Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing oxidative stress

Abstract

Background: Bisphosphonates, including zoledronate, target osteoclasts and are widely used in the treatment of osteoporosis and other bone resorption diseases, despite side effects that include damaging the stomach epithelium. Beneficial and adverse effects on other organ systems, including the cardiovascular system, have also been described and could impact on the use of bisphosphonates as therapeutic agents. Vascular smooth muscle cells (VSMCs) are major constituents of the normal vascular wall and have a key role in intimal thickening and atherosclerosis, in part by secreting MMPs that remodel the extracellular matrix and cleave cell surface proteins or secreted mediators. In this study, we investigated the effects of zoledronate on MMP expression.

Methods: Rat VSMCs were stimulated by PDGF (50 ng/mL) plus TNF-α (10 ng/mL) or left unstimulated for a further 24 hours in serum-free medium. In other series of experiments, cells were pre-treated either with SC-514 (50 μM) or with apocynin (20 nM) for 2 hours, then zoledronate (100 μM) was added into 2% fetal calf serum containing medium for 24 hours.

Results and discussion: Using isolated rat VSMCs in culture, zoledronate (100 μM) increased MMP-9 and -13 mRNA expressions but inhibited MMP-2 expression. MMP-9 and MMP-13 up-regulation was shown to depend on the NF-κB pathway; and this was activated by zoledronate. Furthermore, zoledronate elevated the levels of reactive oxygen species detected by either dichlorofluorescein in isolated VSMCs or lucigenin enhanced chemiluminescence in rat aortic rings in vitro. Apocynin, an inhibitor of NADPH oxidase, reversed NF-κB activation and MMP-9 and MMP-13 up-regulation by zoledronate.

Conclusion: We conclude that zoledronate increases MMP-9 and MMP-13 expressions in rat VSMCs dependent upon stimulation of the NF-κB pathway by reactive oxygen species. Effects on MMP expression may contribute to the pharmacologic profile of bisphosphonates.

Keywords: bisphosphonate; matrix metalloproteinase; reactive oxygen species; vascular smooth muscle cell; zoledronate.

Figure 1

Effects of PDGF plus TNF-α and zoledronate (48 hours) on MMP-2, -9, and -13. Notes: (A) MMP-2 mRNA expression. ***P≤0.001, *P≤0.05 vs Control-Untreated, n=3. (B) MMP-2 and MMP-9 protein expressions. A representative zymogram a: No induction; b: PDGF (50 ng/mL) + TNF-α-induced (10 ng/mL); c: PDGF (50 ng/mL) + TNF-α-induced (50 ng/mL). (C) MMP-9 mRNA expression. **P≤0.01, ***P≤0.001 vs Control-untreated, ^##P≤0.01 vs control-zoledronate (n=3). (D) MMP-13 mRNA expression **P≤0.01, ***P≤0.001 vs control untreated, ^##P≤0.01 vs control-zoledronate (n=3). (E) Protein expression of MMP-13 by Western blotting. Lanes No induction (c); Zoledronate (100 μM) (z); PDGF (50 ng/mL) + TNF-α-induced (50 ng/mL) (p+t); PDGF + TNF-α + Zoledronate (p+t+z). Abbreviation: EDTA, ethylenediaminetetraacetic acid.

Figure 2

Evidence for involvement of the NF-κB pathway. Notes: (A) Effects of IκB kinase 2 inhibitor SC-514 (50 μM) were measured on MMP-9 mRNA expression. ***P≤0.001 untreated vs PDGF plus TNF-α induction, ^###P≤0.001 untreated vs SC-514 (n=4). (B) Effects of SC-514 (50 μM) were measured on MMP-13 mRNA expression. ***P≤0.001 untreated vs PDGF plus TNF-α induction, ^###P≤0.001 untreated vs SC-514 (n=4). (C) Effects of PDGF plus TNF-α induction and zoledronate treatment (48 hours) were measured on IκBα mRNA expression levels. ***P≤0.01 vs control – untreated, ^#P≤0.05 untreated vs SC-514 (n=3).

Figure 3

Effects of zoledronate on ROS levels in rat VSMCs and aortic rings. Notes: (A) ROS levels determined by dichlorofluorescein (DCF) fluorescence were compared in control (c), PDGF (50 ng/mL) plus TNF-α (10 ng/mL) induced (pt) and zoledronate pre-treated (zol, 100 μM) cells. *P≤0.05 control vs zoledronate (n=4). (B) Lucigenin or luminol enhanced chemiluminescence was measured in control and 100 μM zoledronate pre-treated isolated rat aortic rings. *P≤0.05 control vs zoledronate, paired Student’s t-test, (n=5). Abbreviations: ROS, reactive oxygen species; VSMCs, vascular smooth muscle cells; AUC, area under curve; RLU, relative light units.

Figure 4

Effects of apocynin with and without zoledronate on MMP-9, -13, and IκBα. Notes: (A) MMP-9 mRNA expression. **P≤0.01, ^#P≤0.05 vs control – zoledronate. (B) MMP-13 mRNA expression. *P≤0.05, ***P≤0.01 vs control – untreated, ^#P≤0.05 vs control – zoledronate. (C) IκBα mRNA expression. *P≤0.05 vs control – untreated; ^#P≤0.05 control vs apocynin (n=4).