According to the latest Global Burden of Disease Study data, non-communicable diseases in general and cardiovascular disease (CVD) in particular are the leading cause of premature death and reduced quality of life. Demographic shifts, unhealthy lifestyles and a higher burden of adverse environmental factors provide an explanation for these findings. The expected growing prevalence of CVD requires enhanced research efforts for identification and characterisation of novel therapeutic targets and strategies. Cardiovascular risk factors including classical (e.g. hypertension, diabetes, hypercholesterolaemia) and non-classical (e.g. environmental stress) factors induce the development of endothelial dysfunction, which is closely associated with oxidant stress and vascular inflammation and results in CVD, particularly in older adults. Most classically successful therapies for CVD display vasoprotective, antioxidant and anti-inflammatory effects, but were originally designed with other therapeutic aims. So far, only a few 'redox drugs' are in clinical use and many antioxidant strategies have not met expectations. With the present review, we summarise the actual knowledge on CVD pathomechanisms, with special emphasis on endothelial dysfunction, adverse redox signalling and oxidative stress, highlighting the preclinical and clinical evidence. In addition, we provide a brief overview of established CVD therapies and their relation to endothelial dysfunction and oxidative stress. Finally, we discuss novel strategies for redox-based CVD therapies trying to explain why, despite a clear link between endothelial dysfunction and adverse redox signalling and oxidative stress, redox- and oxidative stress-based therapies have not yet provided a breakthrough in the treatment of endothelial dysfunction and CVD.

Background: The impairment of endothelium‐dependent vasodilation, increased endothelial permeability, and glycocalyx degradation are all important pathophysiological components of endothelial dysfunction. However, it is still not clear whether in atherosclerosis, glycocalyx injury precedes other features of endothelial dysfunction or these events coincide. Methods and Results: Herein, we demonstrate that in 4‐ to 8‐week‐old apolipoprotein E/low‐density lipoprotein receptor‐deficient mice, at the stage before development of atherosclerotic plaques, impaired acetylcholine‐induced vasodilation, reduced NO production in aorta, and increased endothelial permeability were all observed; however, flow‐mediated dilation in the femoral artery was fully preserved. In 4‐week‐old mice, glycocalyx coverage was reduced and endothelial stiffness was increased, whereas glycocalyx length was significantly decreased at 8 weeks of age. Early changes in endothelial function were also featured by increased plasma concentration of biomarkers of glycocalyx disruption (endocan), biomarkers of endothelial inflammation (soluble vascular cell adhesion molecule 1), increased vascular permeability (angiopoietin 2), and alterations in hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). In 28‐week‐old mice, at the stage of advanced atherosclerotic plaque development, impaired NO production and nearly all other features of endothelial dysfunction were changed to a similar extent, compared with the preatherosclerotic plaque phase. The exceptions were the occurrence of acetylcholine‐induced vasoconstriction in the aorta and brachiocephalic artery, impaired flow‐mediated vasodilation in the femoral artery, and further reduction of glycocalyx length and coverage with a concomitant further increase in endothelial permeability. Conclusions: In conclusion, even at the early stage before the development of atherosclerotic plaques, endothelial dysfunction is a complex multifactorial response that has not been previously appreciated.

Carbon monoxide (CO) modulates several physiological functions through activation of a cGMP-dependent pathway similar to that of nitric oxide (NO). Here we investigated the possible involvement of soluble guanylate cyclase in the anti-aggregatory effect of micromolar concentrations of CO released by a novel, water-soluble, CO releasing molecule (CORM) in human platelets. Human platelet aggregation was induced by collagen or thrombin, and the effects of CO releasing molecule (CORM-3) and an NO donor on platelet aggregation were compared. CORM-3 liberated CO in a time- and concentration-dependent manner as evidenced by the formation of carbon monoxy myoglobin (MbCO) using a spectrophotometric assay. When added to washed platelets, CORM-3 (10-300 microM) inhibited collagen- and thrombin-induced aggregation in a concentration-dependent manner. The anti-aggregatory effect of CORM-3 was reversed by deoxy-Mb (50 microM). Interestingly, in the presence of an inhibitor of guanylate cyclase (ODQ, 5 microM), inhibition of collagen-induced aggregation by CORM-3 was not blocked but potentiated. Under the same experimental conditions, inhibition of platelet aggregation by an NO donor (SNAP, 1 microM) was prevented by ODQ. In collagen-induced or thrombin-induced platelet aggregation, a stimulator of guanylate cyclase (YC-1, 0.3 microM) did not alter the effect of CORM-3, whereas it markedly potentiated the inhibition of platelet aggregation mediated by SNAP. Notably, CORM-3-induced inhibition of platelet aggregation was of similar degree when platelets were activated by a low (20 mU/ml) or by high concentration of thrombin (100-200 mU/ml), whereas NO donors (SNP and SNAP)- or carbaprostacylin (cPGI(2))-induced effects were considerably attenuated when platelets were activated by high concentrations of thrombin. Inhibition of platelet aggregation by CO released by a novel, water-soluble CORM is not mediated by activation of soluble guanylate cyclase. In contrast to NO and PGI(2), CO effectively inhibits platelets even when cells are activated excessively. We suggest that despite the fact that CO is not a potent inhibitor of platelet activation, it may gain importance when NO and PGI(2) alone are insufficient to overcome excessive platelet activation.

Involvement of phagocyte NADPH oxidase in host defense response is well established. In contrast, little is known about the functional role of NADPH oxidase in platelets. In this study, we analyzed involvement of platelet NADPH oxidase in aggregation of human platelets and in amplification of production of reactive oxygen species (ROS) by activated human neutrophils. Apocynin, a known NADPH oxidase inhibitor, as well as superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-1-pyridyl)porphyrin, inhibited ROS generation by collagen-activated platelets, collagen-induced aggregation of platelets, as well as collagen-induced release of thromboxane B₂. These data suggest the key role of intracellular ROS derived from NADPH oxidase in the control of thromboxane A₂ (TXA₂) production in platelets stimulated by collagen. Apocynin also inhibited thrombin-induced ROS production and thrombin-induced platelet aggregation. Activation of neutrophils with latex resulted in an outburst of ROS that was inhibited by apocynin. ROS production by latex-stimulated platelets was modest and also inhibited by apocynin. However, when a mixture of platelets and neutrophils was stimulated with latex, ROS production was three to six times higher in comparison with activation of neutrophils alone. Platelet-dependent augmentation of neutrophil ROS production was abrogated by TXA₂ synthase inhibitor (furegrelate, 1 μM) or by aspirin (300 μM). In summary, NADPH oxidase in platelets seems to play a major role as an intracellular signaling mechanism in the activation of platelets. However, in host defense response involving neutrophils and platelets, platelets enhance ROS production by neutrophils and possibly their cytotoxic potential via the release of TXA₂, which in turn in platelets is not affected by the extracellular release of free radicals.