Accelerated atherosclerosis is initiated by severe vascular endothelial dysfunction (VED), a phenotype common amongst systemic lupus erythematosus (SLE) patients. Although causes of VED are multifactorial, all pathways converge on the diminished activity of endothelial nitric oxide synthase (eNOS) expression and loss of NO bioavailability important for blood vessel protection. Recent studies indicate that interferon (IFN) -alpha contributes to the development of VED in SLE subjects by impairing endothelial repair mechanisms however, studies are missing that indicate a role for IFN-alpha in vascular damage. We recently showed that treatment of human aortic and umbilical vein endothelial cells (HAECs/HUVECs) with IFN-alpha leads to decreased eNOS expression and phosphorylation. Therefore, the purpose of the proposed study is to tease out mechanisms responsible for type I interferon-mediated inhibition of eNOS expression and activation in an in vitro HUVEC/HAEC cell culture model. We intend to test our hypothesis that IFN-alpha causes diminished eNOS expression/activity by 1) decreasing transcription factor interaction with the eNOS promoter region and by 2) causing increased protein phosphatase 2A activation and/or decreased Akt (protein kinase B) expression. To test this hypothesis we will address the following specific aims: first analyze changes in eNOS promoter region activity, mRNA stability, and transcription factor binding. Secondly, we will quantify changes in different phosphatases and kinases that may attribute to changes in eNOS activation. Finally, we will measure NO and cyclic guanosine monophosphate (cGMP) levels following IFN-alpha treatment to ascertain its impact on functional eNOS outputs. This series of studies are significant because they will provide ample evidence for the role of type I IFNs in compromising endothelial function. The proposed studies accompanied by robust coursework, scientific meeting and seminar presentations, and interactions with other scientist will provide a training plan that will lead tothe applicant's success as an academician. Coursework will address advanced topics in immunology and vascular disease, complementing previous courses including biostatistics, grant writing, scientific ethics, and pharmacology. The student will also work with NO expert, Dr. Philip Shaul to learn techniques currently not available in the laboratory and present work at conferences including the American College of Rheumatology Conference, Society for Free Radical Biology and Medicine Conference, and one of the many Gordon Conferences. Finally, the student will submit work for publication in peer-reviewed journals.
Patients with systemic lupus erythematosus (SLE) have increased interferon signature, a phenotype recently associated with vascular endothelial dysfunction in this patient population. The purpose of this study is to determine how interferon alpha compromises the endothelium to override protective mechanisms innate to the vasculature. Findings from this study will allow researchers to develop novel biomarkers that predict disease occurrence as well as therapeutics that may specifically target vascular disease in SLE and related- autoimmune disease populations.