Identification of Novel Flow Sensitive Transcription Factors in the Endothelium and Their Role in Atherosclerosis
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Demos, Catherine
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Abstract
Atherosclerosis is a chronic inflammatory disease wherein disturbed blood flow induces endothelial dysfunction, inflammation, and plaque accumulation in arterial walls by regulating endothelial gene expression. While Kruppel-like factor (KLF) -2 and KLF4 are known to play a dominant role in flow-dependent gene expression, the role of additional flow-sensitive transcription factors (FSTFs) remains understudied. Here, we tested the hypothesis that there are other FSTFs that play an important role in endothelial function. We identified 30 potential novel FSTFs in arterial endothelial cells in vivo by reanalyzing gene array and single-cell RNAseq datasets generated from our mouse partial carotid ligation model of disturbed flow. These transcription factors were further validated in vivo and in vitro using mice and human aortic endothelial cells (HAECs) at the mRNA and protein levels. From this study we identified seven FSTFs (KLF2, KLF4, SOX4, SOX13, SIX2, ZBTB46, and NFIL3) that are conserved in mouse and human in vivo and in vitro. Of these, we selected SOX13 for further studies because of its robust flow sensitivity and its undefined endothelial function. We found that siRNA-mediated knockdown of SOX13 reduced anti-inflammatory function of endothelial cells in response to the anti-atherogenic flow condition unidirectional high laminar shear stress (ULS) as determined by vascular cell adhesion molecule (VCAM1) expression and monocyte adhesion assay. To define the underlying mechanisms, we carried out RNA sequencing in HAECs treated with SOX13 siRNA and shear stress. We found that 94 genes were downregulated and 40 genes were upregulated in response to shear stress in a SOX13-dependent manner. Interestingly, several chemokines and cytokines including CXCL10, CXCL1, CXCL8, CSF3, CCL2 and CCL5 were found to be the major genes regulated by SOX13 and ULS. These were further validated by qPCR and ELISA. Additionally, both overexpression of SOX13 and treatment with Met-CCL5, a CCL5 competitor that does not activate immune cell adhesion, prevented siSOX13-driven inflammation. Taken together, these data show that SOX13 is a novel flow-sensitive transcription factor regulating endothelial inflammation, a major flow-dependent proatherogenic pathway.
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2022-06-21
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Dissertation