HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro

Abstract

Objectives: Vascular calcification is a major clinical problem and elucidating the underlying mechanism is important to improve the prognosis of patients with cardiovascular disease. We aimed to elucidate the role and mechanism of action of Hepatocyte Growth Factor (HGF)/c-Met signalling in vascular calcification and establish whether blocking this pathway could prevent mineralisation of vascular smooth muscle cells (VSMCs) in vitro.

Methods and results: We demonstrate increased HGF secretion and c-Met up-regulation and phosphorylation during VSMC osteogenic differentiation. Adenoviral-mediated over-expression of HGF (AdHGF) in VSMCs accelerated mineralisation, shown by alizarin red staining, and significantly increased (45)Calcium incorporation (1.96 ± 0.54-fold [P < 0.05]) and alkaline phosphatase (ALP) activity (3.01 ± 0.8-fold [P < 0.05]) compared to controls. AdHGF also significantly elevated mRNA expression of bone-related proteins, Runx2, osteocalcin, BMP2 and osterix in VSMCs. AdHGF-accelerated mineralisation correlated with increased Akt phosphorylation, nuclear translocation of Notch3 intracellular domain (N3IC) and up-regulation of the Notch3 target protein, HES1. In contrast, adenoviral-mediated over-expression of the HGF antagonist, NK4, markedly attenuated VSMC mineralisation, and reduced c-Met phosphorylation, Akt activation and HES1 protein expression compared to AdHGF-treated cells. Furthermore, the Notch inhibitor, DAPT, attenuated N3IC nuclear translocation and AdHGF-induced mineralisation.

Conclusion: We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.

Fig. 1

(A) ELISA showing HGF levels in conditioned medium from SMCs grown in osteogenic or regular media at specific time-points. *P < 0.05, osteogenic vs normal conditions on the same day; ^#P < 0.05, day 21 vs day 2, in osteogenic conditions. (B) Western blot and densitometry analysis showing c-Met expression and phosphorylation in SMCs cultured in osteogenic and regular media.

Fig. 2

(A) VSMCs infected with AdEGFP or AdHGF and cultured in regular media for 21 days showed different growth patterns. (B) Alizarin red staining shows accelerated VSMC mineralisation in osteogenic media at day 8 in AdHGF-infected SMCs vs. AdEGFP-infected SMCs. Bar = 200 μm. (C) ⁴⁵Ca accumulation and (D) ALP activity in VSMCs infected with AdEGFP and AdHGF and grown in osteogenic media. (n = 3) *P < 0.05 AdEGFP vs AdHGF-infected cells. ^#P < 0.005 day 2 vs. day 8. (E) Expression of bone-related genes evaluated 2 days after infection by quantitative PCR. (n = 4) *P < 0.05 AdEGFP vs AdHGF. (F) Representative photomicrographs of AdNK4-infected and control VSMCs cultured in osteogenic media for 21 days (Control) and stained with Alizarin red shows VSMC mineralisation was attenuated in AdNK4-infected cells compared to controls. Bar = 200 μm.

Fig. 3

(A) and (B) Western blot and densitometry analysis show over-expression of HGF significantly increased phosphorylation of c-Met, but little effect on c-Met expression (Ai). In contrast, NK4 over-expression down-regulated c-Met phosphorylation (Aii & Bii). OPG expression was reduced in AdHGF-infected cells but increased in NK4-infected cells, compared to AdEGFP-infected cells (Aii & Biii). (C) Over-expression of HGF up-regulated phosphorylation of Akt at days 2 and 8 (*P < 0.01, ^#P < 0.05, n = 3).

Fig. 4

Representative micrographs show localisation of active Notch3 in SMCs infected with control AdEGFP (A) or AdHGF (B). (C) Western blotting show HES1 expression and β-tubulin expression as a loading control. *P < 0.01. (D) Localisation of Notch3 in VSMCs infected with AdHGF in the presence or absence of DAPT (1 μmol/L). (E) Alizarin red staining showed DAPT reduced mineralisation in VSMC cultured in osteogenic media for 21 days and (F) in SMCs infected with AdHGF and cultured in osteogenic media for 8 days (n = 3). Bar in A & B = 100 μm; D = 25 μm, E & F = 200 μm.

Fig. 5

Model showing elevated calcium and/or phosphate up-regulate HGF secretion and c-Met activation in conjunction with activation of the PI3K/Akt signalling pathway, leading to SMC mineralisation, which can be inhibited by AdNK4 treatment. Signalling molecules and osteogenic transcription factors downstream of PI3K/Akt were shown to be involved in HGF/c-Met-induced mineralisation. We also show HGF/c-Met-induced Notch3 activation and using the Notch inhibitor, DAPT, we demonstrate attenuation of calcification suggesting Notch3 signalling involvement in this process. The dotted arrows between c-Met activation and HGF secretion indicate other factors could be associated with c-Met receptor and HGF ligand interaction.