The heme oxygenase system selectively enhances the anti-inflammatory macrophage-M2 phenotype, reduces pericardial adiposity, and ameliorated cardiac injury in diabetic cardiomyopathy in Zucker diabetic fatty rats.

Cardiac function is adversely affected by pericardial adiposity. We investigated the effects of the heme oxygenase (HO) inducer, hemin on pericardial adiposity, macrophage polarization, and diabetic cardiopathy in Zucker diabetic fatty rats (ZDFs) with use of echocardiographic, quantitative real-time polymerase chain reaction, Western immunoblotting, enzyme immunoassay, and spectrophotometric analysis. In ZDFs, hemin administration increased HO activity; normalized glycemia; potentiated insulin signaling by enhancing insulin receptor substrate 1(IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (PKB)/Akt; suppressed pericardial adiposity, cardiac hypertrophy, and left ventricular longitudinal muscle fiber thickness, a pathophysiological feature of cardiomyocyte hypertrophy; and correspondingly reduced systolic blood pressure, total peripheral resistance, and pro-inflammatory/oxidative mediators, including nuclear factor κB (NF-κB), cJNK, c-Jun-N-terminal kinase (cJNK), endothelin (ET-1), tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1ß, activating protein 1 (AP-1), and 8-isoprostane, whereas the HO inhibitor, stannous mesoporphyrin, nullified the effects. Furthermore, hemin reduced the pro-inflammatory macrophage M1 phenotype, but enhanced the M2 phenotype that dampens inflammation. Because NF-κB activates TNFα, IL-6, and IL-1ß and TNF-α, cJNK, and AP-1 impair insulin signaling, the high levels of these cytokines in obesity/diabetes would create a vicious cycle that, together with 8-isoprostane and ET-1, exacerbates cardiac injury, compromising cardiac function. Therefore, the concomitant reduction of pro-inflammatory cytokines and macrophage infiltration coupled to increased expressions of IRS-1, PI3K, and PKB may account for enhanced glucose metabolism and amelioration of cardiac injury and function in diabetic cardiomyopathy. The hemin-induced preferential polarization of macrophages toward anti-inflammatory macrophage M2 phenotype in cardiac tissue with concomitant suppression of pericardial adiposity in ZDFs are novel findings. These data unveil the benefits of hemin against pericardial adiposity, impaired insulin signaling, and diabetic cardiomyopathy and suggest that its multifaceted protective mechanisms include the suppression of inflammatory/oxidative mediators.

Dark chocolate inhibits platelet isoprostanes via NOX2 down-regulation in smokers.

BACKGROUND: Dark chocolate is reported to decrease platelet activation but the underlying mechanism is still undefined. Dark chocolate is rich in polyphenols that could exert an antiplatelet action via inhibition of oxidative stress. The aim of the present study was to assess if dark chocolate inhibits platelet reactive oxidant species (ROS) formation and platelet activation. METHODS: Twenty healthy subjects (HS) and 20 smokers were randomly allocated to receive 40 g of dark (cocoa > 85%) or milk chocolate (cocoa < 35%) in a cross-over, single-blind study. There was an interval of 7 days between the two phases of the study. At baseline and 2 h after chocolate ingestion, platelet recruitment (PR), platelet ROS, platelet isoprostane 8-ISO-prostaglandin F2α (8-iso-PGF2α), Thromboxane (TxA2) and platelet activation of NOX2, the catalytic sub-unit of NADPH oxidase, and serum epicatechin were measured. RESULTS: Compared with HS, smokers showed enhanced PR, platelet formation of ROS and eicosanoids and NOX2 activation. After dark chocolate, platelet ROS (-48%, P < 0.001), 8-iso-PGF2α (-10%, P < 0.001) and NOX2 activation (-22%, P < 0.001) significantly decreased; dark chocolate did not affect platelet variables in HS. No effect of milk chocolate was detected in both groups. Serum epicatechin increased after dark chocolate in HS (from 0.454 ± 0.3 nm to 118.3 ± 53.7 nm) and smokers (from 0.5 ± 0.28 nm to 120.9 ± 54.2 nm). Platelet incubation with 0.1-10 µm catechin significantly reduced PR, platelet 8-iso-PGF2α and ROS formation and NOX2 activation only in platelets from smokers. CONCLUSIONS: Dark chocolate inhibits platelet function by lowering oxidative stress only in smokers; this effect seems to be dependent on its polyphenolic content.

Grape seed proanthocyanidin extract (GSPE) attenuates collagen-induced arthritis.

To examine whether grape seed proanthocyanidin extract (GSPE) which is known to act as an antioxidant has therapeutic effect on collagen-induced arthritis (CIA) in mice, an animal model of rheumatoid arthritis. Mice were treated with an intraperitoneal injection of GSPE (10, 50, or 100 mg/kg) or saline. Clinical, histological, and biochemical parameters were assessed. The effects of GSPE on osteoclastogenesis were determined by tartrate-resistant acid phosphatase (TRAP) staining of the inflamed joints and bone-marrow cells cultured with the receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Intracellular levels of hydrogen peroxide were determined using carboxy-dichlorodihydrofluorescein diacetate. GSPE treatment significantly attenuated the severity of CIA in a dose-dependent manner and reduced the histology scores for synovial inflammation, cartilage erosion, bone erosion, and the number of TRAP+ osteoclasts. GSPE treatment significantly reduced the numbers of tumor necrosis factor alpha (TNF-alpha)- or interleukin 17 (IL-17)-producing cells in the synovial tissue and the spontaneous production of TNF-alpha and IL-17 by splenocytes compared with those in the control mice. The serum levels of type-II-collagen-specific IgG2a and plasma levels of 8-isoprostane in the GSPE-treated mice were significantly lower than those in the control mice. GSPE dose-dependently suppressed osteoclastogenesis in vitro. GSPE significantly reduced hydrogen peroxide production by anti-CD3-monoclonal-antibody-stimulated CD4+ splenocytes. These results indicate that intraperitoneal injection of GSPE attenuated CIA in mice. GSPE may be useful in the treatment of rheumatoid arthritis.

Expression levels of mRNA for Rho A/Rho kinase and its role in isoprostane-induced vasoconstriction of human placental and maternal vessels.

Preeclampsia is characterized by intense and prolonged vasoconstriction. Rho A-mediated calcium sensitization is central to prolonged contractility of vascular smooth muscle. The aims of this study are (1) to investigate mRNA expression levels of Rho A/Rho kinases in placental tissues from normotensive and preeclamptic women and (2) to investigate the effects of 2 isoprostanes, 8-iso prostaglandin F(2)( alpha) (8-iso PGF(2 alpha) ) and 8-iso prostaglandin E(2) (8-iso PGE(2)), on small placental and myometrial vessel resistance and to determine if their effects were mediated via the Rho kinase pathway. Real-time reverse transcription polymerase chain reaction for Rho A, ROCK I, and ROCK II was performed on total RNA from normotensive and preeclamptic placentae. The effects of 8- iso PGF(2 alpha) and 8-iso PGE(2) (alone and with the specific Rho kinase inhibitor Y-27632) on placental and myometrial vessels (<400 microm) were measured and compared with control recordings. Rho A mRNA expression levels were significantly higher in placentae from preeclamptic women than in placentae from normotensive women (P < .01). There was no significant difference in expression levels of ROCK I and ROCK II between both tissue types (P > .05). Both isoprostanes exerted a significant concentration-dependent vasocontractile effect on both vessel types (P < .001). This effect was antagonized by Y-27632 in placental arteries but not in myometrial arteries. Increased Rho A mRNA expression in placentae from preeclamptic women is suggestive of a role for the Rho kinase pathway in the modulation of the placental vasculature in this condition. Isoprostanes exert their vasocontractile effect, in placental vasculature, in part via the Rho kinase pathway.

Isoprostane formation and inhibition in atherothrombosis.

Enhanced oxidant stress plays a pivotal role in the pathogenesis of several conditions such as atherothrombosis, cancer and neurodegeneration. The availability of reliable assays of isoprostanes in biological fluids has prompted clinical investigations into the pathophysiological role of lipid peroxidation in cardiovascular disease. Established risk factors for coronary heart disease have been associated with enhanced oxidant stress, leading to increased lipid peroxidation and non-enzymatic formation of bioactive isoprostanes. These risk factors include diabetes mellitus, hypercholesterolemia, obesity and hyperhomocysteinemia. Clinical studies support the hypothesis that enhanced lipid peroxidation may contribute, at least in part, to persistent platelet activation.

Determinants of F2-isoprostane biosynthesis and inhibition in man.

Several cardiovascular risk factors are characterized by the coexistence of low-grade inflammation, enhanced oxidative stress and lipid peroxidation. It has been hypothesized that F2-isoprostanes, a product of in vivo lipid peroxidation, may transduce the effects of metabolic and hemodynamic abnormalities into increased cardiovascular risk. Thus, the formation of these compounds, including urinary 8-iso-Prostaglandin (PG) F2alpha, has been investigated in clinical settings putatively associated with oxidant stress. Enhanced lipid peroxidation together with increased in vivo platelet activation have been found in association with the major cardiovascular risk factors. Thus, F2-isoprostanes may transduce the effects of oxidant stress associated with complex metabolic disorders into specialized forms of cellular activation. In particular, the low-grade inflammatory state characterizing metabolic disorders such as obesity, hypercholesterolemia, type 2 diabetes mellitus, and homozygous homocystinuria may be the primary trigger of thromboxane-dependent platelet activation mediated, at least in part, through enhanced lipid peroxidation. Moreover, oxidative stress may promote endothelial dysfunction through increased production of reactive oxygen species that inactivate nitric oxide. Accumulation and activation of leukocytes plays a key role in atherosclerosis and its complications. Interestingly, neutrophil adhesion induced by minimally modified low-density lipoproteins is mainly mediated by F2-isoprostanes. Although epidemiological studies suggest an inverse relationship between antioxidant vitamin intake and cardiovascular disease, several clinical trials have obtained conflicting results on the effects of vitamin E supplementation on the risk of cardiovascular events. On the other hand, the use of F2-isoprostane formation as a biochemical end-point for dose-finding studies of vitamin E supplementation has helped clarifying the unique features of its pharmacodynamic effects on lipid peroxidation. This information could be extremely valuable in the selection of the appropriate patient subgroups that may benefit from antioxidant interventions.

N-acetylcysteine inhibits interleukin-17-induced interleukin-8 production from human airway smooth muscle cells: a possible role for anti-oxidative treatment in chronic lung rejection?

BACKGROUND: Long-term survival of lung transplantation is threatened by obliterative bronchiolitis, or its clinical equivalent, bronchiolitis obliterans syndrome. With a prevalence of >50% at 5 years after transplantation, it has emerged as the most significant long-term complication. Neutrophilic inflammation and increased interleukin (IL)-8 production seem to be part of the basic pathophysiologic mechanism of chronic rejection. Recently, it has been suggested that reactive oxygen species may also play an important role in the pathogenesis because they are known to induce smooth muscle proliferation. METHODS: Human airway smooth muscle cells in vitro were stimulated with IL-17 (0.1 to 10 ng/ml) or with IL-17 (10 ng/ml) and the anti-oxidative agent N-acetylcysteine (1 micromol/liter to 10 mmol/liter). Production of 8-isoprostane was measured with a commercially available enzyme immunoassay kit and production of IL-8 was measured using a standard enzyme-linked immunoassay technique. RESULTS: IL-17 produced a concentration-dependent increase in 8-isoprostane with a maximum of 136.5 +/- 15.5 pg/ml with IL-17 (10 ng/ml, p < 0.001, n = 12, vs unstimulated cells). N-acetylcysteine (NAC) was able to decrease IL-17-induced 8-isoprostane production, with a maximum decrease of 59.3 +/- 9% (p < 0.001, n = 12) with 10 mmol/liter of N-acetylcysteine, which also decreased IL-17-induced IL-8 production in a concentration-dependent manner (with maximum inhibition of 86.3% when combined with NAC 10 mmol/liter as compared with IL-17 alone). CONCLUSIONS: We demonstrated that human airway smooth muscle cells, when stimulated with IL-17, are able to produce 8-isoprostane, which could be inhibited by adding N-acetylcysteline, and which was also able to decrease IL-17-induced IL-8 production. The clinical significance of these in vitro findings for prevention or treatment of chronic rejection after lung transplantation remains to be investigated.

Aspirin insensitive eicosanoid biosynthesis in cardiovascular disease.

Inhibition of platelet cyclooxygenase (COX)-1 is involved in aspirin cardioprotection observed in clinical trials, but, in some patients, aspirin is unable to protect from thrombotic complications. An incomplete suppression of platelet thromboxane (TX) A2 biosynthesis has been assumed to participate in the phenomenon of aspirin resistance, as a consequence of the following possible mechanisms: (i) COX-2 expression in newly formed platelets; (ii) pharmacodynamic interactions between aspirin and coadministered nonsteroidal antiinflammatory drugs (e.g. ibuprofen); (iii) expression of variant isoforms of COX-1 with reduced sensitivity to irreversible inactivation at Ser529. Furthermore, aspirin failure may be due to enhanced formation of vasoactive and/or proaggregatory eicosanoids despite an almost complete suppression of platelet TXA2 biosynthesis by aspirin. Thus, in a subset of patients with unstable angina treated with low-dose aspirin, to almost completely block platelet COX-1 activity, enhanced TXA2 biosynthesis in vivo has been demonstrated, presumably through an increased generation of COX-2-dependent PGH2 in plaque monocytes/macrophages or activated vascular cells. The concomitant increased formation of 8-iso-PGF2alpha, one of the most abundant F2-isoprostanes in humans, generated by free-radical catalyzed arachidonate peroxidation, may be involved in aspirin resistance because of its capacity to induce platelet and vascular smooth muscle cell activation through the interaction with thromboxane receptors (TPs). Finally, enhanced production of vasoactive cysteinyl leukotrienes (cys-LTs) occurs in unstable angina despite conventional antithrombotic and antianginal treatment. The use of selective pharmacological tools (i.e. highly selective COX-2 inhibitors, TP antagonists, cys-LT inhibitors and antagonists) will allow to ascertain the contribution of aspirin insensitive eicosanoid biosynthesis in aspirin cardioprotective failure.

Vasoconstrictor responses, and underlying mechanisms, to isoprostanes in human and porcine bronchial arterial smooth muscle.

We investigated the effects of five different isoprostanes (8-iso PGE1, 8-iso PGE2, 8-iso PGF1alpha, 8-iso PGF2alpha and 8-iso PGF2beta) on vasomotor tone in human and porcine bronchial arterial tissues. In the human bronchial arteries, 8-iso PGE2 and 8-iso PGF2alpha evoked powerful constrictions (magnitudes several fold greater than the responses to high millimolar KCl) with negative log concentration causing 50% excitation (EC50) values of 6.8 and 6.5, respectively; 8-iso PGE1 was less potent (EC50 not calculated, since a clear peak contraction was not obtained), while the other isoprostanes were largely ineffective. In the porcine arteries, on the other hand, all three F-ring isoprostanes as well as 8-iso PGE2 evoked constrictor responses, although the peak magnitudes were approximately 50% of the KCl-evoked response; 8-iso PGE2 and 8-iso PGF2alpha were the most potent, with negative log EC50 values of 6.5. We next sought to characterize the signaling pathways underlying the vasoconstrictor responses to 8-iso PGE2, since this was the most potent of the isoprostanes we tested. These responses were largely reversed by the thromboxane A2-selective (TP) prostanoid receptor antagonist ICI 192605 (10-8 m; 4(Z)-6-[(2,4,5 cis)2-(2-chlorophenyl)-4-(2-hydroxy phenyl)1,3-dioxan-5-yl]hexenoic acid) as well as by the nonspecific tyrosine kinase inhibitor genistein (10-5 and 10-4 m), and were reversed approximately 50% by the Rho-kinase inhibitor Y27632 (10-5 m; (+)-(R)-trans-4-(1-aminoethyl)-N-(pyridyl) cyclohexanecarboxamide dihydrochloride). We conclude, therefore, that 8-iso PGE2 constricts bronchial vasculature through the activation of TP receptors, which in turn trigger tyrosine kinase and Rho-kinase activities, resulting in powerful vasoconstriction. These findings are highly relevant to lung transplantation and to exercise-induced asthma.