Project Summary Cardiovascular disease, primarily driven by atherosclerosis, is the leading cause of death in the U.S. The recent FDA Modernization Act 2.0 allows the use of alternatives to animal models in drug testing, spurring the development of advanced in vitro models like cell-based assays and artificial intelligence-based systems to streamline drug development processes. However, current atherosclerosis assays, mostly based on simple 2D cell cultures, don't adequately replicate the disease's complexity, potentially leading to inaccurate results. To address this issue, Endomimetics has developed innovative nanomatrix cell sheet technology and “cell-as- glue” approach as well as formulated novel atherosclerotic inducing mediums, which can produce an in vitro 3D nanomatrix vascular sheet (VS) and a novel 3D in vitro atherosclerosis model - nanomatrix vascular sheet with atherosclerosis (VSA). Therefore, our aim is to revolutionize the field by developing an automated, highly precise approach for fabricating VS and VSA. This advancement will enable the use of VS and VSA in creating functional assays for efficient, high- throughput drug testing in a 3D human atherosclerosis environment and allow for the evaluation of both the efficacy and cytotoxicity of drugs across extensive sample sizes, while minimizing biological variability. Such an initiative, unprecedented in its scope and precision, has not yet been achieved in any existing in vitro atherosclerosis assays. Specifically, we will develop two innovative assays integrating BODIPY staining and ELISA methods, aimed at determining the efficacy of atherosclerosis treatments by their capacity to suppress foam cell formation and curtail inflammatory cytokine production. Established anti-atherosclerosis and anti-inflammatory compounds will serve as positive controls, validating the accuracy of the assays in detecting reductions in both foam cell formation and cytokine secretion. Simultaneously, negative controls will be employed to evaluate assay specificity, ensuring the precision of our results, and eliminating the potential for false positives. To demonstrate the feasibility of our assays in evaluating new atherosclerosis therapeutics, we will assess the efficacy of two histone deacetylases inhibitors at various doses using our assays. Additionally, we will develop and execute cytotoxicity-based assays to investigate the safety of these inhibitors.

Project Narratives Heart disease, primarily driven by atherosclerosis, is the leading cause of death in the United States. To address this, our proposal focuses on developing an innovative 3D atherosclerosis model using advanced bioprinting technology. This model accurately replicates the complex, multi-layered structure of human arteries and atherosclerotic plaques, incorporating critical atherosclerotic features. By scaling up the in vitro model, we aim to enhance the reliability of the data generated, enabling improved drug efficacy and toxicity assessments. It is also our assertion that this innovative model will more accurately reflect the viability of novel therapeutics proposed for the treatment of atherosclerosis.