| Popis: |
Thrombospondins (TSPs) are conserved, multi-domain, extracellular glycoproteins that participate in many biological processes. TSP-1 is present in low levels in the extracellular matrix (ECM) of healthy vessel walls and is up-regulated during neointima and plaque formation. TSP-1 induces vascular smooth muscle cell (SMC) proliferation and migration upon vascular injury or during atherosclerosis. The aims of this' dissertation were: 1) to identify molecular mechanisms and interactions of the C-terminal, L-Iectin domain that mediates ECM deposition of TSP-1; 2) to examine the functional role of ECM-deposited TSP-1 in vessel wall ECM assembly. From cell culture experiments to identify how TSPs accumulate in ECM, a novel mechanism of trans inter-molecular interactions between TSP molecules within ECM is proposed. This mechanism depends on a specific motif in the L-Iectin domain and can act through either homophilic (TSP-1- TSP-1) or heterophilic (TSP-1-TSP-5) interactions to mediate ECM accumulation of TSPs as puncta. Fibrillar collagen may stabilise these puncta in ECM, as inhibition of fibrillar collagen production in fibroblasts decreased the level of TSP-1 in ECM but did not alter puncta patterning. Because only trimeric TSP-1 is retained in ECM, the basis of trimerisation was investigated by use of synthetic peptides derived from the coil-coiled region of TSP-1. Biophysical methodologies proved that a coiled-coil domain peptide, without any N-terminal cysteines, was stably trimeric in solution. Lastly, the role of TSP-1 in healthy aortae was examined with reference to the organisation of aortic walls in wild-type mice, Thbsl -/- mice and mice with transgenic overexpression of TSP-1 in vascular SMC. 10 to 12-week old mice of both genders were included in each set and measurements were made from the ascending and the thoracic aorta. The total width of elastin layers was increased in Thbs1 -/- mice and layers were more irregular. Similar perturbations were identified in aortae from the TSP-1 transgene overexpression mice. Thus, either increasing or decreasing TSP-1 perturbs the aortic wall ECM. The new mechanisms identified might also have implications for atherosclerosis and cardiovascular disease. |
