Senotherapeutic drugs for human intervertebral disc degeneration and low back pain

  1. Hosni Cherif
  2. Daniel G Bisson  Is a corresponding author
  3. Matthew Mannarino  Is a corresponding author
  4. Oded Rabau  Is a corresponding author
  5. Jean A Ouellet
  6. Lisbet Haglund  Is a corresponding author
  1. Orthopaedic Research Lab, Department of Surgery, McGill University and the Research Institute of the McGill University Health Centre, Canada
  2. McGill Scoliosis and Spine Group, Department of Surgery, McGill University and the Research Institute of the McGill University Health Centre, Canada
  3. Shriner’s Hospital for Children, 1003 Decarie Blvd, Canada
6 figures, 4 tables and 5 additional files

Figures

Figure 1 with 1 supplement
RG-7112 treatment of IVD pellet cultures.

(A) Representative images indicating how we deemed positive and negative p16INK4a (a), Ki-67 (d), and caspase-3 (g) staining. (b, e and h) Magnified images of (a, d and g). Arrow heads indicate …

Figure 1—source data 1

Excel file containing the percentage of p16Ink4a, ki-67 and caspase three positive in NP and AF cells control and treated with RG-7112.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Senolytic activity of RG-7112 on human IVD cells in vitro.

(A) Metabolic activity in control and treated NP and AF pellet cultures from degenerate IVDs (n = 6). (B) Representative photomicrographs of untreated (a–c) and RG-7112 treated (d–f) NP cells …

Figure 1—figure supplement 1—source data 1

Excel file containing the fluorescence values for metabolic and caspase3/7 activity, the number of apoptotic cells, and the concentrations of sGAG in NP (and AF) cells treated and untreated with RG-7112.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Differentially expressed senescence related genes in NP pellets.

(A) Venn diagrams of the differentially up and downregulated genes among the different groups. O-Vanillin, RG-7112-treated NP cells in pellets culture. For Upregulated genes odds ratio (OR) = 2.13 …

Figure 2—source data 1

Excel file containing the list of Differentially Expressed genes significantly up- or downregulated at p < 0.05 in RG-7112 and o-Vanillin conditions and the raw values of the fold change in control and treated groups for all tested genes.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Non differentially expressed genes Heatmap and NP area measure in human IVD.

(A) 47 genes did not show significant expression change in RG-7112 (5 µM) and o-Vanillin (100 µM) treated NP cells when compared with control (CTRL). See methods for statistical calculation and …

Figure 2—figure supplement 1—source data 1

Excel file containing the the list of Non-Differentially Expressed genes in RG-7112 and o-Vanillin conditions and the raw values of the fold change of the 91 genes tested in control and treated groups.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
Profile of SASP factors released from pellet cultures following senolytic treatment.

(A) Culture media was analyzed by a RayBio Human Cytokine Array. Relative mean densitometry units of the 80 factors were normalized to untreated controls with the most 25 downregulated SASP factors …

Figure 3—source data 1

Excel file containing the meausres for the selected and significantly SASPs factors measured using cytokine array and Luminex analysis in NP pellets media following the treatment with RG-7112 and o-Vanilin.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Total cytokine array and Luminex measures in pellet culture media.

(A) Schematic representation of the media analysis by cytokine arrays and Luminex assay. Cytokine/chemokine array quantification of the media in NP cell pellets treated with (B) o-Vanillin (100 μM) …

Figure 3—figure supplement 1—source data 1

Excel file containing the complete list of SASP factors measured by cytokines arrays and the non-significantly regulated factors measured by Luminex assay in treated NP pellets media.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig3-figsupp1-data1-v1.xlsx
RG-7112 and o-Vanillin effects in ex vivo human IVD culture.

(A) Schematic of the ex vivo organ culture experiment. Lumbar spines from organ donors were assessed radiographically for signs of degeneration. Three discs per experiment were isolated from the …

Figure 4—source data 1

Excel file containing the complete list of donors, slices and their respective voxel intensities, p16Ink4a and ki-67-positive cells in control and treated discs.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
Profile of SASP factors released from ex vivo human IVDs cultures following senolytic treatment.

Culture media was analyzed by a RayBio Human Cytokine Array. Relative mean densitometry units of the 80 factors were normalized to pre-treatment media of the same IVD. The percentage change …

Figure 5—source data 1

Excel file containing the meausres of the significantly changed SASP factors measured using cytokine array and Luminex analysis in vehicle, RG-7112 and o-Vanilin disc media before and after the injection.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Complete profiling and measure of growth factors and cytokines in disc cultures media.

Total cytokine/chemokine array quantification of inflammatory factors release in (A) Control, (B) o-Vanillin and (C) RG-7112 discs. Shown in the graphs is the ratio (% change) of the factors …

Figure 5—figure supplement 1—source data 1

Excel file containing the complete list of SASP factors measured by cytokine arrays and the non-significantly regulated factors measured by Luminex assay in the pre and post media of injected discs.

https://cdn.elifesciences.org/articles/54693/elife-54693-fig5-figsupp1-data1-v1.xlsx
Author response image 1

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
AntibodyRabbit polyclonal anti-caspase-3Sigma-AldrichC8487
RRID:AB_476884
IF (1:500)
IHC-Fr (1:500)
AntibodyRabbit polyclonal anti-Ki-67Novus BiologicalsNB500-170
RRID:AB_10001977
IF (1:300)
IHC-Fr (1:500) IHC-P (1:2000)
AntibodyMouse monoclonal Anti- p16Ink4aAbcamab54210
RRID:AB_2059963
IHC-P (1:500)
AntibodyPolyclonal Alexa Fluor 488 (Donkey anti-Rabbit)Invitrogen Thermo FisherA-21202 RRID:AB_141607IF (1:1000)
AntibodyPolyclonal Alexa Fluor 555 (Donkey anti-Mouse)Invitrogen Thermo FisherA-31570 RRID:AB_2536180IF (1:1000)
Antibody ArrayHuman Cytokine Antibody Array C5RayBiotech, IncAAH-CYT-5–2 RRID:AB_10185250
Chemical compound, drugo-VanillinSigma-Aldrich120804
Chemical compound, drugRG-7112Selleck ChemicalsS7030
Chemical compound, drugDMSOSigma-AldrichD8418
Commercial KitApoptosis detectionAbcamab176749 RRID:SCR_018379
Commercial KitCaspase 3/7 activityAAT Bioquest 13503Amplite Fluorimetric Catalog # 13503
Commercial KitqScript cDNA Synthesis KitQuanta Biosciences95047–500
Commercial KitGoat anti-Mouse and Rabbit specific HRP/DAB Detection IHC KitAbcamab64264
Commercial KitMouse monoclonal Anti- p16Ink4aRoche, Ventana LaboratoriesCINtec Plus (9531) RRID:SCR_018380Prediluted
Commercial KitMycoplasma PCR Detection KitZmTech ScientificM209001
Commercial AssayCustom Taqman (R) Gene Expression 96-Well Fast PlatesThermo Fisher4413255See Supplementary file 1 for each gene product ID
Commercial AssayLuminex immunoassay 19-PLEX plate - Human Cytokine PROCARTAPLEXLife TechnologiesMXMFXHX
SoftwareImageJImageJ http://imagej.nih.gov/ij/RRID:SCR_003070
SoftwareGraphPad Prism 8GraphPad Prism https://www.graphpad.comRRID:SCR_015807
SoftwareIngenuity Pathway Analysis (IPA)Ingenuity SystemsRRID:SCR_008653
SoftwareR package ggplot2Wickham, 2016ISBN 978-3-319-24277-4
SoftwareMIPAVNIH Center for Information TechnologyRRID:SCR_007371
SoftwareImage Quant TL array analysisGE HealthcareRRID:SCR_018374
SoftwareFlowCytomix Pro2.2.1eBioscienceRRID:SCR_018375
SoftwareAxioVision LE64ZeissRRID:SCR_002677
OtherDMMBSigma-Aldrich341088
OtherAlamar Blue reagentThermo FisherDAL1025
OtherDAPI stainInvitrogenD1306(1 µg/mL)
OtherSafranin-OSigma-AldrichS2255
OtherTaqMan Fast Universal PCR Master Mix (2×)Thermo Fisher4366073
Cell linePrimary Human Nucleus Pulposus (NP) and Annulus Fibrosis (AF)Primary CellsHuman Biological samples. See Table 1 for demographics.Cell line maintained in Haglund Lab.
Table 1
Donors demographics.
Donor IDAgeSexCause of death
153FAnoxia
253Mbone; cerebral aneurysm rupture
376FAnoxia
452MIschemic cerebral vascular accident
517MBrain death
673FCerebrovascular hemorrhage
768FCerebral hemorrhage
839MGunshot to the neck
966FUnknown
1067MCardiac arrest
1169MCerebral hemorrhage
1261MCardiac arrest
1340FBrain hemorrhage
1453FMedical suicide
1528MSuicide (Hang)
1635MAnoxia
Table 2
Complete donors list per assays and results.
Donor
ID
DiscsThompson grade
and Degeneration
Cell typeType of cultureType of assaysResults
6L1-L53 - (D)NP
&
AF
PelletIHC
Metabolic activity
Figure 1 (A–D)
Donor # 6, 7, 10–15
Figure 1—figure supplement 1(A)
Donor # 10–15
Figure 1—figure supplement 1(G)
Donor # 10–15
7L2-L54 - (D)
8L3-S13 - (D)
10L4-L54 - (D)
11L5-S13 - (D)
12L5-S13 - (D)
13L4-S13 - (D)
14L5-S1
L1-L2
3 - (D)
15L5-S15 - (D)
11L5-S13 - (D)NPPelletRT-qPCR
Cytokine array
Luminex
Figure 2 (A–E)
Figure 3 (A–E)
Figure 2—figure supplement 1(A)
Figure 3—figure supplement 1(A-C)
All Donor # 11–15
12L5-S13 - (D)
13L4-S13 - (D)
14L5-S1
L1-L2
3 - (D)
15L5-S15 - (D)
9T11-L2Table 3Intact discEx vivoMRI
Luminex
IHC
Figure 4 (A–F)
Figure 5 (A–D)
Figure 5—figure supplement 1(A-D)
Donor # 9, 11, 13 and 14
11L1-L4Table 3
13L1-L4Table 3
14L2-L5Table 3
1L1-S14 - (D)NPMonolayerICC
Metabolic activity
Caspase 3/7 activity
Figure 1—figure supplement 1(B, C and D)
Donor # 1–3
Figure 1—figure supplement 1(E and F)
Donor # 1–3 and 11 (D)
Donor # 4, 5, 10 and 11 (ND)
2L4-S13 - (D)
3L2-S14 - (D)
4L1-L51 - (ND)
5T11-S11 - (ND)
10T12-L11 - (ND)
11T12-L1
L5-S1
1 - (ND)
3 - (D)
15T11-L5
L5-S1
1 - (ND)
5 - (D)
Intact discNP Area and position measureFigure 2—figure supplement 1(B-C)
Donor # 15 and 16
16L1-S11 - (ND)
Table 3
Characteristics of discs injected with each treatment.
Donor ID9111314
Age66694053
SexFMFF
CTRLDisc LevelT12/L1L1/2L1/2L2/3
 Grade2222
 Disc Height (cm)0.840.920.920.88
O-VanillinDisc LevelL1/2L2/3L3/4L4/5
 Grade2222
 Disc Height (cm)0.920.890.990.88
RG-7112Disc LevelT11/T12L3/4L2/3L3/4
 Grade2222
 Disc Height (cm)0.820.90.930.99

Additional files

Supplementary file 1

Gene names (aliases) and primers.

https://cdn.elifesciences.org/articles/54693/elife-54693-supp1-v1.xlsx
Supplementary file 2

List of cytokines and synonyms.

https://cdn.elifesciences.org/articles/54693/elife-54693-supp2-v1.docx
Supplementary file 3

List of differentially and non-differentially expressed genes following treatment with o-Vanillin and RG-7112.

(a) Eight Differentially Expressed genes significantly up- or downregulated at p < 0.05 in RG-7112 condition. (b) Forty Differentially Expressed genes significantly up- or downregulated at p < 0.05 in o-Vanillin condition. (c) Non-Differentially Expressed genes in RG-7112. (d) Non-Differentially Expressed genes in o-Vanillin.

https://cdn.elifesciences.org/articles/54693/elife-54693-supp3-v1.docx
Supplementary file 4

O-Vanillin and RG-7112 IPA network scores, molecules and top diseases.

(a) O-Vanillin network scores, molecules and top diseases. Physical function analysis using IPA of o-Vanillin treated NP pellets generated three networks which are ordered by a score denoting significance. The highest-scoring network, which comprises 37 proteins in our list, revealed significant changes in cell death and survival, neurological disease, organismal Injury and abnormalities. In addition, the other networks revealed changes in cancer, cellular movement, connective tissue development and function, cell cycle, organismal injury and abnormalities. (b) RG-7112 network scores, molecules and top diseases. Physical function analysis using IPA of RG-7112 treated NP pellets generated a single network which is ordered by a score denoting significance of 21. This network reveals significant changes in cell cycle, cell death and survival, connective tissue development and function.

https://cdn.elifesciences.org/articles/54693/elife-54693-supp4-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/54693/elife-54693-transrepform-v1.pdf

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