Browsing by Author "Balaoing, Liezl Rae"

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    Evaluation of Valvular Endothelial Cell Hemostatic Behavior in Native Valves and Novel Co-culture Models
    (2014-12-03) Balaoing, Liezl Rae; Grande-Allen, Kathryn J; Moake, Joel L; Kiang, Ching-Hwa; Qutub, Amina; Harrington, Daniel
    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.
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    Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation
    (Public Library of Science, 2015) Balaoing, Liezl Rae; Post, Allison Davis; Lin, Adam Yuh; Tseng, Hubert; Moake, Joel L.; Grande-Allen, K. Jane
    Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.