Amino acid transporter SLC38A5 regulates developmental and pathological retinal angiogenesis
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eLife assessment
This paper will be of interest to those studying retinal angiogenesis and endothelial cell biology. The authors performed rigorous data analysis and presented a logical, well-written report. The key conclusions of the manuscript are supported by the data and uncover a novel factor for retinal endothelial cell growth.
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Abstract
Amino acid (AA) metabolism in vascular endothelium is important for sprouting angiogenesis. SLC38A5 (solute carrier family 38 member 5), an AA transporter, shuttles neutral AAs across cell membrane, including glutamine, which may serve as metabolic fuel for proliferating endothelial cells (ECs) to promote angiogenesis. Here, we found that Slc38a5 is highly enriched in normal retinal vascular endothelium, and more specifically, in pathological sprouting neovessels. Slc38a5 is suppressed in retinal blood vessels from Lrp5 −/− and Ndp y/− mice, both genetic models of defective retinal vascular development with Wnt signaling mutations. Additionally, Slc38a5 transcription is regulated by Wnt/β-catenin signaling. Genetic deficiency of Slc38a5 in mice substantially delays retinal vascular development and suppresses pathological neovascularization in oxygen-induced retinopathy modeling ischemic proliferative retinopathies. Inhibition of SLC38A5 in human retinal vascular ECs impairs EC proliferation and angiogenic function, suppresses glutamine uptake, and dampens vascular endothelial growth factor receptor 2. Together these findings suggest that SLC38A5 is a new metabolic regulator of retinal angiogenesis by controlling AA nutrient uptake and homeostasis in ECs.
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eLife assessment
This paper will be of interest to those studying retinal angiogenesis and endothelial cell biology. The authors performed rigorous data analysis and presented a logical, well-written report. The key conclusions of the manuscript are supported by the data and uncover a novel factor for retinal endothelial cell growth.
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Reviewer #1 (Public Review):
The study focuses on the role of SLC38A5, a neutral amino acid transporter, in retinal angiogenesis. The authors show that Slc38a5 transporter is highly enriched in normal retinal vascular ECs, and upregulated in the ECs in pathogenic neoangiogenesis (the OIR model). Additionally, the authors show that Slc38a5 transcription is regulated by Wnt/β-catenin signaling and deletion of Slc38a5 in mice substantially delays retinal vascular development and suppresses pathological neovascularization in the OIR model by suppressing glutamine uptake and reducing VEGFR2 expression. The authors claim that SLC38A5 is a new metabolic regulator of retinal angiogenesis.
The study is performed carefully and demonstrates clearly an important role for the transporter in retina angiogenesis. However, there are some concerns that …
Reviewer #1 (Public Review):
The study focuses on the role of SLC38A5, a neutral amino acid transporter, in retinal angiogenesis. The authors show that Slc38a5 transporter is highly enriched in normal retinal vascular ECs, and upregulated in the ECs in pathogenic neoangiogenesis (the OIR model). Additionally, the authors show that Slc38a5 transcription is regulated by Wnt/β-catenin signaling and deletion of Slc38a5 in mice substantially delays retinal vascular development and suppresses pathological neovascularization in the OIR model by suppressing glutamine uptake and reducing VEGFR2 expression. The authors claim that SLC38A5 is a new metabolic regulator of retinal angiogenesis.
The study is performed carefully and demonstrates clearly an important role for the transporter in retina angiogenesis. However, there are some concerns that need to be addressed as follows:
The authors show that Slc38a5 is downregulated in the Lrp5-/- and Ndpy/- retinas (Fig 1A, B); however, there is a discrepancy in Slc38a5 expression levels in the control retinas. The expression of Slc38a5 in the WT retina goes down from P8-P12 and then plateaus through P17 (Fig. 1A). In contrast, in Fig.1B, the expression of Slc38a5 in the Ndpy/+ retina plateaus from P8-P12 and then goes up through P17. The authors need to establish better the temporal expression of the transporter in control (WT) retinas.
While it's clear that Slc38a5 mRNA and protein expression is enriched in LCM-isolated retinal vessels, it's unclear whether that expression is exclusively in ECs or also in vessel associated mural cells (Fig.1C, Fig.S1). Although Fig.S1 shows the mining of mouse retinal scRNA-seq database to demonstrate exclusive Slc38a5 expression in ECs, it's necessary to validate that in the tissue using either RNA in situ hybridization or IHC for in combination with an endothelial cell or mural cell marker.
Fig.3: The image qualities are poor. The authors need to enhance image qualities to show the vessels clearly in such low magnification.
Fig.3F: The images in this panel show more than 50% decrease in the vascular area in the deep plexus between WT and Slc38a5-/- retinas. However, the graph shows a far lower (10-15% at best) decrease in the vascular coverage. The authors need to select representative images to match the graph.
The authors show the presence of vessels in the adult Slc38a5-/- retina to claim that vascular abnormalities seen in early development are gone in the adult (Fig. S2). However, the presence of vessels does not mean that there are no vascular abnormalities. The authors should compare established vascular parameters such as branching-density, vascular pruning between adult WT and Slc38a5-/- retinas to justify the claim.
While the authors show that there is a decrease in pathological neovascularization in the Slc38a5-/- retina at P17 in the OIR model (Fig4), they do not mention what happens to the Slc38a5-/- retina at P12 immediately after the hyperoxia phase. Is the vaso-obliteration altered in the Slc38a5-/- retina at that time compared to the WT?
What happens to the neurovascular unit (pericyte, astrocyte, Müller glia etc) in the Slc38a5-/- retina? How do they respond to altered angiogenesis?
Overall, the Discussion needs to emphasize the role of endothelial cell metabolisms in vascular development and maturation and how Slc38a5 may influence these processes.
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Reviewer #2 (Public Review):
Anti-VEGF treatment is currently used to treat patients with pathological retinal angiogenesis, but finding the underlying cause of increased VEGF is a challenge for the field. Wang and colleagues determined the role played by the amino acid transporter, SLC38A5, in retinal angiogenesis. They showed that Slc38a5 mRNA was enriched in retinal blood vessels versus neural retina, supporting previous single cell data that they reanalyzed here. In mouse models of human Retinopathy of Prematurity (ROP; Lrp5-/- and Ndp-/-) with decreased blood vessels, they showed a decrease in SLC38A5 protein. As both LRP5 and NDP encode proteins that work through the Wnt signaling pathway, the authors showed that both Slc38a5 mRNA and protein levels are controlled by Wnt agonists and antagonists in human endothelial cell cultures. …
Reviewer #2 (Public Review):
Anti-VEGF treatment is currently used to treat patients with pathological retinal angiogenesis, but finding the underlying cause of increased VEGF is a challenge for the field. Wang and colleagues determined the role played by the amino acid transporter, SLC38A5, in retinal angiogenesis. They showed that Slc38a5 mRNA was enriched in retinal blood vessels versus neural retina, supporting previous single cell data that they reanalyzed here. In mouse models of human Retinopathy of Prematurity (ROP; Lrp5-/- and Ndp-/-) with decreased blood vessels, they showed a decrease in SLC38A5 protein. As both LRP5 and NDP encode proteins that work through the Wnt signaling pathway, the authors showed that both Slc38a5 mRNA and protein levels are controlled by Wnt agonists and antagonists in human endothelial cell cultures. They further showed that Slc38a5 transcription is affected by Wnt signaling by performing luciferase assays on putative Wnt binding regions that they identified 5' of the Slc38a5 gene. To further characterize the role of SLC38A5 in vivo, they injected a validated si-RNA into mouse eyes and found that formation of retinal vasculature layers was significantly impaired, which they also showed in Slc38a5 knockout mice. Using another mouse model of Retinopathy of Prematurity (oxygen-induced retinopathy), they find that Slc38a5 is required during pathological angiogenesis, and using in vitro cell culture studies show that it is required for endothelial cell viability, migration and tubular formation via its role in transporting glutamine. In part, they find that this may be through the regulation of angiogenesis-promoting receptor, VEGFR2. The authors performed an impressive series of experiments both in vitro and in vivo in studying the role of SLC38A5 in retinal angiogenesis. Their final model also does a nice job of summarizing their manuscript.
While the overall conclusions are supported by the data, some aspects of image acquisition and data analysis need to be clarified and extended.
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Reviewer #3 (Public Review):
The authors showed that SLC38A5, in the retina, was primarily expressed in the vasculature, and its expression is under the direct control of Wnt/beta-catenin signaling. The deficiency of SLC38A5 resulted in delayed retinal vascular growth and reduces neovascularization in OIR model. Additionally, the authors addressed the mechanisms of Slc38a5 as a glutamine transporter regulating retinal vascular development through VEGF receptors.
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