eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin
- Uppsala University, Sweden
- Max Planck Institute for Molecular Biomedicine, Germany
- Yale School of Medicine, United States
Abstract
Background: Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA) promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics may aggravate hypoxia; therefore, therapy development is needed.
Methods: Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) in pathological neovascularization and vessel permeability. Suppression of NO formation was achieved chemically using L-NMMA, or genetically, in endothelial NO synthase (eNOS) serine to alanine (S1176A) mutant mice.
Results: Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remaining vascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions, manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restored the vascular barrier and prevented leakage.
Conclusion: We conclude that NO destabilizes adherens junctions, resulting in vascular hyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway.
Funding: This study was supported by the Swedish Cancer foundation (19 0119 Pj 01 H), the Swedish Research Council (2020-01349), the Knut and Alice Wallenberg foundation (KAW 2020.0057) and a Fondation Leducq Transatlantic Network of Excellence Grant in Neurovascular Disease (17 CVD 03). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275). WCS was supported by Grants R35 HL139945, P01 HL1070205, AHA MERIT Award.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5. Source data files have also been deposited with Dryad: https://doi.org/10.5061/dryad.x69p8czhv
-
eNOS/NO and their role in modifying the vascular barrier in retinopathy - Source dataDryad, 10.5061/dryad.x69p8czhv.
Article and author information
Author details
Lena Claesson-Welsh
Funding
Swedish Cancer Foundation (19 0119 Pj 01 H)
- Lena Claesson-Welsh
Vetenskapsrådet (2020-01349)
- Lena Claesson-Welsh
Knut och Alice Wallenbergs Stiftelse (KAW 2015.0030)
- Lena Claesson-Welsh
Knut och Alice Wallenbergs Stiftelse (KAW.0275)
- Lena Claesson-Welsh
Fondation Leducq (17 CVD 03)
- Lena Claesson-Welsh
National Institutes of Health (R35 HL 139945)
- William C Sessa
National Institutes of Health (P01HL 1070205)
- William C Sessa
American Heart Association (Merit Award)
- William C Sessa
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Mouse husbandry and oxygen-induced retinopathy (OIR) challenge took place at Uppsala University,and the University board of animal experimentation approved all animal work for the studies (ethical permit 5.2.18-8927/16). Animal handling was in accordance to the ARVO statement for the Use of Animals in Ophthalmologic and Vision Research. Professional animal care was provided and overseen by University veterinarians. Every effort was made to minimize suffering of the animal.
Copyright
© 2021, Ninchoji et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 35
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin
eLife 10:e64944.
https://doi.org/10.7554/eLife.64944
Further reading
-
- Cell Biology
Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′-phosphatase and 5′-kinase of DNA ends to promote DNA ligation and repair. Here, we show that cyclin-dependent kinases (CDKs) regulate the phosphorylation of threonine 118 (T118) in PNKP. This phosphorylation allows recruitment to the gapped DNA structure found in single-strand DNA (ssDNA) nicks and/or gaps between Okazaki fragments (OFs) during DNA replication. T118A (alanine)-substituted PNKP-expressing cells exhibited an accumulation of ssDNA gaps in S phase and accelerated replication fork progression. Furthermore, PNKP is involved in poly (ADP-ribose) polymerase 1 (PARP1)-dependent replication gap filling as part of a backup pathway in the absence of OFs ligation. Altogether, our data suggest that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to ssDNA gaps to proceed with OFs ligation and its backup repairs via the gap-filling pathway to maintain genome stability.
-
- Cell Biology
- Neuroscience
The emergence of Aβ pathology is one of the hallmarks of Alzheimer’s disease (AD), but the mechanisms and impact of Aβ in progression of the disease is unclear. The nuclear pore complex (NPC) is a multi-protein assembly in mammalian cells that regulates movement of macromolecules across the nuclear envelope; its function is shown to undergo age-dependent decline during normal aging and is also impaired in multiple neurodegenerative disorders. Yet not much is known about the impact of Aβ on NPC function in neurons. Here, we examined NPC and nucleoporin (NUP) distribution and nucleocytoplasmic transport using a mouse model of AD (AppNL-G-F/NL-G-F) that expresses Aβ in young animals. Our studies revealed that a time-dependent accumulation of intracellular Aβ corresponded with a reduction of NPCs and NUPs in the nuclear envelope which resulted in the degradation of the permeability barrier and inefficient segregation of nucleocytoplasmic proteins, and active transport. As a result of the NPC dysfunction App KI neurons become more vulnerable to inflammation-induced necroptosis – a programmed cell death pathway where the core components are activated via phosphorylation through nucleocytoplasmic shutting. Collectively, our data implicates Aβ in progressive impairment of nuclear pore function and further confirms that the protein complex is vulnerable to disruption in various neurodegenerative diseases and is a potential therapeutic target.
