Zol is the most potent and long-acting N-BP used for the treatment of osteoporosis, Paget's disease, myeloma and cancers to reduce adverse skeletal related events [1]. Change of VEGF levels have been previously reported in cancer patients receiving Zol [17], and decreased VEGF values have been also associated with the BRONJ risk [18].
To the best of our knowledge, this is the first prospective cohort study investigating the early serum changes of VEGF levels in postmenopausal women at high fracture risk receiving Zol. Particularly, VEGF levels significantly raised soon after 3 days from Zol administration, and then decreased after 30 days to a value below the baseline one.
The raise of VEGF at day-3 may fall in the window of the frequent occurring APR after i.v. N-BP administration.
APR is a common side effect observed in 10 to 50% of patients after the first Zol dose. It consists of a systemic immune reaction induced by the stimulation of circulating γδ T cells and the subsequent release of several inflammatory cytokines as interleukin 6 (IL-6), tumor necrosis factor-β (TNF-β) and interferon-γ (IFN-γ) [23,24]. Related symptoms occur within 24-36 hours and usually disappear in about 3 days [25].
VEGF is a specific endothelial cell mitogen and a strong vascular permeability factor; thus, it may be considered as a proinflammatory cytokine. Consequently, in patients receiving Zol, the early increase of VEGF levels could be a biochemical mark of VEGF involvement in the cytokine storm leading to APR [26]. Accordingly, at a post-hoc analysis, the rise of VEGF at day-3 was 13 % higher in the 3 participants who suffered APR.
On the other hand, the decrease of VEGF levels after 30 days could be determined by the well-known Zol related suppression of bone turnover and Zol activity on endothelial cells. Osteoclasts, osteoblasts, osteocytes and endothelial cells are sources of VEGF [27]. In the bone tissue, VEGF stimulates angiogenesis, osteoblast differentiation, and finally bone formation and bone healing. Furthermore, VEGF has been shown to promote osteoclast differentiation and survival [28]. Accordingly, VEGF could orchestrate the crosstalk between endothelial, osteoblastic and hematopoietic cells [29]. Data on VEGF levels in osteoporotic patients are conflicting, and circulating VEGF could be influenced by treatment [30].
In all the participants of this study, at baseline, we observed a significant positive association between VEGF and both BGP and femoral BMD. These findings have not been previously described and add new insights about the possible VEGF key role in bone health; moreover, they are consistent with previous studies showing significantly lower VEGF levels in women with postmenopausal osteoporosis [31].
Several lines of evidence based on both in vitro and in vivo studies [17, 18, 32, 33] suggest the anti-angiogenic and anti-tumor VEGF-related Zol effect. However, few researches involved humans and mostly cancer patients. In 30 patients suffering from solid tumors with bone metastases, treated with a single infusion of Zol 4 mg, Santini et al. reported a significant reduction of VEGF levels after 2, 7 and 21 days, with the largest significance (p <0.001) on day 21 [34]. Moreover, low intermittent doses of Zol (1 mg every 7 days for 4 times followed by 4 mg every 28 days for 3 times), produced significant reductions of the circulating VEGF early after 7 days in cancer patients with bone metastasis from solid tumors. Moreover, after 84 days and a 16 mg cumulative Zol dose, VEGF levels tended to rise, although remaining significantly lower in comparison with baseline [34]. Remarkably, even though the different dose and schedule, the reduction of serum VEGF levels observed in osteoporotic postmenopausal women in our study was of a smaller magnitude than that one seen in the oncology setting.
In women with postmenopausal osteoporosis, only one elegant study by Ishtiaq et al. evaluated the VEGF change in vivo and in vitro after N-BPs. Alendronate (Aln), administered at a weekly dose of 70 mg, produced a VEGF decline after 3 and 6 months, reaching a statistical significance after 12 months (p = 0.02). Consistently, Aln and Zol, in the culture medium of two osteoblastic cell lines, significantly reduced the VEGF production, confirming that N-BP related in vivo modification of this cytokine should be attributed to a direct effect on bone cells [35].
The VEGF reduction obtained in vitro with Zol was more pronounced than that one with Aln and it occurred at a lower dose, in accordance with higher Zol potency [35]. Differently from N-BPs, no published data are available describing the denosumab effects on VEGF. Denosumab is a fully human monoclonal IgG2 antibody against the receptor activator of nuclear factor-kB ligand (RANKL) and it represents a potent antiresorptive agent for osteoporosis [36]. However, as for N-BPs, an antiangiogenic activity of denosumab, possibly mediated by a RANKL-dependent pathway, have been suggested by Girolami et al. [37].
Vitamin D has been proven to influence VEGF levels: calcitriol, the active vitamin D metabolite, promoted VEGF production, and by adding calcitriol to the medium in osteoblastic cell lines, the N-BP induced suppression of VEGF release was reverted [35]. Conversely, few discordant data exist, coming from breast cancer women treated with tamoxifen [38] and chronic spontaneous urticaria patients [39], both showing a significantly vitamin D-induced suppression of VEGF levels.
In line with the largest share of evidences, an inadequate vitamin D status could even amplify the VEGF-mediated suppression of angiogenesis induced by N-BPs, and particularly by Zol, but it represents only a speculation, which needs to be confirmed by further focused researches.
Our findings are consistent with in vitro data by Ishtiaq et al. [35], although the significance of the effect of N-BPs on the production of pro-angiogenic factors to in vivo pharmacology remain uncertain. Moreover, the serum 25(OH)D concentration, indicative of vitamin D status, was significantly associated with the VEGF change in postmenopausal women receiving Zol. Although divergent data exist, the influence of vitamin D on VEGF levels is considered an extraskeletal vitamin D effect [40, 41].
Recent studies have shown an association between vitamin D deficiency and the risk BRONJ [42, 43]. An increased prevalence of BRONJ has been observed in vitamin D deficient rats exposed to Zol in comparison with vitamin D repleted rats [42]. Also in humans, one study showed that patients with secondary hyperparathyroidism due to hypovitaminosis D, under N-BP treatment, were more likely to develop BRONJ [43]. Remarkably, in a case-control study, Bedogni and coauthors, at a histomorphometric analysis, observed that 77% of patients with BRONJ resulted osteomalacic compared with 5% of patients without BRONJ [44].
VEGF has been suggested as a potential biomarker for the development of BRONJ; in fact, Vincenzi et al. [18], in a cohort of 81 cancer patients receiving i.v. N-BPs, observed the development of BRONJ in six patients who showed the largest reduction of circulating VEGF levels after 7 and 21 days after the first N-BP administration.
In light of these data, patients affected by Zol-induced ONJ, was effectively treated with platelet-rich plasma which represents a concentrate of growth factors, including VEGF, obtaining an improvement of the perilesional bone tropism [45].
We must recognize this study has limitations including the small sample size, consisting of only women in postmenopausal age and the observation period not long enough to capture Zol-induced VEGF modifications in the long term. This study is not focused on BRONJ; thus, the observed changes of VEGF levels after Zol administration may have uncertain significance, and their pathophysiological role requires additional investigations. Thus, further studies, focused on osteoporotic postmenopausal women, with a large sample size and with a long follow-up, are needed to confirm our data and to address the association with BRONJ. Additionally, measuring other angiogenic factors could lead to a more in-depth profile of the effect of Zol on angiogenesis.
At the same time, our findings improve the knowledge of cytokine modifications after N-BP administration, suggesting a role for vitamin D on the Zol mediated VEGF perturbancies.
In conclusion, in osteoporotic postmenopausal women, Zol administration at a dose of 5 mg provoked early modifications of circulating VEGF, and vitamin D has been shown to modulate this VEGF change. Since Zol-induced VEGF modifications could contribute to the pathogenesis of BRONJ, further studies focused on VEGF are urgently needed to investigate its role as an early marker of increased risk of BRONJ in patients treated for osteoporosis.