Endurance exercise, plasma oxidation and cardiovascular risk

Objective-Although physical activity is beneficial to health, people who exercise at high intensities throughout their lifetime may have increased cardiovascular risk. Aerobic exercise increases oxidative stress and may contribute to atherogenesis by augmented oxidation of plasma lipoproteins. The aim of this study was to examine the relationship between aerobic power and markers of oxidative stress, including the susceptibility of plasma to oxidation. Methods and results-Aerobic power was measured in 24 healthy men aged 29 9 years (mean +/- SD). Plasma was analysed from subjects of high aerobic power (HAP; VO(2)max, 64.6 +/- 6.1 ml/kg/min) and lower aerobic power (LAP;VO(2)max, 45.1 +/- 6.3 ml/kg/min) for total antioxidant capacity (TAC), malondialdehyde (MDA) and susceptibility to oxidation. Three measures were used to quantify plasma oxidizability: (1) lag time to conjugated diene formation (lag time); (2) change in absorbance at 234 nm and; (3) slope of the oxidation curve during propagation (slope). The HAP subjects had significantly lowerTAC (1.38 +/- 0.04 versus 1.42 +/- 0.06 TEAC units; P < 0.05), significantly higher change in absorbance (1.55 +/- 0.21 versus 1.36 +/- 0.17 arbitrary units; P < 0.05), but no difference in MDA (P = 0.6), compared to LAP subjects. There was a significant inverse association between TAC and slope (r = -0.49; P < 0.05). Lipoprotein profiles and daily intake of nutrients did not differ between the groups. Conclusions-These findings suggest that people with high aerobic power, due to extreme endurance exercise, have plasma with decreased antioxidant capacity and higher susceptibility to oxidation, which may increase their cardiovascular risk.

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473 - 484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.

Pioglitazone increases renal tubular cell albumin uptake but limits proinflammatory and fibrotic responses

Background. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. which are known to be critical factors in lipid metabolism, have also been reported to reduce proteinuria. The mechanism and its relevance to progressive nephropathy have not been determined. The aims of this study were to assess the direct effects of a PPARgamma agonist on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology, using an opossum kidney model of proximal tubular cells. Methods. Cells were exposed to pioglitazone (10 mumol/L) in the presence and absence of low-density lipoprotein (LDL) 100 mug/mL +/- exposure to albumin 1 mg/mL. Results were expressed relative to control (5 mmol/L glucose) conditions. Results. Pioglitazone caused a dose-dependent increase in tubular cell albumin uptake (P < 0.0001). Despite the increase in albumin reabsorption, no concurrent increase in inflammatory or profibrotic markers were observed. Exposure to LDL increased monocyte chemoattractant protein-1 (MCP-1) (P < 0.05) and transforming growth factor-beta1 (TGF-beta1) (P < 0.05) production. which were reversed in the presence of pioglitazone. LDL induced increases in MCP-1 and TGF-β1 were independent of nuclear factor-κB (NF-κB) transcriptional activity. In contrast. tubular exposure to albumin increased tubular protein uptake, in parallel with an increase in MCP-1 (P = 0.05): TGF-β1 (P < 0.02) and NF-kappaB transcriptional activity (P < 0.05). which were unaffected by concurrent exposure to pioglitazone. Conclusion. These findings suggest that dyslipidemia potentiates renal pathology through mechanisms that may be modified PPARγ activation independent of NF-κB transcriptional activitv. In contrast, tubular exposure to protein induces renal damage through NF-κB-dependent mechanisms that are Unaffected by PPARγ activation.

Rho and vascular disease

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Cyclosporine A-induced changes to erythrocyte redox balance is time course-dependent

Cyclosporine A-treated transplant recipients develop pronounced cardiovascular disease and have increased oxidative stress and altered antioxidant capacity in erythrocytes and plasma. These experiments investigated the time-course of cyclosporine A-induced changes to redox balance in plasma and erythrocytes. Rats were randomly assigned to either a control or cyclosporine A-treated group. Treatment animals received 25 mg/kg of cyclosporine A via intraperitoneal injection for either 7 days or a single dose. Control rats were injected with the same volume of the vehicle. Three hours after the final injections, plasma was analysed for total antioxidant status, a-tocopherol, malondialdehyde, and creatinine. Erythrocytes were analysed for reduced glutathione (GSH), alpha-tocopherol, methaemoglobin, malondialdehyde, and the activities of superoxide dismutase, catalase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G6PD). Cyclosporine A administration for 7 days resulted in a significant increase (P < 0.05) in plasma malondialdehyde, methaemoglobin, and superoxide dismutase and catalase activities. There was a significant decrease (P < 0.05) in erythrocyte GSH concentration and G6PD activity in cyclosporine A animals. There were no significant differences (P > 0.05) between groups following a single dose of cyclosporine A in any of the measures. In summary, cyclosporine A alters erythrocyte redox balance after 7 days administration, but not after a single dose.

Evidence for a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as a-lipoic acid and alpha-tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha-lipoic acid and alpha-tocopherol, alone or in combination, prior to incubation with H2O2 or staurosporine. alpha-lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H2O2 and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with a-lipoic acid and/or a-tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of BcI-2, Bax, HSP70 or pERK1/2 or pJNK. alpha-lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha-lipoic acid and alpha-tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha-lipoic acid as an antioxidant.

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), alpha-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, alpha-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, alpha-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.

Are insect immune suppressors driving cellular uptake reactions?

Many insect parasitoids that deposit their eggs inside immature stages of other insect species inactivate the cellular host defence to protect the growing embryo from encapsulation. Suppression of encapsulation by polydnavirus-encoded immune-suppressors correlates with specific alterations in hemocytes, mainly cytoskeletal rearrangements and actin-cytoskeleton breakdown. We have previously shown that the Cotesia rubecula polydnavirus gene product CrV1 causes immune suppression when injected into the host hemocoel. CrV1 is taken up by hemocytes although no receptors have been found to bind the protein. Instead CrV1 uptake depends on dimer formation, which is required for interacting with lipophorin, suggesting a CrV1-lipophorin complex internalisation by hemocytes. Since treatment of hemocytes with oligomeric lectins and cytochalasin D can mimic the effects of CrV1, we propose that some dimeric and oligomeric adhesion molecules are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph.

Ultra-endurance exercise and oxidative damage: Implications for cardiovascular health

At least 30 minutes of moderate-intensity physical activity accumulated on most, preferably all days is considered the minimum level necessary to reduce the risk of developing cardiovascular disease. Despite an unclear explanation, some epidemiological data paradoxically suggest that a very high volume of exercise is associated with a decrease in cardiovascular health. Although ultra-endurance exercise training has been shown to increase antioxidant defences (and therefore confer a protective effect against oxidative stress), an increase in oxidative stress may contribute to the development of atherosclerosis via oxidative modification of low-density lipoprotein (LDL). Research has also shown that ultra-endurance exercise is associated with acute cardiac dysfunction and injury, and these may also be related to an increase in free radical production. Longitudinal studies are needed to assess whether antioxidant defences are adequate to prevent LDL oxidation that may occur as a result of increased free radical production during very high volumes of exercise. In addition, this work will assist in understanding the accrued effect of repeated ultra-endurance exercise-induced myocardial damage.

Vitamin E and alpha-lipoic acid supplementation increase bleeding tendency via an intrinsic coagulation pathway

Vitamin E and a-lipoic acid are potent nutritional antioxidants, and when used together, their antioxidant capabilities are improved as a-lipoic acid recycles vitamin E. Supplementation of vitamin E has been shown to prolong platelet aggregation but the effects of vitamin E and alpha-lipoic acid supplementation on bleeding tendency have yet to be reported. Young, male rats consumed either control diet (n=5) or vitamin E and a-lipoic acid-supplemented diet (n=5) for 14 weeks. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured as markers of intrinsic and extrinsic coagulation pathways respectively in addition to lipid peroxidation (malondialdehyde). Supplementation significantly prolonged APTT (23.8 +/- 1.5 vs 31.4 +/- 1.2s, p < 0.05) compared to the con-trol diet; however, there was no significant difference in PT (27.8 +/- 1.5 vs 26.6 +/- 0.9s, p > 0.05). While vitamin E was increased (p < 0.05), there was no significant difference in plasma levels of malondialdehyde (p > 0.05). Dietary supplementation of vitamin E and alpha-lipoic acid increases bleeding tendency via inhibition of the intrinsic coagulation pathway with no change in markers of lipid peroxidation. Such supplementation could benefit patients with cardiovascular disease who exhibit elevated levels of coagulation and oxidative stress.

Validation of a novel ELISA for measurement of MDA-LDL in human plasma

The involvement of oxidatively modified low density lipoprotein (LDL) in the development of CHD is widely described. We have produced two antibodies, recognizing the lipid oxidation product malondialdehyde (MDA) on whole LDL or ApoB-100. The antibodies were utilized in the development of an ELISA for quantitation of MDA-LDL in human plasma. Intra- and inter-assay coefficients of variation (% CV) were measured as 4.8 and 7.7%, respectively, and sensitivity of the assay as 0.04 μg/ml MDA-LDL. Recovery of standard MDA-LDL from native LDL was 102%, indicating the ELISA to be specific with no interference from other biomolecules. Further validation of the ELISA was carried out against two established methods for measurement of lipid peroxidation products, MDA by HPLC and F2-isoprostanes by GC-MS. Results indicated that MDA-LDL is formed at a later stage of oxidation than either MDA or F2- isoprostanes. In vivo analysis demonstrated that the ELISA was able to determine steady-state concentrations of plasma MDA-LDL (an end marker of lipid peroxidation). A reference range of 34.3 ± 8.8 μg/ml MDA-LDL was established for healthy individuals. Further, the ELISA was used to show significantly increased plasma MDA-LDL levels in subjects with confirmed ischemic heart disease, and could therefore possibly be of benefit as a diagnostic tool for assessing CHD risk. © 2003 Elsevier Inc.

Phospholipid chlorohydrin induces leukocyte adhesion to Apoe(-/-) mouse arteries via upregulation of P-selectin

HOCl-modified low-density lipoprotein (LDL) has proinflammatory effects, including induction of inflammatory cytokine production, leukocyte adhesion, and ROS generation, but the components responsible for these effects are not completely understood. HOCl and the myeloperoxidase-H2O2-halide system can modify both protein and lipid moieties of LDL and react with unsaturated phospholipids to form chlorohydrins. We investigated the proinflammatory effects of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine (SOPC) chlorohydrin on artery segments and spleen-derived leukocytes from ApoE-/- and C57 Bl/6 mice. Treatment of ApoE-/- artery segments with SOPC chlorohydrin, but not unmodified SOPC, caused increased leukocyte-arterial adhesion in a time- and concentration-dependent manner. This could be prevented by pretreatment of the artery with P-selectin or ICAM-1-blocking antibodies, but not anti-VCAM-1 antibody, and immunohistochemistry showed that P-selectin expression was upregulated. However, chlorohydrin treatment of leukocytes did not increase expression of adhesion molecules LFA-1 or PSGL-1, but caused increased release of ROS from PMA-stimulated leukocytes by a CD36-dependent mechanism. The SOPC chlorohydrin-induced adhesion and ROS generation could be abrogated by pretreatment of the ApoE-/- mice with pravastatin or a nitrated derivative, NCX 6550. These findings suggest that phospholipid chlorohydrins formed in HOCl-treated LDL could contribute to the proinflammatory effects observed for this modified lipoprotein in vitro.

Use of narrow mass-window, high-resolution extracted product ion chromatograms for the sensitive and selective identification of protein modifications

Protein modifications, including oxidative modifications, glycosylations, and oxidized lipid-protein adducts, are becoming increasingly important as biomarkers and in understanding disease etiology. There has been a great deal of interest in mapping these on Apo B100 from low density lipoprotein (LDL). We have used extracted ion chromatograms of product ions generated using a very narrow mass window from high-resolution tandem mass spectrometric data collected on a rapid scanning quadrupole time-of-flight (QTOF) instrument, to selectively and sensitively detect modified peptides and identify the site and nature of a number of protein modifications in parallel. We have demonstrated the utility of this method by characterizing for the first time oxidized phospholipid adducts to LDL and human serum albumin and for the detection of glycosylation and kynurenin formation from the oxidation of tryptophan residues in LDL. © 2013 American Chemical Society.

Fatty acid and phospholipid chlorohydrins cause cell stress and endothelial adhesion

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, which is an inflammatory disease involving activation of phagocytic cells. Myeloperoxidase, an enzyme which is able to produce hypochlorous acid (HOCl), is released from these phagocytic cells, and has been found in an active form in atherosclerotic plaques. HOCl can oxidize both the lipid and protein moiety of LDL, and HOCl-modified LDL has been found to be pro-inflammatory, although it is not known which component is responsible for this effect. As HOCl can oxidize lipids to give chlorohydrins, we hypothesized that phospholipid chlorohydrins might have toxic and pro-inflammatory effects. We have formed chlorohydrins from fatty acids (oleic, linoleic and arachidonic acids) and from phospholipids (stearoyl-oleoyl phosphatidylcholine, stearoyl-linoleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine), and investigated various biological effects of these oxidation products. Fatty acid and phospholipid chlorohydrins were found to deplete ATP levels in U937 cells in a concentration-dependent manner, with significant effects observed at concentrations of 25 µM and above. Low concentrations (25 µM) of stearoyl-oleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine chlorohydrins were also found to increase caspase-3 activity. Finally, stearoyl-oleoyl phosphatidylcholine chlorohydrin increased leukocyte adhesion to artery segments isolated from C57Bl/6 mice. These results demonstrate potentially harmful effects of lipid chlorohydrins, and suggest that they may contribute to some of the pro-inflammatory effects that HOCl-modified low density lipoprotein has been found to induce.