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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session D

Poster H12 - Notch Signaling in Stiffness-Mediated Endothelial-to-Mesenchymal Transition and its Association with Atherosclerosis

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Cardiovascular diseases are the leading cause of death globally, with an estimated 17.9 million deaths per year. Among them, atherosclerosis is particularly prevalent, characterized by the stiffening of the vessel and build-up of plaque. In addition, in the atherosclerotic lesions, vascular endothelial cells that normally line the vessel transition to exhibit a mesenchymal phenotype through a process known as endothelial-to-mesenchymal transition (EndoMT). This transition is believed to occur through a series of biological signaling pathways that are affected by mechanical stimulation, but the mechanism remains not fully understood. The exact role of EndoMT in disease progression also remains unclear, with EndoMT being correlated to the worsening of disease pathologies. Notch is a highly conserved signalling pathway critical in cell fate determination, tissue patterning and various developmental processes. The objective of this work is to study how substrate stiffness modulates EndoMT and the role of Notch signaling in mediating the process.

Materials and

Methods:  To study EndoMT, primary human aortic endothelial cells were exposed to polyacrylamide substrates of different stiffnesses (4kPa or 100kPa) or on Tissue Culture Plastic (TC) as a pathologically stiffness substrate. A base media condition with no growth factor supplements was used to help isolate the effects of stiffness. Transforming growth factor-β (TGF-β) treatment was used as a known chemical inducer of EndoMT. Single cell RNA sequencing (scRNAseq) was performed after 14 days. This data was pre-processed and filtered for quality control. Cells then underwent PCA dimensionality reduction, followed by hierarchical clustering using the unsupervised Leiden method. Subsequent sub-clustering was also conducted. Manual cell classification of the clusters was done using key established endothelial, mesenchymal, and EndoMT markers.

Results, Conclusions, and Discussions: Leiden clustering revealed three major cell classifications: Endothelial, Transitory Mesenchymal, and Mesenchymal. Endothelial cells cultured on 4kPa substrates (the lowest, physiological stiffness) demonstrated the highest percentage of endothelial cell phenotypes. Under the influence of TGF-β on 4kPa, there was a relatively higher proportion of mesenchymal and mesenchymal transitory cells. This is expected and reinforces existing literature suggesting that TGF-β progresses EndoMT. In contrast, on higher stiffness substrates, TGF-β seems to exhibit a lesser, more negligible effect on progressing EndoMT. This suggests that at a certain threshold the stiffness becomes the fully dominant force in promoting EndoMT. On the pathological TC substrate, a strong increase in mesenchymal cell states was observed as well, with and without TGF- β treatment. Overall, this suggests that stiffness effectively promotes a further progressed, final state of EndoMT, while TGF-β seemed to progress EndoMT to a more transitory stage. Notch signaling analysis suggests that certain Notch markers are regulated negatively with mesenchymal markers, while sustained Notch signalling is present in the transitory cell state. Notch appears to negatively impact TGF-β and higher stiffness conditions as well. This contrasts previous literature where Notch signalling has been found to induce EndoMT progression and promote the development of atherosclerotic lesions and will be investigated further.

Overall, these findings suggest and reinforce that higher stiffness promotes EndoMT in vascular endothelial cells, and that Notch signaling is partially negatively correlated with EndoMT. These findings establish a base for continued investigation to continue understanding the mechanistic cascade that causes EndoMT. Long-term, this work can help in the development of potential diagnostic and therapeutic technologies for critical cardiovascular diseases, including atherosclerosis, that involve EndoMT.

Acknowledgements (Optional):