(H-295) Three-layer nanomatrix vascular sheets with atherosclerosis for high-throughput therapeutic evaluation

Introduction:: Atherosclerosis is a major cause of cardiovascular disease, and effective therapeutics require reliable in vivo and in vitro models. However, current models have limitations, such as high cost, long induction time, and lack of accurate replication of human atherosclerosis pathogenesis. To overcome these challenges, we developed an innovative in vitro platform called tissue-engineered vascular sheets with atherosclerosis (VSA). The VSA platform features a 3-layered artery structure and replicates disease characteristics such as endothelial dysfunction, macrophages, and foam cells. Additionally, the VSA platform can be easily scaled up for drug evaluation using multi-well plates, allowing for high-throughput screening of potential therapeutics in a cost-effective way. Using pro-atherogenic cytokines, Ox-LDL, and monocytes to induce atherosclerosis in the VSA platform, we can accurately mimic the disease state and evaluate the effectiveness of potential drugs. Overall, the VSA platform offers several advantages over current models and has the potential to improve drug development for atherosclerosis.


Materials and Methods:: VSA are fabricated as follows:1) hAAF and hAoSMC sheets were obtained by encapsulating human aortic adventitial fibroblasts (hAAFs) and aortic smooth muscle cells (hAoSMCs) by nanomatrix thin sheet technology using peptide amphiphile (PA-RGDS) gel; 2) human aortic endothelial cells (hAECs) were seeded on hAoSMC sheet; 3) The hAAF sheet was assembled with hAEC-hAoSMC sheet to form a 3-layered vascular sheet; 4) Atherosclerosis was induced by adding pro-atherogenic cytokines, Ox-LDL, and monocytes. A 48-well plate was used to make the VSA plate of numbers of VSA for effective model drug (e.g., statin) evaluation.

Results, Conclusions, and Discussions:: Fig.1a-h show various images of hAAF and hAoSMC sheets made by nanomatrix thin sheet technology, indicating PA-RGDS gel is biocompatible and does not affect cell phenotype. The cross-sectional fluorescent image of the vascular sheet indicates an excellent three-layered structure composed of hAEC, hAoSMC, and hAAF layers (Fig.1i). Moreover, the positive immunostaining of ICAM-1 and VCAM-1 in the vascular sheets treated with TNF-α and Ox-LDL indicate successful induction of hAEC dysfunction (Fig.1j-k). Good recruitment of monocytes was also achieved (Fig.1l). Large quantity of aggregated foam cells and lipid pool were observed on the VSA, and the VSA can produce various types of inflammatory cytokines observed in human atherosclerotic plaque (Fig.1o). Notably, the production of VSA can be upscaled in a plate for drug evaluation (Fig.1p). Statin was used as a model drug for demonstrating the feasibility of using VSA for drug evaluation. Promisingly, the statin-treated VSA showed significantly more foam cells and inflammatory cytokine production than the untreated VSA (Fig.1r-s), which agrees with the therapeutic effect of statin on atherosclerosis reported in the literature. In summary, we have developed VSA and VSA plates. The VSA plate shows excellent potential for drug evaluation. We are currently working on using the VSA plate to evaluate potential therapeutics for atherosclerosis.

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