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TRAP-seq in 3D Angiogenesis Assays Reveals Distinct Biological Processes Associated with Early and Late Morphogenesis that are Disrupted with PIK3CA-H1047R

ABSTRACT: Endothelial cells (ECs) are often a minority cell type in a tissue, limiting the utility of bulk sequencing approaches. Single cell sequencing lacks sensitivity and requires disruptive tissue digestion techniques. TRAP seq (Translating Ribosome Affinity Purification) or “RiboTag” has been used to overcome these limitations. Co-culture systems allow primary ECs to differentiate and undergo tubular morphogenesis in cell culture, however similar limitations exist with these in vitro assays, as ECs are under-represented by as much as a factor of 10 in many assays. We sought to use TRAP seq to better understand the gene expression landscapes that drive these morphogenic events. We found TRAP seq selectively enriches for endothelial RNA in both the planar and fibrin bead co-culture assays. Intriguingly, there are distinct changes in blood vessel development and in the mitotic cell cycle, unique to early and late phases of morphogenesis. To determine how these biological processes are altered by a known disruptor of vascular morphogenesis, we expressed PIK3CAH1047R in ECs, implicated as a driver in several types of vascular malformations. We found PIK3CAH1047R expression results in profound changes in endothelial cell expression with enrichment in genes associated with blood vessel morphogenesis gene ontology terms. There is a cohort of genes which showed dysregulation in both morphogenesis assays and these include numerous genes known to be important for tip/stalk specification during developmental angiogenesis, with a profound penetrance of NOTCH pathway dysregulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE296693 | GEO | 2025/07/29

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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TRAP-seq in 3D Angiogenesis Assays Reveals Distinct Biological Processes Associated with Early and Late Morphogenesis that are Disrupted with PIK3CA-H1047R

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