Poster BB12 - Contributions of Valve Endothelial Cells on Pathological Remodelling and Biomechanics Driving Calcification in Engineered Aortic Tissue

Introduction: Calcific aortic valve disease (CAVD) is a globally prevalent cardiovascular condition with over 9.4M cases in 2019 [1]. Previous work from our lab indicated that the chronic inflammation and biomineralization observed during CAVD pathogenesis is modulated by NFκB, specifically through the action of transformed osteoblast-like valve endothelial cells (VEC) [2] and activated valve interstitial cells (VIC). These processes influence calcific nodule formation in diseased tissue, resulting in the TNFα-mediated breakdown of the valvular endothelial monolayer and further influenced by changes in hemodynamics affecting bulk mechanical properties. While it has been established that inflammatory conditions influence cardiovascular mechanics, this study aims to perform a dual analysis of bulk mechanical properties of tissue combined with spatial analysis of osteoblastic markers to elucidate the role of VEC on cardiovascular mechanics in conjunction with induced biomineralization under both inflammatory and osteoblastic conditions.

Materials and

Methods: To this end, the Butcher Lab Group employs our “3D spring gel model” in which we coculture VIC/VEC in a collagen hydrogel. Coculture of both cell types replicates the crosstalk important for in vivo pathogenesis. The engineered tissue experiences a spring-generated equibiaxial force, simulating the force felt by the annulus of the aortic valve. To induce osteoblastic conditions, we seed hydrogels with low-crystallinity hydroxyapatite nanoparticles (HAp)3. During a 10-day culture, cells are treated with either general media (GM) or osteogenic media (OGM), creating four experimental conditions: GM (basal conditions), OGM (induced inflammation), HAp GM (osteoblastic conditions), and HAp OGM (induced osteoblastic + inflammatory conditions).

Results, Conclusions, and Discussions: Tissues were analyzed with respect to cellular remodeling, calcification/mineralization, spatial distribution of osteoblastic markers, and cardiovascular mechanics. Area compaction measurements are used as a proxy for cellular remodeling and alizarin red S stains for Ca2+ deposits. We find that OGM and HAp OGM spring gels undergo greater remodeling and that OGM gels specifically have a 2.85-fold increase in calcium when compared to GM gels. Immunohistochemistry results indicate that VE-Cadherin and Runx2 are upregulated in inflammatory/osteogenic conditions. Lastly, we apply a small vacuum force to a tissue to measure bulk tissue stiffness via micropipette aspiration4. We find that osteogenic tissue experiences the greatest increase in tissue stiffness when compared to non-diseased groups. We conclude that inflammatory and osteoblastic conditions exacerbate VEC-mediated processes involving calcification, cellular remodeling, and bulk mechanical properties. This study provides insight into the role of valve endothelial cells and pathological mechanisms to serve as therapeutic targets for the treatment of CAVD and other late-stage valve diseases via our mineral conditions.

References
[1] Liu, Z., Su, Z., Li, W., Zhang, F., Ouyang, W., Wang, S., & Pan, X. (2023). Global, regional,
and national time trends in disability-adjusted life years, mortality, and variable risk factors of non-rheumatic calcified aortic valve disease, 1990–2019: an age-period-cohort analysis of the Global Burden of Disease 2019 study. Journal of Thoracic Disease, 15(4), 2079–2097. https://doi.org/10.21037/jtd-23-480
[2] Gee, T. W., Farrar, E., Wang, Y., Wu, B., Hsu, K., Zhou, B., & Butcher, J. T. (2020). NFκB
(Nuclear Factor κ-Light-Chain Enhancer of Activated B Cells) Activity Regulates Cell-Type–Specific and Context-Specific Susceptibility to Calcification in the Aortic Valve. Arteriosclerosis, Thrombosis, and Vascular Biology, 40(3), 638–655. https://doi.org/10.1161/atvbaha.119.313248
[3] Richards, J. M., Jennie A.M.R. Kunitake, Hunt, H. B., Wnorowski, A. N., Lin, D. W.,
Boskey, A. L., Donnelly, E., Estroff, L. A., & Butcher, J. T. (2018). Crystallinity of hydroxyapatite drives myofibroblastic activation and calcification in aortic valves. Acta Biomaterialia, 71, 24–36. https://doi.org/10.1016/j.actbio.2018.02.024
[4] Buskohl, P. R., Gould, R. A., & Butcher, J. T. (2012). Quantification of embryonic
atrioventricular valve biomechanics during morphogenesis. Journal of Biomechanics, 45(5), 895–902. https://doi.org/10.1016/j.jbiomech.2011.11.032

Acknowledgements (Optional):

Funding: Rawlings Cornell Presidential Research Scholarship