Mechanisms of Angiogenesisand Lymphangiogenesis in Calcific Aortic Valve Stenosis

Aortic valve stenosis (AVS) is the most common valvular disease in Western countries. Pharmacological prevention of AVS having proved unsuccessful, its current treatment is still valve replacement. The etiology of AVS is multifactorial, both genetic and external risk factors predisposing to the active pathological process eventually leading to clinically manifest stenosis. Histological features of the disease resemble those of atherosclerosis, including the accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Furthermore, valvular interstitial cells undergo phenotypic differentiation into actively proliferating myofibroblasts, which contribute to the local inflammatory response as well as extracellular matrix remodeling in stenotic aortic valves. Blood vessels also grow into the normally avascular valve leaflets already in the early stages of the disease. This thesis aimed at elucidating the mechanisms behind the pathological neovascularization of the stenotic aortic valves. Furthermore, we characterized valvular lymphangiogenesis and investigated potential factors contributing to the balance between valvular angiogenesis and lymphangiogenesis, focusing on the role of valvular myofibroblasts and mast cells. For this purpose, we studied a total of 117 stenotic valves obtained at valve replacement surgery, and 49 control valves obtained at cardiac transplantations, at valve replacement surgery due to aortic valve regurgitation, or from deceased organ donors whose hearts were unsuitable for use as grafts. The valve leaflets were either used freshly for myofibroblast cell culture or frozen for e.g. PCR and immunohistochemical analyses. First, we assessed the adverse extracellular matrix remodeling of stenotic aortic valves, a process necessary for angiogenic sprouting to occur. We found that the mRNA expression levels of cathepsins S, K, and V, and their inhibitor cystatin C were higher in stenotic aortic valves than in control valves. Furthermore, the total activity of such cathepsins was increased in AVS. In immunohistochemical stainings, the expressions of cathepsin S, cathepsin V, and cystatin C localized to valvular macrophages, chondroblast-like cells, and endothelial cells lining both the valvular surface and the neovessels in the stenotic valves. Next, we characterized the neovessels and lymphatic vessels in stenotic aortic valves and control valves using immunohistochemistry. We found that in addition to immature microvessels, the stenotic aortic valves contained organized arterioles, indicating an advanced stage of angiogenesis. Lymphatic vessels correlated with valvular blood vessels, but were present in much fewer numbers. Valvular mast cells resided close to neovessels and secreted the angiogenic Vascular endothelial growth factor A (VEGF-A). Furthermore, we showed that the lymphangiogenic growth factors VEGF-C and VEGF-D are locally produced in the aortic valves, and that the receptors