Proteomic analysis and effects on osteogenic differentiation of exosomes from patients with ossification of the spinal ligament

Abstract

Ossification of the spinal ligament (OSL), including ossification of the posterior longitudinal ligament and ossification of the ligamentum flavum (OLF), is a multifactorial disease that includes genetic predisposition. The association between the rate of ossification in the spinal canal and the severity of myelopathy symptoms is well known, but the degree of progression varies widely among patients. Although many candidate genes and biomarkers have been reported, there are no definitive and quantitative conclusions to date, probably because of low reproducibility due to individual differences. In this study, we focused on exosomes secreted by ossified spinal ligament cells. Exosomes are crucial for intercellular communication during development and progression of disease. In a co-culture study of non-OLF cells with OLF cells, there was increased osteogenic differentiation, including Runx2 and Wnt3a expression, with use of exosome-penetrating filters (1.2 μm) compared to exosome-non-penetrating filters (0.03 μm). Dose-dependent increases in alkaline phosphatase activity and mineral deposition were observed in non-OLF cells treated with OLF-derived exosomes. These results support the hypothesis that OLF-derived exosomes are involved in regulation of osteogenic differentiation. In comparative proteomics analysis, 32 factors were increased and 40 were decreased in OLF-derived exosomes compared to non-OLF-derived exosomes. Molecular network analysis of these 72 factors indicated 10 significant pathways, including the matrix metalloproteinase (MMP) signaling, mTOR signaling, Wnt signaling and VDR-associated pathways. Among the upregulated exosomal membrane proteins in OLF samples, COL IV, FMNL3, mTORC2, and PIP4K showed increased expression with greater ossification, suggesting they may serve as biomarkers of disease activity and therapeutic targets. These factors are involved in the PI3K/Akt/mTOR signaling pathway, and particularly mTOR is known to regulate osteogenic and chondrogenic differentiation. In contrast, fatty acid-binding protein 5, several KRT family proteins, S100A8, SERPINB3, and transglutaminase, were significantly downregulated in OLF-derived exosomes. These findings provide novel insights into the molecular mechanisms underlying OSL pathogenesis.

Keywords: exosome; ossification of the ligamentum flavum; ossification of the spinal ligament; osteogenic differentiation; proteomic analysis.

Graphical Abstract

Figure 1

Summary of the study design. (A) Computed tomography classification of thoracic ossification of the ligamentum flavum. (B) A co-culture study was performed to evaluate the effects of exosomes on osteogenic differentiation. (C) Dose-dependent assays were performed to evaluate the direct effects of exosomes on osteogenic differentiation. (D) Comparative exosome analysis (DIA proteome analysis) was performed to detect candidate proteins regulating disease activity of ossification of the ligamentum flavum.

Figure 2

(A) ALP staining at weeks 1, 2 and 3 after culture in osteogenic differentiation medium using exosome-penetrating filters (1.2 μm) and exosome-non-penetrating filters (0.03 μm). (B) Changes in ALP activity of OLF-derived and non-OLF-derived (control) cells in co-culture with 0.03- and 1.2-μm filters. (C) Alizarin Red S staining at wk 2 and 3 after culture in osteogenic differentiation medium using exosome-penetrating (1.2 μm) and exosome-non-penetrating (0.03 μm) filters. (D) Changes in relative expression of OLF-derived and non-OLF-derived (control) cells in co-culture with 0.03- and 1.2-μm filters. Data are expressed as mean ± SD. Scale bar = 200 μm. ^*p<.05, ^**p<.01.

Figure 3

(A) Western blots revealing Runx2 and Wnt3a expression at wk 2 and 3 following culture in osteogenic differentiation medium using exosome-penetrating (1.2 μm) and exosome-non-penetrating (0.03 μm) filters. (B) Expression levels of Runx2 and Wnt3a of OLF-derived and non-OLF-derived (control) cells co-cultured with 0.03- and 1.2-μm filters. (n = 3 each). ^**p<.01.

Figure 4

Direct effects of OLF-derived exosomes on non-OLF-cultured cells. Cultured cells treated with OLF-derived exosomes (Exo (OLF)) exhibited significant dose-dependent (0, 10, 30, and 100 ng/mL) increases in osteogenic differentiation, whereas the addition of non-OLF-derived exosomes (Exo (control)) did not affect osteogenic differentiation. (A) Alkaline phosphatase (ALP) assay. (B) Alizarin Red S staining. (C) Relative expression assay of Alizarin Red S.

Figure 5

The “start points and endpoints” network search algorithm in KeyMolnet produced an intricate network of targets with significant associations. Nodes with higher expression in OLF-derived exosomes than in non-OLF-derived exosomes are highlighted, and nodes with lower expression in OLF-derived exosomes than in non-OLF-derived exosomes are also marked. Molecular relationships are indicated by solid lines with an arrow (direct binding or activation) and without an arrow (complex formation).

Figure 6

Western blots illustrating baseline expression differences in COLIVA1 (A), FMNL3 (B), mTOR (C), and PIP4K2B (D) between OLF- and non-OLF (control)-derived cultured cells. ^*p<.05, ^**p<.01.