The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes

Sabha Asghar, Gary Litherland, Dominic Meek, Carl Goodyear, John Lockhart, Anne Crilly

Research output: Contribution to conferencePoster

Abstract

Purpose. Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but has aspects of low grade inflammation and disruption of synovial homeostasis. Recent insights suggest that 70% of OA patients present with synovial inflammation (synovitis), which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis, such as occurs in OA, results in an altered secretory profile from the synovial tissue, with microvesicles and exosomes contributing to this secretome. Importantly, exosomes have been implicated in cell communication through their ability to carry and transfer a range of potentially modulatory/regulatory cargo, including proteins, lipids and various types of RNA. Protease activated receptor 2 (PAR2) is expressed on OA synovial tissue and cartilage and is known to drive inflammation. Additionally, it has been shown to be a critical receptor in OA pathophysiology, as its knockdown in murine models results in protection from cartilage erosion. The aim of this study was to characterise the PAR2-driven secrtome of OA synovial tissue, including the exosome content. The impact of isolated OA synovial exosomes on catabolic gene expression in primary OA chondrocytes was subsequently evaluated. Methods. Human OA synovial explants (n=35 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 agonist peptide (SLIGKV-NH2) or a reverse peptide (RP) control. Conditioned medium (CM) was harvested after 48h and used for exosome preparation. Dead cells and debris were eliminated by successive centrifugations at increasing speeds, with exosomes isolated by ultracentrifuge at 100,000g. Western blot analysis was used to confirm the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome presence and size between 30-150 nm. Nanoparticle Tracking Analysis (NTA) was also undertaken on exosome preparations to confirm concentration and size. Isolated exosomes were labelled with Exo-Red (stains RNA) or Exo-Green (stains protein) and uptake by primary human OA articular chondrocytes (n=?) evaluated using immunofluorescence imaging. Gene expression changes post exosome uptake by chondrocytes was analysed with real time qPCR. Cytokine and MMP levels in CM were evaluated using ELISA. Results. Exosome preparation were evaluated to determine by the presence of exosome-associated markers CD9, CD81, HSP70 and CD63. NTA and SEM data confirmed 80-90% of microvesicles ranging in exosome size from 30 to 150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter protein concentration. Exosomes carried both protein and RNA cargo and were taken up by primary chondrocytes after 4h exposure. Primary chondrocytes that had taken up exosomes and been stimulated with IL-1β showed significant decreases in COL2A1 (p<0.001) and ACAN expression (p<0.001), and a significant increase in GLUT-1 expression (p<0.1). Stimulation with SLIGKV-NH2 showed similar patterns, with a significant decrease in COL2A1 (p<0.1), ACAN (p<0.1) and SOX9 (p<00.1) expression, as well as a significant increase in MMP-13 expression (p<0.1). Levels of IL-6, TNF and MMP-3 in CM were significantly increased by both IL-1β and SLIGKV-NH2 (p<0.001) stimulated samples compared to controls. Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2. Conclusion. OA synovial tissue has the potential to impact chondrocyte behaviour through the release of a secretome, which includes potentially regulatory exosomes. Future studies into characterisation of cargo, will provide insight into the role of the synovium in OA cartilage pathology.
Original languageEnglish
Publication statusAccepted/In press - 8 Jan 2018
Event2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis - Liverpool, United Kingdom
Duration: 26 Apr 201829 Apr 2018
https://2018.oarsi.org/

Conference

Conference2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis
Abbreviated title2018 OARSI
CountryUnited Kingdom
CityLiverpool
Period26/04/1829/04/18
Internet address

Fingerprint

Exosomes
Osteoarthritis
Cartilage
Pathology
Chondrocytes
PAR-2 Receptor
Interleukin-1
Conditioned Culture Medium
Matrix Metalloproteinases
RNA
Inflammation
Electron Scanning Microscopy
Nanoparticles
Proteins
Homeostasis
Coloring Agents
Joints
Cartilage Diseases
Gene Expression
Peptides

Keywords

  • Exosome
  • Cartilage, Articular
  • Synovitis
  • Osteoarthritis
  • Inflammation

Cite this

Asghar, S., Litherland, G., Meek, D., Goodyear, C., Lockhart, J., & Crilly, A. (Accepted/In press). The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes. Poster session presented at 2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis, Liverpool, United Kingdom.
Asghar, Sabha ; Litherland, Gary ; Meek, Dominic ; Goodyear, Carl ; Lockhart, John ; Crilly, Anne. / The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes. Poster session presented at 2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis, Liverpool, United Kingdom.
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title = "The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes",
abstract = "Purpose. Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but has aspects of low grade inflammation and disruption of synovial homeostasis. Recent insights suggest that 70{\%} of OA patients present with synovial inflammation (synovitis), which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis, such as occurs in OA, results in an altered secretory profile from the synovial tissue, with microvesicles and exosomes contributing to this secretome. Importantly, exosomes have been implicated in cell communication through their ability to carry and transfer a range of potentially modulatory/regulatory cargo, including proteins, lipids and various types of RNA. Protease activated receptor 2 (PAR2) is expressed on OA synovial tissue and cartilage and is known to drive inflammation. Additionally, it has been shown to be a critical receptor in OA pathophysiology, as its knockdown in murine models results in protection from cartilage erosion. The aim of this study was to characterise the PAR2-driven secrtome of OA synovial tissue, including the exosome content. The impact of isolated OA synovial exosomes on catabolic gene expression in primary OA chondrocytes was subsequently evaluated. Methods. Human OA synovial explants (n=35 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 agonist peptide (SLIGKV-NH2) or a reverse peptide (RP) control. Conditioned medium (CM) was harvested after 48h and used for exosome preparation. Dead cells and debris were eliminated by successive centrifugations at increasing speeds, with exosomes isolated by ultracentrifuge at 100,000g. Western blot analysis was used to confirm the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome presence and size between 30-150 nm. Nanoparticle Tracking Analysis (NTA) was also undertaken on exosome preparations to confirm concentration and size. Isolated exosomes were labelled with Exo-Red (stains RNA) or Exo-Green (stains protein) and uptake by primary human OA articular chondrocytes (n=?) evaluated using immunofluorescence imaging. Gene expression changes post exosome uptake by chondrocytes was analysed with real time qPCR. Cytokine and MMP levels in CM were evaluated using ELISA. Results. Exosome preparation were evaluated to determine by the presence of exosome-associated markers CD9, CD81, HSP70 and CD63. NTA and SEM data confirmed 80-90{\%} of microvesicles ranging in exosome size from 30 to 150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter protein concentration. Exosomes carried both protein and RNA cargo and were taken up by primary chondrocytes after 4h exposure. Primary chondrocytes that had taken up exosomes and been stimulated with IL-1β showed significant decreases in COL2A1 (p<0.001) and ACAN expression (p<0.001), and a significant increase in GLUT-1 expression (p<0.1). Stimulation with SLIGKV-NH2 showed similar patterns, with a significant decrease in COL2A1 (p<0.1), ACAN (p<0.1) and SOX9 (p<00.1) expression, as well as a significant increase in MMP-13 expression (p<0.1). Levels of IL-6, TNF and MMP-3 in CM were significantly increased by both IL-1β and SLIGKV-NH2 (p<0.001) stimulated samples compared to controls. Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2. Conclusion. OA synovial tissue has the potential to impact chondrocyte behaviour through the release of a secretome, which includes potentially regulatory exosomes. Future studies into characterisation of cargo, will provide insight into the role of the synovium in OA cartilage pathology.",
keywords = "Exosome, Cartilage, Articular, Synovitis, Osteoarthritis, Inflammation",
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Asghar, S, Litherland, G, Meek, D, Goodyear, C, Lockhart, J & Crilly, A 2018, 'The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes' 2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis, Liverpool, United Kingdom, 26/04/18 - 29/04/18, .

The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes. / Asghar, Sabha; Litherland, Gary; Meek, Dominic; Goodyear, Carl; Lockhart, John; Crilly, Anne.

2018. Poster session presented at 2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis, Liverpool, United Kingdom.

Research output: Contribution to conferencePoster

TY - CONF

T1 - The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes

AU - Asghar, Sabha

AU - Litherland, Gary

AU - Meek, Dominic

AU - Goodyear, Carl

AU - Lockhart, John

AU - Crilly, Anne

PY - 2018/1/8

Y1 - 2018/1/8

N2 - Purpose. Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but has aspects of low grade inflammation and disruption of synovial homeostasis. Recent insights suggest that 70% of OA patients present with synovial inflammation (synovitis), which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis, such as occurs in OA, results in an altered secretory profile from the synovial tissue, with microvesicles and exosomes contributing to this secretome. Importantly, exosomes have been implicated in cell communication through their ability to carry and transfer a range of potentially modulatory/regulatory cargo, including proteins, lipids and various types of RNA. Protease activated receptor 2 (PAR2) is expressed on OA synovial tissue and cartilage and is known to drive inflammation. Additionally, it has been shown to be a critical receptor in OA pathophysiology, as its knockdown in murine models results in protection from cartilage erosion. The aim of this study was to characterise the PAR2-driven secrtome of OA synovial tissue, including the exosome content. The impact of isolated OA synovial exosomes on catabolic gene expression in primary OA chondrocytes was subsequently evaluated. Methods. Human OA synovial explants (n=35 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 agonist peptide (SLIGKV-NH2) or a reverse peptide (RP) control. Conditioned medium (CM) was harvested after 48h and used for exosome preparation. Dead cells and debris were eliminated by successive centrifugations at increasing speeds, with exosomes isolated by ultracentrifuge at 100,000g. Western blot analysis was used to confirm the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome presence and size between 30-150 nm. Nanoparticle Tracking Analysis (NTA) was also undertaken on exosome preparations to confirm concentration and size. Isolated exosomes were labelled with Exo-Red (stains RNA) or Exo-Green (stains protein) and uptake by primary human OA articular chondrocytes (n=?) evaluated using immunofluorescence imaging. Gene expression changes post exosome uptake by chondrocytes was analysed with real time qPCR. Cytokine and MMP levels in CM were evaluated using ELISA. Results. Exosome preparation were evaluated to determine by the presence of exosome-associated markers CD9, CD81, HSP70 and CD63. NTA and SEM data confirmed 80-90% of microvesicles ranging in exosome size from 30 to 150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter protein concentration. Exosomes carried both protein and RNA cargo and were taken up by primary chondrocytes after 4h exposure. Primary chondrocytes that had taken up exosomes and been stimulated with IL-1β showed significant decreases in COL2A1 (p<0.001) and ACAN expression (p<0.001), and a significant increase in GLUT-1 expression (p<0.1). Stimulation with SLIGKV-NH2 showed similar patterns, with a significant decrease in COL2A1 (p<0.1), ACAN (p<0.1) and SOX9 (p<00.1) expression, as well as a significant increase in MMP-13 expression (p<0.1). Levels of IL-6, TNF and MMP-3 in CM were significantly increased by both IL-1β and SLIGKV-NH2 (p<0.001) stimulated samples compared to controls. Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2. Conclusion. OA synovial tissue has the potential to impact chondrocyte behaviour through the release of a secretome, which includes potentially regulatory exosomes. Future studies into characterisation of cargo, will provide insight into the role of the synovium in OA cartilage pathology.

AB - Purpose. Osteoarthritis (OA) is no longer considered to be just a disease of the cartilage but has aspects of low grade inflammation and disruption of synovial homeostasis. Recent insights suggest that 70% of OA patients present with synovial inflammation (synovitis), which significantly correlates with pain and cartilage damage. Disruption of joint tissue homeostasis, such as occurs in OA, results in an altered secretory profile from the synovial tissue, with microvesicles and exosomes contributing to this secretome. Importantly, exosomes have been implicated in cell communication through their ability to carry and transfer a range of potentially modulatory/regulatory cargo, including proteins, lipids and various types of RNA. Protease activated receptor 2 (PAR2) is expressed on OA synovial tissue and cartilage and is known to drive inflammation. Additionally, it has been shown to be a critical receptor in OA pathophysiology, as its knockdown in murine models results in protection from cartilage erosion. The aim of this study was to characterise the PAR2-driven secrtome of OA synovial tissue, including the exosome content. The impact of isolated OA synovial exosomes on catabolic gene expression in primary OA chondrocytes was subsequently evaluated. Methods. Human OA synovial explants (n=35 collected by arthroplasty) were cultured in the presence/absence of IL-1β, PAR2 agonist peptide (SLIGKV-NH2) or a reverse peptide (RP) control. Conditioned medium (CM) was harvested after 48h and used for exosome preparation. Dead cells and debris were eliminated by successive centrifugations at increasing speeds, with exosomes isolated by ultracentrifuge at 100,000g. Western blot analysis was used to confirm the presence of exosome markers (CD9, CD81, HSP70 and CD63), with scanning electron microscopy (SEM) used to verify exosome presence and size between 30-150 nm. Nanoparticle Tracking Analysis (NTA) was also undertaken on exosome preparations to confirm concentration and size. Isolated exosomes were labelled with Exo-Red (stains RNA) or Exo-Green (stains protein) and uptake by primary human OA articular chondrocytes (n=?) evaluated using immunofluorescence imaging. Gene expression changes post exosome uptake by chondrocytes was analysed with real time qPCR. Cytokine and MMP levels in CM were evaluated using ELISA. Results. Exosome preparation were evaluated to determine by the presence of exosome-associated markers CD9, CD81, HSP70 and CD63. NTA and SEM data confirmed 80-90% of microvesicles ranging in exosome size from 30 to 150 nm. Stimulation with IL-1β or SLIGKV-NH2 did not significantly alter protein concentration. Exosomes carried both protein and RNA cargo and were taken up by primary chondrocytes after 4h exposure. Primary chondrocytes that had taken up exosomes and been stimulated with IL-1β showed significant decreases in COL2A1 (p<0.001) and ACAN expression (p<0.001), and a significant increase in GLUT-1 expression (p<0.1). Stimulation with SLIGKV-NH2 showed similar patterns, with a significant decrease in COL2A1 (p<0.1), ACAN (p<0.1) and SOX9 (p<00.1) expression, as well as a significant increase in MMP-13 expression (p<0.1). Levels of IL-6, TNF and MMP-3 in CM were significantly increased by both IL-1β and SLIGKV-NH2 (p<0.001) stimulated samples compared to controls. Interestingly, only SLIGKV-NH2 significantly increased IL-8 (p<0.001), demonstrating differential regulation by PAR2. Conclusion. OA synovial tissue has the potential to impact chondrocyte behaviour through the release of a secretome, which includes potentially regulatory exosomes. Future studies into characterisation of cargo, will provide insight into the role of the synovium in OA cartilage pathology.

KW - Exosome

KW - Cartilage, Articular

KW - Synovitis

KW - Osteoarthritis

KW - Inflammation

M3 - Poster

ER -

Asghar S, Litherland G, Meek D, Goodyear C, Lockhart J, Crilly A. The synovial secretome contributes to cartilage pathology in osteoarthritis: a role for exosomes. 2018. Poster session presented at 2018 Osteoarthritis Research Society International (OARSI) World Congress on Osteoarthritis, Liverpool, United Kingdom.