Endothelium in cardiovascular tissue engineering: studies on exosomes, development, and recruitment

Abstract

Cardiovascular disease is the leading cause of death worldwide. In the Netherlands over 17,000 surgical procedures related to cardiovascular disease are performed annually. About two-thirds of these surgeries are coronary artery bypass surgery procedures, and over 3000 surgeries are related to aortic valve disease. In coronary artery bypass surgery, autologous vessels are used to bypass an occluded vessel. In aortic valve disease-related surgeries, aortic valves are replaced with prosthetic valves, which require life-long treatment of anti-coagulants, or bio-prosthetic valves, which have an average limited lifespan of 15 – 20 years as a result of increased calcification and structural decline. These issues underline the societal need for affordable, autologous, alternatives for both vascular grafts and heart valves. Tissue engineering has the potential to provide such living, autologous replacements. The research in this thesis focuses on three fields of research to search for potential novel therapeutic tools in tissue engineering by drawing inspiration from biological processes: functions and potential applications of endothelial cell-derived extracellular vesicles, regulation of endothelial-to-mesenchymal transition, and tumor-induced endothelial recruitment / angiogenesis. Studying endothelial cell-derived exosomes, we find that they exert an autocrine regulatory role in endothelial cell function and angiogenesis by transfer of microRNA miR-214, which prevents cellular senescence through downregulation of ataxia telangiectasia mutated (ATM) gene and protein expression. Furthermore, we find that culturing conditions of endothelial cells affect both the proteomic and genomic content of their exosomes, demonstrating that cellular stress conditions are reflected in exosomal content. One of the proteins affected by cellular hypoxia, lysyl oxidase-like (LOXL2), caught our attention given its reported role in both extracellular matrix (ECM) remodeling and angiogenesis. We further studied its role in the context of ECM remodeling and showed for the first time that exosome-associated LOXL2 is catalytically active and facilitates crosslinking of collagens and elastin under the regulation of hypoxia, a novel role for exosomes in ECM remodeling. Altogether, these findings show that endothelial cell-derived exosomes are a potential tool to increase vascularization and ECM crosslinking of tissues, and merit additional studies in to the use of exosomes, or components thereof, to promote tissue development in tissue engineering strategies. Furthermore, we show that cellular stress affects both exosomal content and function, underlining their potential value as a source of novel biomarkers in the field of diagnostics. Further studying the role of LOXL2 in endothelial cells, we discover an additional function in intracellular signaling, independent of exosomes or its enzymatic activity. We show that this newly described function of LOXL2 in endothelial cells plays a regulatory role in angiogenesis, ECM production, and cardiovascular development. These data demonstrate a potential role for LOXL2 in cardiovascular tissue engineering as well, given its dual intra- and extracellular functions in endothelial cells, allowing the induction of both tissue vascularization and enzymatic strengthening of the ECM. Altogether, these studies may have implications for future approaches in regenerative medicine, and underline the potential of studying endothelial cell function to discover novel strategies for cardiovascular tissue engineering.