(H-302) Regulation of yes-associated protein activity by serine proteases via topographical micropatterning

Introduction:: Coagulation and inflammation hold innate roles in the pathogenesis of cardiovascular disease, where the two systems have bidirectional activation. Coagulation activation yields the interaction of serine proteases with zymogens, resulting in thrombin and fibrin generation. However, serine proteases have also been shown to bind cell receptors to induce signaling pathways and activation of pro-inflammatory and pro-fibrotic mediators, such as the transcription factor yes-associated protein (YAP). Previous studies have shown thrombin-mediated activation and dephosphorylation of YAP in endothelial cells (ECs) through protease-activated receptor-1, leading to inflammatory target gene expression. However, it is unknown whether other serine proteases of the coagulation cascade effect YAP activity. Activated coagulation factor X (FXa), activated FXI (FXIa), and FXII have been shown to bind endothelial surface receptors and promote EC permeability and/or dysfunction. Additionally, FXa has been shown to upregulate mRNA expression of transcriptional targets of YAP, including CYR61 and CTGF. However, the role between these serine proteases and YAP activity are yet to be elucidated.

Our lab has recently shown anisotropic topography regulates YAP activation and subcellular localization. Topographical micropatterning induces an elongated and aligned EC monolayer in static culture, promoting an anti-inflammatory phenotype. Our previous findings have shown micropatterning downregulates YAP target gene expression and upregulates YAP cytosolic retention. However, this study was only performed with TNFα, and the ability of micropatterns to regulate serine protease-induced YAP activation has not been studied. We hypothesized that thrombin, FXa, and FXII induce YAP dephosphorylation and transcriptional activity, and micropatterning can attenuate these effects.


Materials and Methods:: Human umbilical vein endothelial cells (HUVECs) at passages 5-7 were cultured on non-patterned polyurethane-coated glass coverslips for 24hrs before overnight serum starvation. HUVECs were then incubated with either vehicle, α-thrombin (20-50nM, 0-6hrs), FXa (30nM, 0-6hrs), FXIa (30 nM, 10μM ZnCl₂), or FXII (30nM, 10μM ZnCl₂) in serum-free media before lysing. Total YAP1, pYAP (serine 127), VCAM-1, and β-tubulin protein expression were measured by immunoblotting. HUVECs on topographical micropatterns (3μm width, 1μm depth) or planar polyurethane surfaces were similarly cultured and stimulated with control, α-thrombin (20nM, 4hrs), or FXa (30nM, 6hrs) before fixation using 4% paraformaldehyde for 15mins. Immunofluorescence was used to visualize subcellular localization of YAP by co-staining for YAP1, VE-cadherin, and nuclei. Substrates were imaged at 20X with a Zeiss/Yokogawa CSU-X1 spinning disk confocal microscope, and orthogonal projections were quantified. Relative subcellular localization was classified into three categories based on the intensity of YAP1 in the cytosol (C) or intensity in the nucleus (N): classifications include cytosolic (C >N), cytosol & nucleus (C=N), and nucleus (C< N).

Results, Conclusions, and Discussions:: Stimulation of HUVECs with α-thrombin showed time and concentration-dependent effects of YAP phosphorylation. We found peak dephosphorylation of YAP by concentrations of 20nM or 50nM of α-thrombin at 4hrs. Additionally, VCAM-1 protein expression occurred beginning at 4hrs. Stimulation of HUVECs with FXa resulted in time-dependent effects of pYAP protein expression, with peak dephosphorylation at 6hrs. Lastly, FXII induced the greatest dephosphorylation of pYAP at 12hrs incubation (Fig. 1A). There were no differences in YAP phosphorylation from 0-6hrs when HUVECs were incubated with FXIa. These results show that FXa and FXII activate YAP in HUVECs—a novel finding. Using the optimal incubation times and concentrations, we found micropatterns attenuated YAP nuclear localization and promoted cytosolic retention when stimulated with α-thrombin or FXa (Fig. 1B,C). Ongoing studies will investigate YAP target gene expression and the mechanism by which micropatterning is attenuating YAP activity induced by serine proteases. These findings can be translated to improving cardiovascular device functionality.

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