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The phosphodiesterase 2A regulates lymphatic endothelial development via cGMP-mediated control of Notch signaling

Claudia Carlantoni, Leon Liekfeld, Sandra A. Hemkemeyer, Danny Schreier, Ceren Saygi, Roberta Kurelic, Silvia Cardarelli, Joanna Kalucka, Christian Schulte, Manu Beerens, Reiner Mailer, Tilman Schäffer, Fabio Naro, Manuela Pellegrini, Viacheslav O. Nikolaev, Thomas Renné, Maike Frye

Preprint posted on 20 January 2023 https://www.biorxiv.org/content/10.1101/2023.01.18.524585v1

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An essential function for the phosphodiesterase 2A during regulation of lymphatic vessel maturation

Selected by Andreas van Impel, Sanjay Sunil Kumar

Background:

 The lymphatic system consists of a large, interconnected network of vessels and capillaries lined with endothelial cells (ECs) to perform a variety of vital tasks concerning macromolecule uptake and fluid homeostasis. These lymphatic endothelial cells (LECs), which are mostly of a venous origin, engage in the constant remodeling of cell-cell junctions and contacts during development. This dynamic remodeling process is key to sprout formation and subsequent vessel development. Upon maturation, LECs exit their cell cycle and proliferation is restricted via contact inhibition.  In blood ECs, prior studies have extensively highlighted the contribution of NOTCH and VE-Cadherin signaling toward contact inhibition. Additionally, in blood ECs, Cyclic Nucleotide Phosphodiesterases (PDEs) like PDE2A (and PDE3A) are known to control these endothelial junctions. While the native function of PDEs (eg. PDE2A and PDE3A) lies in the hydrolysis of secondary messengers like cGMP to GMP, their collective contribution towards LEC junctional remodeling has been hitherto in the dark. In the current study, Carlantoni et al., for the first time, provide a comprehensive dissection of the role of PDE2A in lymphatic development via cell-cell contact regulation.

 

Key findings:

  • PDE2A was found to be enriched explicitly in lymphatic cells as compared to venous cells

The extracellular matrix has the ability to dictate (lymphatic) endothelial cell morphology both in vivo and in vitro based on matrix stiffness. In this study, using RNAseq and comparing the expression profiles of lymphatic endothelial cells (HDLECs) versus venous endothelial cells (HUVECs), Carlantoni et al. were able to identify a novel, lymphatic-specific regulator of endothelial cell junctions – PDE2A.

 

  • Endothelial-specific knockout of Pde2a in mice triggers aberrant lymphatic development, resulting in lymphatic dysplasia and the formation of lymphedema

To understand the contribution of PDE2A to lymphatic vessel development in an in vivo context, the authors generated a conditional murine Pde2a knock-out allele. Since the global loss of Pde2a is embryonic lethal, Carlantoni and colleagues analyzed an EC-specific knockout of Pde2a in Tie2-Cre mice. Knockout of Pde2a in ECs resulted in a pronounced enlargement of lymphatic structures in the embryo, which appeared functionally impaired as embryos also displayed a lymphedema phenotype, consistent with a disruption of lymphatic development. Importantly, the loss of Pde2a seemed to predominantly impact the lymphatic vessel beds as the overall blood vessel morphology remained unchanged.

 

  • Loss of PDE2A interferes with the maturation of LEC junctions in vitro, leading to perturbed contact inhibition and increased cell proliferation

To further characterize the cause of these aberrant lymphatic vessels, Carlantoni and colleagues turned to in vitro HDLEC monolayer studies. They observed that PDE2A expression increased with increased monolayer maturity. Loss of PDE2A, however, resulted in decreased junctional stability via downregulation of the adhesion protein CLDN5 in mature cultures. Since junctional maturation in EC typically coincides with the downregulation of proliferation, contact inhibition and cell cycle arrest, the authors addressed the question of whether the loss of PDE2A activity would also interfere with these processes. Using RNAseq, they showed that loss of PDE2A resulted in a junctionally unstable expression profile and a shift towards a more “loose” vessel state. Moreover, the absence of PDE2A gave rise to a highly proliferative expression profile, a finding that was confirmed by KI67-antibody and BrdU stainings of HDLECs.

 

  • Endothelial-specific depletion of Pde2a affects cell cycle arrest and lymphatic maturation during later stages of murine lymphatic development

To investigate whether Pde2a is also involved in lymphatic vessel maturation in vivo, the authors analyzed an EC-specific Cdh5-CreERT2 Pde2a knockout that was induced at later embryonic stages (E10.5-14.5). Consistent with their in vitro data, Pde2a mutant embryos displayed a higher number of LECs, and increased vessel diameter but fewer Prox1-positive valve structures, which are a sign of maturing lymphatic vessels.

 

  • Loss of PDE2A function results in an LEC-specific increase of cGMP levels in vitro

PDE2A encodes for a cyclic nucleotide phosphodiesterase, an enzyme that catalyzes the hydrolysis of the second messenger molecules cAMP and cGMP. For blood ECs, it was reported before that misregulation of cAMP levels impacts the stability of EC junctions. The authors, therefore, assessed the levels of cAMP and cGMP in cultured LECs after deletion of PDE2A and found a specific increase in cGMP levels under these conditions. Importantly, artificially elevating the levels of cGMP by exposing cultured LECs to increased levels of cGMP in the medium resulted in junctional disruption, resembling the defects seen after loss of PDE2A. These results therefore indicate that PDE2A acts via controlling levels of cGMP in LECs, which in turn impacts the stability of LEC junctions.

 

  • Activation of NOTCH signaling in PDE2A KD conditions rescues junctional maturation defects, suggesting PDE2A and cGMP act upstream of NOTCH during lymphatic maturation

Finally, the authors report that in PDE2A KD and KO scenarios, a downregulation of Notch target genes is evident, suggesting that this pathway is misregulated under these conditions. Importantly, the activation of the Notch pathway in LECs depleted of functional PDE2A proved to be sufficient to rescue the cell junction maturation defects indicating that NOTCH acts downstream of PDE2A and cGMP to control lymphatic vessel maturation events during lymphatic development.

 

PDE2A controls LEC contact inhibition and vessel maturation during lymphatic development.

 

What we liked about this preprint:

Functional impairment of the lymphatic vasculature results in the accumulation of interstitial fluids in various tissues, known as lymphedema. In humans, this condition most frequently results from secondary damage of lymphatic vessels by e.g. surgery, trauma or certain parasitic infections but can also be triggered by inherited mutations in genes that are essential for the development of the lymphatic system. The treatment options for lymphedema are very limited in the clinic as no curative treatments are available at present and the standard manual therapies mainly aim at temporarily reducing the fluid accumulations in affected tissues.
A prerequisite to developing treatment strategies for diseases related to the lymphatic vasculature is to better understand the molecular control of lymphangiogenesis and therefore, to identify novel gene functions that are essential for the development and maturation of the lymphatic vasculature.

In this preprint, the authors show for the first time a specific role for PDE2A during lymphatic development. Using an elegant combination of in vivo and in vitro studies, they unravel the function of this enzyme during the regulation of lymphatic vessel maturation and describe a novel PDE2A-cGMP-NOTCH signaling axis that controls cell cycle arrest, contact inhibition and junctional maturation in the lymphatic system. This study therefore adds another important piece to the puzzle on how lymphatic development and maturation are controlled.

 

Questions to the authors:

How does the loss of PDE2A affect other lymphatic beds in vivo? Particularly, the mesentery which generally requires remodeling for valve formation.

What is the link between cGMP levels and Notch signaling?

Does the sDLL4 rescue also reduce the higher proliferation rates in the PDE2A KO cells?

Since Pde2a appears to be more important during lymphatic vessel maturation, what would the impact of postnatal Pde2a knockout be?

 

Further reading:

Petrova TV, Koh GY. Biological functions of lymphatic vessels. Science. 2020 Jul 10;369(6500):eaax4063. doi: 10.1126/science.aax4063. PMID: 32646971.

Surapisitchat J, Beavo JA. Regulation of endothelial barrier function by cyclic nucleotides: the role of phosphodiesterases. Handb Exp Pharmacol. 2011;(204):193-210. doi: 10.1007/978-3-642-17969-3_8. PMID: 21695641; PMCID: PMC4062991.

Netherton SJ, Maurice DH. Vascular endothelial cell cyclic nucleotide phosphodiesterases and regulated cell migration: implications in angiogenesis. Mol Pharmacol. 2005 Jan;67(1):263-72. doi: 10.1124/mol.104.004853. Epub 2004 Oct 8. PMID: 15475573.

Tags: lymphatics, vascular biology

Posted on: 20 February 2023 , updated on: 27 February 2023

doi: https://doi.org/10.1242/prelights.33776

Read preprint (1 votes)

Author's response

Claudia Carlantoni and Maike Frye shared

  1. How does the loss of PDE2A affect other lymphatic beds in vivo? Particularly, the mesentery which generally requires remodeling for valve formation.

We initially decided to focus on dermal embryonic lymphangiogenesis at E17.5 to be able to visualize and study areas of dynamic remodeling (dermal midline) and ongoing maturation (maturing plexus). We agree that this is an interesting question and have begun to analyze junctional maturation and valve formation in Pde2a-deficient mesenteries using the inducible Cdh5-CreERT2 line. Given the fact that dermal and mesenteric lymphatic vessels arise in parts from different lymphatic endothelial progenitor cells, a comparative analysis of both lymphatic beds will provide insights into general and organ-specific principles of PDE2A function in the lymphatic endothelium.

 

  1. What is the link between cGMP levels and Notch signaling?

This is an excellent question and we have already identified potential mechanisms downstream of altered cGMP levels that might regulate NOTCH function in lymphatic contact inhibition. Through a series of in vitro experiments – some of which we already initiated –, we plan to further substantiate the causal link between cGMP levels and these newly identified Notch modulators.

 

  1. Does the sDLL4 rescue also reduce the higher proliferation rates in the PDE2A KO cells?

We did not perform this experiment, but we would predict that indeed activation of NOTCH signaling via sDLL4 would reduce the higher proliferation rates in PDE2A siRNA-treated compared to CTRL siRNA-treated lymphatic endothelial cells. In our opinion, expression of Claudin 5 (CLDN5) is the optimal marker of proper lymphatic junctional integrity and contact inhibition (see also https://elifesciences.org/articles/57732). We have shown that high CLDN5 levels can be rescued using sDLL4 in PDE2A siRNA-treated lymphatic endothelial cells and therefore anticipate low proliferation in these cells.

 

  1. Since Pde2a appears to be more important during lymphatic vessel maturation, what would the impact of postnatal Pde2a knockout be?

This is a very interesting question, and we hypothesize that PDE2A is similarly required in postnatal lymphatic cell contact inhibition and maturation. Therefore, we have begun to delete Pde2a in lymphatic endothelial cells postnatally to uncover (i) if PDE2A function is restricted to the defined process of lymphatic junctional maturation during embryonic and potentially postnatal development; and (ii) if PDE2A is further required to maintain lymphatic junctional integrity of already established lymphatic networks.

In future endeavors, we will explore a potential function for PDE2A in junctional “sealing” at adult stages and specifically in the context of diseases associated with lymphatic dysfunction. We will additionally investigate if we could locally “loosen” established lymphatic junctions via PDE2A modulation in order to achieve improvement of lymphatic regrowth and regeneration.

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