Microarray analysis of bone marrow lesions in osteoarthritis demonstrates upregulation of genes implicated in osteochondral turnover, neurogenesis and inflammation

Anasuya Kuttapitiya, Lena Assi, Ken Laing, Caroline Hing, Philip Mitchell, Guy Whitley, Abiola Harrison, Franklyn A Howe, Vivian Ejindu, Christine Heron, Nidhi Sofat, Anasuya Kuttapitiya, Lena Assi, Ken Laing, Caroline Hing, Philip Mitchell, Guy Whitley, Abiola Harrison, Franklyn A Howe, Vivian Ejindu, Christine Heron, Nidhi Sofat

Abstract

Objective: Bone marrow lesions (BMLs) are well described in osteoarthritis (OA) using MRI and are associated with pain, but little is known about their pathological characteristics and gene expression. We evaluated BMLs using novel tissue analysis tools to gain a deeper understanding of their cellular and molecular expression.

Methods: We recruited 98 participants, 72 with advanced OA requiring total knee replacement (TKR), 12 with mild OA and 14 non-OA controls. Participants were assessed for pain (using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)) and with a knee MRI (using MOAKS). Tissue was then harvested at TKR for BML analysis using histology and tissue microarray.

Results: The mean (SD) WOMAC pain scores were significantly increased in advanced OA 59.4 (21.3) and mild OA 30.9 (20.3) compared with controls 0.5 (1.28) (p<0.0001). MOAKS showed all TKR tissue analysed had BMLs, and within these lesions, bone marrow volume was starkly reduced being replaced by dense fibrous connective tissue, new blood vessels, hyaline cartilage and fibrocartilage. Microarray comparing OA BML and normal bone found a significant difference in expression of 218 genes (p<0.05). The most upregulated genes included stathmin 2, thrombospondin 4, matrix metalloproteinase 13 and Wnt/Notch/catenin/chemokine signalling molecules that are known to constitute neuronal, osteogenic and chondrogenic pathways.

Conclusion: Our study is the first to employ detailed histological analysis and microarray techniques to investigate knee OA BMLs. BMLs demonstrated areas of high metabolic activity expressing pain sensitisation, neuronal, extracellular matrix and proinflammatory signalling genes that may explain their strong association with pain.

Keywords: Chemokines; Chondrocytes; Inflammation; Knee Osteoarthritis; Magnetic Resonance Imaging.

Conflict of interest statement

Competing interests: None declared.

© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Figures

Figure 1
Figure 1
(A) Coronal plane of MRI scan visualising BML and associated cyst. (B) Axial plane of MRI scan presenting BML and associated cyst. (C) Macroscopic view of tibial BML and cystic area. (D) Image of cross section cut through BML and cyst localised by MRI revealing a gelatinous aggregate. (E) H&E staining of cystic region presenting cellular infiltrate in marrow spaces. (F) H&E staining of subchondral cyst forming. (G) H&E staining of BML region with vascular proliferation and cellular infiltration. (H) H&E staining of BML visualising a chondrification centre near the tidemark. (I) H&E staining of adipocyte in bone compartment with a soft tissue infiltrate working through osteoid network. (J) H&E staining of BML showing areas of thickened trabecular adjacent to thinning trabeculae. (K) H&E staining of BML demonstrating areas of fibrotic cartilage formation within the subchondral bone compartment. (L) Quantification of histology analysing 50 BML FOVs and 40 non-BML (NBML) FOVs for blood vessels (BV), cartilage within bone compartment (Cart), cysts (Cys), myxoid/fibrous tissue (M/F), cellular infiltrate (Inf) and trabecular thickening (TT) (n=4). A percentage for the presence of each histological feature was determined for each group. Significance was tested between the groups using Friedman test (*p

Figure 2

(A) Bar chart presenting the…

Figure 2

(A) Bar chart presenting the most significantly upregulated and downregulated entities by fold…

Figure 2
(A) Bar chart presenting the most significantly upregulated and downregulated entities by fold change (FC). One hundred twenty-eight entities were found to be upregulated and 90 were downregulated. The mean WOMAC pain score in the OA microarray group was 61.4, and all subjects in the OA array group had a MOAKS BML score of at least 1, with cartilage and synovitis scores of at least 2. (B) Pearson’s correlation hierarchical clustering of 218 genes clearly segregating the OA BML group from the control group.

Figure 3

(A) Gene ontology analysis of…

Figure 3

(A) Gene ontology analysis of 218 differentially expressed entities found 166 genes associated…

Figure 3
(A) Gene ontology analysis of 218 differentially expressed entities found 166 genes associated with 59 canonical pathways. Pie chart of the 24 predominant pathways identified. The main significant correlation for WOMAC pain with gene correlation was for MMP-13 (p

Figure 4

qPCR validation for stathmin 2…

Figure 4

qPCR validation for stathmin 2 ( STMN2 ), thrombospondin 4 ( THBS4 ),…

Figure 4
qPCR validation for stathmin 2 (STMN2), thrombospondin 4 (THBS4), matrix metalloproteinase 13 (MMP-13) and osteomodulin (OMD) of OA BML compared non-BML tissue and control bone. STMN2, THBS4 and MMP-13 were selected as they were among the most upregulated genes from the microarray. Osteomodulin was selected as a bone-specific marker as it is involved in bone homeostasis (**p<0.005, ***p<0.0005). BML, bone marrow lesion; NBML, non-bone marrow lesion; OA, osteoarthritis.
Similar articles
Cited by
References
    1. Lawrence RC, Felson DT, Helmick CG, et al. . Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum 2008;58:26–35. 10.1002/art.23176 - DOI - PMC - PubMed
    1. Nicholls E, Thomas E, van der Windt DA, et al. . Pain trajectory groups in persons with, or at high risk of, knee osteoarthritis: findings from the knee clinical Assessment Study and the Osteoarthritis Initiative. Osteoarthritis Cartilage 2014;22:2041–50. 10.1016/j.joca.2014.09.026 - DOI - PMC - PubMed
    1. Sofat N, Ejindu V, Kiely P. What makes OA painful? The evidence for peripheral and central pain processing Rheumatology. Rheumatology 2011;50:2157–65. - PubMed
    1. Roemer FW, Kassim Javaid M, Guermazi A, et al. . Anatomical distribution of synovitis in knee osteoarthritis and its association with joint effusion assessed on non-enhanced and contrast-enhanced MRI. Osteoarthritis Cartilage 2010;18:1269–74. 10.1016/j.joca.2010.07.008 - DOI - PubMed
    1. Roy S, Meachim G. Chondrocyte ultrastructure in adult human articular cartilage. Ann Rheum Dis 1968;27:544–58. 10.1136/ard.27.6.544 - DOI - PMC - PubMed
Show all 45 references
MeSH terms
Full text links [x]
[x]
Cite
Copy Download .nbib
Format: AMA APA MLA NLM
Figure 2
Figure 2
(A) Bar chart presenting the most significantly upregulated and downregulated entities by fold change (FC). One hundred twenty-eight entities were found to be upregulated and 90 were downregulated. The mean WOMAC pain score in the OA microarray group was 61.4, and all subjects in the OA array group had a MOAKS BML score of at least 1, with cartilage and synovitis scores of at least 2. (B) Pearson’s correlation hierarchical clustering of 218 genes clearly segregating the OA BML group from the control group.
Figure 3
Figure 3
(A) Gene ontology analysis of 218 differentially expressed entities found 166 genes associated with 59 canonical pathways. Pie chart of the 24 predominant pathways identified. The main significant correlation for WOMAC pain with gene correlation was for MMP-13 (p

Figure 4

qPCR validation for stathmin 2…

Figure 4

qPCR validation for stathmin 2 ( STMN2 ), thrombospondin 4 ( THBS4 ),…

Figure 4
qPCR validation for stathmin 2 (STMN2), thrombospondin 4 (THBS4), matrix metalloproteinase 13 (MMP-13) and osteomodulin (OMD) of OA BML compared non-BML tissue and control bone. STMN2, THBS4 and MMP-13 were selected as they were among the most upregulated genes from the microarray. Osteomodulin was selected as a bone-specific marker as it is involved in bone homeostasis (**p<0.005, ***p<0.0005). BML, bone marrow lesion; NBML, non-bone marrow lesion; OA, osteoarthritis.
Figure 4
Figure 4
qPCR validation for stathmin 2 (STMN2), thrombospondin 4 (THBS4), matrix metalloproteinase 13 (MMP-13) and osteomodulin (OMD) of OA BML compared non-BML tissue and control bone. STMN2, THBS4 and MMP-13 were selected as they were among the most upregulated genes from the microarray. Osteomodulin was selected as a bone-specific marker as it is involved in bone homeostasis (**p<0.005, ***p<0.0005). BML, bone marrow lesion; NBML, non-bone marrow lesion; OA, osteoarthritis.

References

    1. Lawrence RC, Felson DT, Helmick CG, et al. . Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum 2008;58:26–35. 10.1002/art.23176
    1. Nicholls E, Thomas E, van der Windt DA, et al. . Pain trajectory groups in persons with, or at high risk of, knee osteoarthritis: findings from the knee clinical Assessment Study and the Osteoarthritis Initiative. Osteoarthritis Cartilage 2014;22:2041–50. 10.1016/j.joca.2014.09.026
    1. Sofat N, Ejindu V, Kiely P. What makes OA painful? The evidence for peripheral and central pain processing Rheumatology. Rheumatology 2011;50:2157–65.
    1. Roemer FW, Kassim Javaid M, Guermazi A, et al. . Anatomical distribution of synovitis in knee osteoarthritis and its association with joint effusion assessed on non-enhanced and contrast-enhanced MRI. Osteoarthritis Cartilage 2010;18:1269–74. 10.1016/j.joca.2010.07.008
    1. Roy S, Meachim G. Chondrocyte ultrastructure in adult human articular cartilage. Ann Rheum Dis 1968;27:544–58. 10.1136/ard.27.6.544
    1. Felson DT, Chaisson CE, Hill CL, et al. . The association of bone marrow lesions with pain in knee osteoarthritis. Ann Intern Med 2001;134:541–9. 10.7326/0003-4819-134-7-200104030-00007
    1. Sowers MF, Hayes C, Jamadar D, et al. . Magnetic resonance-detected subchondral bone marrow and cartilage defect characteristics associated with pain and X-ray-defined knee osteoarthritis. Osteoarthritis Cartilage 2003;11:387–93. 10.1016/S1063-4584(03)00080-3
    1. Altman R, Asch E, Bloch D, et al. . The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the knee. Arthritis Rheum 1986;29:1039–49.
    1. NICE guidelines ‘Osteoarthritis: Care and Management’.
    1. Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 1957;16:494–502. 10.1136/ard.16.4.494
    1. Bellamy N, Hochberg M, Tubach F, et al. . Development of multinational definitions of minimal clinically important improvement and patient acceptable symptomatic state in osteoarthritis. Arthritis Care Res 2015;67:972–80. 10.1002/acr.22538
    1. Dworkin RH, Turk DC, Farrar JT, et al. . Core outcome measures for chronic pain clinical trials: immpact recommendations. Pain 2005;113:9–19. 10.1016/j.pain.2004.09.012
    1. Bjelland I, Dahl AA, Haug TT, et al. . The validity of the Hospital anxiety and depression Scale. an updated literature review. J Psychosom Res 2002;52:69–77.
    1. RNeasy mini Hand book isolation kit. Fourth Edition Qiagen, 2012.
    1. Microarray-Based Gene Expression Analysis. Version 6.9.1 2015.
    1. Mi H, Poudel S, Muruganujan A, et al. . PANTHER version 10: expanded protein families and functions, and analysis tools. Nucleic Acids Res 2016;44(D1):D336–D342. 10.1093/nar/gkv1194
    1. Garnero P, Piperno M, Gineyts E, et al. . Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage. Ann Rheum Dis 2001;60:619–26. 10.1136/ard.60.6.619
    1. Hunter DJ, Guermazi A, Lo GH, Gh L, et al. . Evolution of semi-quantitative whole joint assessment of knee OA: moaks (MRI Osteoarthritis Knee score). Osteoarthritis Cartilage 2011;19:990–1002. 10.1016/j.joca.2011.05.004
    1. Wilson AJ, Murphy WA, Hardy DC, et al. . Transient osteoporosis: transient bone marrow edema? Radiology 1988;167:757–60. 10.1148/radiology.167.3.3363136
    1. Zanetti M, Bruder E, Romero J, et al. . Bone marrow edema pattern in osteoarthritic knees: correlation between MR imaging and histologic findings. Radiology 2000;215:835–40. 10.1148/radiology.215.3.r00jn05835
    1. Hunter DJ, Gerstenfeld L, Bishop G, et al. . Bone marrow lesions from osteoarthritis knees are characterized by sclerotic bone that is less well mineralized. Arthritis Res Ther 2009;11:R11 10.1186/ar2601
    1. Taljanovic MS, Graham AR, Benjamin JB, et al. . Bone marrow edema pattern in advanced hip osteoarthritis: quantitative assessment with magnetic resonance imaging and correlation with clinical examination, radiographic findings, and histopathology. Skeletal Radiol 2008;37:423–31. 10.1007/s00256-008-0446-3
    1. Leydet-Quilici H, Le Corroller T, Bouvier C, et al. . Advanced hip osteoarthritis: magnetic resonance imaging aspects and histopathology correlations. Osteoarthritis Cartilage 2010;18:1429–35. 10.1016/j.joca.2010.08.008
    1. Roemer FW, Guermazi A, Javaid MK, et al. . Change in MRI-detected subchondral bone marrow lesions is associated with cartilage loss: the MOST study. A longitudinal multicentre study of knee osteoarthritis. Ann Rheum Dis 2009;68:1461–5. 10.1136/ard.2008.096834
    1. Carrino JA, Blum J, Parellada JA, et al. . MRI of bone marrow edema-like signal in the pathogenesis of subchondral cysts. Osteoarthritis Cartilage 2006;14:1081–5. 10.1016/j.joca.2006.05.011
    1. Zhang D, Johnson LJ, Hsu HP, et al. . Cartilaginous deposits in subchondral bone in regions of exposed bone in osteoarthritis of the human knee: histomorphometric study of PRG4 distribution in osteoarthritic cartilage. J Orthop Res 2007;25:873–83. 10.1002/jor.20344
    1. Campbell TM, Churchman SM, Gomez A, et al. . Mesenchymal stem cell alterations in bone marrow lesions in patients with hip osteoarthritis. Arthritis Rheumatol 2016;68:1648–59. 10.1002/art.39622
    1. Jin K, Mao XO, Cottrell B, et al. . Proteomic and immunochemical characterization of a role for stathmin in adult neurogenesis. Faseb J 2004;18:287–99. 10.1096/fj.03-0973com
    1. Liu H, Zhang R, Ko SY, Sy K, et al. . Microtubule assembly affects bone mass by regulating both osteoblast and osteoclast functions: stathmin deficiency produces an osteopenic phenotype in mice. J Bone Miner Res 2011;26:2052–67. 10.1002/jbmr.419
    1. Kim DS, Li KW, Boroujerdi A, et al. . Thrombospondin-4 contributes to spinal sensitization and neuropathic pain states. J Neurosci 2012;32:8977–87. 10.1523/JNEUROSCI.6494-11.2012
    1. Pan B, Guo Y, Wu HE, He W, et al. . Thrombospondin-4 divergently regulates voltage-gated Ca2+ channel subtypes in sensory neurons after nerve injury. Pain 2016;157:2068–80. 10.1097/j.pain.0000000000000612
    1. Foulkes T, Wood JN. Pain genes. PLoS Genet 2008;4:e1000086 10.1371/journal.pgen.1000086
    1. Swaminathan A, Delage H, Chatterjee S, et al. . Transcriptional coactivator and chromatin protein PC4 is involved in hippocampal neurogenesis and spatial memory extinction. J Biol Chem 2016;291:20303–14. 10.1074/jbc.M116.744169
    1. Sofat N. Analysing the role of endogenous matrix molecules in the development of osteoarthritis. Int J Exp Pathol 2009;90:463–79. 10.1111/j.1365-2613.2009.00676.x
    1. Hopwood B, Tsykin A, Findlay DM, et al. . Microarray gene expression profiling of osteoarthritic bone suggests altered bone remodelling, WNT and transforming growth factor-beta/bone morphogenic protein signalling. Arthritis Res Ther 2007;9:R100 10.1186/ar2301
    1. Chou CH, Wu CC, Song IW, et al. . Genome-wide expression profiles of subchondral bone in osteoarthritis. Arthritis Res Ther 2013;15:R190 10.1186/ar4380
    1. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 2014;507:323–8. 10.1038/nature13145
    1. Ramasamy SK, Kusumbe AP, Wang L, et al. . Endothelial notch activity promotes angiogenesis and osteogenesis in bone. Nature 2014;507:376–80. 10.1038/nature13146
    1. Ninomiya K, Miyamoto T, Imai J, et al. . Osteoclastic activity induces osteomodulin expression in osteoblasts. Biochem Biophys Res Commun 2007;362:460–6. 10.1016/j.bbrc.2007.07.193
    1. Zhang YK, Huang ZJ, Liu S, et al. . WNT signaling underlies the pathogenesis of neuropathic pain in rodents. J Clin Invest 2013;123:2268–86. 10.1172/JCI65364
    1. Miller RE, Tran PB, Das R, et al. . CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis. Proc Natl Acad Sci U S A 2012;109:20602–7. 10.1073/pnas.1209294110
    1. Walsh DA, McWilliams DF, Turley MJ, et al. . Angiogenesis and nerve growth factor at the osteochondral junction in rheumatoid arthritis and osteoarthritis. Rheumatology 2010;49:1852–61. 10.1093/rheumatology/keq188
    1. Little CB, Barai A, Burkhardt D, et al. . Matrix metalloproteinase 13-deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development. Arthritis Rheum 2009;60:3723–33. 10.1002/art.25002
    1. Kofuji T, Fujiwara T, Sanada M, et al. . HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival. J Neurochem 2014;130:514–25. 10.1111/jnc.12722
    1. Vaudry D, Gonzalez BJ, Basille M, et al. . Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development. Proc Natl Acad Sci U S A 1999;96:9415–20. 10.1073/pnas.96.16.9415

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe