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P300 Modulates Endothelial Mechanotransduction of Fluid Shear Stress

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Abstract

Purpose

P300 is a lysine acetyltransferase that plays a significant role in regulating transcription and the nuclear acetylome. While P300 has been shown to be required for the transcription of certain early flow responsive genes, relatively little is known about its role in the endothelial response to hemodynamic fluid stress. Here we sought to define the role of P300 in mechanotransduction of fluid shear stress in the vascular endothelium.

Methods

To characterize cellular mechanotransduction and physical properties after perturbation of P300, we performed bulk RNA sequencing, confocal and Brillouin microscopy, and functional assays on HUVEC.

Results

Inhibition of P300 in HUVEC triggers a hyper-alignment phenotype, with cells aligning to flow sooner and more uniformly in the presence of the P300 inhibitor A-485 compared to load controls. Bulk transcriptomics revealed differential expression of genes related to the actin cytoskeleton and migration in cells exposed to A-485. Scratch wound and bead sprouting assays demonstrated that treatment with A-485 increased 2D and 3D migration of HUVEC. Closer examination of filamentous actin revealed the presence of a perinuclear actin cap in both P300 knockdown HUVEC and HUVEC treated with A-485. Interrogation of cell mechanical properties via Brillouin microscopy demonstrated that HUVEC treated with A-485 had lower Brillouin shifts in both the cell body and the nucleus, suggesting that P300 inhibition triggers an increase in cellular and nuclear compliance.

Conclusions

Together, these results point to a novel role of P300 in modulating endothelial cell mechanics and mechanotransduction of hemodynamic shear stress.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon resonable request. Bulk RNA sequencing data has been submitted to the gene expression omnibus (GEO).

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Acknowledgements

This work was supported by the National Institutes of Health (R35GM142944 to W.J.P.), the American Heart Association (CDA857738 to W.J.P.), and the Lymphatic Malformation Institute (to W.J.P.). E.L.D. and C.P.W. acknowledge financial support of the National Institutes of Health through the Integrative Vascular Biology Training Program (T32HL69768), and E.L.D. acknowledges a Ruth L. Kirchstein predoctoral individual fellowship (F31HL162462). W.Y.A. is supported by a grant from the CLOVES Syndrome Community. Mechanical testing was performed in the Chapel Hill Analytical and Nanofabrication Laboratory, CHANL, and sample preparation for scanning electron microscopy was performed in part at the Analytical Instrumentation Facility (AIF) at North Carolina State University. CHANL and AIF are members of the North Carolina Research Triangle Nanotechnology Network, RTNN, and are supported by the National Science Foundation (ECCS-2025064), as part of the National Nanotechnology Coordinated Infrastructure (NNCI). We would like to thank Jacqueline Brinkman (UNC-CH Curriculum in Genetics and Molecular Biology, supported in part by a grant from the National Institute of General Medical Sciences under award 1T32GM135128) for her assistance with computational analysis and visualization of bulk transcriptomic data.

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Whitworth, C.P., Aw, W.Y., Doherty, E.L. et al. P300 Modulates Endothelial Mechanotransduction of Fluid Shear Stress. Cel. Mol. Bioeng. (2024). https://doi.org/10.1007/s12195-024-00805-2

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