BLNK/BTK: Novel Components in the NF-kappa B Pathways of Endothelial Cells in Diabetic Condition

Abstract Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart that most commonly affects the lower extremities. The effects of PAD-related ischemia are exacerbated under diabetic hyperglycemic conditions. Under ischemic conditions, an adaptive induction of the NFkB pathways is in vascular endothelial cells is required for recovery. We have recently shown that prolonged exposure of cells to high glucose before ischemia resulted in impairment of the canonical NFkB pathway through decrease in IkBa degradation. However, the signaling pathways involved in hyperglycemia and ischemia mediated effects on the NFkB pathways are not well understood. Since the NFkB signaling pathways propagate through a cascade of phosphorylation events, 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 (LG) or high glucose (HG) were subjected to ischemia for 24 hours (LGI and HGI, respectively). Cell lysates were then incubated with the array of antibodies printed on glass slides (Full Moon Biosystems, Sunnyvale, CA) 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 LG and LGI samples, and HG and HGI were calculated by dividing the phosphorylation ratio of the LGI and HGI with that of the LG and HG controls, respectively. A threshold of 1.5-fold increase or decrease was used to determine changes. Compared to the LG, LGI samples had 26 protein sites with increased phosphorylation whereas 36 sites had decreased phosphorylation. Similarly, compared to HG, HGI samples increased phosphorylation of 25 protein sites and decreased phosphorylation of 40 sites. A Venn-diagram analysis of LGI and HGI sites revealed 8 sites with an increase and 12 sites with a decrease in phosphorylation were specific to HGI. Pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK) that are critical for B cell antigen receptor (BCR)-coupled signaling. BTK expression in EC was confirmed by immunoblotting. Inhibition of BTK by a specific inhibitor terreic acid restored IkBa degradation in EC grown in high glucose suggesting a critical role of BLNK/BTK in diabetic ischemia. Thus, we have identified BLNK/BTK as potentially new components of the NFkB pathway in endothelial cells that contributes to the poor recovery during hyperglycemic ischemia.