Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

Abstract

Introduction: The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.

Methods: Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.

Results: Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.

Conclusions: The release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.

Figure 1

Identification of neurotrophic factors and their receptors within native intervertebral disc tissue. Percentage expression is represented as the number of disc samples in each cohort that expressed the target gene, shown by the number above the bars. Bars represent the levels of mRNA expression seen in these samples. Expression of neurotrophic nerve growth factor (NGF) (A) and its receptor tropomyosin kinase (Trk)A (B), of brain-derived neurotrophic factor (BDNF) (C) and its receptor TrkB (D), and of neurotrophin 3 (NT3) (E) and its receptor TrkC (F) in human intervertebral discs graded histologically: grade 0 to 3, n = 8; grade 4 to 6, n = 12; grade 7 to 12, n = 20; infiltrated, n = 12 samples of intervertebral disc tissue. *Statistical significance between levels of gene expression: P ≤ 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Neuropeptide and angiogenic factor expression within native intervertebral disc tissue. Percentage expression is represented as the number of disc samples in each cohort that expressed the target gene, shown by the number above the bars. Bars represent the levels of mRNA expression seen in these samples. Expression of pain-related peptides substance P (A) and calcitonin gene-related peptide (CGRP) (B) and angiogenic factors vascular endothelial growth factor (VEGF) (C) and pleiotrophin (D) in human intervertebral discs graded histologically: grade 0 to 3, n = 8; grade 4 to 6, n = 12; grade 7 to 12, n = 20; infiltrated, n = 12. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *Statistical significance: P ≤ 0.05.

Figure 3

Cytokine regulation of neurotrophic factors NGF, BDNF and NT3 in human nucleus pulposus cells, SH-SY5Y cells and HDMEC cells. Neurotrophic factor mRNA expression in human nucleus pulposus (NP) cells stimulated by interleukin (IL)-1 (A), tumour necrosis factor alpha (TNFα) (B) and IL-6 (C). Neurotrophic factor mRNA expression in SH-SY5Y neuronal cells following cytokine stimulation (D). Neurotrophic factor mRNA expression in human dermal microvascular endothelial cells (HDMEC; endothelial cell line) following cytokine stimulation (E). *Statistical significance: P ≤ 0.05. BDNF, brain-derived neurotrophic factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NGF, nerve growth factor; NT3, neurotrophin 3.

Figure 4

Cytokine regulation of neuropeptides substance P and CGRP within human nucleus pulposus cells, SH-SY5Y and HDMEC cells. Substance P (SP) and calcitonin gene-related peptide (CGRP) mRNA expression in human nucleus pulposus (NP) cells following stimulation with interleukin (IL)-1β (A1), tumour necrosis factor alpha (TNFα) (B) and IL-6 (C). SP protein was also determined in human NP cells following stimulation with IL-1β (A2). SP and CGRP mRNA expression in SH-SY5Y neuronal cells following cytokine stimulation (D). SP mRNA was not expressed by human dermal microvascular endothelial cells (HDMEC)-1 and cytokines did not significantly regulate CGRP mRNA expression (E). *Statistical significance: P ≤ 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Cytokine regulation of angiogenic factors VEGF and pleiotrophin in human nucleus pulposus cells, SH-SY5Y and HDMEC cells. Vascular endothelial growth factor (VEGF) and pleiotrophin mRNA expression in human nucleus pulposus (NP) cells following stimulation with interleukin (IL)-1β (A), tumour necrosis factor alpha (TNFα) (B) and IL-6 (C). VEGF and pleiotrophin mRNA expression in human NP cells following cytokine stimulation (D). Human dermal microvascular endothelial cells (HDMEC) cells did not express pleiotrophin mRNA, and cytokines nonsignificantly upregulated VEGF mRNA expression (E). *Statistical significance: P ≤ 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.