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High Shear Stress Enhances Endothelial Permeability in the presence of a CAD Risk Locus

ABSTRACT: Single nucleotide polymorphisms are exceedingly common in non-coding loci, yet their impact on cellular dysfunction–especially under stressed conditions associated with coronary artery disease (CAD)–is still unclear. Here we show that when exposed to external stressors, the presence of polymorphisms in the non-coding 9p21.3 locus increases endothelial monolayer and microvessel dysfunction; endothelial cells (ECs) derived from induced pluripotent stem cells of patients homozygous for this risk haplotype (R/R WT) differentiated similarly to their non-risk (N/N) and isogenic knockout (R/R KO) counterparts; monolayers exhibited greater permeability and ROS signaling when the risk locus was present. Addition of the inflammatory cytokine TNFa further enhanced EC monolayer permeability but independent of risk haplotype. When wall shear stress was applied to ECs in a microfluidic vessel, R/R WT vessels were more permeable at lower shear stresses than R/R KO vessels. Transcriptomes of sheared cells clustered more by risk haplotype than by patient or clone, resulting in significant differential regulation of EC function and junction genes versus static conditions. A subset of previously identified CAD risk genes invert expression patterns in the presence of high shear concomitant with aberrant cell-cell adhesion, vessel permeability, and the risk haplotype, suggesting that shear stress could be a unique regulator of non-coding loci and its impact on CAD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE167861 | GEO | 2021/06/08

REPOSITORIES: GEO

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Project description:Development and homeostasis of blood vessels critically depend on the regulation of endothelial cell-cell junctions. Perturbations in cell-cell junction organization and function results in developmental defects and vascular pathologies including chronic inflammation, edema and atherosclerosis. Although many aspects of blood vessel formation and homeostasis depend on cell-cell junctions, the molecular mechanisms that regulate their dynamic rearrangement are not fully understood. The VEcad-catenin complex, which constitute the molecular basis of the adherens junctions (AJ), is connected to the actin cytoskeleton and its function is regulated by cytoskeletal contraction and actin-driven plasma membrane protrusions. Junction-associated intermitted lamellipodia (JAIL) are small actin-driven protrusions at cell-cell junctions controlled by the actin related protein 2/3 (Arp2/3)-complex that contribute to the regulation of cell-cell junctions. JAIL drive VEcad dynamics within the cell-cell junction thereby being critical for monolayer integrity, cell migration and angiogenesis. The molecular mechanisms regulating JAIL during vessel development are not completely understood. Coronin 1B (Coro1B) is an actin binding protein that controls actin networks at classical lamellipodia via both Arp2/3 complex and cofilin-mediated pathways. The role of Coro1B in endothelial cell (ECs) is not fully understood. In this study we demonstrate that Coro1B is a novel component and regulator of cell-cell junctions in ECs. Immunofluorescence studies show that Coro1B colocalizes with VEcad at cell-cell junctions in monolayers of ECs. Live-cell imaging reveal that Coro1B is recruited to, and operated at, actin-driven membrane protrusions at the cell-cell junctions. Coro1B recruitment to cell-cell junctions is regulated by cytoskeleton tension. By analyzing the Coro1B interactome, we identify integrin linked kinase (ILK) as new Coro1B-associated protein. Coro1B colocalizes with α-parvin, an interactor of ILK, at the leading edge of lamellipodia protrusions. Finally, functional experiments reveal that depletion of Coro1B causes defects in actin cytoskeleton and cell-cell junctions. In matrigel vessel network assays, depletion of Coro1B results in reduced network complexity, vessel number and vessel length. Together, our findings point towards a critical role for Coro1B in the dynamic remodeling of endothelial cell-cell junction and the assembly of vessel network.

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High Shear Stress Enhances Endothelial Permeability in the presence of a CAD Risk Locus

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