(H-299) Valve-on-Chip Platform Reveals Novel Insights into the Early Calcific Disease Progression

Introduction::
Calcific aortic valve disease (CAVD) has been increasingly prevalent in the world with an aging population. Unfortunately, early diagnosis and intervention have not been clinically viable. Elucidating early disease mechanisms is pivotal in the development of early intervention strategies. In this study, we developed a physiologically relevant 3D valve-on-chip (VOC) model and utilized non-invasive NADH lifetime imaging, protein sequencing, and principal component analysis to assess cellular mechanisms during early CAVD progression.




Materials and Methods:: VOC consisted of collagen and glycosaminoglycan (GAG) based hydrogel that mimicked the healthy and diseased compositions of the fibrosa and spongiosa layers of the aortic valve. Porcine aortic valve endothelial cells VECs were seeded in a monolayer on the collagen-GAG-based hydrogel with VICs embedded within the hydrogel. These gels were mounted on a custom cyclic stretcher to apply physiological strain and cultured under quiescent (healthy) or osteogenic (diseased) conditions. After 2 days the constructs were assessed by fluorescence lifetime imaging to quantify NADH lifetime. After imaging protein sequencing was performed on the constructs to correlate label-free quantitative metrics with differentially expressed proteins. Proteomic data from the porcine VOCs were then compared with proteomic data from human aortic valves to elucidate the biological processes and pathways implicated during early CAVD progression.

Results, Conclusions, and Discussions:: The lifetime of NADH revealed decreased A1/A2 ratio suggestive of increased oxidative phosphorylation in diseased VOCs. The proteomic assessment suggested that diseased VOCs had decreased expression of NSDHL, FDPS, EIF3L, UFM1, and PSMD1 and increased expression of ASNS, GCN1, ACAVDL, HSPH1, and EEA1 (Figure 1A). Proteomic data suggested that diseased VOCs exhibited decreased cholesterol biosynthesis, increased oxidative phosphorylation, amino acid stress response, and protein folding response. Intriguingly, comparison of human aortic valve proteomic assessment with VOCs using gene ontology (Figure 1B) and principal component analysis (Figure 1C) revealed that healthy and diseased VOCs were closer to healthy human aortic valves in phenotype and function.
Assessment of early CAVD processes using our novel VOCs provided new insights into the CAVD initiation process. Diseased VOCs represented an early disease initiation stage marked by altered cholesterol biosynthesis and altered protein homeostasis. Ours is one of the first few studies to elucidate altered protein homeostasis in response to stress in the context of early CAVD. Our VOCs mimicked the human disease progression and may provide a powerful tool for gaining further insights into the CAVD initiation process.

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