Obesity is a major risk factor for cardiovascular (CV) disease, in large part due to an altered metabolic state. An early precursor to CV disease is the loss of endothelial function and impaired nitric oxide (NO) signaling. NO is vital to many processes, but is best known as the mediator of endothelium-dependent relaxation. NO is synthesized by endothelial nitric oxide synthase (eNOS) and dysregulation of eNOS has been shown to occur, at least in part, through changes in its phosphorylation status. The biological actions of NO can be further compromised via inactivation by superoxide generated by NADPH oxidases (NOX) and other enzymes. Our lab has previously shown that an imbalance between NO and superoxide-generating pathways is responsible for loss of endothelial function in obesity. However, the signals coordinating loss of NO and increased superoxide with altered metabolism remain poorly defined. We have found that expression of the glycolytic enzyme PFKFB3 is increased in endothelial cells (EC) of obese mice. Further, increased expression of PFKFB3 via EC-specific adenoviral transduction of mouse blood vessels is accompanied by the loss of endothelial function and decreased NO production in cultured cells. Loss of NO is accompanied by increased eNOS phosphorylation of T495, an inhibitory PKC site, blunted Akt-S473 phosphorylation and reduced eNOS phosphorylation of S1177. In addition, PFKFB3 increased the activity and mRNA level of NOX1, a major contributor to endothelial dysfunction (ED) in obesity. Based on our preliminary findings we hypothesize that in obesity, increased PFKFB3 expression contributes to impaired vascular function by suppressing vascular protective signaling mechanisms and increasing damaging ROS production. Thus, we are proposing to determine whether high glucose is responsible for PFKFB3 upregulation and to define its role in regulating PKC, Akt and in eNOS phosphorylation. In CRISPR-Cas9 modified human ECs lacking eNOS expression, we will use mutants of eNOS phosphorylation sites to confirm the functional importance of these sites to PFKFB3 mediated decreases in NO. We will also investigate the role of glucose and PKC in the regulation of NOX1 activity and expression and test whether PFKFB3 is a mediator of obesity-driven ED in vivo. This will be achieved via the generation of a novel EC-specific PFKFB3 knockout mouse on a leptin receptor deficient background and analysis of endothelial function and ROS production.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|