Dissecting the Roles of Reactive Oxygen Species in Cardiovascular Diseas

Cardiovascular disease remains the leading cause of death in the USA. While much has been learned about the root causes, the underlying mechanisms remain incompletely understood. In particular, elevated levels of reactive oxygen species (ROS) have been observed in the vasculature of blood vessels from animal models and humans with hypertension, atherosclerosis and diabetes. The importance of ROS to cardiovascular disease and the mechanisms by which it alters the function of cells of the cardiovascular system are the goals of this dissertation.;The NADPH oxidases (NOX) represent a family of 7 transmembrane enzymes that share a basic structural paradigm and the common ability to utilize NADPH to synthesize superoxide and other reactive oxygen species (ROS). NOX isoforms are distinguished by their amino acid sequences, expression in different cell types, how production of superoxide is initiated and the type of ROS that are generated. Cardiovascular roles of Nox1, Nox2 and Nox4 have been described in atherosclerosis, heart failure, hypertension and diabetes. NOX5 was the last NOX family member identified and it is unique for the presence of 4 helix-loop-helix structural domains which is also called EF-hands, that confer calcium-dependent regulation of reactive oxygen species (ROS) generation. Altered expression and activity of Nox5 has been reported in cardiovascular diseases and cancers, but a functional role in cardiovascular disease has been hampered by the lack of appropriate genetic models, as Nox5 is missing from rodent genomes, and the absence of selective inhibitors. Multiple polymorphisms have been identified within the coding sequence of human Nox5, but whether this translates into altered enzyme function is unknown.;Herein, our first aim is to investigate the relationship between SNPs in the coding region of Nox5 and possible changes in enzyme activity. We have generated 15 novel mutants of Nox5beta to evaluate the effect of exonic SNPs on basal and stimulated enzyme activity. Compared to the WT enzyme, ROS production was unchanged or slightly modified in the majority of mutants, but significantly decreased in 7. Focusing on M77K, Nox5 activity was dramatically reduced in unstimulated cells and following challenge with both calcium- and phosphorylation-dependent stimuli despite equivalent levels of expression. The M77K mutation did not influence the Nox5 phosphorylation or the ability to bind Hsp90, but in cell-free assays with excess co-factors and calcium, ROS production was dramatically reduced. A more conservative substitution M77V arising from another SNP, yielded a different profile of enzyme activity and suggests a critical role of M77 in calcium-dependent ROS production. Two C-terminal mutants, R530H and G542R were observed that had little to no activity and relatively MAF. In conclusion we have identified 7 missense SNPs in Nox5 that result in little or no enzyme activity. Whether humans with dysfunctional Nox5 variants have altered physiology or disease remains to be determined.;The cardiovascular system is subject to circadian regulation. A molecular clock exists in both human and animal cells that orchestrates the 24 hour timing of biological processes. One of the best known examples is the daily oscillation of blood pressure which in humans, peaks during the day and dips at night. These rhythms are important to the health of the organism and have evolved to anticipate the timing of physiological demands and stress. Disruption of circadian rhythms results in increased cardiovascular diseases in animals and is associated with increased risk of death in humans including exaggerated vascular remodeling, atherosclerosis and impaired blood pressure control. The mechanisms underlying dysfunction of circadian regulation in cardiovascular disease are not known.;The goal of our second aim is to determine whether the circadian pattern of gene expression might be influenced by inflammatory stimuli and that loss of circadian function in inflammatory cells can predispose to cardiovascular disease. To investigate circadian rhythms in inflammatory cells, peritoneal macrophages were isolated from Per2-luciferase transgenic mice and circadian oscillations were studied in response to stimuli. Using Cosinor analysis, we found that LPS significantly altered the circadian period in peritoneal macrophages from Per2-luc mice while real-time PCR data suggested that the pattern of expression of the core circadian genes (Bmal1 and Per2) was disrupted. Inhibition of the TLR4 offered protection from the LPS induced impairment in rhythm, suggesting that the LPS effect occurred through the toll 4 receptor pathway. To explore the mechanisms involved, we inhibited LPS-stimulated NO and superoxide. Inhibition of NO synthesis with L-NAME had no effect on circadian rhythms. In contrast, inhibition of superoxide with TEMPOL or PEG-SOD mitigated LPS-induced changes in circadian periodicity. In gain of function experiments, we found that overexpression of Nox5, a source of ROS, could significantly disrupt circadian function in U2OS cells (a useful circadian reporting cell line) whereas iNOS overexpression, a source of NO, was ineffective. To assess whether alteration of circadian rhythms influences macrophage function, peritoneal macrophages were isolated from Bmal1-KO and Per-TKO mice. Compared to WT macrophages, macrophages from circadian knockout mice exhibited altered balance between NO and ROS release, increased uptake of oxLDL and increased adhesion and migration. These results suggest that pro-inflammatory stimuli can disrupt circadian rhythms in macrophages and that impaired circadian rhythms may contribute to cardiovascular diseases by altering macrophage behavior.;The overall conclusion of these studies is that inactivating polymorphisms in the Nox5 gene are relatively common in certain human populations. Whether loss of Nox5 activity alters susceptibility to cardiovascular disease is not yet known. We also identify superoxide as a potent mechanism that can disrupt circadian rhythms in immune cells. Loss of circadian rhythms in macrophages promotes a bias towards increased superoxide production, increased adhesion and lipid accumulation which may contribute to the development of cardiovascular disease.