Search bioRxiv⌕ Search

bioRxiv · 10.1101/2023.04.19.537575

Impaired H19 lncRNA expression contributes to the compromised developmental angiogenesis in EVL-deficient mice

Abstract

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. Endothelial-specific deletion of EVL, a member of the mammalian Ena/VASP protein family, reduced the expression of the tip cell marker protein endothelial cell specific molecule-1 (Esm1) and compromised the radial sprouting of the vascular plexus in the postnatal mouse retina. The latter effects could at least partly be attributed to reduced VEGF receptor 2 (VEGFR2) internalization and signaling but the underlying mechanisms(s) are not fully understood. In the present study, we revealed that the expression of the long non-coding RNA H19 was significantly reduced in endothelial cells from postnatal EVL-/- mice and in siRNA-transfected human endothelial cells under hypoxic conditions. H19 was recently shown to promote VEGF expression and bioavailability via Esm1 and hypoxia inducible factor 1 (HIF-1). Similar to EVL-/- mice, the radial outgrowth of the vascular plexus was significantly delayed in the postnatal retina of H19-/- mice. In summary, our data suggests that loss of EVL not only impairs VEGFR2 internalization and downstream signaling, but also impairs VEGF expression and bioavailability in the hypoxic retina via downregulation of lncRNA H19.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Zink, J., Froemel, T., Boon, R. A., Fleming, I., Benz, P. M.. 2023-04-19. Impaired H19 lncRNA expression contributes to the compromised developmental angiogenesis in EVL-deficient mice. https://doi.org/10.1101/2023.04.19.537575

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

HUH-tag conjugation enables accessible, site-specific functionalization of DNA origami with signaling-competent proteins

DNA origami enables precise nanometer scale control over the number, spacing, and geometry of biomolecules, making it a powerful tool for dissecting spatially organized biological processes such as receptor signaling. However, functionalizing origami with proteins remains a bottleneck restricting the technology to specialized laboratories. Here we show that viral-derived HUH endonuclease tags, small (~10 kDa) domains that form a covalent phosphotyrosine bond with short unmodified ssDNA, provide a simple and cost-effective route to decorating DNA origami with functional proteins. Using interleukin-1{beta} (IL-1{beta}) as a model ligand, we generated recombinant IL-1{beta}-HUH, achieved efficient site-specific conjugation to unmodified ssDNA within minutes, and coupled the conjugates to a DNA origami pegboard with tunable stoichiometry. IL-1{beta}-functionalized origami triggered ligand-dependent signalling in living cells and allowed visualization of the recruitment of downstream signaling molecules, confirming that HUH-tag conjugation preserves functionality. Because this strategy requires only standard molecular biology equipment and unmodified DNA, it lowers a technical and financial barrier to DNA origami-based experimentation, opening the approach to a broader range of biological questions and laboratories outside the DNA nanotechnology field.

cell biology↗

Global mapping of VEGF-A driven changes in the RNA bound phospho-proteome reveals a molecular switch to coordinate angiogenesis in endothelial cells

RNA binding proteins (RBPs) serve as upstream master regulators of cellular fate transitions at the post-transcriptional level. RBP-RNA interactions are dynamically regulated by post-translational modifications to coordinate cellular responses. We hypothesised that changes in RBP phosphorylation will play a key role in coordinating angiogenesis, the formation of new blood vessels, in endothelial cells (ECs). Therefore, we used phospho-proteomic analysis of the mRNA bound proteome to identify global phosphorylation changes in mouse cardiac ECs during acute and prolonged VEGF-A stimulation. Of the differentially phosphorylated RBPs, we found that the phosphorylation of serine 199 within mRNA bound Phf6 (PHD finger protein 6) was consistently increased following VEGF-A stimulation. Phf6 phosphorylation at this specific site was sufficient and required to induce a pro-angiogenic phenotype, facilitating a change in Phf6 conformation promoting interaction with distinct RNAs. We show here that Phf6 phosphorylation at serine 199, downstream of VEGF-A, acts as a molecular switch to differentially coordinate a decrease of anti-angiogenic RNAs (i.e. extracellular matrix genes) and an increase in pro-angiogenesis related RNAs, in part by differentially regulating their stability, to promote angiogenesis.

cell biology↗

Metaplasia Orchestrates Tissue Recovery and Immune Remodeling Through CD73 in Chronic Pancreatitis

Chronic inflammatory injury promotes epithelial metaplasia, and progression from metaplasia to dysplasia is a recognized initiating event in carcinogenesis, as metaplastic cells exhibit heightened susceptibility to genetic alterations. In the pancreas, persistent acinar-to-ductal metaplasia (ADM) can progress to pancreatic intraepithelial neoplasia (PanIN), yet the mechanisms that determine whether ADM resolves or persists with fibrosis and sustained inflammation remain poorly defined. Here, we identify CD73 as a central regulator of ADM fate, tissue repair, and immune microenvironment remodeling during chronic pancreatic injury. Analysis of previously published single-cell RNA sequencing dataset from chronic pancreatitis (CP) tissues identified enrichment of Nt5e, which encodes CD73, within Hnf1{beta} ADM populations. Hnf1bCreERT2 and CD73fl/fl;Hnf1bCreERT2 mice were treated with caerulein for 4 weeks to induce pancreatitis and ADM prior to tamoxifen-induced deletion of CD73 in HNF1B-expressing metaplastic epithelial cells. Histologic phenotyping and immune profiling were performed using immunohistochemistry and imaging mass cytometry. Metaplasia-specific CD73 deletion exacerbated pancreatic injury and fibrosis, impaired epithelial proliferation and recovery of the acinar compartment, and altered the abundance and spatial organization of inflammatory cells. Ex vivo ADM assays further demonstrated that CD73 loss increased ADM formation and the production of MCP-1 and TIMP-1. These findings identify CD73 as a context-dependent checkpoint linking epithelial plasticity and immune modulation during chronic pancreatic injury and tissue repair.

cell biology↗