Wnt/beta-catenin signaling regulates Vascular Endothelial Growth Factor (VEGF) receptors in central nervous system endothelial cells
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eLife Assessment
This study provides important novel insight into how Wnt/beta-catenin signaling may regulate the responsiveness of blood vessels in the brain and retina to vascular endothelial growth factor, with potential relevance to Wnt surrogate therapies aimed at restoring vascular barriers. Solid evidence that Wnt/beta-catenin signaling changes the protein abundance and relative balance of two vascular endothelial growth factor receptors in brain blood vessels is provided by informative genetic mosaic experiments; in the retina, however, regulation of VEGFR2 was difficult to establish because expression by neighboring cells obscured its detection in blood vessels. Physiological implications are discussed, but evidence remains incomplete because the observed changes in receptor abundance were not directly linked to altered signaling or vascular function in living animals. A second mechanism, in which Wnt/beta-catenin signaling suppresses signaling through VEGFR2 with little or no change in receptor abundance, was inferred from reporter experiments in cell-based systems.
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Abstract
In CNS endothelial cells (ECs), VEGF signaling promotes vascular permeability, and Wnt/beta-catenin signaling reduces vascular permeability by controlling the gene expression program for the blood-brain barrier. Here we show, using genetic mosaics, that, in mouse brain ECs in vivo , an increase in Wnt/beta-catenin signaling produces an increase in VEGFR1 levels and a decrease in VEGFR2 levels, and a decrease in Wnt/beta-catenin signaling produces a decrease in VEGFR1 levels and an increase in VEGFR2 levels. As VEGFR1 functions as a decoy receptor to reduce VEGF signaling through VEGFR2, these data imply that Wnt/beta-catenin signaling acts at the receptor level to reduce VEGF signaling. In HEK/293T cells, VEGF signaling is suppressed by Wnt/beta-catenin signaling in a dose-dependent manner, with nearly complete suppression observed at levels of Wnt/beta-catenin signaling that produce little or no change in VEGF receptor levels. These data reveal two mechanisms by which VEGF signaling is regulated by Wnt/beta-catenin signaling.
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eLife Assessment
This study provides important novel insight into how Wnt/beta-catenin signaling may regulate the responsiveness of blood vessels in the brain and retina to vascular endothelial growth factor, with potential relevance to Wnt surrogate therapies aimed at restoring vascular barriers. Solid evidence that Wnt/beta-catenin signaling changes the protein abundance and relative balance of two vascular endothelial growth factor receptors in brain blood vessels is provided by informative genetic mosaic experiments; in the retina, however, regulation of VEGFR2 was difficult to establish because expression by neighboring cells obscured its detection in blood vessels. Physiological implications are discussed, but evidence remains incomplete because the observed changes in receptor abundance were not directly linked to altered …
eLife Assessment
This study provides important novel insight into how Wnt/beta-catenin signaling may regulate the responsiveness of blood vessels in the brain and retina to vascular endothelial growth factor, with potential relevance to Wnt surrogate therapies aimed at restoring vascular barriers. Solid evidence that Wnt/beta-catenin signaling changes the protein abundance and relative balance of two vascular endothelial growth factor receptors in brain blood vessels is provided by informative genetic mosaic experiments; in the retina, however, regulation of VEGFR2 was difficult to establish because expression by neighboring cells obscured its detection in blood vessels. Physiological implications are discussed, but evidence remains incomplete because the observed changes in receptor abundance were not directly linked to altered signaling or vascular function in living animals. A second mechanism, in which Wnt/beta-catenin signaling suppresses signaling through VEGFR2 with little or no change in receptor abundance, was inferred from reporter experiments in cell-based systems.
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Reviewer #1 (Public review):
Summary:
Wang et al. report that changes in the levels of canonical Wnt signaling in CNS ECs modulate protein expression levels of VEGFR2 and VEGFR1. An increase in Wnt/beta-catenin signaling results in a decrease in VEGFR2 and an increase in the decoy receptor VEGFR1; conversely, reduction of Wnt signaling leads to the opposite changes in VEGF receptors in brain CVOs (less clear in the retina). Next to the solid quantitative immunostaining of VEGFR2 and VEGFR1 in multiple CNS regions using genetic mosaics and loss-of-function mouse mutants, the authors employ HEK293T cells to investigate the effect of activation of Wnt/beta-catenin signaling on multiple RTK signaling systems and on the level of overexpressed VEGFR2. The principal conclusions are that Wnt/beta-catenin regulates VEGF signaling. Two distinct …
Reviewer #1 (Public review):
Summary:
Wang et al. report that changes in the levels of canonical Wnt signaling in CNS ECs modulate protein expression levels of VEGFR2 and VEGFR1. An increase in Wnt/beta-catenin signaling results in a decrease in VEGFR2 and an increase in the decoy receptor VEGFR1; conversely, reduction of Wnt signaling leads to the opposite changes in VEGF receptors in brain CVOs (less clear in the retina). Next to the solid quantitative immunostaining of VEGFR2 and VEGFR1 in multiple CNS regions using genetic mosaics and loss-of-function mouse mutants, the authors employ HEK293T cells to investigate the effect of activation of Wnt/beta-catenin signaling on multiple RTK signaling systems and on the level of overexpressed VEGFR2. The principal conclusions are that Wnt/beta-catenin regulates VEGF signaling. Two distinct levels of regulation are reported: one acts in vivo on receptor protein expression levels, the other acts in 293T cells, and the precise mechanism remains to be identified.
Strengths:
Strengths of the study include the use of genetic mosaics that allow for quantification of immuno-staining intensity with an internal reference and the significance of understanding the interplay of beta-catenin-dependent and VEGF pathways.
There are also some weaknesses noted:
(1) Experiments to restore the VEGFR2/VEGFR1 ratio in vivo, or to modulate VEGF signaling in vivo, and assess to what extent Wnt/beta-catenin LOF phenotypes can be restored by modulation of VEGF signaling have not been conducted at this point. In addition, it remains to be determined to what degree an experimental recreation of the aberrant VEGFR2/VEGFR1 ratio is sufficient to recreate (partially) some of the phenotypes observed with loss of Wnt/beta-catenin signaling in ECs.
(2) It would be useful to consider and discuss other essential components of the VEGF signaling system, especially Neuropilins. Wang et al. (2012, Figure 2, PMID: 23217714) reported that CNS ECs with loss of Norrin signaling gain Sema3F binding while not displaying VEGF-B binding sites, suggesting changes in VEGF/semaphorin co-receptor biology.
(3) Martowicz et al. (2019) PMID: 31533473 also investigated the influence of Wnt/beta-catenin signaling in CNS ECs on VEGFR2 and VEGFR3 expression and reported findings regarding VEGFR2 regulation that appear to differ from the present study. This should be discussed.
(4) Norrin/beta-catenin and Wnt/beta-catenin signaling have a critical role in CNS angiogenesis, and Wnt surrogates counteract neovascularization in the oxygen-induced retinopathy model. These findings highlight context-dependent roles of Wnt/beta-catenin signaling in normal and pathological ECs. How the modulation of VEGFR2/VEGFR1 may influence developmental and pathological angiogenesis could be discussed.
(5) When looking up the publicly available scRNA-seq datasets from Heng et al. (2019, PMID: 30988181) from Norrin KO mice, it appears that there is transcriptional regulation of VEGFR1/VEGFR2 consistent with the protein regulation reported here. If transcriptional regulation is important, CMV-driven VEGFR2 expression in Fig.5 supplement 2 may not model the regulatory mechanisms observed in vivo. Does CHIR99021 modulate VEGFR2/VEGFR1 transcripts and proteins in HRMVECs?
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Reviewer #2 (Public review):
The manuscript by Wang, Nathans, and colleagues presents a compelling investigation into the regulatory crosstalk between Wnt/beta-catenin and VEGF signaling within central nervous system (CNS) endothelial cells. Overall, it is a very solid paper from a very respected research lab. The use of in vivo genetic mosaics to directly compare endothelial cells (ECs) with differing beta-catenin states within the same tissue is a particularly elegant approach to mapping receptor dynamics.
Major Comments:
(1) Distinguishing Receptor Abundance from Functional Signaling:
The study relies heavily on immunohistochemistry to assess total VEGFR1 and VEGFR2 levels, inferring that changes in receptor abundance directly equate to altered VEGF signaling. Because immunofluorescence does not establish plasma membrane …
Reviewer #2 (Public review):
The manuscript by Wang, Nathans, and colleagues presents a compelling investigation into the regulatory crosstalk between Wnt/beta-catenin and VEGF signaling within central nervous system (CNS) endothelial cells. Overall, it is a very solid paper from a very respected research lab. The use of in vivo genetic mosaics to directly compare endothelial cells (ECs) with differing beta-catenin states within the same tissue is a particularly elegant approach to mapping receptor dynamics.
Major Comments:
(1) Distinguishing Receptor Abundance from Functional Signaling:
The study relies heavily on immunohistochemistry to assess total VEGFR1 and VEGFR2 levels, inferring that changes in receptor abundance directly equate to altered VEGF signaling. Because immunofluorescence does not establish plasma membrane localization, receptor phosphorylation, or downstream pathway activation, the definitive conclusion that beta-catenin "acts at the receptor level to reduce VEGF signaling" is currently overstated.
Recommendation: The authors should tone down the functional claims throughout the text (e.g., state that the receptor changes are "predicted to reduce VEGF signaling") or provide direct functional readouts of VEGFR2 activation in vivo, such as phospho-VEGFR2, phospho-PLCγ, ERK, or AKT.
(2) Limitations of the In Vitro HEK293T Model:
The HEK293T STF reporter system is utilized to argue for a "post-receptor physiological suppression" of VEGF signaling. While experimentally practical, this non-endothelial system does not definitively establish a physiological mechanism for CNS ECs. Furthermore, NFAT-luciferase is not uniquely specific to VEGFR2 and can be activated by broader calcium/calcineurin pathways. The authors also show that high Wnt1 expression reduces total VEGFR2 protein in these cells, but only at plasmid concentrations much higher than those required to suppress the VEGFR2/NFAT reporter.
Recommendation: Explicitly decouple the in vitro observations from the physiological in vivo claims. Remove the term "physiological" unless you validate post-receptor suppression in a primary endothelial system under endogenous signaling conditions.
(3) Regional Heterogeneity (Brain vs. Retina):
The retinal vascular mosaic data reveal that while VEGFR1 is reduced in BRB- ECs, VEGFR2 shows no obvious difference. The authors correctly acknowledge that Müller glial VEGFR2 expression complicates endothelial quantification, rendering Figure 4C a negative result with uncertain sensitivity. Consequently, the broad generalization that an "increase in Wnt/beta-catenin signaling produces an increase in VEGFR1 levels and a decrease in VEGFR2 levels" across all CNS ECs is not entirely accurate.
Recommendation: Embrace this biological nuance. Revise the generalized claims to reflect that beta-catenin regulates VEGFR1 consistently across the brain and retina, but VEGFR2 regulation appears context-dependent and is not clearly demonstrated in the retina. To resolve the Müller glia interference, consider orthogonal approaches to isolate the EC signal, such as EC-specific RNAscope or flow cytometry on FACS-sorted ECs.
(4) Statistical Rigor and Experimental Units:
The figure legends (e.g., Figure 1) explicitly state that data points represent the mean values from single confocal images. If multiple images were obtained from the same animal, treating them as independent units. Furthermore, the authors utilized unpaired statistics (Wilcoxon rank-sum) to compare BBB+ and BBB- ECs within the same mosaic tissue sections.
Recommendation: Reanalyze the data using the animal as the primary biological replicate if such data are available (averaging all image measurements within each animal). For mosaic comparisons within the same tissue image/animal, utilize paired statistical tests (e.g., paired t-test or paired Wilcoxon signed-rank test). Figure legends should clearly report the exact N (animals) and n (images/cells), the specific statistical tests used, and confidence intervals.
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Reviewer #3 (Public review):
Summary:
The manuscript by Wang et al. describes the identification of a novel mechanism by which the canonical Wnt/beta-catenin pathway could regulate endothelial permeability in the central nervous system (CNS). Specifically, they show that in circumventricular organs (CVOs), in which the blood-brain barrier (BBB) is not established in vessels and endothelial cells are highly permeable, the previously reported low levels of Wnt/beta-catenin coincide with high levels of the vascular endothelial growth factor receptor 2 (VEGFR2, Flk-1), whereas the decoy receptor VEGFR1 (flt-1) is expressed at low levels. In order to test the hypothesis in vivo that the Wnt/beta-catenin pathway controls VEGF signaling in CNS vessels by regulating VEGF receptors, the authors analyzed mosaic cerebellar vessels of mice …
Reviewer #3 (Public review):
Summary:
The manuscript by Wang et al. describes the identification of a novel mechanism by which the canonical Wnt/beta-catenin pathway could regulate endothelial permeability in the central nervous system (CNS). Specifically, they show that in circumventricular organs (CVOs), in which the blood-brain barrier (BBB) is not established in vessels and endothelial cells are highly permeable, the previously reported low levels of Wnt/beta-catenin coincide with high levels of the vascular endothelial growth factor receptor 2 (VEGFR2, Flk-1), whereas the decoy receptor VEGFR1 (flt-1) is expressed at low levels. In order to test the hypothesis in vivo that the Wnt/beta-catenin pathway controls VEGF signaling in CNS vessels by regulating VEGF receptors, the authors analyzed mosaic cerebellar vessels of mice deficient for the Wnt receptor frizzled 4 (Fzd4), showing that BBB+ (normal condition) vessels show low VEGFR2 and high VEGFR1, whereas BBB- (Fzd4ko) vessels show high VEGFR2 and low VEGFR1. Similarly, although not as conclusively due to strong background from Müller cells, these effects were also visible in the retina of these mice. Conversely, dominant genetic activation of beta-catenin signaling in endothelial cells in vivo resulted in upregulation of VEGFR1 and downregulation of VEGFR2 in vessels of the CVOs as well as of the choroid plexus.
In order to better understand the direct relevance of Wnt/beta-catenin signaling for this regulatory theme, the authors activated the pathway in HEK293 cells, stably harboring the Super-Top-FLASH reporter construct in the presence of VEGFR2 and VEGF. Moreover, by transfecting Wnt1 into HEK293 cells, the authors analysed the effect of TGFbeta, EGF, and FGF signaling. Together, these experiments showed that Wnt/beta-catenin signaling potently suppressed VEGF signaling at low concentrations of Wnt1 transfection, whereas VEGFR2 suppression was only visible at relatively high concentrations. Also, the other pathways were suppressed, although to a lower extent.
In conclusion, the authors suggest a mechanism of VEGF signaling suppression by Wnt/beta-catenin 1) at the post-receptor level of VEGF receptors and 2) at the transcriptional level of VEGFR1 and VEGFR2, independent of VEGF signaling suppression.
Strengths:
(1) The present manuscript provides interesting additional information on top of the published work on the mechanistic function of vascular permeability control by Wnt/beta-catenin, specifically by the highlighted interaction with the VEGF pathway and the opposed regulation of the two VEGF receptors VEGFR1 and VEGFR2.
(2) They have used state-of-the-art animal models, high-resolution imaging, and appropriate in vitro analysis.
(3) This finding has potentially strong implications for future therapeutic strategies, aiming at Wnt/beta-catenin activation in various diseases of the CNS in which vascular repair is desired.
Weaknesses:
A detailed analysis of the post-receptor and transcriptional mechanisms remains largely unexplored and needs to be explored in the future.
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