Heightened maternal inflammation is linked to placental oxidative and nitrosative stress associated with fetal growth restriction in the rat

Deficient trophoblast invasion and spiral artery remodeling are associated with pregnancy complications such as pre-eclampsia (PE) and fetal growth restriction (FGR). Using a model in which pregnant Wistar rats are given daily, low-dose, injections of bacterial lipopolysaccharide (LPS; 10 – 40 µg/kg) on gestational days (GD) 13.5 – 16.5, our group has shown that abnormal maternal inflammation is causally linked to shallow trophoblast invasion, deficient spiral artery remodeling, and altered utero-placental hemodynamics leading to FGR/PE; these alterations were shown to be mediated by TNF-a. The present research evaluated certain consequences of decreased placental perfusion; this was accomplished by examining placental alterations indicative of decreased placental perfusion. Additionally, the role of glyceryl trinitrate (GTN) was determined as a potential therapeutic to prevent the consequences of decreased placental perfusion. Results indicated that dams experiencing heightened maternal inflammation showed significantly greater expression of hypoxia-inducible factor-1a (HIF-1a) and nitrotyrosine, both of which are markers of decreased perfusion and oxidative/nitrosative stress. Contrary to expectations, inflammation did not appear to affect nitric oxide (NO) bioavailability, as revealed by a lack of change in placental or plasma levels of cyclic guanosine monophosphate (cGMP). However, continuous transdermal administration of GTN (25 µg/hr) on GD 12.5 – 16.5 prevented the accumulation of HIF-1a and nitrotyrosine in placentas from LPS-treated rats. These results support the concept that maternal inflammation contributes to placental hypoxia and oxidative/nitrosative stress. Additionally, they indicate that GTN has potential applications in the treatment and/or prevention of pregnancy complications associated with abnormal maternal inflammation.

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 105 molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 105 molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

Role of creatine supplementation on exercise-induced cardiovascular function and oxidative stress

Many degenerative diseases are associated with increased oxidative stress. Creatine has the potential to act as an indirect and direct antioxidant; however, limited data exist to evaluate the antioxidant capabdities of creatine supplementation within in vivo human systems. This study aimed to investigate the effects of oral creatine supplementation on markers of oxidative stress and antioxidant defenses following exhaustive cycling exercise. Following preliminary testing and two additional familiarization sessions, 18 active males repeated two exhaustive incremental cycling trials (T1 and T2) separated by exactly 7 days. The subjects were assigned, in a double-blind manner, to receive either 20 g of creatine (Cr) or a placebo (P) for the 5 days preceding T2. Breath-by-breath respiratory data and heart rate were continually recorded throughout the exercise protocol and blood samples were obtained at rest (preexercise), at the end of exercise (postexercise), and the day following exercise (post24 h). Serum hypdroperoxide concentrations were elevated at postexercise by 17 +/- 5% above preexercise values (p = 0.030). However, supplementation did not influence lipid peroxidation (serum hypdroperoxide concentrations), resistance of low density lipoprotein to oxidative stress (t(1/2max) LDL oxidation) and plasma concentrations of non-enzymatic antioxidants (retinol, alpha-carotene, beta-carotene, alpha-tocopherol, gamma-tocopherol, lycopene and vitamin Q. Heart rate and oxygen uptake responses to exercise were not affected by supplementation. These findings suggest that short-term creatine supplementation does not enhance non-enzymatic antioxidant defence or protect against lipid peroxidation induced by exhaustive cycling in healthy males.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

Background: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD’s actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). Aim/method: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. Results: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. Conclusion: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.

Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

The effect of increased dietary fruit and vegetable consumption on endothelial activation, inflammation and oxidative stress in hypertensive volunteers

Background and aims

Public health campaigns recommend increased fruit and vegetable (FV) consumption as an effective means of cardiovascular risk reduction. During an 8 week randomised control trial among hypertensive volunteers, we noted significant improvements in endothelium-dependent vasodilatation with increasing FV consumption. Circulating indices of inflammation, endothelial activation and insulin resistance are often employed as alternative surrogates for systemic arterial health. The responses of several such biomarkers to our previously described FV intervention are reported here.
Methods and results

Hypertensive volunteers were recruited from medical outpatient clinics. After a common 4 week run-in period during which FV consumption was limited to 1 portion per day, participants were randomised to 1, 3 or 6 portions daily for 8 weeks. Venous blood samples for biomarker analyses were collected during the pre and post-intervention vascular assessments. A total of 117 volunteers completed the 12 week study. Intervention-related changes in circulating levels of high sensitivity C-reactive protein (hsCRP), soluble intracellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) did not differ significantly between FV groups. Similarly, there were no significant between group differences of change in homeostasis model assessment (HOMA) scores.
Conclusions

Despite mediating a significant improvement in acetylcholine induced vasodilatation, increased FV consumption did not affect a calculated measure of insulin resistance or concentrations of the circulating biomarkers measured during this study. Functional indices of arterial health such as endothelium-dependent vasomotion are likely to provide more informative cardiovascular end-points during short-term dietary intervention trials.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.