Association of Endothelin-1 Expression and Cartilaginous Endplate Degeneration in Humans

Wei Yuan,Yi Liu,Feng-Lai Yuan,Wu Che,Jian Dong,Ming-Dong Zhao,Ping-Guo Duan,Pranela Rameshwar

doi:10.1371/journal.pone.0060062

Abstract

BackgroundInflammatory cytokines are involved in intervertebral disc (IVD) degeneration. Endothelin-1 (ET-1), a 21-amino-acid cytokine implicated with cartilage degradation, is secreted by vascular endothelial cells and also by many other cell types. The expression of ET-1 in human IVD cartilage endplate (CEP) and its role in disc degeneration have not been explored.Methods and FindingsThe expression of ET-1 in degenerated CEP was analyzed by immunohistochemical staining and Western blotting; ET-1 was demonstrated in cartilaginous endplate cells (CECs) by immunofluorescent staining. The ET-1 mRNA expression and protein production by CECs stimulated by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, were determined by real-time PCR analysis and Western blotting, respectively. The matrix metalloprotease-1 (MMP-1), MMP-13 and tissue inhibitor of metalloproteases-1 (TIMP-1) levels in the supernatant of cultured CECs treated with ET-1 were determined using enzyme-linked immunosorbent assays. Nitric oxide (NO) release and nitric oxide synthase (NOS) activity were measured using a spectrophotometric assay. The apoptosis of CECs by ET-1 was measured by an Annexin V-FITC detection assay. The production of ET-1 in degenerated cartilage endplate was significantly higher than normal CEP. The results showed that ET-1 was expressed by CECs and modulated by TNF-α in a dose-dependent manner. ET-1 increased production of MMP-1 and MMP-13, decreased TIMP-1 production, and induced NO and NOS release by cultured CECs. The direct stimulation of CECs by ET-1 did not promote cell apoptosis.ConclusionThe study results suggest that ET-1 played a pivotal role in human CEP degeneration, and may be a new target for development of therapies for this condition.

Highlights

Vertebral endplates form the superior and inferior boundaries of the vertebral bodies, which articulate with intervertebral discs (IVD)
The activity of matrix metalloproteinases (MMPs) is high in degenerative discs, and the balance between production of tissue inhibitors of metalloproteinase (TIMP) and MMPs appears to be altered [4]. This is accompanied by the induction of collagenases (MMP-1 and MMP-13) that are known to be involved in disc degeneration
Expression of ET-1 in Human Degenerated cartilage endplate (CEP) With hematoxylin and eosin (H&E) staining, normal CEP appeared as hyaline cartilage (Fig. 1A) after being cleared of both annulus fibrosus and nucleus pulposus

Summary

Introduction

Vertebral endplates form the superior and inferior boundaries of the vertebral bodies, which articulate with intervertebral discs (IVD). They are composed of a thin layer of cortical bone covered by hyaline cartilage produced by chondrocytes. The activity of matrix metalloproteinases (MMPs) is high in degenerative discs, and the balance between production of tissue inhibitors of metalloproteinase (TIMP) and MMPs appears to be altered [4]. This is accompanied by the induction of collagenases (MMP-1 and MMP-13) that are known to be involved in disc degeneration. The expression of ET-1 in human IVD cartilage endplate (CEP) and its role in disc degeneration have not been explored

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