R.H.M.J. Stassen (1), G.G.H van den Akker (1), M.M.C Caron (1), T.J.M. Welting 1,2)
Affiliation(s):
1. Laboratory for Experimental Orthopedics, Maastricht University, Maastricht, The Netherlands,
2. Department of Orthopedic surgery, Maastricht University Medical Center, Maastricht, The Netherlands
Objectives: Characterizing pCPP- and sCPP-induced changes of the human OA articular chondrocyte protein secretome, in order to study their pathological role in OA and compare this to the BCP-induced protein secretome.
Status: The pathobiological role of calcium-containing crystals has become inseparable from osteoarthritis (OA). In order to prevent ectopic calcification, proteins such as alpha-2 HS glycoprotein (AHSG) bind amorphous calcium phosphate particles to facilitate their clearance. These protein-bound calcium phosphate species can be categorized into primary (pCPP) and secondary calciprotein particles (sCPP), based on their distinct maturation characteristics. Presence of hydroxyapatite and increased AHSG4 in OA synovial fluid strongly hints towards the presence of CPPs. However, their pathobiological contribution remains yet to be elucidated.
Methodology: BCP crystals were produced as previously described5. Calciprotein particles were prepared in calcium and phosphate-enriched culture medium as previously described3. pCPP formation was allowed for 24 hours and sCPP for 7 days, both in the presence of 10% FCS. Characterization was performed by transmission electron microscopy and nanoparticle tracking analysis. Human OA articular chondrocytes (OA-HACs) were isolated from total knee replacement surgical waste material (METC 2017-0183). OA-HACs from 8 individual donors were stimulated with 50 μg/ml of either of the three particle species for 24 hours. Conditioned medium samples from these cultures were analyzed for expression of soluble proteins using antibody arrays (Raybiotech L1000) or ELISA. Differential expression was determined considering fold-change (FC) > 1.25 and < 0.8 with P≤0.05.
Findings: BCP crystals showed a well-defined crystal structure of approximately 123 nm in size, while pCPPs presented as spherical particles with an average size of 144.5 nm. sCPPs were characterized by spikey protrusions with needle-like shapes with an average size of 143.3 nm (Figure 1A). This is in line with earlier reported characteristics2. Exposure of OA-HACs to BCP crystals increased their IL-6 and PGE2 secretion. pCPPs did not increase secretion of IL-6. However, sCPPs did induce secretion of these factors, but were less potent provoking this compared to BCP crystals (Figure 1B). Protein secretome analysis unveiled the full extent of differential OA-HAC responses between the three different particles. BCP crystal stimulation resulted in 26 differentially expressed (DE) proteins, of which 4 were up- and 22 downregulated with respect to the non-treated control condition. The highest upregulated protein was Activin A (FC = 3.35), and the most downregulated protein CBR1 (FC = 2,08). Although pCPPs did not induce secretion of IL-6 or PGE2, we found 56 DE proteins of which 42 proteins were significantly up- and 14 downregulated by pCPPs compared to control. Furthermore, 49 DE proteins were found in response to sCPP crystal stimulation of OA-HACs. Of these proteins, 22 were upregulated and 27 downregulated by sCPPs. In both CPP datasets, FGF-BP was the most upregulated protein (pCPP FC = 2.36 and sCPP FC = 5.23). For pCPP we found CA125 (FC = 3.89) and for sCPP Fibrinogen (FC = 3.61) to be the most down regulated proteins. Other DE proteins that we identified were, amongst others, involved in cell chemotaxis, ossification and angiogenesis.
Significance: This study for the first time characterized CPP-driven chondrocyte secretory responses. The characterization of the OA-HAC protein secretomes uncovered divergent responses between the different particle types. These results help to gain a better understanding of the diverse roles of calcium-containing crystals in OA pathobiology and potentially aid the development of DMOADs.
References:
1. Yavorskyy et al R. Soc. Chem. 2008
2. Jahnen-Dechent and Smith. Kidney International 2020.
3. Aghagolzadeh et al. Atherosclerosis 2016.
4. Timur et al OAC 2020 5. Stassen et al OAC 2023