The overall goal of this proposal is to dissect the role of the endothelial glucocorticoid receptor (GR) in states of vascular inflammation, such as atherosclerosis. Exciting preliminary data generated through genomic sequencing experiments shows that endothelial GR interacts with a number of genes present in the Wnt signaling pathway. The Wnt pathways have recently been implicated in the pathogenesis of atherosclerosis and other states of vascular inflammation though the mechanisms remain unstudied thus far. To investigate the regulation of Wnt signaling by endothelial GR we propose three Specific Aims.
In Aim 1 we will take advantage of novel data created in the course of GR-ChIP-seq experiments performed in mouse lung endothelial cells which show a relative enrichment of GR binding in genes relevant to the Wnt signaling pathway as well as a novel motif containing a putative glucocorticoid response element (GRE) which has not previously been described. To further characterize these findings we propose RNA-seq of GR in endothelial cells to gain direct information about gene expression patterns. We will also use cloning techniques followed by luciferase assays to definitively identify GREs in endothelial cells.
In Aim 2 we plan to evaluate the in vitro phenotypes of endothelial cells exposed to Wnt ligands that induce both the canonical and non-canonical Wnt signaling pathways. Assays to be employed include cell permeability assays, expression of pro-inflammatory cytokines and adhesion molecules and measurement of reactive oxygen species. By testing cells in the presence and absence of dexamethasone, a synthetic steroid acting through GR, the regulation of Wnt by the endothelial GR in the context of inflammation can be examined in depth. Finally, in Aim 3 we will use our established mouse model of atherosclerosis in which mice lacking endothelial GR have been bred onto an ApoE knockout background. We have previously shown that these mice develop a more severe atherosclerosis phenotype compared to Apo E knock out mice alone. We will directly measure levels of several Wnt related proteins in atherosclerotic lesions and in serum. In addition we propose the generation of a novel mouse model that allows direct visualization of canonical Wnt signaling after Xgal staining by crossing our mice with the Bat gal reporter strain.
Atherosclerosis and other vascular inflammatory disorders are a significant and widespread public health problem. This proposal seeks to improve understanding of the molecular mechanisms leading to vascular inflammation by examining the role of the endothelial glucocorticoid receptor in this process. The proposed work has the potential to influence development of new therapeutic options for these conditions.
|Srivastava, Swayam Prakash; Li, Jinpeng; Kitada, Munehiro et al. (2018) SIRT3 deficiency leads to induction of abnormal glycolysis in diabetic kidney with fibrosis. Cell Death Dis 9:997|