Influence of zoledronic acid on proliferation, migration, and apoptosis of vascular endothelial cells
Introduction
Bisphosphonates have been used in medicine for over 30 years, 1 and their beneficial effect in the treatment of patients with malignant bone neoplasias such as multiple myeloma and bony metastases, or metabolic bone diseases such as Paget's disease and severe osteoporosis, is accepted. However, in recent years, as their use has become more widespread, the number of cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) has increased, and over two-thirds of them are in the mandible.
The American Association of Oral and Maxillofacial Surgeons has defined BRONJ as exposed bone in the maxillofacial region that has persisted for more than eight weeks, together with current or previous treatment with a bisphosphonate, and no history of radiation to the head and neck.2 The natural history of osteonecrosis of the jaw generally begins with a superficial mucosal ulcer in either jaw, which progresses to detectable exposure of bone, and extension of the ulcerated area in breadth and depth with bony necrosis and sequestration. In some serious cases exposed bone is accompanied by pain, infection, and one or more of the following: pathological fracture, extraoral fistula, or osteolysis that extends to the inferior border or sinus floor.3
Since 2003, BRONJ has increasingly become the focus of clinical and preclinical investigations, but the mechanism is still not clear. Vascular injury is possible. Studies have shown that zoledronic acid induced a 50% reduction of angiogenesis in the prostate gland in rats, 4 but after three years of daily bisphosphonates given orally to beagles, the necrotic regions showed no patent canaliculi but had retained their vasculature.5
In patients with cancer, a single infusion of zoledronate 4 mg induced considerable and long-lasting modifications to circulating angiogenic factors such as vascular endothelial growth factor and platelet-derived growth factor, 6 but histological examination of samples obtained from patients diagnosed with BRONJ showed patent vessels in most cases.7 The effect of antiangiogenesis and impaired vascularity of bisphosphonates is not therefore delineated at present, and more research is necessary. To understand the mechanisms more clearly we investigated the effect of zoledronate on vascular endothelial cells in vitro.
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Cell culture
Cell cultures were prepared and maintained according to standard procedures. Human umbilical vein endothelial cells line ECV-304 (American Type Culture Collection) were cultured in Dulbecco's Modified Eagle's Medium (Gibco®) supplemented with 1% penicillin–streptomycin–neomycin antibiotic mixture (Gibco®), and 10% fetal calf serum (Gibco®).
Test of cell viability by MTT assays
The effect of zoledronate on the viability of human umbilical vein endothelial cells was measured using the
Zolendronate-induced morphological change
Human umbilical vein endothelial cells were stained with crystal violet staining solution (Beyotime Institute of Biotechnology) after 24 hours treatment with zoledronate at concentrations of 0, 15, 50, and 150 μmol. As seen under microscopy (Nikon), the cells were rounded and showed the typical cobblestone appearance of endothelial cells in the control group. However, the zoledronate resulted in considerable cell shrinkage with varied shapes and rough edges compared with the control group
Discussion
There are several theories about the development of necrosis in BRONJ. First, accumulation of microdamage secondary to low bone turnover has been suggested as a contributory factor.8 A role for low bone turnover has been supported by case reports that have documented the healing of BRONJ after treatment with teriparatide,9 which suggests that the lesions resolve when turnover is pharmacologically increased. Secondly, infection is an almost universal finding in histological descriptions of BRONJ,
Conclusion
Zolendronate can inhibit the proliferation and migratory ability of human umbilical vein endothelial cells, so we suggest that it may have an effect of antiangiogenesis. Increased cellular apoptosis was also found, so zoledronate may also directly impair vascularity.
Conflict of interest
We have no conflicts of interest.
Ethics statement/confirmation of patients’ permission
No ethics approval required.
Acknowledgements
The authors thank Zhifeng Huang for technical assistance. Medical and Health Research Project of Zhejiang province, China financially supported this study.
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