Poster T16 - A Stiffness-Tunable ECM-Based Vascular Model Reveals Survivin/YAP-Mediated Mechanotransduction in Human VSMCs

Introduction: Arterial stiffness is a principal risk factor in cardiovascular diseases, prominently featured in pathologies such as atherosclerosis, vascular injury, and coronary artery disease. It is associated with aberrant behaviors in vascular smooth muscle cells (VSMCs), such as increased migration and proliferation, contributing to neointimal hyperplasia and subsequent neointima formation [1]. However, the specific mechanisms by which changes in the local microenvironment induce VSMC hyperplasia and neointima formation are unclear. Our previous research has revealed that survivin, an inhibitor of apoptosis protein, is significantly upregulated in VSMCs post-vascular injury in vivo and under conditions mimicking the stiffness of diseased arteries [2, 3]. Interestingly, inhibition of survivin reduces stiffness induced VSMC proliferation and migration, while also diminishing cellular stiffness and extracellular matrix protein secretion, critical for arterial stiffness and remodeling. These findings highlight a novel biological function for survivin in VSMC mechanobiology and pathology. To better replicate the in vivo diseased arterial conditions, in this study, we further develop an in vitro vascular model system that includes a stiffness-tunable nanofibrous scaffold derived from decellularized porcine aorta ECM.

Materials and

Methods: Adult porcine aortic tissue from four biological replicates was finely minced into small pieces. The tissues then underwent a series of treatments for decellularization, including washing, and sequential exposure to SDS, Triton-X, and DNase. Following decellularization, the tissues were sterilized and lyophilized. The resultant decellularized extracellular matrix (dECM) then underwent cryogenic milling to obtain a fine powder and was prepared for mass spectrometry analysis. To fabricate tunable fibrous scaffold systems, the dECM powder was solubilized in a mixture of acetic acid, ethyl acetate, and water and then blended with methacrylated hyaluronic acid (MeHA) at modification rates of either 20% (soft) or 100% (stiff), along with polyethylene oxide and Irgacure. The mixture was then electrospun onto glass slides to form stiffness-tunable dECM scaffolds, which were crosslinked under UV exposure. Post-fabrication, fibrous scaffold morphology was examined using brightfield microscopy. The stiffness of the scaffolds was quantitatively assessed using atomic force microscopy (AFM). Prior to cell seeding, the scaffolds were hydrated in phosphate-buffered saline and sterilized. Serum-starved human VSMCs were then seeded onto these scaffolds. After 24 hours of culture, the cells were fixed with an ethanol-methanol mixture, stained for survivin, actin, and DAPI, and then imaged for analysis.

Results, Conclusions, and Discussions: Mass spectrometry analysis and principal component analysis (PCA) depicted distinct clustering between the native tissue and dECM groups, with greater variability observed within the dECM samples (Fig. 1A). This marked separation of the native and dECM groups was further confirmed by the heatmap (Fig. 1B). The upset plot (not included) revealed that approximately 90% of the proteins in the native tissue, including key proteins such as collagens, were retained during the decellularization process (Fig. 1C). These findings demonstrate the preservation of important ECM components in the decellularized tissues. Brightfield microscopy images of the electrospun dECM scaffold highlighted the well-defined fiber formations in the scaffolds (Fig. 1D). AFM measurements showed that the elastic modulus of the stiff dECM group was nearly four-fold greater than that of the soft group (Fig. 1E). To elucidate cellular responses to varying substrate stiffnesses, VSMCs were cultured on the stiffness-tunable nanofibrous scaffolds and cells on the soft dECM had nearly a two-fold increase in cell area and exhibited a more elongated morphology on the stiff dECM (Fig. 2A, B). Correspondingly, there was a modest increase in the survivin and YAP nuclear to cytoplasmic ratio in the VSMCs on the stiff dECM (Fig. 2C, D), consistent with previous findings of enhanced survivin and YAP nuclear localization in response to increased substrate stiffness [4]. These results indicate the mechanosensitive regulatory role of survivin and YAP in VSMCs within this in vitro vascular model system. Current research focuses on exploring how this stiffness-tunable dECM scaffold influences gene expression and epigenetic profiling, along with the manipulation of survivin expression, to unravel the complex mechanisms underlying cardiovascular diseases. These efforts aim to contribute to the development of therapeutic strategies for cardiovascular pathologies.

Acknowledgements (Optional): This work was supported by the NIH (K01 AR077087, P50 AR080581, R01 HL163168).

References: [1] Ross+ 1999, N Engl J Med; [2] Bae+ 2023, APL Bioeng; [3] Bae+ 2023, APL Bioeng, [4] Dupont+ 2011, Nature.