Lay summary
Background: Coronary artery disease (CAD) is the leading cause of death among adults in industrialized countries. CAD is based on an inflammatory process with subsequent formation of atherosclerotic plaques, a process called atherogenesis, which leads to narrowing and occlusion of coronary arteries. Established options to treat CAD include angioplasty and bypass surgery, both of which are not suitable in 1/5 to 1/3 of patients in whom the extent of atherosclerosis is especially severe. An alternative treatment strategy is therefore warranted both to control symptoms as well as to alter the course of CAD. Among the population of CAD patients, one third does have well developed collateral vessels. Hence, the therapeutical promotion of coronary collateral arteries, a process called arteriogenesis, in patients with poorly developed collateral vessels is an interesting option. Vascular endothelial cells (ECs) play a switch-board function in athero- and, likely, also in arteriogenesis. Both, athero- and arteriogenesis, develop in response to a complex interaction of fluid shear stress at the vascular endothelium followed by recruitment of blood cells (primarily monocytes) with subsequent activation of inflammatory processes. However, they result in completely different outcomes. The study of cell biology of ECs derived from patients with well or poorly developed collateral arteries may elucidate biological mechanisms necessary to induce arteriogenesis.Working hypothesis: In an in-vitro flow model, ECs derived from patients with sufficient collateral blood flow are more competent in their ability to induce arteriogenesis-like processes, as determined by the ability to home monocytes and degrade extracellular matrix proteins, than ECs from patients with insufficient collateral vessels.Aims:1) To establish cell cultures of ECs, differentiated from circulating endothelial progenitor cells (ECPs) which are found in the peripheral blood, from CAD patients having well and poorly developed collateral arteries.2) To establish an in-vitro flow model with EC cultures.3) To test the ability of different EC cultures to initiate primary arteriogenesis-like processes in response to fluidic shear stress activation as quantified by the number of homed monocytes.4) To quantify the arteriogenic potential of laminar shear stress activated ECs from both study groups in an in-vitro cell co-culture system by the release of proteolytic fragments of extracellular matrix proteins.5) To characterize the protein expression profile of ECs from both study groups cultured under steady fluidic shear stress conditions.Experimental design: Coronary collateral flow index (CFI) has been proposed and extensively validated for measuring coronary collateral flow.
EC’s are derived from ECP’s present in peripheral blood. Blood samples will be collected from patients who were admitted to the Cardiology Clinic at the University Hospital Bern due to angina pectoris symptoms, and who have given written consent to participate in this study. CFI will be determined by measuring simultaneously aortic pressure and central venous pressure in the right femoral vein during angioplasty balloon inflation in a proximal vessel segment. It is the aim to establish five EC cultures and monocyte preparations, each from CAD patients with well (CFI ≥ 0.25) and poorly (CFI < 0.25) developed collateral arteries, respectively. Cell cultures will be made anonymous and stored frozen at -150°C until use in in-vitro experiments. The number of ECPs per unit blood will be counted based on formed ECP colonies during the initial cell differentiation phase in culture. In vitro, the potential of shear stress activated ECs to induce arteriogenesis-like processes will be determined by their ability to home monocytes, and the degree by which extracellular matrix proteins are degraded. Both parameters represent the degree to which a vessel wall can be remodeled by shear stress activated ECs, i.g. the initiation phase of arteriogenesis. By means of proteomics methods, with the use of mass spectrometry, it is the aim to characterize proteins which are CFI-dependent differentially expressed.
Expected value: The results of this project should reveal if genetic predisposition or the number of circulating ECP’s are associated with the development of collateral arteries. The comparative protein expression study can provide valuable clues about biochemical pathways and regulatory mechanisms involved in collateral artery growth.