Date of Award

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Jim C. Oates

Second Advisor

Gary Gilkeson

Third Advisor

Samar Hammad

Fourth Advisor

Robin Muise-Helmericks

Fifth Advisor

Bruce Hollis

Sixth Advisor

Zihai Li

Abstract

Patients with systemic lupus erythematosus (SLE) have an increased propensity for developing endothelial dysfunction. Declines in endothelial derived NO production as evidenced by decreases in brachial artery flow mediated dilation, suggest that inadequate NO levels may be one mechanism whereby vascular homeostasis is perturbed in SLE patients. NO modifies progression to atherogenesis through inhibition of platelet aggregation, smooth muscle cell proliferation, adhesion molecule expression, and prevention of oxLDL. Recent studies suggest that Type I interferons and oxidative stress, leading to endothelial nitric oxide synthase uncoupling, are critical for accelerated vasculopathies observed in SLE. We postulate that two potential mechanisms are responsible for abnormalities observed in SLE patients, as chronic inflammation has pleiotropic affects. (1) Due to the chronic immune response, increases in oxidative stress persist within the endothelial milieu in vivo. Inflammatory and injurious states enhance vascular permeability, cytotoxicity, and inflammatory cell infiltration. While previous reports in Framingham risk populations have implicated a role for eNOS uncoupling in advanced atherogenesis, studies examining these mechanisms in SLE are limited. Analysis of the effects of SLE sera on eNOS in vitro revealed an SLE-mediated induction of eNOS that paralleled patient age and protein kinase R expression. Induction of eNOS expression in non-autoimmune populations implicated a role for increased hydrogen peroxide associated with eNOS uncoupling. To support this hypothesis, flow cytometry analysis of NO production in SLE sera cultured HUVECs demonstrated a profound reduction in NO that was partially restored with the addition of L-sepiapterin, an analog of the eNOS co-factor, tetrahydrobiopterin. We investigated correlations between NO production and eNOS mRNA levels in vitro and found no relationship between basal NO, however, there was a strong positive association between NO from cultures supplemented with L-sepiapterin and eNOS. Thus, enhanced NO production in response to L-sepiapterin may explain a role for eNOS uncoupling and therapeutic possibilities for recoupling in SLE endothelial dysfunction. Changes in NO also coincided with enhanced neutrophil adhesion to the endothelial cell surface and neutrophil chemotaxis. (2) Despite evidence that SLE sera modulates eNOS and NO production, specific inflammatory factors responsible for this phenomenon remain unclear. Previous reports showing that Type I interferons negatively associated with endothelial dysfunction in concert with our preliminary studies showing a correlation between eNOS and PKR gene expression incites the hypothesis that IFNα may negatively modulate eNOS and NO production. Inhibition of eNOS expression with the addition of IFNα was both time and dose dependent that correlated with PKR and MX1 induction. As a downstream consequence, we observed that protein, NO, and insulin induced NO levels were decreased in response to IFNα treatment. Additional pro-atherogenic changes occurred in response to IFNα stimulation in endothelial cells including decreased cGMP and ICAM-1, VCAM-1, and MCP-1 induction. However, leukocyte adhesion did not change. Finally, we examined used PKR and NFκB knockdown studies to demonstrate that the removal of NFκB but not PKR, mediates IFNα effects on eNOS mRNA. In lieu of IFNα mediated declines in eNOS stability, our investigation into microRNA 155, a known eNOS gene de-stabilizer, revealed the novel finding that IFNα enhances miR155 expression which may promote eNOS dysfunction and subsequent increases in endothelial dysfunction. Taken together, these results indicate that SLE sera and factors previously identified to be associated with endothelial dysfunction negatively impact NO production and eNOS expression in vitro. Due to differential expression of eNOS modulated by rIFNα and SLE serum, we hypothesize that crosstalk between oxidative stress and IFNα downstream signals may promote the accelerated phenotype observed in SLE patients. Further, therapeutic targets that alleviate oxidative stress and prevent IFNα signaling may be beneficial in improving cardiovascular disease outcomes.

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