Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction. Modic type 1 changes (MC1) are vertebral bone marrow signal intensity changes found predominantly in patients with degenerated intervertebral discs and damaged cartilage endplates. Discs adjacent to MC1 (MC1 discs) degenerate faster and release more pro-inflammatory and pro-fibrotic factors compared to other degenerated discs. Yet, it remains unknown if the degenerative process of MC1 discs differ from non-MC discs. The aim of this study was to identify MC1-specific mechanisms of disc degeneration. We hypothesize that the MC1 discs contain more cleaved extracellular matrix (ECM) fragments and that they are generated through a MC1-specific mechanism.

Methods. 23 degenerated non-MC discs and 30 MC1 discs from patients undergoing lumbar spinal fusion were analyzed. First, shotgun mass spectrometry (MS) was used for proteome analysis, in particular to identify overexpressed proteases. Second, the degradome (all cleaved proteins) was determined with N-terminal amine isotopic labeling of substrates (N-TAILS) MS. The proteins with the greatest number of de-novo fragments were identified. Third, to reproduce the identified protein fragments, the full-length recombinant proteins were digested with a protease identified to be overexpressed in MC1 (high temperature requirement A serine peptidase 1 (HTRA1)) or with an inactive mutant of HTRA1. Cleavage was confirmed with SDS-PAGE followed by Coomassie Blue staining. To test if the in-vitro generated protein fragments are the same as found in human disc samples, the in-vitro generated protein fragments were analysed with N-TAILS, and the identify cleavage sites was compared against the cleavage sites identified in the human samples.

Results. Shotgun proteomics found more HTRA1 in MC1 discs. With N-TAILS, we found that overall, the newly cleaved fragments were more abundant in MC1 discs (Figure 1A), indicating a difference in degeneration patterns in MC1 discs. The proteins with the most fragments found in greater abundance in MC1 were fibronectin (FN), type 1 collagen alpha-1 (COL1A1), cartilage intermediate layer protein 1 (CILP1), and COMP (Figure 1B). In-vitro, HTRA1 cleaved recombinant CILP1, COMP and FN but not COL1A1. N-TAILS of the cleaved proteins identified 20 N-terminally labeled peptides of which nine were also found in the previously analyzed degenerated discs and were significantly more abundant in MC1 discs (Figure 2). FN fragments identified were part of the 29kDa N-terminal FN fragment, a known damage-associated molecular pattern (DAMP).

Discussion. We identified HTRA1 cleavage of the disc as a MC1-specific disc degradation mechanism contributing to increased ECM fragmentation in MC1 discs. Interestingly HTRA1 is also highly relevant in osteoarthritis progression. The fragments of the proteins that were most cleaved in MC1 discs can be pro-inflammatory and pro-fibrotic. This highlights the importance of further investigating HTRA1 cleavage to comprehend the breakdown mechanism of MC1 discs and the role of the HTRA1 generated fragments in inducing MC1.