Elevated Netrin-4 Expression and Its Action in Infrapatellar Fat Pad

Abstract

Knee osteoarthritis (KOA) is a degenerative joint disease characterized by inflammation and cartilage degradation. The infrapatellar fat pad (IFP), located beneath the patella within the knee joint, serves as a key anatomical structure involved in cushioning and supporting the knee. It is also an active endocrine organ that secretes various bioactive substances, potentially influencing the local inflammatory environment and contributing to KOA pathogenesis. Netrin-4 (NTN4), a protein primarily known for its role in neuronal guidance, has been implicated in various non-neuronal functions, including inflammatory processes and tissue remodeling. This study aims to explore the involvement of NTN4 in KOA, focusing on its expression in the IFP and its potential impact on disease progression. This study involved 82 patients with radiographically confirmed KOA undergoing total knee arthroplasty (TKA). The correlation between NTN4 expression and OA pathology, including Kellgren-Lawrence (K/L) grades, was investigated. NTN4-expressing cells were identified in the stromal vascular fraction, including fibroblastic, hematopoietic, and endothelial cells of the IFP. To elucidate the molecular effects of NTN4, RNA sequencing (RNA-seq) was performed on fibroblastic cells treated with recombinant NTN4. Subsequent quantitative PCR (qPCR) was used to validate the RNA-seq findings. NTN4 expression was significantly elevated in the IFP of patients with advanced KOA (K/L grades 3 and 4) compared to those with early-stage disease (K/L grade 2). Higher NTN4 expression was found in fibroblastic cells, and RNA-seq analysis revealed upregulation of genes associated with pro-inflammatory pathways, including IL-17 and TNF-α signaling, and matrix degradation. Notably, genes including IL6, MMP1, CXCL1, and CXCL8 were significantly elevated, as confirmed by qPCR, indicating NTN4's role in promoting an inflammatory and catabolic environment. Our findings suggest that NTN4 plays a significant role in the pathogenesis of KOA by promoting inflammation and matrix degradation within the IFP. Although NTN4 expression was not directly correlated with clinical symptoms, its elevated expression in fibroblastic cells and influence on inflammatory and degradative pathways suggest a potential mechanism for exacerbating joint damage. Targeting NTN4 could offer a novel therapeutic approach to mitigating inflammation and slowing disease progression in KOA, ultimately improving patient outcomes. Further research is needed to clarify NTN4's specific roles and therapeutic potential in OA management.

Keywords: Netrin-4; infrapatellar fat pad; osteoarthritis.

Figure 1

Relationship between NTN4 expression and radiographic OA grades. * indicates a significant difference compared to the KL2 group (p < 0.05).

Figure 2

Relationship between NTN4 expression and pain score. Correlation analysis between NTN4 expression levels and pain scores, as measured by the Visual Analog Scale (VAS) at rest (VAS-R) (A) and on movement (VAS-M) (B).

Figure 3

NTN4 expression in stromal vascular fractions. Expression levels of NTN4 in hematopoietic, endothelial, and stromal cell fractions are derived from the infrapatellar fat pad (IFP). * indicates significant differences between groups (p < 0.05).

Figure 4

RNA-sequencing of vehicle and recombinant Netrin-4 treated fibroblastic cells derived from the IFP. (A) Common upregulated genes in Netrin-4 (NTN4)-treated cells compared to vehicle-treated cells from two patient-derived cell samples were identified and visualized using a Venn diagram. (B) KEGG pathway analysis of the commonly upregulated genes in NTN4-treated cells.

Figure 5

qPCR analysis of vehicle and recombinant Netrin-4 treated fibroblastic cells derived from the IFP. (A) C3 (B) CXCL1, (C) CXCL6, (D) CXCL8, (E) IL6, (F) MMP1, and (G) VCAM1. * indicates significant differences between groups at each time point (p < 0.05).