Brenda Bakker (Presenter)
University of Twente
Bio: Ms Brenda Bakker is a PhD student at the University of Twente. In collaboration with Maastricht University she aims to discover novel markers for early osteoarthritis that may provide new clues for therapeutic intervention.
Authorship: Brenda Bakker(1), Gert Eijkel(2), Ronny Mohren(2), Ron Heeren(2), Marcel Karperien(1), Janine Post(1), Berta Cillero-Pastor(2)
1) Developmental BioEngineering, Faculty of Science and Technology, University of Twente, 7522 NB Enschede, The Netherlands (2)The Maastricht Multimodal Molecular Imaging Institute (M4I), Maastricht University, 6229 ER Maastricht, The Netherlands
In this study we employed several mass spectrometry modalities to identify molecular changes by oxygen in chondrocytes. We found changes in the lipidome, proteome and metabolome that all signify alterations in mitochondria. These mitochondrial changes may explain why chondrocytes perform poorly and lose their phenotype in supraphysiological oxygen levels and cartilage degenerative disease such as osteoarthritis. Targeting these specific molecular changes may be relevant for retaining the chondrogenic phenotype which has important implications for the treatment of bone and cartilage diseases.
Osteoarthritis is the most common joint disease and its main characteristic is the breakdown of articular cartilage, resulting in joint pain and impaired mobility. During cartilage degenerative diseases, such as osteoarthritis (OA), the inflammatory processes result in more reactive oxygen species (ROS) and oxygen levels within the joint are altered. The chondrocyte is the only resident cell type in cartilage and plays a unique role in the maintenance of the extracellular matrix (ECM) by keeping a fine balance between anabolic and catabolic activities. Chondrocytes are generally exposed to low oxygen levels (1-6%). When they are exposed to supraphysiological oxygen levels they produce less ECM, shifting the balance to ECM breakdown. Using time-of-flight secondary ion mass spectrometry (ToF-SIMS) we have previously shown that cholesterol levels were lower in physiological oxygen levels compared the generally used supraphysiological oxygen levels. Additionally, matrix-assisted laser desorption ionization (MALDI) imaging revealed elevated levels of phosphatidylglycerol and cardiolipins in supraphysiological oxygen conditions. To understand the biological implications of these changes in the lipidome we studied changes in the proteome and in the metabolic distribution of 3D chondrocyte cultures in these oxygen levels to ultimately map all molecular changes and find targets for therapy.
Human chondrocytes were isolated from articular cartilage of three patients undergoing total knee replacement and cultured in 3D pellets in high or physiological oxygen levels (20% and 2.5%, respectively). To detect protein changes 3 to 5 cell pellets were pooled for protein extraction followed by trypsin digestion. Nano LC-MS was performed on a Thermo Scientific (Dionex) Ultimate 3000 RSLCnano system equipped with an Acclaim PepMap C18 analytical column (2 um, 100Å, 75 um x 150 mm) coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). Proteins were identified using data dependent acquisition (DDA) and protein fold changes between culture conditions analyzed with Proteome Discoverer. Additionally, succinate dehydrogenase (SDH) activity was assessed on chondrocyte monolayer cultures.
To detect metabolic changes MALDI mass spectrometry imaging (MSI) was employed. Chondrocyte cell pellets were cryosectioned and sprayed with 9-aminoacridine using the TM-sprayer (HTX Imaging). Mass spectra of each pixel (40 μm raster size) were acquired in the negative ion mode between 100 and 1000 Da with a Bruker 9.4T SolariX Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to search for spectral similarities and differences between the conditions. Human metabolome database was employed for metabolite identification.
Bottom-up proteomics revealed differences in the protein composition of the chondrocytes cultured in physiological and high oxygen levels. Several mitochondrial proteins were elevated in high oxygen conditions, including proteins part of the electron transport chain complex III and V, superoxide dismutase and several mitochondrial proteins involved in lipid metabolism. SDH protein levels were not altered by oxygen. However, the enzyme activity showed a negative correlation with oxygen levels in monolayer cultures of chondrocytes. MALDI-MSI followed by LDA clearly distinguished physiological and high oxygen cultures. Amongst others we could identify adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP).
Conclusions & Discussion
Using various mass spectrometry modalities we show that human chondrocytes have a distinct, oxygen-dependent molecular profile. These changes in the lipidome, metabolome and proteome signify changes in mitochondria and may be signs of elevated levels of ROS in high oxygen conditions. These mitochondrial changes may explain why chondrocytes perform poorly and lose their phenotype in supraphysiological oxygen levels and cartilage degenerative disease such as osteoarthritis. Targeting these mitochondrial changes may restore the balance between anabolic and catabolic activities and thereby halt cartilage degeneration.
References & Acknowledgements:
(1) Georgi, N.; Cillero-Pastor, B.; Eijkel, G. B.; Periyasamy, P. C.; Kiss, A.; van Blitterswijk, C.; Post, J. N.; Heeren, R. M.; Karperien, M. Analytical chemistry 2015, 87, 3981-3988.
(2) Bakker, B.; Eijkel, G. B.; Heeren, R. M. A.; Karperien, M.; Post, J. N.; Cillero-Pastor, B. Analytical chemistry 2017, 89, 9438-9444.
|Grants||yes||Dutch arthritis association|
|Salary||yes||University of Twente|
IP Royalty: no
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