RF04 | PSUN163 In High Glucose Exposed Endothelial Cells BTK is a Novel Component of the Nuclear Factor-Kappa B Pathway

Abstract

Abstract Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFkB signaling in ischemic endothelial cells (EC). We have shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFkB pathway through activation of protein kinase C β (PKC β). However, the signaling pathways upstream of PKC β involved in this high glucose induced desensitization of NFkB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFkB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spot by the signal intensity of the corresponding non-phosphorylated spot for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton's tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling but not known to be expressed in ECs or play a role in EC function. Here we confirmed BTK expression in ECs by immunoblotting. Inhibition of BTK by a specific inhibitor terreic acid or by shRNA mediated knockdown decreased PKCβ-S661 phosphorylation and restored NFkB activation in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as a potentially new component of the NFkB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.