The inner ear is dense in vascular beds. Microvascular networks are situated in different locations of the cochlea (Shi, 2011). The major microvascular network is located in the cochlear lateral wall, receiving ~80% of cochlear blood flow (Gyo, 2013). Blood flow to the cochlear lateral wall is essential for cochlear homeostasis and is particularly important for generating the endocochlear potential necessary for sensory hair cell (HC) transduction (Hibino et al., 2010). The next largest microvascular network is situated in the region of the spiral ganglion neurons (SGNs), comprising ~19–24% of cochlear blood flow. The network is critical for neural activity (Angelborg et al., 1984; Gyo, 2013; Nakashima et al., 2001), in particular, the vascular network of the spiral ganglion directly delivers nutrients and growth hormones to SGNs (as illustrated in Appendix 1—figure 1). Defects in vascular structure and function would significantly and quickly affect the viability of vulnerable neural tissue. Loss of SGNs is commonly associated with different types of hearing loss (Leake et al., 2020; Viana et al., 2015).

Pericytes are a type of mural cell that encircle endothelial cells of capillaries, pre-capillary arterioles, and post-capillary venules (Armulik et al., 2011). They extend their processes along and around these vessels and play a critical role in regulating vascular morphogenesis and function (Attwell et al., 2016; Birbrair, 2018). Normal function of pericytes and adequate blood supply is crucial for providing vital oxygen, ions, and glucose to meet organ metabolic needs (Shaw et al., 2018). Pericyte degeneration or loss has been identified as a major pathology in many diseases, including ischemic stroke (Greif and Eichmann, 2014), brain trauma (Zehendner et al., 2015), myocardial infarction (O’Farrell and Attwell, 2014), diabetic retinopathy (Pfister et al., 2008), and neurodegeneration diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (Sweeney et al., 2018). Pericytes are known to display phenotypic and functional heterogeneity, which is highly associated with organ function (Dias Moura Prazeres et al., 2017). While little is known about the role of pericytes in the inner ear; nothing is known about how pericyte loss affects peripheral neuron health.

SGN cell bodies, located in Rosenthal’s canal, extend distal processes radially outward into the spiral lamina toward sensory HCs in the organ of Corti and central processes project into the auditory nerve (as illustrated in Appendix 1—figure 1; Nayagam et al., 2011). The microvascular network, located in the spiral ganglion region, forms radial vascular twigs that directly supply nutrients to the SGNs. Although the volume of blood flow to the spiral lamina and SGN is lower than to the cochlear lateral wall, it is critical for neuronal activity. Constant sound stimulation to the inner ear imposes a high energy demand on neurons, requiring rapid delivery of oxygen and glucose. Previously, we demonstrated the microvascular network in the spiral ganglion region is richly populated by pericytes (Jiang et al., 2019). Pericytes actively communicate with SGNs as evidenced by the pericyte-released particles observed in the soma of SGNs (Jiang et al., 2019). However, the role of pericytes in SGN viability and stability of vascular beds is not yet known. In this study, we use an inducible and conditional genetic pericyte ablation model, in combination with two newly established co-culture models, to demonstrate that pericyte loss in vivo causes loss of vessel volume and SGNs in adult mice. In vitro, we demonstrate vigorous vascular and neuronal growth in the neonatal SGN explants in the presence of exogenous pericytes. Increased SGN survival and neurite growth were also observed in adult SGNs. Most interestingly, we find the promotion of vascular and neuronal growth is mediated by pericyte-released exosomes, nano-sized 50–150 nm extracellular vesicles (EVs), containing vascular endothelial growth factor-A (VEGF-A). VEGF-A binding to VEGFR2 (Flk1) expressed in recipient cells, including endothelial cells and SGNs, controlled vascular and neuronal growth. Our data demonstrate for the first time that loss of pericytes leads to a reduction in vascular density and degeneration of SGNs, underscoring the vital role of pericytes in vascular and auditory peripheral neural health.

Pericytes are prevalent on microvessels in the cochlea, but they have not received much research attention. In this investigation, we used an inducible and conditional pericyte depletion mouse model (PDGFRB-CreER^T2 ROSA26iDTR) to demonstrate that pericytes in the adult ear are critical for the stability of mature vessel beds and viability of SGNs in the peripheral spiral ganglion region. Depletion of pericytes causes loss of vascular volume and SGNs, with concomitant loss of hearing sensitivity. We report on a pericyte-released growth factor, VEGF-A, conveyed by exosomes, shown to strongly mediate and promote SGN survival and growth by binding to VEGFR2 (Flk-1). This study provides the first clear evidence that pericytes have a critical role in vascular and neuronal health in the adult ear. Without the normal disposition of pericytes, animals lose hearing.

Recent research has highlighted the extent to which pericytes play a critical role in vascular function and neuronal protection in the CNS (Bergers and Song, 2005; Brown et al., 2019). Studies, however, on the role of pericytes in the peripheral nervous system, as in the ear, are limited. In the CNS, loss of pericytes causes brain infarction with diminished capillary perfusion and blood flow (Armulik et al., 2010; Bell et al., 2010). In the mouse brain, damaged pericytes effectively constrict capillaries and induce a long-lasting reduction in cerebral blood flow, even after recanalization of the larger vessels, and it causes a no-reflow condition (Hall et al., 2014; Kloner et al., 2018; Yemisci et al., 2009). Loss of pericytes also interrupts blood-brain-barrier integrity (Armulik et al., 2010; Bell et al., 2010; Nikolakopoulou et al., 2019). In peripheral organs such as the ear, we previously reported that pericytes control perfusion of cochlear blood flow (Dai et al., 2010). Loss of pericytes leads to increased cochlear permeability and reduced endocochlear potential (Zhang et al., 2021). In this study, we depleted pericytes in the cochlea in a genetically inducible/conditional adult murine model in which the PDGFRB-CreER^T2; ROSA26iDTR mice express DTR under pericyte marker Pdgfrb-Cre control. The Cre-recombined PDGFR-β⁺ pericytes expressing DTR are selectively susceptible to ablation on exposure to the DT, while the Cre-negative murine cells, lacking DTR expression, are unaffected. We found depletion of PDGFR-β+ cells reduces pericyte coverage in vascular beds in the spiral ganglion region, where the microvascular network is also densely populated by pericytes (Jiang et al., 2019), as shown in Figure 1. The depletion has a direct effect on vascular density (Figure 2). The results obtained in this study further demonstrate the important role of pericytes in maintaining vascular stability in peripheral systems such as auditory SGNs in the cochlea. However, PDGFR-β is a general membrane protein not only expressed by all cochlear pericytes (Canis and Bertlich, 2019; Shi et al., 2008) but also by pericytes in other organs. Thus, our PDGFR-β depletion model should deplete all pericytes in different organs and in different regions of the cochlea, including in the region of the blood-labyrinth barrier in the stria vascularis. This causes blood-barrier dysfunction of the adult mouse cochlea, leading to increased cochlear permeability, reduced endocochlear potential, and consequent hearing dysfunction, as we reported in a previous study (Zhang et al., 2021). However, with these caveats taken into consideration, pericytes are also well-known to be heterogeneous, organ-oriented, and tissue-specific (Dias Moura Prazeres et al., 2017; Hirschi and D’Amore, 1996). As one caveat and consistent with a study from Park et al., 2017, we have not observed obvious abnormalities of retina blood vessels in our adult pericyte depletion model (Appendix 1—figure 2). The different results obtained from pericyte depletion does, however, support the notion that pericytes display considerable phenotypic and functional heterogeneity in different tissues and organs (Sims, 2000).

SGNs are neural elements transmitting localized electrical activity from HCs to second-order neurons in the cochlear nucleus (Coate et al., 2019). By labeling SGNs with β-III tubulin, a specific marker for neuronal cytoskeleton is commonly used to distinguish neurons from other cell types (Katsetos et al., 2003), we found that loss of pericytes affects SGN viability and causes significant loss of SGNs across all turns. Pericyte loss also significantly decreases expression of β-III tubulin protein in SGNs in pericyte-depleted animals, particularly at the middle and basal turns, as shown in Figure 4. Tubulin is the primary structural protein in microtubules. In fact, the tubulin assembly is essential to the structure, stability, and function of the neurons. A gradual loss of neuronal microtubule mass is seen in many neuro-degenerative diseases (Baas et al., 2016). In ABR functional studies, delay in latency and low amplitude is first seen at the basal turn and moves to regions corresponding to middle and low frequencies, as shown in Figure 3. The mechanism underlying the vulnerability of SGNs at the basal turn is not known. However, the significantly decreased expression of β-III tubulin protein in SGNs at the basal and middle turns may indicate that SGN damage is more severe than at the apical turn. In addition, a recent single-cell analysis study of mouse SGNs showed there were three functionally distinct subtypes of SGN, each displaying molecular variation along the tonotopic axis (Shrestha et al., 2018). Sensitivity at the different turns to pericyte loss could also be due to SGN diversity and the variance in SGN vulnerability to environmental perturbation. Further study is needed to define the mechanisms of SGN vulnerability.

What is the underlying mechanism of pericyte-loss-related damage to SGNs? Our data suggest both blood flow dysfunction and direct disruption of pericyte-SGN communication are at issue. It is well-documented that pericyte loss triggers primary vascular dysfunction, leading to neurodegeneration (Winkler et al., 2011). For example, in the CNS, pericyte loss induces vascular leakage, insufficient blood perfusion, and tissue hypoxia, ultimately leading to neuron degeneration (Quaegebeur et al., 2010; Sagare et al., 2013; Winkler et al., 2011; Zlokovic, 2011). SGNs are vulnerable to interruption of the blood supply since the energy demand of SGNs is directly met by the surrounding blood circulation (Jiang et al., 2019; Mei et al., 2020). In this study, our data demonstrate reduction in vascular density and change in vessel diameter in an adult PDGFRβ+ pericyte depletion model. Our data is supported by Kramann et al., where it was found pericyte loss induces capillary rarefaction in acute kidney injury (Guzmán-Hernández et al., 2014). Detachment of pericytes from endothelial cells can interrupt cross-talk signaling and destabilize capillaries, as we earlier reported (Hou et al., 2020). Without the presence of pericytes, tight junction adhesive proteins such as ZO-1 are significantly reduced (Neng et al., 2013b). The increased vascular permeability and loss of vascular volume cause cochlear tissue edema and hypoxia, damaging neurons (SGNs) and sensory HCs, as we found in both this study and a previous study of sensory HCs (Zhang et al., 2021), although we did not measure vascular permeability in this study. In addition to the effect of blood flow on SGNs, we previously demonstrated active communication between pericytes, endothelial cells, and SGNs in the cochlea (Jiang et al., 2019), also shown in Figure 1. Pericytes release many growth factors relevant to maintenance of organ homeostasis (Gaceb et al., 2018a; Gaceb et al., 2018b; Gaceb and Paul, 2018c). In this study, we used bulk-RNA-seq analysis on the purified cochlear pericytes to identify the different vascular- and neuronal-growth factors in our mouse cochlear pericyte dataset. We found pericyte-related angiogenesis and neuroprotective pathways overrepresented (Figure 5A). In pericyte co-culture models of neonatal SGN explants and adult SGNs, we found pericytes to induce robust vascular and neuronal dendritic growth in the neonatal SGN explants and to increase cell survival and neurite growth in the adult SGNs, as shown in Figure 5B–E. The results suggest direct intercellular communication between pericytes and SGNs in addition to direct effects exerted by pericytes on vascular function.

How do pericytes communicate with endothelial cells and SGNs? Many cells release exosomes, nano-sized EVs (50–150 nm diameter), which transport cargo such as nucleic acids, proteins, and lipids. These exosomes have significant physiological effects in the recipient cells (Bang and Thum, 2012; Colombo et al., 2014; Zhang et al., 2019). Pericyte-released exosomes were first reported by Gaceb and Paul, 2018c. Pericyte-derived exosome physiology, including characterization of size and morphology, remains limited. However recent studies have amply demonstrated that pericyte-derived exosomes participate in regulation of microvascular function under different pathological conditions (Ye et al., 2021; Yuan et al., 2019). In our study, we observed NG2-positive particles (likely released by pericytes) in the soma of SGNs (Figure 1E). The pericyte-derived particles displayed the classic cup-shaped morphology (Figure 6B and C). Further proteomic analysis of these particles identified 580 proteins enriched in the GO of cellular components in extracellular exosomes, including proteins relating to angiogenesis, neurodegeneration, and neuroprotection (Figure 6E–F). Our findings are strong indication of participation of cochlear pericyte-derived exosomes in intercellular communication with spiral ganglion.

How do pericyte-derived exosomes affect angiogenesis and neurogenesis? VEGF/VEGFR2 signaling is a well-defined, classical pathway that plays a critical role in both angiogenesis and neuroprotection (Storkebaum and Carmeliet, 2004). VEGFR2 is typically expressed in both vascular cells and SGNs (Figure 8A–D). In this study, we used in vitro neonatal SGN explant and adult SGN tissue culture models to specifically investigate the VEGF-A controlled angiogenesis signaling pathway in relation to vascular and neuronal growth. Our results show blockage of the VEGFR2 receptor with a specific VEGFR2 inhibitor, SU5408 (Roskoski, 2017), dramatically halts angiogenesis and neuronal growth (Figure 9), indicating that pericyte-derived VEGF-A-containing exosomes promote both vascular and neuronal growth through a VGFR2 signaling pathway. However, the exact mechanism of the exosome-mediated VEGF-A/VEGFR2 signaling pathway remains unclear. For example, studies have shown the shedding of VEGFA-165 from the cell surface (together with other membrane components) appears to be a unique mechanism by which some VEGF is delivered to the surroundings to exert its known biological actions (Guzmán-Hernández et al., 2014). The interaction of isoform VEGF189 with the EV surface membrane profoundly increased the ligand half-life and stability (Ko et al., 2019). Although we have determined VEGF-A is predominant in the exosomes of cultured PC media, suggesting the VEGF-A is delivered by exosomes, it still needs further investigation to better understand the difference between the pericyte-released VEGFA in diffusible isoform and exosomes. It is also unclear how VEGF-A contained in exosomes binds to Flk1 receptors in recipient cells. Studies demonstrate EVs interface with neighboring cells through different modes, including direct receptor-binding without internalization of EVs, phagocytosis, macropinocytosis, internalization by clathrin-caveolae- and lipid raft-mediated endocytosis (Mulcahy et al., 2014). Once internalized, the EVs release their cargo in the cytoplasm, and the cell-internalized cargo then regulates the cell at the transcription or translation level (Abels and Breakefield, 2016; Berumen Sánchez et al., 2021). It has also been reported that VEGF presents on the surface of EVs through binding to surface proteins such as heparan and heat shock protein 90 (HSP90), which can directly stimulate the cellular domain of Flk1 and induce phosphorylation (Ko et al., 2019; Ko and Naora, 2020). Overall, however, the data clearly indicate cochlear angiogenesis and neurogenesis require VEGF-A signaling. A recent study by Okabe et al. reports that neuron-derived VEGF contributes to cortical and hippocampal development but is independent of VEGFR1/2-mediated neurotropism (Okabe et al., 2020). They conclude that neuron-derived VEGF contributes to cortical and hippocampal development likely through angiogenesis independent of any direct neurotrophic effects mediated by VEGFR1 and 2. Although there is still no consensus on whether cochlear angiogenesis affects auditory peripheral neurogenesis, our co-culture model of pericyte-SGN explants and adult SGNs does provide evidence the VEGF-A/VEGFR2 signal strongly promotes auditory SGN dendritic growth, as shown in Figure 9. In addition, cochlear pericyte-derived exosomes contain numerous other proteins, as we identified in our proteomic analysis. For example, they contain messenger RNA and micro-RNAs not investigated in this study. Some of these may also be playing an important role in the regulation of angiogenesis, SGN development, and neurite growth. However, it must be emphasized that treatment with the VEGFR2 inhibitor almost completely blocked the effect of the exosomes relative to control groups (see Figure 9). This suggests the VEGF-A/VEGFR2 signaling is the dominant factor regulating vascular and neuronal growth. However, this does not obviate the possibility the underlying mechanism of pericyte-related neuroprotection is more complicated than what we have discovered in this study. A future in vivo study in a conditional SGN-specific VEGFR2 knockdown animal model will give more conclusive results.

In summary, our data provide the first clear-cut experimental evidence that pericytes are essential for the maintenance of cochlear vascular stability and SGN viability in adults. Loss of pericytes disrupts vascular structure and damages SGNs. The damage is related to the impairment of VEGF-A mediated communication between pericytes, endothelial cells, and SGNs, as illustrated in Figure 11. These findings demonstrate for the first time that pericyte-released exosomes have a major role in vascular and neuronal health.

Figure 11

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Table 1 - Source Data 1 contains the RNA sequencing data used to generate Table 1 and Figure 5A. Figure 6 - Source Data 1 contains the proteomics data used to generate Figure 6 and Table 2. Figure 7 - Source Data 1 contains the original uncropped blots of VEGFA and PDGFRβ, and raw gel data of whole protein staining with the relevant bands clearly labelled.

1. 1. Abels ER
   2. Breakefield XO

   (2016) Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake

   Cellular and Molecular Neurobiology 36:301–312.

   https://doi.org/10.1007/s10571-016-0366-z

   • PubMed
   • Google Scholar
2. 1. Abhinand CS
   2. Raju R
   3. Soumya SJ
   4. Arya PS
   5. Sudhakaran PR

   (2016) Vegf-A/vegfr2 signaling network in endothelial cells relevant to angiogenesis

   Journal of Cell Communication and Signaling 10:347–354.

   https://doi.org/10.1007/s12079-016-0352-8

   • PubMed
   • Google Scholar
3. 1. Angelborg C
   2. Axelsson A
   3. Larsen HC

   (1984) Regional blood flow in the rabbit cochlea

   Archives of Otolaryngology 110:297–300.

   https://doi.org/10.1001/archotol.1984.00800310021004

   • PubMed
   • Google Scholar
4. 1. Armulik A
   2. Genové G
   3. Mäe M
   4. Nisancioglu MH
   5. Wallgard E
   6. Niaudet C
   7. He L
   8. Norlin J
   9. Lindblom P
   10. Strittmatter K
   11. Johansson BR
   12. Betsholtz C

   (2010) Pericytes regulate the blood-brain barrier

   Nature 468:557–561.

   https://doi.org/10.1038/nature09522

   • PubMed
   • Google Scholar
5. 1. Armulik A
   2. Genové G
   3. Betsholtz C

   (2011) Pericytes: developmental, physiological, and pathological perspectives, problems, and promises

   Developmental Cell 21:193–215.

   https://doi.org/10.1016/j.devcel.2011.07.001

   • PubMed
   • Google Scholar
6. 1. Attwell D
   2. Mishra A
   3. Hall CN
   4. O’Farrell FM
   5. Dalkara T

   (2016) What is a pericyte?

   Journal of Cerebral Blood Flow and Metabolism 36:451–455.

   https://doi.org/10.1177/0271678X15610340

   • PubMed
   • Google Scholar
7. 1. Baas PW
   2. Rao AN
   3. Matamoros AJ
   4. Leo L

   (2016) Stability properties of neuronal microtubules

   Cytoskeleton 73:442–460.

   https://doi.org/10.1002/cm.21286

   • PubMed
   • Google Scholar
8. 1. Bang C
   2. Thum T

   (2012) Exosomes: new players in cell-cell communication

   The International Journal of Biochemistry & Cell Biology 44:2060–2064.

   https://doi.org/10.1016/j.biocel.2012.08.007

   • PubMed
   • Google Scholar
9. 1. Bell RD
   2. Winkler EA
   3. Sagare AP
   4. Singh I
   5. LaRue B
   6. Deane R
   7. Zlokovic BV

   (2010) Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging

   Neuron 68:409–427.

   https://doi.org/10.1016/j.neuron.2010.09.043

   • PubMed
   • Google Scholar
10. 1. Bellon A
    2. Luchino J
    3. Haigh K
    4. Rougon G
    5. Haigh J
    6. Chauvet S
    7. Mann F

    (2010) Vegfr2 (KDR/flk1) signaling mediates axon growth in response to semaphorin 3E in the developing brain

    Neuron 66:205–219.

    https://doi.org/10.1016/j.neuron.2010.04.006

    • PubMed
    • Google Scholar
11. 1. Bergers G
    2. Song S

    (2005) The role of pericytes in blood-vessel formation and maintenance

    Neuro-Oncology 7:452–464.

    https://doi.org/10.1215/S1152851705000232

    • PubMed
    • Google Scholar
12. 1. Berumen Sánchez G
    2. Bunn KE
    3. Pua HH
    4. Rafat M

    (2021) Extracellular vesicles: mediators of intercellular communication in tissue injury and disease

    Cell Communication and Signaling 19:104.

    https://doi.org/10.1186/s12964-021-00787-y

    • PubMed
    • Google Scholar
13. 1. Birbrair A

    (2018) Pericyte biology: development, homeostasis, and disease

    Advances in Experimental Medicine and Biology 1109:1–3.

    https://doi.org/10.1007/978-3-030-02601-1_1

    • PubMed
    • Google Scholar
14. 1. Breglio AM
    2. May LA
    3. Barzik M
    4. Welsh NC
    5. Francis SP
    6. Costain TQ
    7. Wang L
    8. Anderson DE
    9. Petralia RS
    10. Wang YX
    11. Friedman TB
    12. Wood MJ
    13. Cunningham LL

    (2020) Exosomes mediate sensory hair cell protection in the inner ear

    The Journal of Clinical Investigation 130:2657–2672.

    https://doi.org/10.1172/JCI128867

    • PubMed
    • Google Scholar
15. 1. Brown LS
    2. Foster CG
    3. Courtney JM
    4. King NE
    5. Howells DW
    6. Sutherland BA

    (2019) Pericytes and neurovascular function in the healthy and diseased brain

    Frontiers in Cellular Neuroscience 13:282.

    https://doi.org/10.3389/fncel.2019.00282

    • PubMed
    • Google Scholar
16. 1. Buch T
    2. Heppner FL
    3. Tertilt C
    4. Heinen T
    5. Kremer M
    6. Wunderlich FT
    7. Jung S
    8. Waisman A

    (2005) A cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

    Nature Methods 2:419–426.

    https://doi.org/10.1038/nmeth762

    • PubMed
    • Google Scholar
17. 1. Canis M
    2. Bertlich M

    (2019) Cochlear capillary pericytes

    Advances in Experimental Medicine and Biology 1122:115–123.

    https://doi.org/10.1007/978-3-030-11093-2_7

    • PubMed
    • Google Scholar
18. 1. Coate TM
    2. Scott MK
    3. Gurjar M

    (2019) Current concepts in cochlear ribbon synapse formation

    Synapse 73:e22087.

    https://doi.org/10.1002/syn.22087

    • PubMed
    • Google Scholar
19. 1. Colombo M
    2. Raposo G
    3. Théry C

    (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles

    Annual Review of Cell and Developmental Biology 30:255–289.

    https://doi.org/10.1146/annurev-cellbio-101512-122326

    • PubMed
    • Google Scholar
20. 1. Dai M
    2. Yang Y
    3. Omelchenko I
    4. Nuttall AL
    5. Kachelmeier A
    6. Xiu R
    7. Shi X

    (2010) Bone marrow cell recruitment mediated by inducible nitric oxide synthase/stromal cell-derived factor-1α signaling repairs the acoustically damaged cochlear blood-labyrinth barrier

    The American Journal of Pathology 177:3089–3099.

    https://doi.org/10.2353/ajpath.2010.100340

    • PubMed
    • Google Scholar
21. 1. Dai J
    2. Su Y
    3. Zhong S
    4. Cong L
    5. Liu B
    6. Yang J
    7. Tao Y
    8. He Z
    9. Chen C
    10. Jiang Y

    (2020) Exosomes: key players in cancer and potential therapeutic strategy

    Signal Transduction and Targeted Therapy 5:145.

    https://doi.org/10.1038/s41392-020-00261-0

    • PubMed
    • Google Scholar
22. 1. D’Amore PA

    (2007) Vascular endothelial cell growth factor-A: not just for endothelial cells anymore

    The American Journal of Pathology 171:14–18.

    https://doi.org/10.2353/ajpath.2007.070385

    • PubMed
    • Google Scholar
23. 1. Dias Moura Prazeres PH
    2. Sena IFG
    3. Borges IDT
    4. de Azevedo PO
    5. Andreotti JP
    6. de Paiva AE
    7. de Almeida VM
    8. de Paula Guerra DA
    9. Pinheiro Dos Santos GS
    10. Mintz A
    11. Delbono O
    12. Birbrair A

    (2017) Pericytes are heterogeneous in their origin within the same tissue

    Developmental Biology 427:6–11.

    https://doi.org/10.1016/j.ydbio.2017.05.001

    • PubMed
    • Google Scholar
24. 1. Dragovic RA
    2. Gardiner C
    3. Brooks AS
    4. Tannetta DS
    5. Ferguson DJP
    6. Hole P
    7. Carr B
    8. Redman CWG
    9. Harris AL
    10. Dobson PJ
    11. Harrison P
    12. Sargent IL

    (2011) Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis

    Nanomedicine 7:780–788.

    https://doi.org/10.1016/j.nano.2011.04.003

    • PubMed
    • Google Scholar
25. 1. Gaceb A
    2. Barbariga M
    3. Özen I
    4. Paul G

    (2018a) The pericyte secretome: potential impact on regeneration

    Biochimie 155:16–25.

    https://doi.org/10.1016/j.biochi.2018.04.015

    • PubMed
    • Google Scholar
26. 1. Gaceb A
    2. Ozen I
    3. Padel T
    4. Barbariga M
    5. Paul G

    (2018b) Pericytes secrete pro-regenerative molecules in response to platelet-derived growth factor-BB

    J Cereb Blood Flow Metab 38:45–57.

    https://doi.org/10.1177/0271678X17719645

    • Google Scholar
27. 1. Gaceb A
    2. Paul G

    (2018c) Pericyte secretome

    Advances in Experimental Medicine and Biology 1109:139–163.

    https://doi.org/10.1007/978-3-030-02601-1_11

    • PubMed
    • Google Scholar
28. 1. Greenhalgh SN
    2. Iredale JP
    3. Henderson NC

    (2013) Origins of fibrosis: pericytes take centre stage

    F1000prime Reports 5:37.

    https://doi.org/10.12703/P5-37

    • PubMed
    • Google Scholar
29. 1. Greif DM
    2. Eichmann A

    (2014) Vascular biology: brain vessels squeezed to death

    Nature 508:50–51.

    https://doi.org/10.1038/nature13217

    • PubMed
    • Google Scholar
30. 1. Guzmán-Hernández ML
    2. Potter G
    3. Egervári K
    4. Kiss JZ
    5. Balla T

    (2014) Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane

    Molecular Biology of the Cell 25:1061–1072.

    https://doi.org/10.1091/mbc.E13-07-0418

    • PubMed
    • Google Scholar
31. 1. Gyo K

    (2013) Experimental study of transient cochlear ischemia as a cause of sudden deafness

    World Journal of Otorhinolaryngology 3:1.

    https://doi.org/10.5319/wjo.v3.i1.1

    • Google Scholar
32. 1. Hall CN
    2. Reynell C
    3. Gesslein B
    4. Hamilton NB
    5. Mishra A
    6. Sutherland BA
    7. O’Farrell FM
    8. Buchan AM
    9. Lauritzen M
    10. Attwell D

    (2014) Capillary pericytes regulate cerebral blood flow in health and disease

    Nature 508:55–60.

    https://doi.org/10.1038/nature13165

    • PubMed
    • Google Scholar
33. 1. Hibino H
    2. Nin F
    3. Tsuzuki C
    4. Kurachi Y

    (2010) How is the highly positive endocochlear potential formed? the specific architecture of the stria vascularis and the roles of the ion-transport apparatus

    Pflugers Archiv 459:521–533.

    https://doi.org/10.1007/s00424-009-0754-z

    • PubMed
    • Google Scholar
34. 1. Hirschi KK
    2. D’Amore PA

    (1996)

    Pericytes in the microvasculature

    Cardiovascular Research 32:687–698.

    • PubMed
    • Google Scholar
35. 1. Hou Z
    2. Neng L
    3. Zhang J
    4. Cai J
    5. Wang X
    6. Zhang Y
    7. Lopez IA
    8. Shi X

    (2020) Acoustic trauma causes cochlear pericyte-to-myofibroblast–like cell transformation and vascular degeneration, and transplantation of new pericytes prevents vascular atrophy

    The American Journal of Pathology 190:1943–1959.

    https://doi.org/10.1016/j.ajpath.2020.05.019

    • PubMed
    • Google Scholar
36. 1. Jiang H
    2. Wang X
    3. Zhang J
    4. Kachelmeier A
    5. Lopez IA
    6. Shi X

    (2019) Microvascular networks in the area of the auditory peripheral nervous system

    Hearing Research 371:105–116.

    https://doi.org/10.1016/j.heares.2018.11.012

    • PubMed
    • Google Scholar
37. 1. Jung MK
    2. Mun JY

    (2018) Sample preparation and imaging of exosomes by transmission electron microscopy

    Journal of Visualized Experiments 1:e82.

    https://doi.org/10.3791/56482

    • PubMed
    • Google Scholar
38. 1. Kalluri R
    2. LeBleu VS

    (2020) The biology, function, and biomedical applications of exosomes

    Science 367:6478.

    https://doi.org/10.1126/science.aau6977

    • Google Scholar
39. 1. Katsetos CD
    2. Legido A
    3. Perentes E
    4. Mörk SJ

    (2003) Class III β-tubulin isotype: a key cytoskeletal protein at the crossroads of developmental neurobiology and tumor neuropathology

    Journal of Child Neurology 18:851–866.

    https://doi.org/10.1177/088307380301801205

    • PubMed
    • Google Scholar
40. 1. Kloner RA
    2. King KS
    3. Harrington MG

    (2018) No-reflow phenomenon in the heart and brain

    American Journal of Physiology. Heart and Circulatory Physiology 315:H550–H562.

    https://doi.org/10.1152/ajpheart.00183.2018

    • PubMed
    • Google Scholar
41. 1. Ko SY
    2. Lee W
    3. Kenny HA
    4. Dang LH
    5. Ellis LM
    6. Jonasch E
    7. Lengyel E
    8. Naora H

    (2019) Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

    Communications Biology 2:386.

    https://doi.org/10.1038/s42003-019-0609-x

    • PubMed
    • Google Scholar
42. 1. Ko SY
    2. Naora H

    (2020) Extracellular vesicle membrane-associated proteins: emerging roles in tumor angiogenesis and anti-angiogenesis therapy resistance

    International Journal of Molecular Sciences 21:15.

    https://doi.org/10.3390/ijms21155418

    • PubMed
    • Google Scholar
43. 1. Leake PA
    2. Akil O
    3. Lang H

    (2020) Neurotrophin gene therapy to promote survival of spiral ganglion neurons after deafness

    Hearing Research 394:107955.

    https://doi.org/10.1016/j.heares.2020.107955

    • PubMed
    • Google Scholar
44. 1. Luck R
    2. Urban S
    3. Karakatsani A
    4. Harde E
    5. Sambandan S
    6. Nicholson L
    7. Haverkamp S
    8. Mann R
    9. Martin-Villalba A
    10. Schuman EM
    11. Acker-Palmer A
    12. Ruiz de Almodóvar C

    (2019) Vegf/vegfr2 signaling regulates hippocampal axon branching during development

    eLife 8:e49818.

    https://doi.org/10.7554/eLife.49818

    • PubMed
    • Google Scholar
45. 1. Mei X
    2. Glueckert R
    3. Schrott-Fischer A
    4. Li H
    5. Ladak HM
    6. Agrawal SK
    7. Rask-Andersen H

    (2020) Vascular supply of the human spiral ganglion: novel three-dimensional analysis using synchrotron phase-contrast imaging and histology

    Scientific Reports 10:5877.

    https://doi.org/10.1038/s41598-020-62653-0

    • PubMed
    • Google Scholar
46. 1. Meyer W
    2. Godynicki S
    3. Tsukise A

    (2008) Lectin histochemistry of the endothelium of blood vessels in the mammalian integument, with remarks on the endothelial glycocalyx and blood vessel system nomenclature

    Annals of Anatomy = Anatomischer Anzeiger 190:264–276.

    https://doi.org/10.1016/j.aanat.2007.11.004

    • PubMed
    • Google Scholar
47. 1. Mi H
    2. Muruganujan A
    3. Casagrande JT
    4. Thomas PD

    (2013) Large-scale gene function analysis with the panther classification system

    Nature Protocols 8:1551–1566.

    https://doi.org/10.1038/nprot.2013.092

    • PubMed
    • Google Scholar
48. 1. Mi H
    2. Muruganujan A
    3. Huang X
    4. Ebert D
    5. Mills C
    6. Guo X
    7. Thomas PD

    (2019) Protocol update for large-scale genome and gene function analysis with the panther classification system (v.14.0)

    Nature Protocols 14:703–721.

    https://doi.org/10.1038/s41596-019-0128-8

    • PubMed
    • Google Scholar
49. 1. Miners JS
    2. Kehoe PG
    3. Love S
    4. Zetterberg H
    5. Blennow K

    (2019) Csf evidence of pericyte damage in alzheimer’s disease is associated with markers of blood-brain barrier dysfunction and disease pathology

    Alzheimer’s Research & Therapy 11:81.

    https://doi.org/10.1186/s13195-019-0534-8

    • PubMed
    • Google Scholar
50. 1. Montgomery SC
    2. Cox BC

    (2016) Whole mount dissection and immunofluorescence of the adult mouse cochlea

    Journal of Visualized Experiments 1:e61.

    https://doi.org/10.3791/53561

    • PubMed
    • Google Scholar
51. 1. Mulcahy LA
    2. Pink RC
    3. Carter DRF

    (2014) Routes and mechanisms of extracellular vesicle uptake

    Journal of Extracellular Vesicles 3:24641.

    https://doi.org/10.3402/jev.v3.24641

    • PubMed
    • Google Scholar
52. 1. Nakashima T
    2. Suzuki T
    3. Iwagaki T
    4. Hibi T

    (2001) Effects of anterior inferior cerebellar artery occlusion on cochlear blood flow – a comparison between laser-Doppler and microsphere methods

    Hearing Research 162:85–90.

    https://doi.org/10.1016/S0378-5955(01)00372-0

    • PubMed
    • Google Scholar
53. 1. Nayagam BA
    2. Muniak MA
    3. Ryugo DK

    (2011) The spiral ganglion: connecting the peripheral and central auditory systems

    Hearing Research 278:2–20.

    https://doi.org/10.1016/j.heares.2011.04.003

    • PubMed
    • Google Scholar
54. 1. Neng L
    2. Zhang W
    3. Hassan A
    4. Zemla M
    5. Kachelmeier A
    6. Fridberger A
    7. Auer M
    8. Shi X

    (2013a) Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear

    Nature Protocols 8:709–720.

    https://doi.org/10.1038/nprot.2013.033

    • PubMed
    • Google Scholar
55. 1. Neng L
    2. Zhang F
    3. Kachelmeier A
    4. Shi X

    (2013b) Endothelial cell, pericyte, and perivascular resident macrophage-type melanocyte interactions regulate cochlear intrastrial fluid–blood barrier permeability

    Journal of the Association for Research in Otolaryngology 14:175–185.

    https://doi.org/10.1007/s10162-012-0365-9

    • PubMed
    • Google Scholar
56. 1. Neng L
    2. Zhang J
    3. Yang J
    4. Zhang F
    5. Lopez IA
    6. Dong M
    7. Shi X

    (2015) Structural changes in thestrial blood–labyrinth barrier of aged C57BL/6 mice

    Cell and Tissue Research 361:685–696.

    https://doi.org/10.1007/s00441-015-2147-2

    • PubMed
    • Google Scholar
57. 1. Nikolakopoulou AM
    2. Montagne A
    3. Kisler K
    4. Dai Z
    5. Wang Y
    6. Huuskonen MT
    7. Sagare AP
    8. Lazic D
    9. Sweeney MD
    10. Kong P
    11. Wang M
    12. Owens NC
    13. Lawson EJ
    14. Xie X
    15. Zhao Z
    16. Zlokovic BV

    (2019) Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss

    Nature Neuroscience 22:1089–1098.

    https://doi.org/10.1038/s41593-019-0434-z

    • PubMed
    • Google Scholar
58. 1. O’Farrell FM
    2. Attwell D

    (2014) A role for pericytes in coronary no-reflow

    Nature Reviews Cardiology 11:427–432.

    https://doi.org/10.1038/nrcardio.2014.58

    • PubMed
    • Google Scholar
59. 1. Ogunshola OO
    2. Antic A
    3. Donoghue MJ
    4. Fan SY
    5. Kim H
    6. Stewart WB
    7. Madri JA
    8. Ment LR

    (2002) Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system

    The Journal of Biological Chemistry 277:11410–11415.

    https://doi.org/10.1074/jbc.M111085200

    • PubMed
    • Google Scholar
60. 1. Okabe K
    2. Fukada H
    3. Tai-Nagara I
    4. Ando T
    5. Honda T
    6. Nakajima K
    7. Takeda N
    8. Fong GH
    9. Ema M
    10. Kubota Y

    (2020) Neuron-derived VEGF contributes to cortical and hippocampal development independently of VEGFR1/2-mediated neurotrophism

    Developmental Biology 459:65–71.

    https://doi.org/10.1016/j.ydbio.2019.11.016

    • PubMed
    • Google Scholar
61. 1. Park DY
    2. Lee J
    3. Kim J
    4. Kim K
    5. Hong S
    6. Han S
    7. Kubota Y
    8. Augustin HG
    9. Ding L
    10. Kim JW
    11. Kim H
    12. He Y
    13. Adams RH
    14. Koh GY

    (2017) Plastic roles of pericytes in the blood–retinal barrier

    Nature Communications 8:15296.

    https://doi.org/10.1038/ncomms15296

    • PubMed
    • Google Scholar
62. 1. Pfister F
    2. Feng Y
    3. vom Hagen F
    4. Hoffmann S
    5. Molema G
    6. Hillebrands JL
    7. Shani M
    8. Deutsch U
    9. Hammes HP

    (2008) Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy

    Diabetes 57:2495–2502.

    https://doi.org/10.2337/db08-0325

    • PubMed
    • Google Scholar
63. 1. Quaegebeur A
    2. Segura I
    3. Carmeliet P

    (2010) Pericytes: blood-brain barrier safeguards against neurodegeneration?

    Neuron 68:321–323.

    https://doi.org/10.1016/j.neuron.2010.10.024

    • PubMed
    • Google Scholar
64. 1. Roskoski R

    (2017) Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas

    Pharmacological Research 120:116–132.

    https://doi.org/10.1016/j.phrs.2017.03.010

    • PubMed
    • Google Scholar
65. 1. Sagare AP
    2. Bell RD
    3. Zhao Z
    4. Ma Q
    5. Winkler EA
    6. Ramanathan A
    7. Zlokovic BV

    (2013) Pericyte loss influences alzheimer-like neurodegeneration in mice

    Nature Communications 4:2932.

    https://doi.org/10.1038/ncomms3932

    • PubMed
    • Google Scholar
66. 1. Shaw I
    2. Rider S
    3. Mullins J
    4. Hughes J
    5. Péault B

    (2018) Pericytes in the renal vasculature: roles in health and disease

    Nature Reviews Nephrology 14:521–534.

    https://doi.org/10.1038/s41581-018-0032-4

    • PubMed
    • Google Scholar
67. 1. Shi X
    2. Han W
    3. Yamamoto H
    4. Tang W
    5. Lin X
    6. Xiu R
    7. Trune DR
    8. Nuttall AL

    (2008) The cochlear pericytes

    Microcirculation 15:515–529.

    https://doi.org/10.1080/10739680802047445

    • PubMed
    • Google Scholar
68. 1. Shi X

    (2011) Physiopathology of the cochlear microcirculation

    Hearing Research 282:10–24.

    https://doi.org/10.1016/j.heares.2011.08.006

    • PubMed
    • Google Scholar
69. 1. Shrestha BR
    2. Chia C
    3. Wu L
    4. Kujawa SG
    5. Liberman MC
    6. Goodrich LV

    (2018) Sensory neuron diversity in the inner ear is shaped by activity

    Cell 174:1229–1246.

    https://doi.org/10.1016/j.cell.2018.07.007

    • PubMed
    • Google Scholar
70. 1. Sims DE

    (2000) Diversity within pericytes

    Clinical and Experimental Pharmacology & Physiology 27:842–846.

    https://doi.org/10.1046/j.1440-1681.2000.03343.x

    • PubMed
    • Google Scholar
71. 1. Storkebaum E
    2. Carmeliet P

    (2004) VEGF: a critical player in neurodegeneration

    The Journal of Clinical Investigation 113:14–18.

    https://doi.org/10.1172/JCI20682

    • PubMed
    • Google Scholar
72. 1. Supek F
    2. Bošnjak M
    3. Škunca N
    4. Šmuc T

    (2011) REVIGO summarizes and visualizes long lists of gene ontology terms

    PLOS ONE 6:e21800.

    https://doi.org/10.1371/journal.pone.0021800

    • PubMed
    • Google Scholar
73. 1. Sweeney MD
    2. Sagare AP
    3. Zlokovic BV

    (2018) Blood-brain barrier breakdown in alzheimer disease and other neurodegenerative disorders

    Nature Reviews. Neurology 14:133–150.

    https://doi.org/10.1038/nrneurol.2017.188

    • PubMed
    • Google Scholar
74. 1. Teran M
    2. Nugent MA

    (2019) Characterization of receptor binding kinetics for vascular endothelial growth factor-A using SPR

    Analytical Biochemistry 564–565:21–31.

    https://doi.org/10.1016/j.ab.2018.10.001

    • PubMed
    • Google Scholar
75. 1. Trapnell C
    2. Roberts A
    3. Goff L
    4. Pertea G
    5. Kim D
    6. Kelley DR
    7. Pimentel H
    8. Salzberg SL
    9. Rinn JL
    10. Pachter L

    (2012) Differential gene and transcript expression analysis of RNA-seq experiments with tophat and cufflinks

    Nature Protocols 7:562–578.

    https://doi.org/10.1038/nprot.2012.016

    • PubMed
    • Google Scholar
76. 1. Viana LM
    2. O’Malley JT
    3. Burgess BJ
    4. Jones DD
    5. Oliveira C
    6. Santos F
    7. Merchant SN
    8. Liberman LD
    9. Liberman MC

    (2015) Cochlear neuropathy in human presbycusis: confocal analysis of hidden hearing loss in post-mortem tissue

    Hearing Research 327:78–88.

    https://doi.org/10.1016/j.heares.2015.04.014

    • PubMed
    • Google Scholar
77. 1. Vieira M
    2. Christensen BL
    3. Wheeler BC
    4. Feng AS
    5. Kollmar R

    (2007) Survival and stimulation of neurite outgrowth in a serum-free culture of spiral ganglion neurons from adult mice

    Hearing Research 230:17–23.

    https://doi.org/10.1016/j.heares.2007.03.005

    • PubMed
    • Google Scholar
78. 1. Winkler EA
    2. Bell RD
    3. Zlokovic BV

    (2011) Central nervous system pericytes in health and disease

    Nature Neuroscience 14:1398–1405.

    https://doi.org/10.1038/nn.2946

    • PubMed
    • Google Scholar
79. 1. Xie L
    2. Wang M
    3. Liao T
    4. Tan S
    5. Sun K
    6. Li H
    7. Fang Q
    8. Tang A

    (2018) The characterization of auditory brainstem response (ABR) waveforms: a study in tree shrews (tupaia belangeri)

    Journal of Otology 13:85–91.

    https://doi.org/10.1016/j.joto.2018.05.004

    • PubMed
    • Google Scholar
80. 1. Ye L
    2. Guo H
    3. Wang Y
    4. Peng Y
    5. Zhang Y
    6. Li S
    7. Yang M
    8. Wang L
    9. Lloret A

    (2021) Exosomal circehmt1 released from hypoxia-pretreated pericytes regulates high glucose-induced microvascular dysfunction via the NFIA/NLRP3 pathway

    Oxidative Medicine and Cellular Longevity 2021:8833098.

    https://doi.org/10.1155/2021/8833098

    • PubMed
    • Google Scholar
81. 1. Yemisci M
    2. Gursoy-Ozdemir Y
    3. Vural A
    4. Can A
    5. Topalkara K
    6. Dalkara T

    (2009) Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery

    Nature Medicine 15:1031–1037.

    https://doi.org/10.1038/nm.2022

    • PubMed
    • Google Scholar
82. 1. Yuan X
    2. Wu Q
    3. Wang P
    4. Jing Y
    5. Yao H
    6. Tang Y
    7. Li Z
    8. Zhang H
    9. Xiu R

    (2019) Exosomes derived from pericytes improve microcirculation and protect blood–spinal cord barrier after spinal cord injury in mice

    Frontiers in Neuroscience 13:319.

    https://doi.org/10.3389/fnins.2019.00319

    • PubMed
    • Google Scholar
83. 1. Zehendner CM
    2. Sebastiani A
    3. Hugonnet A
    4. Bischoff F
    5. Luhmann HJ
    6. Thal SC

    (2015) Traumatic brain injury results in rapid pericyte loss followed by reactive pericytosis in the cerebral cortex

    Scientific Reports 5:13497.

    https://doi.org/10.1038/srep13497

    • PubMed
    • Google Scholar
84. 1. Zhang Y
    2. Liu Y
    3. Liu H
    4. Tang WH

    (2019) Exosomes: biogenesis, biologic function and clinical potential

    Cell & Bioscience 9:19.

    https://doi.org/10.1186/s13578-019-0282-2

    • PubMed
    • Google Scholar
85. 1. Zhang J
    2. Hou Z
    3. Wang X
    4. Jiang H
    5. Neng L
    6. Zhang Y
    7. Yu Q
    8. Burwood G
    9. Song J
    10. Auer M
    11. Fridberger A
    12. Hoa M
    13. Shi X

    (2021) VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss

    JCI Insight 6:285.

    https://doi.org/10.1172/jci.insight.143285

    • PubMed
    • Google Scholar
86. 1. Zlokovic BV

    (2011) Neurovascular pathways to neurodegeneration in alzheimer’s disease and other disorders

    Nature Reviews. Neuroscience 12:723–738.

    https://doi.org/10.1038/nrn3114

    • PubMed
    • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "Pericytes control vascular stability and auditory spiral ganglion neuron survival" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Gou Young Koh as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Barbara Shinn-Cunningham as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The authors are required to carefully address all of the comments point-by-point in a data-driven manner or with further analyses. I believe the authors could revise the manuscript successfully given their expertise but please let us know if it takes more than 3 months, and provide the reasons for not implementing the suggested changes if that applies.

Reviewer #1 (Recommendations for the authors):

1. Deletion of PDGFRβ+ pericytes using the iDTR system is an artificial approach to understanding the roles of the pericytes in each organ. What are the circumstances of pericyte loss or pericyte drop-out in the cochlea? It would be constructive if the authors show such a loss or dropout in pathophysiologic conditions including diabetic condition, aging, and other hearing loss conditions.

2. The authors are required to distinguish the phenotypes of the pericyte deletion, at least the inner ear versus the outer ear (or any organs as negative and positive controls), to strengthen the findings in the inner ear. The phenotype of the pericyte deletion is organ- and tissue-specific. For example, the study by DY Park et al. (Plastic roles of pericytes in the blood-retinal barrier, Nature Comm. 2017) did not show any alterations in the adult retinal blood vessels in the absence of the PDGFRβ+ pericytes. This paper needs to be cited and discussed in this study.

3. Are there any PDGFRβ+ cells (or fibroblasts) rather than the vascular pericytes in the SGNs? There are more PDGFRβ+ fibroblasts than PDGFRβ+ vascular pericytes in most organs. It is required to provide images supporting the selective distribution of PDGFRβ+ pericytes in the SGNs.

Reviewer #2 (Recommendations for the authors):

Several points should be clarified through discussion or supported by additional experiments as listed below:

1. Figure 1 is helpful for readers outside the field, but I don't think such introductory illustrations are appropriate to be shown in regular articles.

2. (Figure 2C, D) "Exclusive co-localization" of tdTomato and PDGFRb is not clear to me. Especially, in areas outside the box in C, they look mostly separated. Better images or accurate interpretations should be presented.

3. (Figure 7A) How did you set the PCR primers? Which isoform did you detect? Please clarify.

4. (Figure 11) Red particles might be PC-derived exosomes as described. Is it difficult to show these particles contain VEGF histologically?

5. Pericytes are depleted systemically, making the interpretation of the data difficult. This point should be discussed as a limitation.

6. Is it appropriate to use PdgfrgCreERT2-/- as controls? The toxicity of Cre, in particular for inducible lines, is well known (Brash et al., Circ Res 2020). Did you exclude non-specific damages to pericytes in PdgfrgCreERT2+/- mice after tamoxifen injection?

7. Functional significance of direct neurotrophic effects of VEGF versus indirect effects mediated by angiogenesis has long been debated. Former effects have been mostly supported by in vitro culture, but not in vivo genetic evidence. Ideally, Flk1 deletion in neurons should be performed. In most cases using neuronal Flk1 CKO, the difference is very small in spite of statistical significance.

8. Why do you think pericytes need exosomes to deliver VEGF? People may think diffusible isoforms or truncation would be feasible. This point should be discussed.

9. Related to the above, how does VEGF in exosomes bind Flk1? Are they once internalized or ruptured around neurons? Please discuss.

Reviewer #3 (Recommendations for the authors):

- Images in Figure 1 show increased permeability of vessels in pericyte-ablated mice. As pericytes are important for controlling vessel permeability, it would be important that the authors comment on it.

- It could be that VEGF is not only expressed by pericytes but also but other cell types in the inner ear (i.e. endothelial cells and neurons). Thus, it would be important that the authors complement the analysis of VEGF expression not only via Elisa but also via in situ hybridization or immunohistochemistry.

- In Figure 10 the authors show reduced neurite length when explants are cultured with pericyte conditioned medium. However, is this effect due to VEGF acting directly on neurons or indirectly in endothelial cells? Also of note: the title of Figure 10 is not easy to understand.

- Figure 11 should be presented in the results section and not in the discussion. Also, with the presented images it is not clear what the claim that SGN takes up particles released by pericytes is. Better evidence is recommended.

Reviewer #1 (Recommendations for the authors):

1. Deletion of PDGFRβ+ pericytes using the iDTR system is an artificial approach to understanding the roles of the pericytes in each organ. What are the circumstances of pericyte loss or pericyte drop-out in the cochlea? It would be constructive if the authors show such a loss or dropout in pathophysiologic conditions including diabetic condition, aging, and other hearing loss conditions.

Reviewer #1 makes a significant and insightful point regarding the translation potential of these findings. We agree the iDTR/ PDGFRβ+ depletion animal model is not a disease model, but rather an artificial biological model. The advantage of this model is it allows us to study the direct role of pericytes in cochlear health while avoiding other complications which occur in a disease model. Reduction in pericyte number, migration of pericytes, and pericyte trans-differentiation are well-defined in aging and noise-induced hearing loss, demonstrated in both our early work (Neng et al., 2015) and more recent work (Hou et al., 2020; Hou et al., 2018).

2. The authors are required to distinguish the phenotypes of the pericyte deletion, at least the inner ear versus the outer ear (or any organs as negative and positive controls), to strengthen the findings in the inner ear. The phenotype of the pericyte deletion is organ- and tissue-specific. For example, the study by DY Park et al. (Plastic roles of pericytes in the blood-retinal barrier, Nature Comm. 2017) did not show any alterations in the adult retinal blood vessels in the absence of the PDGFRβ+ pericytes. This paper needs to be cited and discussed in this study.

Yes, pericytes are organ-oriented and tissue specific. In the revision, we have added supplemental data demonstrating that while pericyte depletion in our model affects retinal pericytes, we have not observed obvious abnormalities of retina blood vessels in adult animals (Appendix-Figure 2), consistant with the previous report by DY Park et al. (2017). We also added discussion on the effect of pericyte depletion in different organs on page 7, lines 286-297.

3. Are there any PDGFRβ+ cells (or fibroblasts) rather than the vascular pericytes in the SGNs? There are more PDGFRβ+ fibroblasts than PDGFRβ+ vascular pericytes in most organs. It is required to provide images supporting the selective distribution of PDGFRβ+ pericytes in the SGNs.

Great Question! Yes, PDGFRβ can be expressed cells other than pericytes, including fibroblasts and glia (Hewitt et al., 2012). In the cochlea, we find PDGFRβ is mostly expressed by pericytes. However, there is a small population of PDGFRβ positive cells in non-vascular regions that are not pericytes. The population is small and insignificant, however, compared to the majority of PDGFRβ+ pericytes. We have now provided better images on the distribution of PDGFRβ+ pericytes in the spiral ganglion region (see Figures2C and D).

Reviewer #2 (Recommendations for the authors):

Several points should be clarified through discussion or supported by additional experiments as listed below:

1. Figure 1 is helpful for readers outside the field, but I don't think such introductory illustrations are appropriate to be shown in regular articles.

Thanks for the suggestion. We moved Figure 1 to appendix 1- Figure 1 to better inform non-auditory scientists on the location of vascular structure in the cochlea.

2. (Figure 2C, D) "Exclusive co-localization" of tdTomato and PDGFRb is not clear to me. Especially, in areas outside the box in C, they look mostly separated. Better images or accurate interpretations should be presented.

We apologize for the definitive statement. We have revised the text to accurately reflect the findings. Reviewer #1 might agree that immunostaining in the area is challenging due to the complexity of the tissue structure. We have replaced the original image with better images (see Figures 2C and D). But, overall, the Reviewer is correct. While some PDGFRβ+ cells are outside of blood vessels and are of unknown cell type, most PDGFRβ+ cells are vascular perciytes. This could be due to that the underside of vessels is not captured because of the thickness of the spiral lamina. Visualization of vascular structure in large-scale volumetric models in this region is often difficult and limited by the optical scattering and absorption of light by bone. In our 2019 publication (Jiang et al., 2019), we showed that details of vascular architecture in the spiral limbus and SGN in whole-mounted mouse preparations are not fully revealed without tissue clearing. However, when we do use tissue clearance, the procedure damages membrane proteins and immunolabeling for PDGFRβ fails. In addition, some of the tdTomato-positive structures apparently not co-labeled by PDGFRβ antibodies could also be the result of a common signal-leakage side-effect in transgenic mouse models (Song and Palmiter, 2018; Stifter and Greter, 2020). Nevertheless, since we only deplete partial of the PDGFRβ cells in the vessel beds, we believe the small number of ‘non-pericytes’ is insignificant.

3. (Figure 7A) How did you set the PCR primers? Which isoform did you detect? Please clarify.

Thanks for the question. We designed the PCR primers using Primer-BLAST to specifically detect for VEGFA expression. Details have been added to the Methods and Materials section on page 16, lines 608-610.

4. (Figure 11) Red particles might be PC-derived exosomes as described. Is it difficult to show these particles contain VEGF histologically?

We apologize for the confusion; our description of Figure 11 was unclear. The purpose of Figure 11 in the initial submission was to demonstrate active communication between pericytes and the spiral ganglion (material from one cell interacting with another cell). We are not certain what those red particles are. They could be particles released by pericytes. But it is very difficult to identify the content of exosomes without high-resolution EM combined with immunochemical binding for VEGFA. The current image resolution used is by far insufficient to visualize exosomes and their contents. To provide the reader with on this issue, and follow the Reviewer’s suggestion, we moved Figure 11 to Figure 1. The original Figure 11 has also been replaced with a better data set to demonstrate pericyte-SGN cellular communication.

5. Pericytes are depleted systemically, making the interpretation of the data difficult. This point should be discussed as a limitation.

Thanks to reviewer #2 for the comments. We have added discussion of the limitations of our study to the Discussion section on page 7, lines 284-297.

6. Is it appropriate to use PdgfrgCreERT2-/- as controls? The toxicity of Cre, in particular for inducible lines, is well known (Brash et al., Circ Res 2020). Did you exclude non-specific damages to pericytes in PdgfrgCreERT2+/- mice after tamoxifen injection?

Great point. We used PdgfrbCreERT2-/- mice as controls to test the toxicity of both tamoxifen and DT in PdgfrbCreERT2-/-;DTR+/- mice. To test the toxicity of Cre, we assessed the ABR in PdgfrbCreERT2+/-;DTR+/- mice after tamoxifen injection and before injection of DT. We did not observe any ABR threshold difference between the PdgfrbCreERT2-/- and PdgfrbCreERT2+/- mice (Figure 3A). Therefore the dose of tamoxifen we used did not affect hearing function.

7. Functional significance of direct neurotrophic effects of VEGF versus indirect effects mediated by angiogenesis has long been debated. Former effects have been mostly supported by in vitro culture, but not in vivo genetic evidence. Ideally, Flk1 deletion in neurons should be performed. In most cases using neuronal Flk1 CKO, the difference is very small in spite of statistical significance.

Yes, we fully agree! Our study has shown Flk1 strongly expressed in peripheral auditory neurons (as shown in Figures8C and D) and in isolated adult SGNs. Our in vitro data also demonstrated SGNs directly respond to VEGF-A containing vesicles, as shown in Figure 9C. The exosome-treated adult SGNs showed more SGN dendritic growth (red, labeled for β-III tubulin) relative to control and VEGFR2-inhibited groups. Taken together the data indicates the direct effect of VEGF-A in SGN growth. However, it would be ideal if further evidence could be provided in a conditional SGN-Flk1 KO mouse model, as the Reviewer has suggested. We have mentioned this in the Discussion on page 9, lines 403-406.

8. Why do you think pericytes need exosomes to deliver VEGF? People may think diffusible isoforms or truncation would be feasible. This point should be discussed.

Great point! Yes, we agree that VEGF-A is directly released by cells in diffusible isoforms. However, in our early study, we observed that cochlear pericytes release particles, and cellular communication between pericytes and SGNs in vivo, as shown in the new Figure 1. In vitro PC culture medium is rich in exosomes. With protein immunoblots, we have determined VEGF-A is predominant in the exosomes, suggesting the VEGF-A is delivered by exosomes. That said, why pericytes need to deliver VEGF-A in exosomes is not clear. Studies in other organ systems do show shedding of VEGFA-165 from the cell surface, together with other membrane components; this appears to be a vehicle by which some VEGF is delivered to the surroundings to exert its known biological actions (Guzman-Hernandez et al., 2014). However, the exact mechanism by which pericytes release VEGFA in the form of exosomes and affect SGN health is unknown and needs further investigation. Thanks to the reviewer for pointing this out. We have mentioned this in the Discussion on page 9, lines 372-379.

9. Related to the above, how does VEGF in exosomes bind Flk1? Are they once internalized or ruptured around neurons? Please discuss.

This is an excellent question, one we asked ourselves during the study. In general, exosome-driven mechanisms are complicated and poorly understood, which we have pointed out in a recent review article (Sharma et al., 2022). For example, studies demonstrate that EVs interface with neighboring cells through different modes, including direct receptor-binding without internalization of EVs, phagocytosis, macropinocytosis, internalization by clathrin-caveolae- and lipid raft-mediated endocytosis (Mulcahy et al., 2014). Once internalized, the EVs release their cargo in the cytoplasm and the cell-internalized cargo then regulates the cell at the transcription or translation level (Abels & Breakefield, 2016; Berumen Sanchez et al., 2021). VEGF also presents on the surface of EVs by binding to surface proteins such as heparin and heat shock protein 90 (HSP90), which can directly stimulate the cellular domain of Flk1 and induce phosphorylation (Ko et al., 2019; Ko & Naora, 2020). We have now added discussion of these mechanisms on page 9, lines 379-388. It is our future goal to identify the underlying mechanisms in collaboration with cell biologists with expertise in this area of research.

Reviewer #3 (Recommendations for the authors):

- Images in Figure 1 show increased permeability of vessels in pericyte-ablated mice. As pericytes are important for controlling vessel permeability, it would be important that the authors comment on it.

Great point! Yes, pericytes play a significant role in blood vessel integrity, particularly in the blood labyrinth and blood-brain barriers, as we showed in our early studies (Hou et al., 2018; Zhang et al., 2021). Pericytes stabilize vascular integrity by releasing signaling factors that control the expression of tight junction proteins in endothelial cells. Without pericytes, tight junction protein is significantly downregulated in vascular systems of the cochlea (Neng et al., 2013). We have now added comments on this in the Discussion section on page 8, lines 328-334.

- It could be that VEGF is not only expressed by pericytes but also but other cell types in the inner ear (i.e. endothelial cells and neurons). Thus, it would be important that the authors complement the analysis of VEGF expression not only via Elisa but also via in situ hybridization or immunohistochemistry.

Yes, the reviewer is correct. Earlier studies by Pasqualina et al., 2004 demonstrated that VEGF is expressed in supporting cells, hair cells, stria vascularis, and spiral ganglia (Picciotti et al., 2004). It is likely that VEGF signaling in these cells in part plays a role in regulation of cochlear and hearing function. Our study has shown for the first time that VEGF is expressed in pericytes and secretion of VEGF participates in regulation of vascular and neuronal function. In accord with Reviewer#3’s suggestion, we added new images of the VEGF immunostaining to demonstrate that VEGFA is expressed in pericytes (Fig.7D). We appreciate the reviewer’s suggestion to conduct an in situ hybridization, which could indeed confirm mRNA expression for VEGF in the pericytes. However, since our RNA seq data already show expression of VEGF mRNA in pericytes, the in situ hybridization would be redundant; the new immunostaining data should therefor be sufficient for demonstrating expression in pericytes.

- In Figure 10 the authors show reduced neurite length when explants are cultured with pericyte conditioned medium. However, is this effect due to VEGF acting directly on neurons or indirectly in endothelial cells? Also of note: the title of Figure 10 is not easy to understand.

Thanks for pointing out this problem. To ensure that we are assessing the direct effect of VEGF in neurons, we treated isolated adult neurons with pericyte-derived exosomes with and without the specific VEGFR2 inhibitor, SU5408, as shown in Figs. 9C & E. This method eliminated the possible neighborhood effects of endothelial and supporting cells. Fig. 10 shows results from a preliminary experiment to optimize the dose of the VEGFR2 inhibitor in SGN explants. The title of Fig. 10 has been changed for clarity.

- Figure 11 should be presented in the results section and not in the discussion. Also, with the presented images it is not clear what the claim that SGN takes up particles released by pericytes is. Better evidence is recommended.

Great suggestion. In accord with the Reviewer’s suggestion, we have moved Fig.11 to the Results section and redubbed it Fig.1. The original Fig.11 is also replaced with a better image. The purpose of this Figure is to demonstrate cellular communication between pericytes and SGNs in vivo. We have correspondingly revised our interpretation in the Results section and Figure legend.

References

Abels, E. R., and Breakefield, X. O. (2016). Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake. Cell Mol Neurobiol, 36(3), 301-312. https://doi.org/10.1007/s10571-016-0366-z

Berumen Sanchez, G., Bunn, K. E., Pua, H. H., and Rafat, M. (2021). Extracellular vesicles: mediators of intercellular communication in tissue injury and disease. Cell Commun Signal, 19(1), 104. https://doi.org/10.1186/s12964-021-00787-y

Deyama, S., Li, X. Y., and Duman, R. S. (2020). Neuron-specific deletion of VEGF or its receptor Flk-1 impairs recognition memory. Eur Neuropsychopharmacol, 31, 145-151. https://doi.org/10.1016/j.euroneuro.2019.11.002

Guzman-Hernandez, M. L., Potter, G., Egervari, K., Kiss, J. Z., and Balla, T. (2014). Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane. Mol Biol Cell, 25(7), 1061-1072. https://doi.org/10.1091/mbc.E13-07-0418

Hewitt, K. J., Shamis, Y., Knight, E., Smith, A., Maione, A., Alt-Holland, A., Sheridan, S. D., Haggarty, S. J., and Garlick, J. A. (2012). PDGFRbeta expression and function in fibroblasts derived from pluripotent cells is linked to DNA demethylation. J Cell Sci, 125(Pt 9), 2276-2287. https://doi.org/10.1242/jcs.099192

Hou, Z., Neng, L., Zhang, J., Cai, J., Wang, X., Zhang, Y., Lopez, I. A., and Shi, X. (2020). Acoustic Trauma Causes Cochlear Pericyte-to-Myofibroblast-Like Cell Transformation and Vascular Degeneration, and Transplantation of New Pericytes Prevents Vascular Atrophy. Am J Pathol, 190(9), 1943-1959. https://doi.org/10.1016/j.ajpath.2020.05.019

Hou, Z., Wang, X., Cai, J., Zhang, J., Hassan, A., Auer, M., and Shi, X. (2018). Platelet-Derived Growth Factor Subunit B Signaling Promotes Pericyte Migration in Response to Loud Sound in the Cochlear Stria Vascularis. J Assoc Res Otolaryngol, 19(4), 363-379. https://doi.org/10.1007/s10162-018-0670-z

Jiang, H., Wang, X., Zhang, J., Kachelmeier, A., Lopez, I. A., and Shi, X. (2019). Microvascular networks in the area of the auditory peripheral nervous system. Hear Res, 371, 105-116. https://doi.org/10.1016/j.heares.2018.11.012

Ko, S. Y., Lee, W., Kenny, H. A., Dang, L. H., Ellis, L. M., Jonasch, E., Lengyel, E., and Naora, H. (2019). Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake. Commun Biol, 2, 386. https://doi.org/10.1038/s42003-019-0609-x

Ko, S. Y., and Naora, H. (2020). Extracellular Vesicle Membrane-Associated Proteins: Emerging Roles in Tumor Angiogenesis and Anti-Angiogenesis Therapy Resistance. Int J Mol Sci, 21(15). https://doi.org/10.3390/ijms21155418

Mulcahy, L. A., Pink, R. C., and Carter, D. R. (2014). Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles, 3. https://doi.org/10.3402/jev.v3.24641

Neng, L., Zhang, F., Kachelmeier, A., and Shi, X. (2013). Endothelial cell, pericyte, and perivascular resident macrophage-type melanocyte interactions regulate cochlear intrastrial fluid-blood barrier permeability. J Assoc Res Otolaryngol, 14(2), 175-185. https://doi.org/10.1007/s10162-012-0365-9

Neng, L., Zhang, J., Yang, J., Zhang, F., Lopez, I. A., Dong, M., and Shi, X. (2015). Structural changes in thestrial blood-labyrinth barrier of aged C57BL/6 mice. Cell Tissue Res, 361(3), 685-696. https://doi.org/10.1007/s00441-015-2147-2

Picciotti, P., Torsello, A., Wolf, F. I., Paludetti, G., Gaetani, E., and Pola, R. (2004). Age-dependent modifications of expression level of VEGF and its receptors in the inner ear. Exp Gerontol, 39(8), 1253-1258. https://doi.org/10.1016/j.exger.2004.06.003

Sharma, K., Zhang, Y., Paudel, K. R., Kachelmeier, A., Hansbro, P. M., and Shi, X. (2022). The Emerging Role of Pericyte-Derived Extracellular Vesicles in Vascular and Neurological Health. Cells, 11(19). https://doi.org/10.3390/cells11193108

Song, A. J., and Palmiter, R. D. (2018). Detecting and Avoiding Problems When Using the Cre-lox System. Trends Genet, 34(5), 333-340. https://doi.org/10.1016/j.tig.2017.12.008

Stifter, S. A., and Greter, M. (2020). STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems. Eur J Immunol, 50(3), 338-341. https://doi.org/10.1002/eji.202048546

Zhang, J., Hou, Z., Wang, X., Jiang, H., Neng, L., Zhang, Y., Yu, Q., Burwood, G., Song, J., Auer, M., Fridberger, A., Hoa, M., and Shi, X. (2021). VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss. JCI Insight, 6(8). https://doi.org/10.1172/jci.insight.143285

Author details

1. Yunpei Zhang

   Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, Portland, United States

   Contribution

   Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Lingling Neng

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5254-796X

2. Lingling Neng

   Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, Portland, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft

   Contributed equally with

   Yunpei Zhang

   Competing interests

   No competing interests declared

3. Kushal Sharma

   Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, Portland, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

4. Zhiqiang Hou

   Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, Portland, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5154-7474

5. Anatasiya Johnson

   Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, Portland, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Junha Song

   Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States

   Contribution

   Visualization

   Competing interests

   No competing interests declared

7. Alain Dabdoub

   1. Biological Sciences, Sunnybrook Research Institute, Toronto, Canada
   2. Department of Otolaryngology-Head & Neck Surgery, University of Toronto, Toronto, Canada
   3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

   Contribution

   Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4259-2425

8. Xiaorui Shi

   Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, Portland, United States

   Contribution

   Resources, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   shix@ohsu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6068-1413

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