Assessment of anti-inflammatory synthetic vascular grafts in a novel mouse model

Abstract

Cardiovascular disease is the largest cause of mortality in the world with coronary artery disease (CAD) accounting for half of these mortalities. One of the main treatment options for CAD is the surgical bypass of the blocked vessels, in a procedure known as coronary artery bypass grafting (CABG). The best performing conduits used for CABG are autologous arteries and veins harvested from the patient. However, for a large portion of patients requiring CABG, these autologous grafts are not available due to comorbidity affecting the viability or prior use. Current synthetic vascular grafts uniformly fail in low diameter applications (< 6mm) including for bypass of the coronary arteries. Significant and ongoing research in regenerative medicine and tissue engineering aims to develop new synthetic conduits, effective for low diameter revascularisation to meet this need. This thesis focuses on two important deficits in the development of synthetic vascular grafts: 1) Lack of an appropriate animal model for screening novel materials that enables the use of transgenic tools widely available in other fields of biology. 2) Synthetic graft functionalisation that addresses chronic inflammation, a key driver of failure. The first part of this thesis characterises a mouse model of small diameter vascular grafting implanted at the carotid artery position. A cuff anastomosis technique was used to implant electrospun polycaprolactone (PCL) synthetic vascular grafts in the right common carotid artery of mice. Histological analysis of explanted grafts at three timepoints up to 28 days showed significant neointimal hyperplasia at the anastomoses, decreasing towards the middle, reflective of human pathology. Further analysis showed temporal increases in smooth muscle cell (SMC) and collagen content within the neointima, demonstrating its maturation. Endothelisation assessed by scanning electron microscopy and immunohistochemistry showed near complete coverage within 28 days. Abstract xvi The second part of thesis explores two novel functionalisation strategies to address inflammation in synthetic vascular grafts. Viral mimicry of inflammation suppression was trialled using a broadspectrum CC chemokine inhibitor known as 35K. An in vitro assay designed to recapitulate inflammation driven SMC proliferation did not show the expected anti-inflammatory effects. 35K was functionalised on electrospun PCL scaffolds via two methods, immobilised by plasma immersion ion implantation (PIII) or passively adsorbed, and assessed in a mouse subcutaneous implant model to measure the inflammatory response in vivo. Macrophage response was only significantly different at early timepoints for the passively adsorbed scaffold, but this did not translate to a functional benefit when assessing the fibrotic response. Next IL-4 was investigated as an immunomodulation agent for biomaterial functionalisation. In vitro assays were performed to confirm immobilisation on electrospun PCL scaffolds and activity. Similarly, in vivo assessment in mouse subcutaneous model was carried out. PIII+IL-4 was found to have striking effects to the macrophage response in terms of number of macrophage and polarisation to M2 phenotype, which lead to significant effects on the fibrotic response. Examining PIII+IL-4 further in the mouse grafting model, histological analysis reflected results of the subcutaneous model, with decreases of macrophage and M2 polarisation. Cytokine expression surrounding the PIII+IL-4 grafts showed increased anti-inflammatory cytokines and decreased inflammatory cytokines relative to PCL and PIII controls. Finally, functional benefits of PIII+IL4 were clearly observed compared to PCL control, where both neointimal hyperplasia and fibrotic response was markedly reduced. Finally, to further demonstrate the potential of this mouse model, two pilot studies were conducted with transgenic mice. Longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain, FVB L2G, with a dual reporter construct encoding both luciferase and green fluorescent protein. This enabled characterisation of mononuclear cell homing and engraftment to PCL grafts using bioluminescence imaging and histological staining over time. Peak luminescence was observed at 7 days post implantation that persisted until sacrifice at 28 days. GFP staining Abstract xvii confirmed the presence and localisation of the cells. To assess vascular graft in the context of disease states, apolipoprotein E knockout mice were grafted to reflect dyslipidaemia, the most common comorbidity of CABG patients.