Abstract 337: Inhibiting Thrombospondin-1 Activation Of Transforming Growth Factor Beta Under Oscillatory Shear Stress Decreases Connective Tissue Growth Factor And Neurogenic Locus Notch Homolog Protein 1

Abstract

Peripheral arterial disease (PAD) is the 3 rd cause of atherosclerotic disease morbidity with an incidence of 12-20% in individuals over 65yrs. PAD arteries are stiffer than healthy ones and they present disturbed flow, defined by low and oscillatory shear stress (OS), in the vicinity of obstructions. Previous work from our group discovered the upregulation of thrombospondin 1 (TSP-1) and profibrotic genes in HAECs exposed to OS. Furthermore, TSP-1 is an activator of the transforming growth factor beta (TGF- β), which has shown to regulate connective tissue growth factor (CTGF) expression (Fig. 1A). CTGF has been associated with profibrotic signaling. There is a pressing need to understand the involvement of CTGF in the TSP-1 mediated profibrotic signaling occurring in HAECs under disturbed flow. We hypothesize that, inhibiting the activation of TGF-β via TSP-1 mediation will lead to a decrease in CTGF expression in HAECs exposed to disturbed flow. Female HAECs were treated to block the TSP-1-mediated activation of TGF-β with LSKL, a competitive inhibitor of TSP-1, while SLLK, a scrambled protein, was used as the control treatment. Then, the HAECs were subjected to low shear stress for 24 hrs followed by gene expression quantification using qPCR. Blocking the activation of TGF-β via TSP-1 mediation led to the significant downregulation of CTGF and NOTCH1 (Fig. 1B and 1C) in HAECs subjected to low shear stress when compared to the control group. Similarly, there was a decrease in the expression of collagen genes in the group treated with LSKL (not statistically significant). In conclusion, using an in vitro system for applying low shear to HAECs, we discovered that CTGF expression was significantly reduced when TSP-1 mediation was inhibited. The same occurred with NOTCH1, a gene associated with protection against inflammation. Future studies will investigate if these gene expression patterns are observed at later time points or if it is part of an early response to low shear.