Thu0451 cell-Matrix Adhesion of Bone Marrow Stromal Cells in Modic Type 1 Changes Is Increased and Relates to Increased Expression of Integrin Β1

Abstract

Background:Modic type 1 changes (MC1) are vertebral bone marrow lesions associated with non-specific low back pain (LBP). The pathophysiology of MC1 includes inflammation, fibrosis, and high bone turnover. Bone marrow stromal cells (BMSCs) are key regulators of these processes: BMSCs contribute to inflammation by regulating myelopoiesis/osteoclastogenesis; BMSCs can differentiate into osteoblasts contributing to high bone turnover, and BMSCs can differentiate into pro-fibrotic myofibroblasts.Objectives:To identify dysregulated biological processes in MC1 BMSCs contributing to the pathobiology of MC1.Methods:Bone marrow aspirates were obtained from LBP patients with MC1 undergoing lumbar spinal fusion. Aspirates were taken prior to screw insertion. From each patient, a MC1 and a healthy control (HC) aspirate from the adjacent vertebral body was collected. BMSCs were isolated by plastic adherence and expanded. RNA from BMSCs passage 3 was sequenced (n=3 + 3) (Illumina Novaseq) and gene ontology of significantly dysregulated genes (p-value < 0.05) was analyzed. Specificity and rate of BMSC matrix adhesion were quantified: BMSCs (n=8 + 8) were seeded on fibronectin-coated, collagen-I-coated, and non-coated plastic dishes. BMSC adhesion was evaluated from 15min to 30min (Δ 30min - 15min). Percentage of adherent cells of MC1 and HC BMSC was compared with paired t-test. In order to identify integrins responsible for dysregulated cell-matrix adhesion, gene expression of 15 relevant integrins was measured by quantitative real-time PCR (qPCR). Normalized expressions were compared between MC1 and HC BMSC with paired t-test. Integrin β1 protein level was semi quantitatively analyzed by Western Blot (n = 5 + 5) and normalized to β-Actin expression.Results:By RNA sequencing, 154 genes were differentially expressed between MC1 and HC BMSCs (p-value ≤ 0.01; log2-ratio ≥ 0.5). Gene ontology enrichment analysis revealed an overrepresentation of the biological process “cell-matrix adhesion” among all significantly regulated genes (p-value < 9.3e-13). A change in cell adhesion was corroborated with adhesion assay. Binding (Δ 30min - 15min) to collagen I (MC1 + 16%, HC +10%, p-value = 0.10), fibronectin (MC1 + 17%, HC +6%, p-value = 0.03), and non-coated surface (MC1 + 46%, HC +35%, p-value = 0.05) was increased in MC1 (Figure 1). Integrin gene expression analysis revealed significant upregulation of integrin beta-1 gene (ITGB1) in MC1 vs. HC (fold change = 1.24, p-value = 0.047), whereas there was no significant difference between the other integrins tested. On protein level, integrin β1 was upregulated in MC1 in four out of five patients (Figure 2).Figure 1.Adhesion assay.Figure 2.Western Blot analysis.Conclusion:Adhesion of BMSCs to matrix and integrin β1 expression are increased in MC1. Integrin β1 is essential for cell-matrix adhesion and an important contributor to the initiation and progression of tissue fibrosis, a hallmark of MC1. Therefore, BMSCs and integrin β1 might be relevant novel targets for the treatment of MC1.Disclosure of Interests: :Irina Heggli: None declared, Susanne Epprecht: None declared, Tamara Mengis: None declared, Astrid Juengel: None declared, Michael Betz: None declared, Jose Spirig: None declared, Florian Wanivenhaus: None declared, Florian Brunner: None declared, Mazda Farshad: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Stefan Dudli: None declared