Arterial stiffness and inflammation are associated with atherosclerosis, and each have individually been shown to increase endothelial monolayer tension and permeability. The objective of this study was to determine if substrate stiffness enhanced endothelial monolayer tension and permeability in response to inflammatory cytokines. Porcine aortic endothelial cells were cultured at confluence on polyacrylamide gels of varying stiffness and treated with either tumor necrosis factor-α (TNFα) or thrombin. Monolayer tension was measured through vinculin localization at the cell membrane, traction force microscopy, and phosphorylated myosin light chain quantity and actin fiber colocalization. Cell permeability was measured by cell-cell junction confocal microscopy and a dextran permeability assay. When treated with TNFα or thrombin, endothelial monolayers on stiffer substrates showed increased traction forces, vinculin at the cell membrane, and vinculin phosphorylation, suggesting elevated monolayer tension. Interestingly, VE-cadherin shifted toward a smaller molecular weight in endothelial monolayers on softer substrates, which may relate to increased VE-cadherin endocytosis and degradation. Phosphorylated myosin light chain colocalization with actin stress fibers increased in endothelial monolayers treated with TNFα or thrombin on stiffer substrates, indicating elevated cell monolayer contractility. Endothelial monolayers also developed focal adherens intercellular junctions and became more permeable when cultured on stiffer substrates in the presence of the inflammatory cytokines. Whereas each of these effects was likely mitigated by Rho/ROCK, Rho/ROCK pathway inhibition via Y27632 disrupted cell-cell junction morphology, showing that cell contractility is required to maintain adherens junction integrity. These data suggest that stiff substrates change intercellular junction protein localization and degradation, which may counteract the inflammation-induced increase in endothelial monolayer tension and thereby moderate inflammation-induced junction loss and associated endothelial monolayer permeability on stiffer substrates.

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

Endothelial cell interactions with normal and cancerous breast epithelial cells have been widely studied in tissue growth and development, as well as in angiogenesis and metastasis. Despite the understanding that 3D multicellular architecture is critical to the cell phenotype, 3D vascular structures have not yet been cocultured with 3D breast spheroids in vitro. The objective of this study was therefore to create a hierarchical, multiscale model of vascular endothelial-breast epithelial cell interactions in which both cell types were assembled into their 3D architectures. The model was successfully fabricated by adding preformed breast spheroids onto preformed endothelial tube-like networks. Through this model, we observed that breast spheroids maintain vascular tube-like networks. Over time, breast epithelial cells migrate out of the spheroid structure along the endothelial networks. This research shows that 3D cell structures serve as an important building block for creating multicellular coculture models to study physiologically relevant cell−cell interactions.

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.