunction in chronic heart failure (CHF), and second, we sought to investigate the role of neutrophils as a cause of oxidative stress in CHF. BACKGROUND Oxidative stress may contribute to endothelial dysfunction in CHF. Vitamin C ameliorates endothelial dysfunction in CHF, presumably by reducing oxidative stress, but this is unproven. METHODS We studied 55 patients with CHF (ischemic and nonischemic etiologies) and 15 control subjects. Flow-mediated dilation (FMD) in the brachial artery was measured by ultrasound wall-tracking, neutrophil superoxide anion (O2-) generation by lucigenin-enhanced chemiluminescence and oxidative stress by measurement of free radicals (FRs) in venous blood using electron paramagnetic resonance (EPR) spectroscopy and plasma thiobarbituric acid reactive substances (TBARS). Measurements were performed at baseline in all subjects. The effects of short-term (intravenous) and long-term (oral) vitamin C therapy versus placebo were tested in patients with nonischemic CHF. RESULTS At baseline, FRs were higher in patients with CHF than in control subjects (p < 0.01), TBARS were greater (p < 0.005), neutrophil O2- -generating capacity was enhanced (p < 0.005) and FMD was lower (p < 0.0001). Compared with placebo, short-term vitamin C therapy reduced FR levels (p < 0.05), tended to reduce TBARS and increased FMD (p < 0.05), but did not affect neutrophil O2- -generating capacity. Long-term vitamin C therapy reduced FR levels (p < 0.05), reduced TBARS (p < 0.05) and improved FMD (p < 0.05), but also reduced neutrophil O2- -generating capacity (p < 0.05). Endothelial dysfunction was not related to oxidative stress, and improvements in FMD with vitamin C therapy did not relate to reductions in oxidative stress. CONCLUSIONS Oxidative stress is increased in ischemic and nonischemic CHF, and neutrophils may be an important cause. Vitamin C reduces oxidative stress, increases FMD and, when given long term, decreases neutrophil O2- generation, but the lack of a correlation between changes in endothelial function and oxidative stress with vitamin C implies possible additional non-antioxidant benefits of vitamin C.

Exercise, free radicals, and lipid peroxidation in type 1 diabetes mellitus.

Indirect biochemical techniques have solely been used to ascertain whether type 1 diabetes mellitus patients are more susceptible to resting and exercise-induced oxidative stress. To date there is no direct evidence to support the contention that type 1 diabetic patients have increased levels of free radical species. Thus, the aim of this study was to use electron spin resonance (ESR) spectroscopy in conjunction with alpha-phenyl-tert-butylnitrone (PBN) spin trapping to measure pre- and postexercise free radical concentration in the venous blood of young male patients with type 1 diabetes mellitus (HbA(1c) = 8.2 +/- 1%, n = 12) and healthy matched controls (HbA(1c) = 5.5 +/- 0.2%, n = 13). Supporting measures of lipid peroxidation (malondialdehyde and lipid hydroperoxides), ambient blood glucose and selected antioxidants were also measured. The diabetic patients presented with a comparatively greater concentration of free radicals as measured by ESR and lipid hydroperoxides (LH) compared to the healthy group (p <.05, pooled rest and exercise data), although there was no difference in malondialdehyde (MDA) concentration. alpha-Tocopherol was comparatively lower in the healthy group (p <.05, pooled rest and exercise data vs. diabetic group) due to a selective decrease during physical exercise (p <.05 vs. rest). The hyperfine coupling constants recorded from the ESR spectra (a(Nitrogen) = 1.37 mT and abeta(Hydrogen) = 0.17 mT) are suggestive of either oxygen or carbon-centered species and are consistent with literature values. We suggest that the greater concentration of oxidants seen in the diabetic group may be due to increased glucose autoxidation as a function of this pathology and/or a lower exercise-induced oxidation rate of the major lipid soluble antioxidant alpha-tocopherol. We suggest that the ESR-detected radicals are secondary species derived from decomposition of LH because these are the major initial reaction products of free radical attack on cell membranes.

Electron paramagnetic spectroscopic evidence of exercise-induced free radical accumulation in human skeletal muscle

The present study determined if acute exercise increased free radical formation in human skeletal muscle. Vastus lateralis biopsies were obtained in a randomized balanced order from six males at rest and following single-leg knee extensor exercise performed for 2 min at 50% of maximal work rate (WRMAX) and 3 min at 100% WRMAX. EPR spectroscopy revealed an exercise-induced increase in mitochondrial ubisemiquinone [0.167 ± 0.055 vs. rest: 0.106 ± 0.047 arbitrary units (AU)/g total protein (TP), P < 0.05] and α-phenyl-tert-butylnitrone-adducts (112 ± 41 vs. rest: 29 ± 9 AU/mg tissue mass, P < 0.05). Intramuscular lipid hydroperoxides also increased (0.320 ± 0.263 vs. rest: 0.148 ± 0.071 nmol/mg TP, P < 0.05) despite an uptake of α-tocopherol, α-carotene and β-carotene. There were no relationships between mitochondrial volume density and any biomarkers of oxidative stress. These findings provide the first direct evidence for intramuscular free radical accumulation and lipid peroxidation following acute exercise in humans.

Endotoxin-induced changes in intrarenal pO2, measured by in vivo electron paramagnetic resonance oximetry and magnetic resonance imaging.

Electron Paramagnetic Resonance (EPR) oximetry was used to measure tissue oxygen tension (pO2-partial pressure of oxygen) simultaneously in the kidney cortex and outer medulla in vivo in mice. pO2 in the cortex region was higher compared to that in the outer medulla. An intravenous injection of endotoxin resulted in a sharp drop in pO2 in the cortex and an increase in the medulla region, resulting in a transient period of equal pO2 in both regions. In control kidneys, functional Magnetic Resonance (MR) images showed the cortex region to have high signal intensity (T2*-weighted images), indicating that this region was well supplied with oxygenated hemoglobin, whereas the outer medulla showed low signal intensity. After administration of endotoxin, we observed an immediate increase in signal intensity in the outer medulla region, reflecting an increased level of oxygenated blood in this region. Pretreatment of mice with NG-monomethyl-L-arginine prevented both the changes in tissue pO2 and distribution of oxygenated hemoglobin, suggesting that localized production of nitric oxide has a critical role to play in renal medullary hemodynamics. In combining in vivo EPR with MR images of kidneys, we demonstrate the usefulness of these techniques for monitoring renal pO2 and changes in the distribution of oxygen.

Long-term viability and proliferation of alginate-encapsulated 3-D HepG2 aggregates formed in an ultrasound trap.

We report proof of principle here of a gel encapsulation technique that departs from the minimum surface area to volume restriction of spherical microcapsules and allows gelation of preformed high-density (>or=2x10(4) cells/aggregate) 3-D HepG2 cell aggregates. The process involves forming a discoid 3-D cell aggregate in an ultrasound standing wave trap (USWT), which is subsequently recovered and encapsulated in alginate/CaCl2 hydrogel. The size of the ultrasound-formed aggregates was dependent upon the initial cell concentration, and was in the range of 0.4-2.6 mm in diameter (for cell concentrations ranging between 10(4) and 5x10(6)/ml). At low cell concentrations (<or=5x10(5)/ml), aggregates were 2-D, while at concentrations of >or=10(6)/ml, 3-D aggregates were generated. Cells in non- and encapsulated 3-D HepG2 aggregates remained 70-80% viable over 10 days in culture. The proliferative activity of the aggregates resulted in the doubling of the aggregate cell number and a subsequent increase in the aggregate thickness, while albumin secretion levels in encapsulated aggregates was 4.5 times higher compared to non-encapsulated, control aggregates. The results reported here suggest that the ultrasound trap can provide an alternative, novel approach of hydrogel cell encapsulation and thus rapidly (within 5 min) produce in vitro models for hepatocyte functional studies (for example, toxicity studies particularly if primary hepatocytes are used) in a tissue-mimetic manner.