Atherosclerotic three-layer nanomatrix vascular sheets for high-throughput therapeutic evaluation

Introduction: : Cardiovascular diseases (CVDs) are the leading cause of death worldwide, primarily driven by atherosclerosis, which narrows and stiffens arteries through lipid accumulation and chronic inflammation. In vitro models are essential for preclinical evaluation of novel therapeutics, offering human-relevant data, lower cost, and higher throughput than in vivo studies. However, conventional two-dimensional (2D) cultures lack three-dimensional (3D) architecture and multicellular interactions, while existing 3D systems—organ-on-chips, spheroids, and tissue-engineered blood vessels—often lack key plaque features such as a layered vessel structure, extracellular matrix remodeling, calcification, and foam cell formation, and are not readily scalable for high-throughput screening. To overcome these limitations, we engineered a 3D three-layer nanomatrix vascular sheet (VS) that replicates the intima, media, and adventitia in a reproducible and scalable format. We then induced atherosclerotic pathology to generate vascular sheet with atherosclerosis (VSA) by exposing VSs to monocytes, pro-atherogenic cytokines, and oxidized low-density lipoprotein, triggering endothelial dysfunction, monocyte recruitment, macrophage and foam cell formation, cytokine secretion, and focal calcification. By arranging arrays of these VSA, we developed high-throughput foam cell and inflammation assays to evaluate therapeutics—including statins and sirolimus. The assays produced drug efficacy profiles consistent with in vivo studies, demonstrating this platform’s potential to accelerate preclinical screening, improve translation to clinical outcomes, and streamline therapeutic development.

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. For drug treatment, VSAs were fabciated and cultured in 48-well plates for 35 days, followed by treatment on day 35 with rosuvastatin (10 μM) and sirolimus (10 nM) in customized medium containing monocytes, with media refreshed on days 38 and 42. On day 45, VSAs were subjected to a foam-cell assay by staining with BODIPY and imaging by fluorescence microscopy or quantifying by plate reader. Inflammation assays involved harvesting VSAs for RNA extraction, reverse-transcribing total RNA for TaqMan qRT-PCR, and analyzing culture supernatants by RayBiotech multiplex ELISA per manufacturer’s instructions.

Results, Conclusions, and Discussions:: Results
The VSA successfully recapitulated human atherosclerosis, as demonstrated by its three-layered structure mimicking native arterial architecture (Fig. 1A-C). Immunostaining confirmed healthy endothelial (hAEC), fibroblast cells, and smooth muscle cell (hAoSMC) phenotypes (Fig. 1D-F), while ICAM-1 expression indicated endothelial dysfunction (Fig. 1G). Key atherosclerotic features, including monocyte recruitment, macrophage formation, foam cell aggregation, and lipid pool development, were replicated (Fig. 1H-L). The VSA secreted pro-inflammatory cytokines (e.g., IL-1β, IFN-γ) and anti-inflammatory IL-10, reflecting dynamic macrophage formation and polarization (Fig. 1M). Gene expression analysis (Fig. 1N) revealed endothelial dysfunction (reduced PECAM-1, eNOS), SMC phenotypic switching (decreased MYH11, elevated PDGF-B), and inflammation/oxidative stress markers (elevated TGF-β1, NOX4; reduced CDKN2A). ECM remodeling was evident through upregulated MMP-2 and COL3A1, while elevated RUNX2 and calcification staining confirmed osteogenic SMC transition. In addition, the results demonstrated that statin and sirolimus treatment significantly reduced foam cell formation and inflammatory cytokine production in VSAs compared to untreated VSAs (Fig. 2A,B,E,F), demonstrating its utility for therapeutic evaluation. In addition, the VSA inflammation gene expression assay (Fig.2C-D) showed that statin suppressed angiogenesis, evidenced by reduced VEGF-A expression, and modulated ECM remodeling via elevated COL17A1 and suppressed MMP-2. It also demonstrated anti-inflammatory effects of statin with markedly decreased IL-6. Sirolimus promoted inflammation resolution, significantly upregulating IL-10 while reducing IL-6 and VEGF-A, consistent with its known mTOR pathway inhibition to block angiogenesis.

Discussion and Conclusions
We have developed VSA and VSA based functional assays for drug testing. The VSA recapitulates early-to-intermediate atherosclerosis, including calcification, foam cell formation, SMC transition, and ECM remodeling. VSA based functional assays demonstrated therapeutic efficacy: rosuvastatin suppressed TNF-α and IL-6, mirroring its anti-atherosclerotic effects in prior in vivo studies, while sirolimus reduced pro-inflammatory mediators (MCP-1, IFN-γ, TNF-α) and elevated autophagy-related cytokines (IL-4, IL-13), consistent with its known autophagy-enhancing and mTOR-inhibiting mechanisms. Both drugs inhibited foam cell formation, indicating potential to curb early plaque development. These findings validate the VSA’s ability to recapitulate disease mechanisms and drug responses observed in animal models, bridging preclinical and human relevance. This study establishes a transformative tool for atherosclerosis research and drug development.

Acknowledgements and/or References (Optional)::