Association of Inflammatory Transcription Factors in Human Blood Outgrowth Endothelial Cells and Development of Stroke in Sickle Cell Disease

Abstract

The pathogenesis of Sickle Cell Disease (SCD) is understood to be due to systemic inflammation and vaso-occlusion. To better understand the endothelial cell biology of stroke in SCD, Blood Outgrowth Endothelial Cells (BOEC) were harvested from donors at risk (AR) and not at risk (NAR) of developing ischemic stroke due to circle of Willis (CoW) occlusive disease. BOEC RNA array data has recently revealed transcription factors (TF) involved in inflammation to be among the top genes differentially expressed in BOEC from patients with SCD. We assessed levels of pro-inflammatory NFkB-RelA and Ets Variant gene 5 (ETV5) as well as the anti-inflammatory transcription factors KLF2 and RFX1 in BOEC. After IL1β/TNFα stimulation at various timepoints, TF levels were then assessed by flow cytometry or Western Blot. Levels of activated RelA (phosphorylated S276) are higher in AR than in normal and NAR patients by flow cytometry, and ETV5 levels higher in AR than in normal or NAR by western blot. ICAM-1, a key surface molecule upregulated during inflammation, increased greater than 2 fold in AR and NAR compared to normal over 24 hours by flow cytometry. These results suggest AR patients have higher levels of active RelA, ETV5 and ICAM-1 compared to normal after stimulation. Conversely, levels of anti-inflammatory KLF2 and RFX1 are lower in AR and NAR by Western Blot, and the ratio of KLF2 to RelA in AR and NAR is lower than in normal BOEC. Decreased KLF2 and RFX1 activity in AR and NAR patients further suggests an imbalance of pro and anti-inflammatory programs in SCD endothelium, and an inability to properly dampen the exaggerated inflammatory response in patients AR for stroke.