Evaluation of Valvular Endothelial Cell Hemostatic Behavior in Native Valves and Novel Co-culture Models
Balaoing, Liezl Rae
Grande-Allen, Kathryn J
Doctor of Philosophy
The endothelial cell-mediated process of hemostasis is critical in all living heart valve tissues. As these tissues undergo changes with age and disease, the ability for valvular endothelial cells (VECs) to manage anti- and pro-thrombotic mechanisms may also change. Furthermore, degeneration- and thrombosis-related failures in artificial valves emphasizes the need to understand the anti-thrombotic mechanisms of VECs in order to develop effective strategies to endothelialize implants and tissue-engineered heart valves. Therefore, a study was performed to evaluate the regulation and function of von Willebrand Factor (VWF), ADAMTS-13 (VWF cleaving enzyme), and other thrombotic and anti-thrombotic mediators secreted from VECs from different aged valves. This work identified age-related differences in VEC hemostatic protein regulation, and an increased capacity of specific proteins to aggregate within regions of elderly valves, which are known to have age-associated loss of extracellular matrix (ECM) organization that are linked to calcific aortic valve disease. With the knowledge that ECM can influence hemostasis, we then studied changes in VEC hemostatic regulation using synthetic culture conditions that modulated substrate stiffness and adhesive ligands. RKRLQVQLSIRT (RKR), a syndecan binding cell adhesive peptide derived from laminin-α1 G-domain, was optimal for promoting strong VEC adhesion and balanced hemostatic function on hydrogel constructs of various stiffness in comparison to the commonly used integrin binding peptide RGDS. Next, to evaluate interactions between valve cells, magnetic levitation technology was used to co-culture VECs with valvular interstitial cells (VICs) in a 3D scaffoldless aortic valve co-culture (AVCC). The cell-based AVCC design allowed for synthesis of multiple constructs within a few hours. AVCCs had regional localization of CD31 positive VECs at construct surface. Cells in the AVCC interior (including VECs) expressed low levels of α-smooth muscle actin (αSMA), suggesting maintenance of quiescent VIC phenotype, but potential endothelial to mesenchymal differentiation in interior-localized VECs. In addition, AVCCs produced ECM and expressed hemostatic proteins such as endothelial nitric oxide synthase (eNOS) and VWF. In light of the VEC localization within the AVCC potentially affecting healthy phenotype, a more physiologically organized and customizable scaffold model was needed for further evaluation of direct interactions between VECs and VICs. Therefore, previous RKR-functionalization work was combined with strategies for VIC encapsulation in biofunctionalized-MMP degradable hydrogels to develop a 3D adhesive ligand localized hydrogel scaffold for an endothelialized aortic valve co-culture model. The resulting hydrogel-based endothelialized aortic valve model (HEAVM) promoted the formation of a stable VEC monolayer at the scaffold surface, and supported the maintenance of VIC quiescent phenotypes within the scaffold, thereby mimicking physiological valve cell organization in aortic valves. Platelet adhesion and nitric oxide functional assays confirmed healthy VEC cell behavior, while immunohistochemistry and qRT-PCR were used to asses VIC and VEC phenotype and extracellular matrix (ECM) production. Overall, by utilizing principles from cell and extracellular matrix biology, biomechanics, and biomaterials, this work was able to improve the understanding of the VEC roles in valve homeostasis and the pathogenesis of valvular disease. Furthermore, new biomaterial-based models were designed to enhance the field’s understanding of VEC functions and communication with VICs. The knowledge learned from these models may be applied to future evaluation of various valve diseases, as well as endothelialization strategies for valve implants.
Aortic heart valve; Valvular endothelial cell; Hemostasis; Co-culture