Bisphosphonate and geranylgeraniol regulate angiogenic genes in human gingival fibroblasts

Previous studies have indicated that the mevalonate pathway (MVP) and an anti-angiogenic effect of bisphosphonates may play a role in the pathogenesis of bisphosphonate related osteonecrosis of the jaw (BRONJ). Objectives: To determine the effects of the bisphosphonate, zoledronic acid (ZA) and replenishment of the MVP by geranylgeraniol (GGOH), on human gingival fibroblasts (HGFs) at the cellular and genetic levels. Methods: Primary cell lines of HGFs (n=5) were cultured from gingival tissue excised during gingivectomy/crown-lengthening surgeries. All assays were conducted at 24, 48, 72 and 96hrs post treatment with and without ZA and GGOH (72hrs only).  Cell viability and apoptosis were determined using the CellTiter-Blue viability assay, the caspase3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative Real-Time PCR (qRT²-PCR) gene expression assays were conducted for VEGFA, BMP2, RHOB, EREG and IFNA1 with ELISAs for VEGF-A and BMP-2. Results: Cellular viability increased in the presence of 20-50μM ZA at 24hrs and then decreased rapidly. The simultaneous addition of ZA and GGOH (at 72hrs) restored cell viability to control levels. Caspase 3/7 was detected in ZA treated HGF. TEM revealed dilation of the rER with ZA and multiple lipid-like vesicles following the addition of GGOH. ZA significantly (p<0.05, Fold Induction>±2) up-regulated VEGFA, RHOB, BMP2 and EREG at one or more time points but not IFNA1. Addition of GGOH resulted in a reduction in the expression of all genes compared with ZA treated HGFs. The protein concentration of VEGFA was higher in ZA treated HGFs compared to controls, however BMP2 proteins were undetected. Conclusions: ZA increased expression of four key angiogenesis genes in HGFs. GGOH partially reversed the effects of ZA in HGFs both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the MVP. This study was funded by the MPPT and NZDARF.