Reactive Oxygen Species
Finding the Right Balance
See related article, pages 598–605
Appropriate regulation of reactive oxygen species (ROS) has a significant impact on health and disease. ROS includes oxygen ions (O2·) free radicals (superoxide [O,2] and hydroxyl radicals), and peroxides (hydrogen peroxide [H2O2]) and are the products of normal oxygen consuming metabolic process in the body. ROS are small and highly reactive molecules with important cell signaling roles when maintained at proper cellular concentrations. During times of cell stress ROS levels can greatly increase. Because of their highly reactive nature, ROS can modify other oxygen species, proteins, or lipids, a situation often termed oxidative stress. Maintaining normal cellular ROS concentrations is, therefore, vital to the proper physiological function of numerous cell types throughout the body. An excess production or decreased scavenging of ROS has been implicated in the pathogenesis of diverse diseases such as neurodegeneration, diabetes, cancer, and atherosclerosis.
Kisucka et al now demonstrate that peroxiredoxin1 (Prdx1) has an important role in the maintenance of endothelial ROS.1 Prdx1 is an antioxidant enzyme that reduces H2O2, lipid peroxides, and peroxynitrite. Prior studies have shown that Prdx1−/− mice develop late onset hemolytic anemia and have increased frequency of cancer2 caused by an increase in ROS (such as H2O2), emphasizing the importance of Prdx1 in normal vascular homeostasis. Like many ROS, H2O2 can have disparate effects depending on the cell type and its local concentration. H2O2 can have normal regulatory functions as a second messenger molecule in signal transduction such as in the mitogen-activated protein kinase pathway. H2O2 makes a good signaling molecule because of its reactive nature and its ease of scavenging by antioxidant enzymes such as Prdx1 making for rapid signaling activation and inactivation. H2O2 can also be a source of oxidative stress and vascular injury. Excess H2O2 has been implicated in nitric oxide (NO) dysregulation and mitogenic activities that lead to intimal hyperplasia. In this study, the authors demonstrate that a loss of Prdx1 accelerates the development of atherosclerosis in part by disrupting normal regulation of Weibel–Palade body release resulting in an increase in white blood cell (WBC) interactions with the vasculature.1
ROS in Vascular Biology
Endothelial cells form the vital interface between blood constituents and the vessel wall. A loss of endothelial cells leads to thrombus formation and vessel occlusion. Alterations in maintaining endothelial cells in a quiescent state leads to an increase in endothelial cell interactions with platelets and WBC, stimulating more endothelial activation and leukocyte trafficking. Dysregulation of ROS homeostasis can lead to endothelial cell dysfunction. Vascular cells themselves are sources of ROS production. The primary enzymatic sources for vascular oxygen species production are xanthine oxidase, NADH/NADPH oxidase isoforms, and endothelial NO synthase.3 There must be a balance maintained between the production of ROS and the scavenging of ROS. Some of the important scavengers include superoxide dismutase, glutathione, thioredoxin, and peroxiredoxins.3
Oxygen species, such as NO, are important players in maintaining normal physiology. NO helps to maintain vascular tone, inhibits endothelial cell stimulation, and is a regulator of platelet activation.4,5 NO also has a key physiological role as a second messenger in cell signaling in neurons and macrophages. A lack of NO therefore can have significant physiological effects, and excessive NO can also contribute to cell injury by combining with superoxide to produce damaging peroxynitrite.
Kisucka et al demonstrated that Prdx1 has a functional role in endothelial cells and a lack of Prdx1 leads to an increase in WBC interactions with endothelial cells.1 This implies that Prdx1−/− mice have an endothelial dysfunction, including a loss of normal regulation of endothelial cell degranulation as reflected by an increase in plasma von Willebrand factor and endothelial P-selectin expression. NO has an important role in regulating Weibel–Palade body exocytosis by S-nitrosylation of the key regulatory protein N-ethylmaleimide–sensitive factor (NSF).4,5 NO modification of NSF decreases NSF ATPase activity and, thus, blocks a critical function of NSF in promoting SNARE complex disassembly and sustaining exocytosis.4 Prdx1−/− mice may therefore have an endothelial dysfunction by direct ROS effects or secondary to alterations in NO availability.
A lack of proper ROS scavenging can lead to a decrease in bioavailable NO and perhaps unchecked exocytosis. ROS can decrease NO bioactivity by directly interacting with and inactivating NO or by modifying other protein sites where NO may react, therefore decreasing its physiological influence.3 With a loss of antioxidant activity, there may be a reduction in bioavailable NO, increased endothelial exocytosis, and, with it, increased P-selectin expression and von Willebrand factor release, such as in Prdx1−/− mice. Increased endothelial exocytosis leads to increased WBC localization and vascular inflammation. Alternatively, a loss in Prdx1 and increased H2O2 may drive nuclear factor κB stimulation in endothelial cells and the elaboration of endothelial derived cytokines and adhesion molecules. It is noteworthy that Kisucka et al did not find an increase in the expression of the vascular adhesion molecules intercellular adhesion molecule and vascular cell adhesion molecule, suggesting a loss of Prdx1 has its most significant effects on the regulation of exocytosis.1 NO target protein regulation must also be maintained for normal cellular function. Recently, Benhar et al have demonstrated that nitrosylated proteins in lymphocytes are reduced by thioredoxin.6 The role of thioredoxin in endothelial cell biology and its effects on the state of NO target proteins remains to be determined.
Platelets also produce ROS and platelet physiology can be affected by ROS produced at the interface with endothelial cells. ROS produced by endothelial cells or during thrombus formation have the potential to influence platelet function locally and to lead to a further prothrombotic response.7 With agonist stimulation, platelets also produce significant amounts of ROS and the enzymatic sources in platelets include NAD/NADPH oxidase and superoxide dismutase.7 In the study by Kisucka et al, Prdx1−/− mice had no change in platelet function in vitro and in vivo.1 This does not exclude the possibility of Prdx having a role in platelet physiology. There are other Prdx family members expressed throughout the body. Platelets may express an alternative family member or its function made redundant by the expression of other reducing enzymes in platelets.
ROS and Atherosclerosis
Atherosclerosis is an inflammatory disease that begins at the vascular interface with an endothelial cell dysfunction. Many pathophysiologic processes contribute to the initiation of endothelial dysfunction, including elevated plasma cholesterol, hypertension, and diabetes. Each of these risk factors either result in an increase in vascular ROS or are exacerbated by an increase in ROS. Endothelial dysfunction results in increased endothelial cell activation and proinflammatory molecule expression culminating in inflammatory cell adhesion and migration into the developing lesion. Macrophages have a key role in atherosclerotic development and elaborate even more ROS production within the lesion. With a lack of proper ROS scavenging, the cycle of inflammation and leukocyte trafficking is accelerated.
With a sustained increase in plasma LDL and ROS species production there is an increase in oxidized (Ox)-LDL. Ox-LDL is readily removed by macrophages but, in turn, drives more macrophage stimulation. Ox-LDL also stimulates platelet activation in a platelet CD36 dependent manner8 and platelet dependent atherothrombosis is an important part of myocardial infarction. Although Kisucka et al did not investigate whether Prdx1−/− mice on an ApoE−/− background have increased thrombotic risk, it seems reasonable to propose that, unlike in Prdx1−/− mice on a normal cholesterol background, the Prdx1−/−/ApoE−/− mice may have an increase in platelet activation and thrombosis secondary to an increase in Ox-LDL.
Kisucka et al demonstrate that Prdx1−/− mice bred onto an ApoE−/− background have an exacerbation of atherosclerotic lesion development and an increase in macrophage infiltrates into the lesion.1 The results of their studies suggest that in Prdx1−/− mice persistent endothelial cell degranulation results in monocyte recruitment, trafficking, and activation. This perhaps leads to further production of ROS species that is not held in check. Interestingly, the effect of Prdx1 on atherosclerotic lesion development is more pronounced in females than males. This may suggest a difference in mechanisms of ROS scavenging in males and females or that other gender specific variables are relevant in Prdx1 function.
ROS have both beneficial and harmful effects in the vasculature. Because of their highly reactive nature ROS make rapid and transient second messenger molecules. However, this highly reactive property means that the loss of proper ROS scavenging can have deleterious effects. To minimize this potential, cells have developed multiple redundant enzymes to remove ROS, each of which may have a greater or lesser role in an individual cell type. Vascular biologists are beginning to understand the antioxidant systems such as perioredoxin that plays an important role in maintaining proper levels of oxidants within the vasculature.
Sources of Funding
Supported by NIH grants 5K08HL74945 and 1R01HL093179.
C.N.M. receives financial research support from TorreyPines Therapeutics.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
Kisucka J, Chauhan AK, Patten IS, Yesilaltay A, Neumann C, Van Etten RA, Krieger M, Wagner DD. Peroxiredoxin1 prevents excessive endothelial activation and early atherosclerosis. Circ Res. 2008; 103: 598–605.
Morrell CN, Matsushita K, Chiles K, Scharpf RB, Yamakuchi M, Mason RJ, Bergmeier W, Mankowski JL, Baldwin WM III, Faraday N, Lowenstein CJ. Regulation of platelet granule exocytosis by S-nitrosylation. Proc Natl Acad Sci U S A. 2005; 102: 3782–3787.
Benhar M, Forrester MT, Hess DT, Stamler JS. Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science. 2008; 320: 1050–1054.
Freedman JE. Oxidative stress and platelets. Arterioscler Thromb Vasc Biol. 2008; 28: s11–s16.