Pericytes control vascular stability and auditory spiral ganglion neuron survival

  1. Yunpei Zhang
  2. Lingling Neng
  3. Kushal Sharma
  4. Zhiqiang Hou
  5. Anatasiya Johnson
  6. Junha Song
  7. Alain Dabdoub
  8. Xiaorui Shi  Is a corresponding author
  1. Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University, United States
  2. Life Sciences Division, Lawrence Berkeley National Laboratory, United States
  3. Biological Sciences, Sunnybrook Research Institute, Canada
  4. Department of Otolaryngology-Head & Neck Surgery, University of Toronto, Canada
  5. Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada
14 figures, 3 tables and 1 additional file

Figures

Cochlear vascular networks in the spiral ganglion region are densely populated by pericytes, and spiral ganglion neurons (SGNs) contains particles released by pericytes.

(a and b) Illustrations of pericyte containing microvascular networks in the spiral ganglion region. (A) A confocal projection image of the spiral lamina from an NG2DsRedBAC mouse shows pericytes (red) situated on microvessels labeled with Lectin-Alexa Fluor 488 conjugate (green) around SGNs and their peripheral fibers labeled with an antibody for β-III tubulin (blue). The pericyte distribution can be better visualized under high magnification (A, lower). (B–D) Cross-talk between SGNs and pericytes is suggested by the red fluorescent particle (arrows) observed in SGNs (green). Further evidenced by 3D reconstructive images showing the particles are inside the soma of SGNs.

Pericyte-depletion induces vascular regression in the region of spiral ganglion neuron (SGN) peripheral nerve fibers in adult mice.

(A) Schematic of a pericyte-depletion mouse model incorporating an inducible Cre-loxP system. (B) The diagram shows the timeline of tamoxifen and diphtheria toxin (DT) administration and the time point of auditory brainstem response (ABR) test and tissue harvest. (C) Co-localization of PDGFRβ-Cre (tdTomato) and immune-labeled PDGFRβ (green) signals in the pericytes of PDGFRB-CreERT2; ROSA26tdTomato mice. (D) A high-magnification image further shows co-localization of the Cre and PDGFRβ fluorescence signals (arrows). (E) Representative figures show pericyte coverage in the region of the spiral limbus in DT-treated control inducible diphtheria toxin receptor (iDTR; left) and DT-treated PDGFRB-CreERT2; ROSA26iDTR mice (right) 2 wk after DT injection. DT injection significantly leads to loss of pericyte coverage. (F) Pericyte density was significantly reduced in the PDGFRB-CreERT2; ROSA26iDTR mice at the apical, middle, and basal turn relative to density in the control of iDTR mice (n=5, pApex=0.0295, pMid=0.0361, pBase=0.005, unpaired t test). (G) Representative figures show the capillaries of the spiral lamina in control ROSA26iDTR (left) and PDGFRB-CreERT2; ROSA26iDTR (right) mice, with the distribution of vessel diameter shown in π charts, and the location of SGNs shown in ellipses. (H) Total vascular density in the spiral limbus and lamina is significantly reduced in the pericyte-depleted mice 2 wk after DT injection (n=5, pApex=0.027, pMid=0.0108, pBase=0.0194, unpaired t test). Loss of vascular volume with pericyte depletion is better seen in the high-magnification image inserts in panel G (a) and (b). Data are presented as the mean ± SEM. Scale bars: C, 50 µm; D, 20 µm; E, 20 µm; F, 200 µm.

The depletion of pericytes led to hearing loss, increased latency and reduced amplitude of wave I.

(A) The control mice showed no significant hearing threshold change after diphtheria toxin (DT) injection (n=10, p=0.9651). In contrast, the hearing threshold in pericyte-depleted animals was significantly elevated at 1 wk after DT injection and persisted to 2 wk after injection (n=10, p<0.0001). Two-way ANOVA, followed by Dunnett’s multiple comparison test, and individual p values of different time points versus before DT injection are labeled on the graph. (B) The control mice showed no significant wave I latency change after DT injection (n=10, p=0.3576). In pericyte-depleted animals, the latency was significantly delayed at high frequency (32 kHz) starting with the fourth DT injection, and at low frequency (8 kHz) 2 wk after DT injection (n=10, p<0.0001). Two-way ANOVA, followed by Dunnett’s multiple comparison test, and individual p values of different time points versus before DT injection are labeled on the graph. (C) Although changed wave I amplitude was randomly observed in control mice after DT injection, changes rarely persisted to 2 wk after the injection (n=10, p16kHz<0.0001, p24kHz<0.0001). In contrast, significant reduction in wave I amplitude was observed in pericyte-depleted animals at 2 wk after DT injection, particularly at high frequency (32 kHz) (n=10, p8kHz<0.0001, p16kHz<0.0001, p24kHz<0.0001, p32kHz<0.0001). Two-way ANOVA, followed by Dunnett’s multiple comparison test, and individual p values at 2 wk after DT injection versus before DT injection at different sound pressure levels are labeled on the graph. Data are presented as the mean ± SEM.

The depletion of pericytes led to spiral ganglion neuron (SGN) loss and decreased expression of β-III tubulin in SGNs.

(A) Representative confocal images from control and pericyte-depleted animals, labeled with antibody for β-III tubulin. (B) Significant SGN loss at all turns 2 wk after diphtheria toxin (DT) treatment (n=9, pApex=0.0085, pMid=0.0099, pBase=0.0127, unpaired t-test). (C) Significantly decreased β-III tubulin expression in SGNs at middle and basal turns 2 wk after DT treatment (pApex=0.1074, pMid<0.0001, pBase<0.0001, unpaired t-test). Data are presented as the mean ± SEM. Scale bar: D, 50 µm.

Pericytes promote both vascular and neuronal growth in spiral ganglia in vitro.

(A) The 20 most significant overrepresented PANTHER pathways for genes (RPKM>0.5) identified in cochlear pericytes. (B) Neonatal spiral ganglion neuron (SGN) explants co-cultured with pericytes show robust SGN dendritic growth (green, labeled for β-III Tubulin) and new vessel growth (red, labeled for CD31). (C) There are significant differences in number of new vessel branches and in number and length of dendritic fibers in the two groups (n=6, pVascular branch #/area=0.0018, pNeurites #/area<0.0001, pNeurite length/explant=0.0393, unpaired t test). (D) Adult SGNs co-cultured with pericytes show robust SGN dendritic growth (red, labeled for β-III Tubulin). (E) There are significant differences in cell survival, and in average neurite number and length, in the two groups (n=3 wells per group, 25 cells per well, pneuron survival=0.0186, pNeurites #/cell<0.0001, plongest neurite length/cell<0.001, unpaired t test). Data are presented as the mean ± SEM. Scale bars: B, 300 µm (top), 150 µm (middle), 50 µm (bottom); D, 50 µm.

Identification of pericyte-derived exosomes.

(A) Schematic of the exosome purification procedure used to isolate exosomes from pericyte-conditioned culture medium for nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and proteomics analysis. Exosomes were isolated via ultracentrifugation followed by size-exclusion separation. (B) NTA shows a rich population of exosome-sized (~50–150 nm) particles, 96.4% of which are labeled with Exoglow (a membrane extracellular vesicle [EV] marker kit). (C) TEM images show a classic cup-shaped structure membrane vesicle with diameter around 100 nm. (D) Proteomics analysis of exosomes identified a total of 570 unique protein families in the exosomes, 496 of which overlap with genes identified in pericyte isolated total RNA. (E) The 22 most significantly enriched Gene Ontology (GO) cellular component terms for proteins identified in the pericyte-derived exosomes. (F) The relative PANTHER pathways identified in pericyte-derived exosome proteins. Scale bars: C, 500 nm (top) and 200 nm (bottom).

Figure 6—source data 1

Protein families of cochlear pericyte-derived exosomes identified by proteomics analysis.

https://cdn.elifesciences.org/articles/83486/elife-83486-fig6-data1-v2.xlsx
Pericytes release VEGFA through exosomes.

(A) mRNA expression of VEGF-A in primary cochlear pericytes. (B) VEGF-A production assessed by ELISA at day 3 in the control and pericyte containing culture medium (n=6, p<0.0001, unpaired t test). (C) VEGFA expression level assessed by ELISA in the cochlea of control and pericyte-depleted mice (ncontrol = 5, npericyte depletion=8, p=0.0387, unpaired t test). (D) Representative confocal images showing VEGF-A expression in pericytes. (E) Exosomes and the non-exosomal fraction (supernatant) were purified from pericyte-conditioned media using differential ultracentrifugation for western blot analysis. (F) Western blot showing the expression of VEGFA in exosomes. Similarly, PDGFR-β (pericyte membrane marker) was detected exclusively in exosomes. Data are presented as the mean ± SEM. Scale bars: D, 5 µm.

Figure 7—source data 1

Original uncropped blots of VEGFA and PDGFR-β, and raw gel data of whole protein staining with the relevant bands clearly labeled.

https://cdn.elifesciences.org/articles/83486/elife-83486-fig7-data1-v2.zip
Figure 7—source data 2

Original uncropped blots of VEGFA and PDGFR-β, and raw gel data of whole protein staining with the relevant bands clearly labeled.

https://cdn.elifesciences.org/articles/83486/elife-83486-fig7-data2-v2.pdf
VEGFR2 expression in the spiral ganglion region.

(A–C) Representative confocal images of a cochlear whole mount under low (A) and high (B and C) magnification. (D) Cross section showing VEGFR2 (red) is positively expressed in both spiral ganglion neurons (SGNs; labeled for β-III Tubulin) and blood vessels (labeled for lectin). Scale bars: E, 100 µm; F, 30 µm; G, 20 µm; H, 50 µm.

Pericyte-derived exosomes promote angiogenesis and spiral ganglion neuron (SGN) growth through VEGFR2 signaling.

(A) Exosomes were purified from pericyte-conditioned media using total exosome isolation reagent for in vitro treatment. (B) Compared to the control group, exosome treated SGN explants showed robust SGN dendritic growth (green, labeled for β-III Tubulin) and new vessel growth (red, labeled for CD31). In contrast, both neurogenic and angiogenetic activity were decreased when a VEGFR2 inhibitor, SU5408, was presented in the medium. (C) Exosome treated adult SGNs showed more SGN dendritic growth (red, labeled for β-III Tubulin) compared to control and VEGFR2 blocked groups. (D) There are significant differences in new vessel branch number and in dendritic fiber number and length in the three groups (n=6, p=0.0017). (E) There are significant differences in cell survival and in average neurite number and length, in the three groups (n=4 wells per group, 25 cells per well, p=0.0048). One-way ANOVA followed by Tukey’s multiple comparison test, individual p values of different group comparisons are labeled on the graph. Data are presented as the mean ± SEM. Scale bars: B, 300 µm (left), 150 µm (center), 50 µm (right); C, 50 µm.

Neuronal dendritic growth is reduced when VEGFR2 is blocked in a pericyte-spiral ganglion neuron (SGN) explant co-culture model.

(A) Representative images showing the pattern of SGN dendritic growth under different experimental conditions. (B and C) Number and length of dendritic fibers when the VEGFR2 receptor is blocked. A dose-dependent relationship is shown (n=3, pnumber<0.0001, plength<0.0001, one-way ANOVA followed by a Tukey’s multiple comparison test, individual p values between different groups labeled on the graph). Data are presented as the mean ± SEM. Scale bar: A, 150 µm.

Hypothesized mechanisms of pericyte-mediated effects on spiral ganglion neuron (SGN) growth and survival.

(A) Under normal conditions, pericytes participate in the maintenance of SGN health through two parallel pathways: (1) One maintains vascular stability and function and (2) the other ‘nourishes’ SGNs through release of exosomes. (B) Pericyte depletion causes reduction in vascular volume as well as dysfunction, including loss of SGNs and hair cells (Buch et al., 2005), and, in consequence, hearing loss. (C) VEGF-A-carrying exosomes interact with VEGFR2 on the SGNs to stimulate growth and promote survival, which can be arrested by the specific VEGFR2 inhibitor, SU5408. (D) The schematic model shows the molecular structure of exosomes and includes mention of common exosome markers such as CD81, CD63, CD9, and Tsg101 and exosome cargo such as proteins, DNAs, RNAs, lipids, and metabolites.

Schematic of dissection and culture of spiral ganglion neuron (SGN) explants and method of quantitative analysis of neural dendritic growth in a P2 neonatal mouse cochlea over the course of 5 d in culture.

(A and B) The cochlear middle turns were dissected out, stria vascularis and organ of Corti discarded, and the remaining SGN cut into three 90° fan-shaped pieces. These were attached to a coated 6-well glass bottom plate and cultured. (C) A representative confocal projection image of an SGN explant labeled with an antibody for β-III tubulin (green). (D–F) Illustration showing the method used for quantification of SGN neurite number and length. N: the number of neurites; L: the length of the neurites. (F) High magnification of the SGN explant showing new vessel growth labeled for CD31 (red). Arrows indicate new vessel branches.

Appendix 1—figure 1
The anatomy and blood supply of cochlea.

Two major microvascular networks in the cochlea include the network in the cochlear lateral wall and network in the region of spiral ganglion neurons (SGNs)-blood vessels penetrate the SGNs and directly supply nutrients to the neurons. SV, stria vascularis; SL, spiral ligament; HCs, hair cells; BM, basilar membrane.

Appendix 1—figure 2
Pericyte depletion in the retina does not affect the blood vessels in adult animals.

Scale bar: 50 µm.

Tables

Table 1
Overrepresented PANTHER pathways for genes (RPKM>0.5) identified in cochlear pericytes.
No.PANTHER pathways% of gene in the list−Log10 (FDR (false discovery rate))
Mus musculus (REF)Pericyte
1Inflammation mediated by chemokine and cytokine signaling pathway1.1819795.07246414.88941
2CCKR signaling map0.741013.80434813.30277
3Angiogenesis0.8137473.71376811.61261
4Heterotrimeric G-protein signaling pathway-Gq alpha and Go alpha mediated pathway0.5546212.7173918.9914
5Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway0.7228262.989138.364516
6Wnt signaling pathway1.4183754.2572468.073658
7Gonadotropin-releasing hormone receptor pathway1.0683273.6231888.066513
8T cell activation0.4091472.1739137.876148
9Apoptosis signaling pathway0.5637132.4456527.314258
10Endothelin signaling pathway0.3773241.9927547.218245
11B cell activation0.3227711.8115946.931814
12VEGF signaling pathway0.3045871.6304355.958607
13Oxytocin receptor mediated signaling pathway0.2727641.4492755.242604
14Histamine H1 receptor mediated signaling pathway0.2091191.2681165.136677
15PI3 kinase pathway0.2454881.3586965.135489
16Thyrotropin-releasing hormone receptor signaling pathway0.2864031.4492755.086716
17Alzheimer disease-presenilin pathway0.5773511.9927544.640165
18Integrin signaling pathway0.8637542.4456524.315155
19Alzheimer disease-amyloid secretase pathway0.3000411.3586964.308919
205HT2 type receptor mediated signaling pathway0.3091331.3586964.194499
21Muscarinic acetylcholine receptor 1 and 3 signaling pathway0.2727641.2681164.154902
22PDGF signaling pathway0.6500891.9927544.017277
23Heterotrimeric G-protein signaling pathway-rod outer segment phototransduction0.1772970.9963773.886057
24EGF receptor signaling pathway0.6182661.9021743.882729
25Interleukin signaling pathway0.3955081.4492753.721246
26Muscarinic acetylcholine receptor 2 and 4 signaling pathway0.2727641.1775363.636388
27FGF signaling pathway0.5546211.7210143.603801
28Cytoskeletal regulation by Rho GTPase0.3636861.3586963.595166
29Nicotinic acetylcholine receptor signaling pathway0.4500611.4492753.19382
30p53 pathway feedback loops 20.2273040.9963773.157391
31ATP synthesis0.0318230.4528993.123782
32Axon guidance mediated by netrin0.1591130.8152173.010105
33Beta1 adrenergic receptor signaling pathway0.2091190.9057972.886057
34Enkephalin release0.1682050.8152172.882729
35GABA-B receptor II signaling0.1682050.8152172.869666
36Metabotropic glutamate receptor group II pathway0.2136650.9057972.832683
37Insulin/IGF pathway-protein kinase B signaling cascade0.1772970.8152172.772113
38Blood coagulation0.2363960.9057972.551294
39FAS signaling pathway0.1545670.7246382.522879
40Nicotine pharmacodynamics pathway0.1591130.7246382.462181
41Dopamine receptor mediated signaling pathway0.254580.9057972.376751
42Beta2 adrenergic receptor signaling pathway0.2091190.8152172.366532
43Alpha adrenergic receptor signaling pathway0.104560.5434782.095284
44Axon guidance mediated by Slit/Robo0.1136520.5434781.978811
45TGF-beta signaling pathway0.4591541.1775361.978811
46p53 pathway0.4000551.0869571.974694
47Androgen/estrogene/progesterone biosynthesis0.0772830.4528991.970616
48Endogenous cannabinoid signaling0.1136520.5434781.970616
49Cortocotropin releasing factor receptor signaling pathway0.1545670.6340581.970616
50Histamine H2 receptor mediated signaling pathway0.1181980.5434781.931814
515HT1 type receptor mediated signaling pathway0.2091190.7246381.910095
52Opioid proopiomelanocortin pathway0.1682050.6340581.853872
53Hypoxia response via HIF activation0.127290.5434781.821023
54TCA cycle0.0545530.3623191.754487
55Cadherin signaling pathway0.7364641.5398551.686133
56Pyruvate metabolism0.0590990.3623191.669586
57Triacylglycerol metabolism0.0090920.1811591.446117
58Opioid prodynorphin pathway0.1636590.5434781.407823
59Opioid proenkephalin pathway0.1727510.5434781.321482
Table 1—source data 1

Gene list of cochlear pericytes identified by RNA-seq analysis.

https://cdn.elifesciences.org/articles/83486/elife-83486-table1-data1-v2.xlsx
Table 2
Common protein markers for exosomes were identified in the pericyte-derived exosomes.
ProteinGene names# PSMsLog2 LFQ (label-free quantitation) intensity
CD81 antigenCd812629.6153
CD63 antigenCd63326.1908
CD9 antigenCd91629.2576
Tumor susceptibility gene 101Tsg101525.7598
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6JThe Jackson LaboratoryRRID:IMSR_JAX:000664
Strain, strain background (M. musculus)B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/JThe Jackson LaboratoryRRID:IMSR_JAX:007909
Strain, strain background (M. musculus)B6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/JThe Jackson LaboratoryRRID:IMSR_JAX:029684
Strain, strain background (M. musculus)C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/JThe Jackson LaboratoryRRID:IMSR_JAX:007900
Strain, strain background (M. musculus)Tg(Cspg4-DsRed.T1)1Akik/JThe Jackson LaboratoryRRID:IMSR_JAX:008241
Antibodyanti-Desmin [Y66] (Rabbit monoclonal)AbcamCat#: ab323621:50
Antibodyanti-β-III Tubulin [EP1569Y] (Rabbit monoclonal)AbcamCat#: ab526231:200
Antibodyanti-CD31 [MEC 7.46] (Rat monoclonal)AbcamCat#: ab73881:100
Antibodyanti-VEGFR2 [EPR21884-236] (Rabbit monoclonal)AbcamCat#: ab2336931:500
Antibodyanti-VEGFA (Rabbit polyclonal)AbcamCat#: ab517451:400
Antibodyanti-PDGFRβ [Y92] (Rabbit monoclonal)AbcamCat#: ab325701:1000-1:5000
Sequence-based reagentB6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/J FThe Jackson LaboratoryPCR primersGAA CTG TCA CCG GGA GGA
Sequence-based reagentB6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/J RThe Jackson LaboratoryPCR primersAGG CAA ATT TTG GTG TAC GG
Sequence-based reagentB6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/J internal positive control FThe Jackson LaboratoryPCR primersCAA ATG TTG CTT GTC TGG TG
Sequence-based reagentB6.Cg-Tg(Pdgfrb-cre/ERT2)6096Rha/J internal positive control RThe Jackson LaboratoryPCR primersGTC AGT CGA GTG CAC AGT TT
Sequence-based reagentMouse VEGFA FIDTPCR primersGCAGCGACAAGGCAGACTA
Sequence-based reagentMouse VEGFA RIDTPCR primersGGTCCGATGCAAGATCCCAA
Peptide, recombinant proteinDiphtheria toxinSigmaCat#: D056410 ng/g body weight
Peptide, recombinant proteinDispase IISigmaCat#: D4693
Peptide, recombinant proteinCollagenase IThermoFisherCat#: 17018029
Peptide, recombinant proteinDNase ISigmaCat#: 10104159001
Peptide, recombinant protein100× Penicillin-Streptomycin SolutionInvitrogen/GibcoCat#: 15140–122
Commercial assay or kitInvitrogen Total Exosome Isolation Reagent (from cell culture media)ThermoFisherCat#: 4478359
Commercial assay or kitSuperSignal West Femto Duration SubstrateThermo Fisher ScientificCat#: A38554
Commercial assay or kitClontech SMARTer cDNA kitClontech LaboratoriesCat#: 634925
Commercial assay or kitNEBNext reagentsNew England BiolabsCat#: E6040
Commercial assay or kitRNeasy micro kitQiagenCat#: 74004
Commercial assay or kitSuperScript IV First-Strand Synthesis kitThermoFisherCat#: 18091050
Commercial assay or kitVEGFA ELISA KitAbcamCat#: ab119565
Commercial assay or kitBCA protein assay kitAbcamCat#: ab102536
Commercial assay or kitExoGlow-Protein EV Labeling Kit (Green)SBICat#: EXOGP300A-1
Chemical compound and drugTamoxifenSigmaCat#: T564875 mg/kg body weight
Chemical compound and drugSU5408AbcamCat#: ab145888
Software and algorithmSample Size CalculatorN/Ahttps://clincalc.com/stats/samplesize.aspx
Software and algorithmImageJNIHhttps://imagej.nih.gov/ij/
Software and algorithmPANTHER classification systemN/Ahttp://www.pantherdb.org/
Software and algorithmREVIGORuđer Bošković Institutehttp://revigo.irb.hr/
OtherLectin-DyLight 488Vector LaboratoriesCat#: DL-117420 μg/ml
OtherLectin-DyLight 649Vector LaboratoriesCat#: DL-117820 μg/ml
OtherDecal Stat DecalcifierStatLabCat#: 1212–32Use directly (contains Hydrogen Chloride, Acid mists, strong inorganic)
OtherAntifade Mounting Medium with DAPIVector LaboratoriesCat#: H-1200Use directly (contains 1 μg/ml of DAPI)

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  1. Yunpei Zhang
  2. Lingling Neng
  3. Kushal Sharma
  4. Zhiqiang Hou
  5. Anatasiya Johnson
  6. Junha Song
  7. Alain Dabdoub
  8. Xiaorui Shi
(2023)
Pericytes control vascular stability and auditory spiral ganglion neuron survival
eLife 12:e83486.
https://doi.org/10.7554/eLife.83486