(F-213) Yes-associated protein partially mediates the anti-inflammatory transcriptome of endothelial cells induced by topographical micropatterning

Introduction:: Anisotropic topography alters endothelial cell (EC) morphology and function in vitro. Endothelial monolayers in static culture elongate and align in response to topographical micropatterns. Micropatterning has also been shown to modify other aspects of EC behavior, including directional migration, adhesion, and attenuation of inflammatory adhesion molecule expression. These responses have also been shown to be influenced by EC exposure to unidirectional, pulsatile fluid shear stress (UFSS) that produces an anti-inflammatory phenotype and elongated morphology. Conversely, a pro-inflammatory EC phenotype and cobblestone morphology is observed when ECs experience oscillatory FSS (OFSS). Previous studies of EC phenotype under various flow profiles have established the partial mediation by the transcription factor yes-associated protein (YAP). Under UFSS, YAP remains inactive and is retained within the cytosol. OFSS induces dephosphorylation, nuclear translocation, and transcriptional activity of YAP. However, despite the similar impacts on morphology and function, it is unknown if the morphological change induced via micropatterning or UFSS share molecular mechanisms. We hypothesized that transcriptional reprogramming induced by micropatterning affects EC phenotype and is similar to that of ECs exposed to UFSS.

Materials and Methods:: To investigate alterations in the transcriptome induced by changes in morphology, bulk RNA-sequencing (RNA-seq) was utilized for ECs seeded on either planar or micropatterned (3μm width, 1μm depth) polyurethane surfaces for 24hrs in static culture. RNA-seq results were validated using qRT-PCR for select YAP target genes. Informatic analyses were conducted on differentially expressed genes (DEGs) for KEGG signaling pathways using Enrichr. To investigate the time-dependent effects of TNFα (10ng/mL), a pro-inflammatory YAP agonist, on YAP phosphorylation activity, protein expression for YAP1 and β-tubulin were performed with Western blot after 0-24hrs of stimulation. Effects of 4hrs of TNFα stimulation on YAP1 phosphorylation activity between micropatterned and planar surfaces was performed by Western blot. Additionally, to visualize relative YAP subcellular localization, immunocytochemistry was utilized for ECs seeded for 44 hours on either planar or micropatterned substrates followed by 4hrs of control or TNFα stimulation. ECs were fixed and co-stained for VE-cadherin, YAP1, and nuclei. Functional IL8 chemokine secretion was quantified in cell supernatant after 4hrs of ±TNFα stimulation using a ProQuantum Immunoassay kit.

Results, Conclusions, and Discussions:: EC monolayers on anisotropic topographical micropatterned surfaces significantly elongated and aligned relative to the pattern direction compared to ECs on planar surfaces in static culture (p< 0.001, Fig.1A). Using an unbiased approach via bulk RNA-seq, the cellular morphological change induced an altered endothelial transcriptome with 1802 total DEGs altered by ECs on micropatterned versus planar surfaces, with 914 or 888 genes significantly upregulated or downregulated, respectively (p< 0.05, Fig.1B). KEGG pathway analysis identified the IL17, lipid and atherosclerosis, NFκB, and TNF pro-inflammatory signaling pathways were significantly downregulated by ECs on the micropatterned surfaces compared to planar surfaces (p< 0.05, Fig.1C). Importantly, YAP target genes (ANKRD1, CCN1, CCN2, VCAM1, ICAM1, IL6, IL8) were significantly downregulated, which were further verified with qPCR. We, therefore, hypothesized that micropatterning was inducing YAP cytosolic retention. Relative YAP1 subcellular localization was significantly upregulated within the cytosol for micropatterned ECs under both basal and inflammatory conditions, indicating YAP phosphorylation and inactivation induced by the micropatterns. Additionally, using TNFα as a pro-inflammatory agonist, ECs on micropatterned surfaces significantly attenuated nuclear YAP and promoted cytosolic retention compared to ECs on planar surfaces (p< 0.001, Fig.1D). Functional testing revealed that IL8, a YAP transcriptional target, secretion in the cell supernatant was significantly attenuated by micropatterned ECs under TNFα stimulation compared to planar (p< 0.05, Fig.1E). Thus, topographical micropatterning induces an anti-inflammatory EC phenotype, which is partially mediated by YAP activity.

This study identified that micropattern-induced morphological changes in cellular monolayers alters the endothelial transcriptome. EC elongation and alignment, similar to that under UFSS, induces an anti-inflammatory phenotype by downregulating select inflammatory signaling pathways and is partially mediated by YAP subcellular location and activity. Our findings are a significant step in our understanding of the molecular underpinning of endothelial remodeling that will be required to successfully translate topographical surface modifications for more biocompatible vascular biomaterials.


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