Melatonin prevents the binding of vascular endothelial growth factor to its receptor and promotes the expression of extracellular matrix-associated genes in nucleus pulposus cells

Abstract

The mechanisms of intervertebral disc degeneration (IDD) involve numerous factors, including loss of the extracellular matrix (ECM) and vascular ingrowth. Melatonin has been reported to protect intervertebral discs (IVDs) from degeneration and to exert a potential anti-angiogenic effect. The aim of the present study was to investigate the anti-angiogenic and anabolic effects of melatonin in IVDs. Human nucleus pulposus (NP) and degenerative nucleus pulposus (DNP) cells were isolated and treated with melatonin. The results indicated that melatonin promoted ECM synthesis and NP cell proliferation. In addition, an NP/DNP and human umbilical vein endothelial cell (HUVEC) co-culture model was used to investigate the anti-angiogenesis effect of melatonin. Melatonin was indicated to suppress tube formation and migration of HUVECs in culture with NP cell-conditioned medium, as well as in an NP cell co-culture model. Fluorescence-labeled vascular endothelial growth factor (VEGF) was used to study the binding between VEGF and its receptor. The results of the present study indicated that melatonin exerts an angiogenic effect via inhibition of the binding of VEGF to its receptor in HUVECs. Taken together, these results suggest that melatonin is a potential agent to prevent IDD.

Keywords: extracellular matrix; intervertebral disc degeneration; melatonin; nucleus pulposus.

Figure 1

Extracellular matrix-associated gene expression and proliferation in NP cells. NP cells were treated with melatonin (10 µM) or TGF-β1 (10 ng/ml) for 1-6 days. After 24 h, gene expression was measured by reverse transcription-quantitative PCR. (A-C) Gene expression of (A) COL2A1, (B) ACAN and (C) SOX9. (D) At days 1-6, cell proliferation was evaluated with a CCK-8. Values are expressed as the mean ± standard deviation. ^*P<0.05. ^#P<0.05, the melatonin group compared with the control group. ACAN, aggrecan; CCK-8, Cell Counting Kit-8; COL2A1, type II collagen A1; NP, nucleus pulposus; TGF-β1, transforming growth factor-β1.

Figure 2

Collagen-II protein expression in NP cells. NP cells were treated with melatonin (10 µM) or TGF-β1 (10 ng/ml) for 3 or 7 days. (A) At days 3 and 7, immunohistochemistry was performed to determine the collagen-II expression levels (scale bar, 20 µm). Black arrow, extracellular staining. Red arrow, intracellular staining. (B) The immunohistochemistry results were quantified as the IOD. Values are expressed as the mean ± standard deviation. ^*P<0.05. DNP, degenerative nucleus pulposus cells; IOD, integrated optical density; NP, nucleus pulposus cells; TGF-β1, transforming growth factor-β1.

Figure 3

Tube formation and cell migration of HUVECs. HUVECs were pretreated with melatonin (10 µM) or TGF-β1 (10 ng/ml) for 4 h and then incubated with NP-conditioned medium and 25 ng/ml VEGF for 6 h. (A) Tube formation was observed under a light microscope. Scale bar, 50 µm. (B) HUVECs were co-cultured with NP or DNP and incubated with melatonin (10 µM) or TGF-β1 (10 ng/ml) for 24 h. After fixation and staining, HUVEC migration was observed under a light microscope. Scale bar, 50 µm). (C) Tube formation and (D) cell migration of HUVECs was quantified. Values are expressed as the mean ± standard deviation. ^*P<0.05. DNP, degenerative nucleus pulposus cells; HUVECs, human umbilical vein endothelial cells; M, melatonin; NP, nucleus pulposus cells; T/TGF-β1, transforming growth factor-β1; V/VEGF, vascular endothelial growth factor.

Figure 4

VEGF binding assay. HUVECs were pretreated with melatonin ((10 µM) or TGF-β1 (10 ng/ml) for 4 h then incubated with fluorescence-labeled VEGF with for 4 h. The VEGF binding rate was investigated by immunofluorescence. (A) Representative fluorescence microscopy images (scale bar, 100 µm). (B) Mean density of positive staining. Values are expressed as the mean ± standard deviation. ^*P<0.05. HUVECs, human umbilical vein endothelial cells; IOD, integrated optical density; TGF-β1, transforming growth factor-β1; VEGF, vascular endothelial growth factor.