Pericytes control vascular stability and auditory spiral ganglion neuron survival
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eLife assessment
This study presents a valuable finding in identifying the roles of the pericytes in maintaining vascular volume and integrity of spiral ganglion neurons (SGNs) in the cochlea, the main hearing organ. The evidence supporting the authors' claims is solid using an inducible and conditional pericyte depletion mouse model and the co-culture models. While the study provides a modest translational contribution, understanding the roles of organ-specific pericytes is paramount, making this study timely and significant. The work will be interesting for biomedical biologists working on hearing, blood vessels, signaling, and cell-to-cell interactions.
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Abstract
The inner ear has a rich population of pericytes, a multi-functional mural cell essential for sensory hair cell heath and normal hearing. However, the mechanics of how pericytes contribute to the homeostasis of the auditory vascular-neuronal complex in the spiral ganglion are not yet known. In this study, using an inducible and conditional pericyte depletion mouse (PDGFRB-CreER T2 ; ROSA26iDTR) model, we demonstrate, for the first time, that pericyte depletion causes loss of vascular volume and spiral ganglion neurons (SGNs) and adversely affects hearing sensitivity. Using an in vitro trans-well co-culture system, we show pericytes markedly promote neurite and vascular branch growth in neonatal SGN explants and adult SGNs. The pericyte-controlled neural growth is strongly mediated by pericyte-released exosomes containing vascular endothelial growth factor-A (VEGF-A). Treatment of neonatal SGN explants or adult SGNs with pericyte-derived exosomes significantly enhances angiogenesis, SGN survival, and neurite growth, all of which were inhibited by a selective blocker of VEGF receptor 2 (Flk1). Our study demonstrates that pericytes in the adult ear are critical for vascular stability and SGN health. Cross-talk between pericytes and SGNs via exosomes is essential for neuronal and vascular health and normal hearing.
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Author Response
Reviewer #1 (Public Review):
Like other sensory organs, the inner ear has a rich population of pericytes, essential for sensory hair cell heath and normal hearing. In this study, using an inducible and conditional pericyte depletion mouse (PdgfrbCreERT2/iDTR) model, the authors demonstrate that the pericytes play critical roles in maintaining vascular volume and integrity of spiral ganglion neurons (SGNs) in the cochlea. Moreover, using the coculture models, they show vigorous vascular and neuronal growth in neonatal SGN explants in the presence of exogenous pericytes. Mechanistically, this study demonstrates that these roles are achieved mainly through the interactions between pericyte-released exosomes containing VEGF-A and VEGFR2-expressing the vessels and SGNs.
Overall, the data are analyzed thoroughly, and the …
Author Response
Reviewer #1 (Public Review):
Like other sensory organs, the inner ear has a rich population of pericytes, essential for sensory hair cell heath and normal hearing. In this study, using an inducible and conditional pericyte depletion mouse (PdgfrbCreERT2/iDTR) model, the authors demonstrate that the pericytes play critical roles in maintaining vascular volume and integrity of spiral ganglion neurons (SGNs) in the cochlea. Moreover, using the coculture models, they show vigorous vascular and neuronal growth in neonatal SGN explants in the presence of exogenous pericytes. Mechanistically, this study demonstrates that these roles are achieved mainly through the interactions between pericyte-released exosomes containing VEGF-A and VEGFR2-expressing the vessels and SGNs.
Overall, the data are analyzed thoroughly, and the conclusions are novel and convincing. It is mechanistically solid. The study is somewhat translationally limited. Nevertheless, understanding the roles of organ-specific pericytes is paramount, making this study timely and significant.
We thank Reviewer #1 for the positive comment. We agree the pericyte depletion model is not a translational disease model. However, pericyte pathologies, including the decline in pericyte number, pericyte migration, and pericyte trans-differentiation, are frequently seen in aging and noise-induced hearing loss animal models. Moreover, hearing dysfunction due to pericyte pathology has been demonstrated in recent studies (Hou et al., 2020; Hou et al., 2018; Neng et al., 2015).
Reviewer #2 (Public Review):
The present study from Xiaorui Shi's lab investigated the effect of pericyte depletion on spiral ganglion neurons and auditory function. Results in vitro culture system proposed that pericyte-derived exosomes contain VEGF, and promote not just vascular stability but neuronal survival through Flk1. This study is an extension of their previous study showing pericyte depletion causes auditory dysfunction, which is ameliorated by VEGF gene therapy (Zhang et al., JCI insight 2021). Overall, the data are clear and sophisticated and promote our understanding of the biological roles of pericytes in neuronal function. Several points should be thoroughly discussed or supported by definitive experiments like analysis of neuron-specific Flk1 KO mice.
We thank Reviewer #2 for the encouraging positive comments on our study. We especially appreciated the reviewer’s view that there would be value in using neuron-specific Flk1 KO mice to consolidate the results. However, since our in vitro adult SGN neuron cell culture model cearly demonstrates the direct role of exosome-VEGF-A signaling on adult SGN health, as shown in Figs. 5D & E and Figs. 9C & E, we are confident our conclusion is valid. A recent study used neuron-specific Flk1 conditional KO mice to demonstrate neuronal atrophy and dysfunction in memory impairment (Deyama et al., 2020). We do presume disruption of neuronal VEGF/FLK1 signaling in a specific neuronal Flk-1 deletion animal model would cause similar spiral ganglion death and subsequent hearing loss. To test this possibility, we are seeking a Cre-SGN driver animal model from the auditory community and Flk1 floxed mice from the larger research community. Of course, obtaining these models and setting up for a future study will require some time. Nevertheless, reviewer #2’s suggestion is excellent, we have added discussion of the suggestion to the Discussion section.
Reviewer #3 (Public Review):
Zhang et al focus on investigating the role of pericytes in the vasculature of the inner ear. They propose that pericyte-derived VEGF is required for vessels and SGN survival. Functionally, they show that pericyte ablation leads to hearing loss.
This work is interesting to the scientific community. It describes a very specific organ vasculature and its potential crosstalk with the neuronal compartment in the peripheral nervous system.
Major strengths and weaknesses:
- The study is well explained, written, and discussed;
- The design of the experiments is adequate;
- The study is performed in vivo, in vitro, and with functional readouts;
- Results are convincing.
We thank the reviewer for the positive comments on our study. We especially appreciate the reviewer’s suggestions for improving the soundness and quality of the study. We address Review#3’s specific concerns below.
The main conclusion of the study is that pericyte-derived VEGF acts on inner ear vessels and SGNs to maintain their functionality and survival. While all presented data supports this model, there could be other potential interpretations that should be tested and validated with further evidence:
The in vitro experiments are performed with SGN explants. Using this system the authors see that pericyte-derived conditioned medium or exosomes lead to increase vessel branching and SGN neurite outgrowth. As explants contain vessels and neurons, there is the possibility that VEGF is primarily acting on endothelial cells, which then in turn signal to neurons (independent of VEGF, even when neurons express VEGFR2). This should be tested. Perhaps by targeting VEGFR2 specifically in neurons, or by culturing isolated SGN neurons and testing the effect of pericyte-derived exosomes.
This is a great point. To confirm the effect of exosome VEGF-A on SGN neurite outgrowth, we treated isolated adult SGNs with exosomes. As shown in Figs.9C & E, we found much greater SGN dendrite and branch growth in the treated than in the untreated groups.
- Pericyte ablation via DTA might result in the activation of the immune system, which could also influence vessel and neuronal survival. It should be checked whether there is immune activation upon pericyte ablation.
Excellent point. We checked on macrophage activation at two weeks after pericyte depletion. We didn’t see any obvious signs of macrophage activation, but we did notice a decrease in macrophage number. We presume the reduction in macrophage number results from insufficiency blood flow and nutrient availability.
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eLife assessment
This study presents a valuable finding in identifying the roles of the pericytes in maintaining vascular volume and integrity of spiral ganglion neurons (SGNs) in the cochlea, the main hearing organ. The evidence supporting the authors' claims is solid using an inducible and conditional pericyte depletion mouse model and the co-culture models. While the study provides a modest translational contribution, understanding the roles of organ-specific pericytes is paramount, making this study timely and significant. The work will be interesting for biomedical biologists working on hearing, blood vessels, signaling, and cell-to-cell interactions.
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Reviewer #1 (Public Review):
Like other sensory organs, the inner ear has a rich population of pericytes, essential for sensory hair cell heath and normal hearing. In this study, using an inducible and conditional pericyte depletion mouse (PdgfrbCreERT2/iDTR) model, the authors demonstrate that the pericytes play critical roles in maintaining vascular volume and integrity of spiral ganglion neurons (SGNs) in the cochlea. Moreover, using the co-culture models, they show vigorous vascular and neuronal growth in neonatal SGN explants in the presence of exogenous pericytes. Mechanistically, this study demonstrates that these roles are achieved mainly through the interactions between pericyte-released exosomes containing VEGF-A and VEGFR2-expressing the vessels and SGNs.
Overall, the data are analyzed thoroughly, and the conclusions are …
Reviewer #1 (Public Review):
Like other sensory organs, the inner ear has a rich population of pericytes, essential for sensory hair cell heath and normal hearing. In this study, using an inducible and conditional pericyte depletion mouse (PdgfrbCreERT2/iDTR) model, the authors demonstrate that the pericytes play critical roles in maintaining vascular volume and integrity of spiral ganglion neurons (SGNs) in the cochlea. Moreover, using the co-culture models, they show vigorous vascular and neuronal growth in neonatal SGN explants in the presence of exogenous pericytes. Mechanistically, this study demonstrates that these roles are achieved mainly through the interactions between pericyte-released exosomes containing VEGF-A and VEGFR2-expressing the vessels and SGNs.
Overall, the data are analyzed thoroughly, and the conclusions are novel and convincing. It is mechanistically solid. The study is somewhat translationally limited. Nevertheless, understanding the roles of organ-specific pericytes is paramount, making this study timely and significant.
-
Reviewer #2 (Public Review):
The present study from Xiaorui Shi's lab investigated the effect of pericyte depletion on spiral ganglion neurons and auditory function. Results in in vitro culture system proposed that pericyte-derived exosomes contain VEGF, and promote not just vascular stability but neuronal survival through Flk1. This study is an extension of their previous study showing pericyte depletion causes auditory dysfunction, which is ameliorated by VEGF gene therapy (Zhang et al., JCI insight 2021). Overall, the data are clear and sophisticated and promote our understanding of the biological roles of pericytes in neuronal function. Several points should be thoroughly discussed or supported by definitive experiments like analysis of neuron-specific Flk1 KO mice.
-
Reviewer #3 (Public Review):
Zhang et al focus on investigating the role of pericytes in the vasculature of the inner ear. They propose that pericyte-derived VEGF is required for vessels and SGN survival. Functionally, they show that pericyte ablation leads to hearing loss.
This work is interesting to the scientific community. It describes a very specific organ vasculature and its potential crosstalk with the neuronal compartment in the peripheral nervous system.
Major strengths and weaknesses:
- The study is well explained, written, and discussed;
- The design of the experiments is adequate;
- The study is performed in vivo, in vitro, and with functional readouts;
- Results are convincing.The main conclusion of the study is that pericyte-derived VEGF acts on inner ear vessels and SGNs to maintain their functionality and survival. …
Reviewer #3 (Public Review):
Zhang et al focus on investigating the role of pericytes in the vasculature of the inner ear. They propose that pericyte-derived VEGF is required for vessels and SGN survival. Functionally, they show that pericyte ablation leads to hearing loss.
This work is interesting to the scientific community. It describes a very specific organ vasculature and its potential crosstalk with the neuronal compartment in the peripheral nervous system.
Major strengths and weaknesses:
- The study is well explained, written, and discussed;
- The design of the experiments is adequate;
- The study is performed in vivo, in vitro, and with functional readouts;
- Results are convincing.The main conclusion of the study is that pericyte-derived VEGF acts on inner ear vessels and SGNs to maintain their functionality and survival. While all presented data supports this model, there could be other potential interpretations that should be tested and validated with further evidence:
- The in vitro experiments are performed with SGN explants. Using this system the authors see that pericyte-derived conditioned medium or exosomes lead to increase vessel branching and SGN neurite outgrowth. As explants contain vessels and neurons, there is the possibility that VEGF is primarily acting on endothelial cells, which then in turn signal to neurons (independent of VEGF, even when neurons express VEGFR2). This should be tested. Perhaps by targeting VEGFR2 specifically in neurons, or by culturing isolated SGN neurons and testing the effect of pericyte-derived exosomes.
- Pericyte ablation via DTA might result in the activation of the immune system, which could also influence vessels and neuronal survival. It should be checked whether there is immune activation upon pericyte ablation.
-