About the Project
The long saphenous vein (LSV) is the most commonly used conduit in cardiac surgery to treat patients with coronary artery disease; however, its use is complicated by high failure rates due to the development of vascular inflammation, intimal hyperplasia (IH) and accelerated atherosclerosis. It has been reported that 50% of vein grafts become occluded within 10 years after surgery and the remaining 50% have variable but significant degrees of IH. Consequently, vein graft failure is a serious problem which has a major impact on patient’s outcomes and health economics. No treatment has yet been identified that can effectively modulate the development of IH in vein grafts. We are interested in the important initial adaptation of the vein after grafting and the impact of sudden changes flow patterns and environment on endothelial cell (EC) function, integrity and interaction with circulating monocytes. Veins are usually subjected to low shear stress in the body; however, when grafted into arterial circulation they suddenly become exposed to high levels of shear stress which results in endothelial cell (EC) activation and monocyte adhesion. We have recently demonstrated that monocyte adhesion and initiation can be suppressed by inhibition of Wnt/b-catenin signalling.
Aims and Objectives
We hypothesize that the exposure of venous ECs to acute high shear stress results in the activation and adherence of circulating monocytes which is regulated by Wnt/b-catenin signalling. Inhibition of this signalling in vivo has the capacity to reduce acute inflammation and improve vein graft failure. Furthermore, we will address whether pharmaceutical suppression of Wnt/b-catenin signalling attenuates monocyte adhesion to EC in vitro and in our novel human ex vivo bioreactor model of acute high shear stress and ultimately in vivo using our established large animal model.
The research will be performed at the Bristol Heart Institute. Human venous ECs will be exposed to laminar, unidirectional high shear stress (at 12 dyn/cm2) for varying times, using established parallel plate flow chambers designed in-house or maintained in static conditions. Following application of shear stress, venous ECs will be analysed by proteomic analysis, Western blotting, RT-qPCR, Luminex multi-plex ELISA, and immunocytochemistry and confocal microscopy. Monocyte adhesion to venous ECs will be assessed in real-time using the specialised Bioflux 200 microfluidic channel system (Fluxion) which enable live imaging of monocyte adherence and rolling. Ex vivo experiments will use surplus segments of human LSV retrieved from coronary artery bypass patients. These will be cultured under static or HSS (12±0.2dyn/cm2) using an in-house designed bioreactor. Segments of human LSV will be treated with different pharmaceutical inhibitors or infected with recombinant viral vectors to modify gene expression prior to exposure to high shear stress or maintenance under static condition then incubated with Calcein AM-labelled monocytes then imaged to determine monocyte invasion and adherence to vein segments. Optimisation of delivery approaches will be determined by comparing microspheres, nanoparticles and hydrogels to enable optimal translation to our in vivo study.
Ultimately, we will deliver the optimal pharmaceutical inhibitors to vein grafts in our porcine vein graft in vivo model and quantify suppression of monocyte adhesion and EC activation at several timepoints after implantation. This work will determine the feasibility of translating this approach to patients undergoing bypass surgery.
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