(E-180) P300 inhibition modulates endothelial cytoskeletal dynamics in response to flow

Introduction:: Endothelial cells (EC) regulate the transport of molecules and cells between blood and tissue¹. EC also respond to hemodynamic forces². When exposed to laminar shear stress, healthy endothelial cells undergo cytoskeletal remodeling to align their junctions and actin cytoskeleton parallel to the flow vector. Multiple groups have demonstrated that Notch signaling is required for EC alignment in vitro^3,4. Canonical Notch signaling culminates in the translocation of the Notch receptor intracellular domain (NICD) to the nucleus, where it acts as a transcriptional coregulator to modulate gene expression⁵. Importantly, the histone acetyltransferase (HAT) P300 has been shown to acetylate the NICD, thereby contributing to its stability within the nucleus⁶. Despite this important role of P300 in Notch signaling, little is known about the contribution of P300 to EC alignment. Here we investigate a potential role of P300 as a modulator of cytoskeletal remodeling during the flow response in EC.


Materials and Methods:: Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) were cultured continuously with drug or DMSO load control under static, orbital shaking, or laminar flow (parallel plate with positive pressure pump to generate 15 Dyn/cm² wall shear stress) conditions for 24-72h. A485 (SelleckChem), a potent and selective small molecule catalytic inhibitor of P300⁷ was used at 5uM to inhibit P300 activity, and the gamma secretase inhibitor (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) (DAPT, SelleckChem), was used at 20uM to inhibit Notch receptor cleavage. Immunofluorescence for VE-Cadherin and counterstaining with DAPI and phalloidin was performed to interrogate features of cell alignment, cell axis ratio and nuclear shape index. Relative protein expression was assayed by Western blot.


Results, Conclusions, and Discussions:: Here, we show that inhibition of P300 with the small molecule A485 drives a cytoskeletal phenotype in EC which causes them to become hyperaligned under laminar flow conditions. As expected, both cells treated with A485 for and cells treated with DAPT for 72 hours demonstrate reduced levels of NICD compared to controls under static and orbital shaking conditions (Figure 1A). However, under laminar flow conditions, EC treated with A485 demonstrate reduced variance in the major angles of their nuclei and cell bodies relative to the flow vector when compared to DAPT treated cells and DMSO controls (Figure 1B). EC exposed to A485 have significantly lower nuclear shape indices and higher cell axis ratios compared to DMSO controls under both static and flow conditions (Figure 1C and 1D).



The ability of EC to respond to fluid shear stress is absolutely crucial for early vascular remodeling and for postnatal vascular maintenance. The role of Notch signaling in the EC flow response has been traditionally attributed to downstream effects of NICD-mediated transcription. Recent reports suggest that Notch signaling contributes to EC barrier function through activity of its transmembrane domain (TMD) in a manner that is independent of NICD activity⁸. Despite the fact that both DAPT-treated cells and A485-treated cells demonstrate drastically reduced levels of NICD, A485-treated cells successfully align to the flow vector in vitro. Given that this hyperalignment phenotype is observed in A485-treated cells, but not DAPT-treated cells, these data suggest that EC alignment may also occur independently of the NICD. Because flow sensing is a critical part of endothelial biology, these findings have important implications for future studies of both vascular development and disease.


Acknowledgements (Optional): : This work has been supported by NIH R35GM142944 (WJP) and NIH T32HL69768 (CPW).

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