In vitro model of distinct catabolic and inflammatory response patterns of endothelial cells to intervertebral disc cell degeneration

Min Ho Hwang,Hyeong-Guk Son,Hyuk Choi,Joohan Kim

doi:10.1038/s41598-020-77785-6

Min Ho Hwang, Hyeong-Guk Son + Show 2 more

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https://doi.org/10.1038/s41598-020-77785-6

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Abstract

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. We analysed IL-6 and -8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and -3, nerve growth factor (NGF)-β, and brain-derived neurotrophic factors (BDNFs) with qRT-PCR and ELISA. We implement a microfluidic platform to analyse migration properties of AF and NP cells and ECs in 3D cultures. Our results show that IL-1β-stimulated AF cells produced significantly higher levels of IL-6 and -8, VEGF, and MMP-1 than IL-1β-stimulated NP cells. However, production of IL-6 and -8, VEGF, and MMP-3 was significantly higher in NP cells than in AF cells, under the presence of ECs conditioned medium. We observed considerable migration of NP cells co-cultured with ECs through the microfluidic platform. These results suggest that AF cells may play a major role in the initial degeneration of intervertebral disc. Furthermore, it was found that interactions between NP cells and ECs may play a significant role in the development or progression of diseases.

Highlights

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NP-conditioned medium (NPCM), respectively) and vice versa
We hypothesised that the human AF and NP cells exhibit different responses to pro-inflammatory cytokines IL-1β or soluble factors derived from ECs, respectively With regard to the interactions between each Intervertebral disc (IVD) cell and the ECs, we investigated the effects of potential contributing factors, including inflammatory mediators and catabolic enzymes derived from IL-1β-stimulated IVD cells on ECs and vice versa, in order to explore the development of IVD degeneration through the secretion of inflammatory mediators and neurotrophins
To examine the regulation of neurotrophic factors in HMEC-1 exposed to AFconditioned medium (AFCM) or NPCM, we investigated the expressions of β-nerve growth factor (NGF) and brain-derived neurotrophic factors (BDNFs) using qRT-PCR and enzyme-linked immunosorbent assay (ELISA)

Summary

Introduction

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. Several clinical studies have observed blood vessels within the inner IVD region in patients experiencing L BP3–5 This phenomenon results from several processes, including inflammatory reactions, matrix degeneration, and angiogenesis, which can be mediated by augmenting the catabolic reactions and immoderate expressions of inflammatory mediators. In the first phase of IVD degeneration, immune cells such as monocytes and macrophages infiltrate the area of the lesion through the vascular structure and secrete pro-inflammatory cytokines including IL-1β9–11 In this inflammatory environment, AF cells stimulated by pro-inflammatory cytokines express several catabolic enzymes, including MMPs, inflammatory mediators, and angiogenic factors, such as IL-6, IL-8, and VEGF2,12,13. Invasive EC can interact with NP cells located in inner IVD tissues Variations in this microenvironment due to invasive ECs or cytokine-mediated inflammatory response influence the IVD cell behaviour, such as cellular mobility and migration. These can induce an expression of neuronal pain-associated cation channels related to pain d evelopment[4,15,17]

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