Are We Over Oxidized? Oxidative Stress, Cardiovascular Disease, and the Future of Intervention Studies with Antioxidants

A number of recent clinical trials with antioxidants, notably vitamin C and E, have provided no support for the commonly held view that increasing our intake of antioxidants will offset the ravages of cardiovascular disease as well as other diseases (for extensive critical reviews see: Kritharides and Stocker 2002; Antoniades et al 2003; Touyz 2004). Is this conclusion justified? The role of antioxidant dietary adjuncts and therapy in prevention and treatment remains a highly important clinical question. In this opinion article we address the question: Is there a future for antioxidant therapy in the treatment and prevention of cardiovascular disease? We conclude that there is a need for better-designed studies as well as a re-thinking of the choice of antioxidants

Effects of aggregation on the blood flow velocity field measured by a μPIV based technique

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.The flow of red blood cells is investigated by means of a micro-PIV based technique at physiological hematocrit levels and in the presence of aggregation. The technique developed differs from typical micro-PIV as the RBCs are used as tracer particles and illumination is provided by a simple halogen light source. Changes in the microstructure of blood caused by aggregation were observed to affect the RBC flow characteristics in a narrow-gap plate-plate geometry. At low shear rates, high aggregation caused the RBC motion to become essentially two-dimensional and network formation lead to the flow deviating from the expected radial profile. The accuracy of the micro-PIV technique was shown to be dependent on aggregation, illustrating the need to take aggregation into account in future RBC flow studies.This work was supported in part by the EPSRC Life Sciences Interface program (EP/F007736/1) and by the Leverhulme Trust(F/07 040/X)

Flow field characterisation of aggregating human blood in bifurcating microchannels

This paper was presented at the 3rd Micro and Nano Flows Conference (MNF2011), which was held at the Makedonia Palace Hotel, Thessaloniki in Greece. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, Aristotle University of Thessaloniki, University of Thessaly, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute.Erythrocyte aggregation is a shear dependent physiological phenomenon that modifies local properties of blood flow. Blood flow characteristics in microvascular bifurcations are dependent on many parameters; however the influence of erythrocyte aggregation has not been investigated previously in vitro. In the present study, micro-PIV is used to provide high spatial resolution velocity data for both erythrocytes and suspending medium for aggregating and non-aggregating human blood samples in a microchannel with a T-bifurcation geometry on the scale of the microcirculation. Simultaneous hematocrit distributions are inferred from brightfield images. Full field shear distributions are described for an evenly split flow and single flow rate. Velocity profiles of cells upstream of the bifurcation are found to be less blunt than those of the suspended particles. Daughter branch velocity profiles downstream of the bifurcation are skewed towards the wall closest to the parent branch, and non-aggregating cell velocities are significantly less blunted than those of the aggregating case. The local hematocrit is increased at the channel wall opposite the parent branch and a cell-depleted layer is observed near the channel wall closest to the parent branch. Thus, it is shown that aggregation influences both hematocrit and velocity distributions around and downstream of a bifurcation

Quantifying local characteristics of velocity, aggregation and hematocrit of human erythrocytes in a microchannel flow

The effect of erythrocyte aggregation on blood viscosity and microcirculatory flow is a poorly understood area of haemodynamics, especially with relevance to serious pathological conditions. Advances in microfluidics have made it possible to study the details of blood flow in the microscale, however, important issues such as the relationship between the local microstructure and local flow characteristics have not been investigated extensively. In the present study an experimental system involving simple brightfield microscopy has been successfully developed for simultaneous, time-resolved quantification of velocity fields and local aggregation of human red blood cells (RBC) in microchannels. RBCs were suspended in Dextran and phosphate buffer saline solutions for the control of aggregation. Local aggregation characteristics were investigated at bulk and local levels using statistical and edge-detection image processing techniques. A special case of aggregating flow in a microchannel, in which hematocrit gradients were present, was studied as a function of flowrate and time. The level of aggregation was found to strongly correlate with local variations in velocity in both the bulk flow and wall regions. The edge detection based analysis showed that near the side wall large aggregates are associated with regions corresponding to high local velocities and low local shear. On the contrary, in the bulk flow region large aggregates occurred in regions of low velocity and high erythrocyte concentration suggesting a combined effect of hematocrit and velocity distributions on local aggregation characteristics. The results of this study showed that using multiple methods for aggregation quantification, albeit empirical, could help towards a robust characterisation of the structural properties of the fluid

Quantifying local characteristics of velocity, aggregation and hematocrit of human erythrocytes in a microchannel flow

The effect of erythrocyte aggregation on blood viscosity and microcirculatory flow is a poorly understood area of haemodynamics, especially with relevance to serious pathological conditions. Advances in microfluidics have made it possible to study the details of blood flow in the microscale, however, important issues such as the relationship between the local microstructure and local flow characteristics have not been investigated extensively. In the present study an experimental system involving simple brightfield microscopy has been successfully developed for simultaneous, time-resolved quantification of velocity fields and local aggregation of human red blood cells (RBC) in microchannels. RBCs were suspended in Dextran and phosphate buffer saline solutions for the control of aggregation. Local aggregation characteristics were investigated at bulk and local levels using statistical and edge-detection image processing techniques. A special case of aggregating flow in a microchannel, in which hematocrit gradients were present, was studied as a function of flowrate and time. The level of aggregation was found to strongly correlate with local variations in velocity in both the bulk flow and wall regions. The edge detection based analysis showed that near the side wall large aggregates are associated with regions corresponding to high local velocities and low local shear. On the contrary, in the bulk flow region large aggregates occurred in regions of low velocity and high erythrocyte concentration suggesting a combined effect of haematocrit and velocity distributions on local aggregation characteristics. The results of this study showed that using multiple methods for aggregation quantification, albeit empirical, could help towards a robust characterisation of the structural properties of the fluid

Gene Therapy with Endogenous Inhibitors of Angiogenesis for Neovascular Age-Related Macular Degeneration: Beyond Anti-VEGF Therapy

Age-related macular degeneration (AMD) is the leading cause of substantial and irreversible vision loss amongst elderly populations in industrialized countries. The advanced neovascular (or “wet”) form of the disease is responsible for severe and aggressive loss of central vision. Current treatments aim to seal off leaky blood vessels via laser therapy or to suppress vessel leakage and neovascular growth through intraocular injections of antibodies that target vascular endothelial growth factor (VEGF). However, the long-term success of anti-VEGF therapy can be hampered by limitations such as low or variable efficacy, high frequency of administration (usually monthly), potentially serious side effects, and, most importantly, loss of efficacy with prolonged treatment. Gene transfer of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization and/or excessive vascular leakage in the eye. Preclinical studies of gene transfer in a large animal model have provided impressive preliminary results with a number of transgenes. In addition, a clinical trial in patients suffering from advanced neovascular AMD has provided proof-of-concept for successful gene transfer. In this mini review, we summarize current theories pertaining to the application of gene therapy for neovascular AMD and the potential benefits when used in conjunction with endogenous antiangiogenic proteins

Role of NADPH Oxidase-4 in human endothelial progenitor cells

Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization

Nitrite is produced by elicited but not by circulating neutrophils

The generation of nitrite (NO2-) was used as an index of the production of nitric oxide by human and rat polymorphonuclear leukocytes (PMN) and rat peritoneal macrophages. Human peripheral blood PMN did not produce significant levels of NO2-. Attempts to induce NO2- generation in human PMN by incubation with GM–CSF (1 nM), TNFα (0.3 nM), endotoxin (1 μg/ml) or formyl-Met-Leu-Phe (100 nM) for up to 16 h were not successful. Addition of human PMN primed by GM–CSF (1 nM) to rabbit aortic ring preparations precontracted with phenylephrine had no effect on tone. In contrast to these observations, PMN, isolated from the peritoneum of oyster glycogen treated rats, generated NO2- via a pathway sensitive to inhibition by the nitric oxide synthase inhibitor, NG-monomethyl L-arginine. However, peripheral blood rat PMN obtained from the same animals did not produce NO2-, even during prolonged incubation for periods of up to 16 h. It is suggested that detectable NO production by PMN requires NO synthase activity to be induced either by the process of PMN migration or by exposure to certain cytokines produced locally at the site of inflammation