Identification of BMP-7-mimicking peptides that reverse the katabolic-chondrocyte phenotype in OA chondrocytes

Abstract

Purpose: Osteoarthritis (OA) is the most common degenerative joint disease worldwide causing joint immobility and chronic pain in patients. To this date treatment is mainly based on alleviating chronic pain and reducing progression of the disease to postpone the inevitable joint replacement surgery. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, resulting in the active degradation of the ECM. However, cell biological-based therapies to slow down or reverse this katabolic phenotype towards a healthy chondrocyte phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7, OP-1) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and its bioactivity is promising to target the chondrocyte phenotype in OA. Although promising, the intra-articular use of BMP-7 is at risk due to the highly proteolytic and hydrolytic joint-environment. For clinical relevance, weekly intra-articular injections were necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, the production of GMP-grade BMP-7 is challenging with accompanying high costs. To enable the clinical use of the OA disease-modifying BMP-7 bioactivity, we sought for BMP-7-mimicking peptides better compatible with the joint-environment, while maintaining BMP-7 bioactivity and which potentially can be incorporated in an intra-articular controlled drug-delivery system. We hypothesized that human BMP-7 harbors peptide sequences that are able to mimic the disease-modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. Methods: A BMP-7 peptide library was custom-made consisting of overlapping peptides covering the full-length mature BMP-7 amino acid sequence. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE2 levels were measured by EIA. Results: The human BMP-7 peptide library was screened for the presence of bioactive peptides that represent full-length BMP-7 bioactivity. Peptides were tested at different concentrations (1, 10, 100 or 1000nM) on a validated pool of human OA articular chondrocytes. Two distinct regions in the BMP-7 protein were identified from which peptides originate that have identical chondrocyte phenotype-improving bioactivity as compared to full-length BMP-7. This was based on increased COL2A1, ACAN, SOX9, BAPX1.NKX.3.2 mRNAs and GAG; and reduced levels of COL10A1, ALP, RUNX2, ADAMTS5, IL-6, MMP13, COX2 mRNAs and PGE2. The two most potent peptides were further analysed for their OA chondrocyte phenotype-modifying properties in the presence of OA synovial fluid. Gene expression analyses showed similar OA phenotype-suppressive activity of the selected peptides in the presence of OA synovial fluid. Additionally, after a single exposure, the phenotype-improving bioactivity of the selected peptides could be detected up to 8 days in chondrocyte culture. Conclusions: Taken together, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA chondrocyte phenotype-suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in a peptide-based cartilage-targeting experimental OA treatment, addressing the diseased chondrocyte phenotype in OA.