Effect And Mechanisms Of Fluvastatin On Vegf Induced Calcification Of Pulmonary Valve Endothelial Cell

(Part I)Background:Aortic valve is the most sufferable area in the pathologic process of vascular calcification, which is charaterized by calcium deposits and osteogenesis. Calcific aortic stenosis, previously thought of as a degenerative disease, is now confirmed as an active inflammatory process involving cell proliferation and mineralization. The central role of neoangiogenesis and bone formation in the pathobiology of calcific aortic stenosis suggest the involvement of vascular endothelial growth factor (VEGF).Objective:To investigate the effects of VEGF on calcific changes of human pulmonary valve endothelial cells and the signaling pathway.Methods:Human pulmonary valve endothelial cells (HPVEC) were isolated from human pulmonary valve leaflets obtained from patients undergoing cardiovascular surgical procedures. Fura-2were used for intracellular calcium detection, transfected fusion NFAT-GFP for localizing NFAT nuclear transportation, real-time PCR for detecting osteoblast-like gene expression and Von kossa staining for calcification. Results:100ng/ml VEGF stimulated calcium oscillation in HPVEC with frequency of0.3±0.03/min and signal wave widths of80±3.5S (n=7). VEGF also stimulate the cytoplasmic-to-nuclear translocation of a GFP-NFAT fusion construct, upregulated expression of osteoblast and bone matrix markers including BMP-2, cbfal, osteocalcin and osteoprotegerin and induced calcium deposits in monolayer HPVEC.Conclusions:VEGF caused osteogenesis gene expression and calcium deposits in valve endothelial cells, which may related with calcium oscillation dependent NFAT signaling. (Part II)Background:Fluvastatin is an inhibitor of HMG-CoA reductase. Inhibition of HMG-CoA reductase by statins decrease the production of mevalonic acid and other products of mevalonate in the cholesterol biosynthetic pathway, so as to protect cardiovascular system from a wide spectrum of diseases.Objective: To investigate the effects of fluvastatin on VEGF induced valve endothelial cells calcification and the correlates with intracellular calcium signaling.Methods:Pretreated HPVEC with10nM fluvastatin for24hours before stimulating with100ng/ml VEGF. Fura-2were used for intracellular calcium detection, transfected fusion NFAT-GFP for localizing NFAT nuclear transportation, real-time PCR for detecting osteoblast-like gene expression and Von kossa staining for calcification.Results:Pretreatment of fluvastatin inhibited VEGF induced calcium oscillation and NFAT nuclear translocation in HPVEC. RNA level of BMP-2, cbfal, osteocalcin and osteoprotegerin decreased (50.61±10.88)%,(82.23±22.11)%,(85.58±19.03)%and (85.57±8.42)%respectively (n=3for each),and VEGF induced calcium deposits in monolayer HPVEC was abolished.Conclusions:Fluvastatin inhibited VEGF induced valvular calcification through inhibition of calcium oscillation dependent NFAT signaling in HPVEC. (Part III)Background:geranylgeranyl pyrophosphate (GGPP), which could be blocked by statins, is isoprenoid involved in membrane localization and cell signaling of the small GTP-binding proteins. Statins induced inhibition of GGPP has been suggest as a important mechanism that statins exert protective effects.Objective:To investigate the mechanism of fluvastatin on VEGF induced valve endothelial cells calcification.Methods:Pretreated HPVEC with10nM GGPP and fluvastatin for24hours before stimulating with100ng/ml VEGF. Fura-2were used for intracellular calcium detection, transfected fusion NFAT-GFP for localizing NFAT nuclear transportation, real-time PCR for detecting osteoblast-like gene expression and Von kossa staining for calcification.Results:Calcium oscillation with frequency of0.074±0.02/min and signal wave widths of65.7±8.8S (n=9) and NFAT nuclear translocation were observed in HPVEC. RNA level of BMP-2, cbfal, osteocalcin and osteoprotegerin increased1.88±0.16,8.44±3.56,6.59±3.7and2.34±0.16folds respectively compared with fluvastatin treatment (n=3for each). Calcium deposits were found in monolayer HPVEC after72hours of VEGF treatment.Conclusions:GGPP reversed the inhibition effect of fluvastatin on VEGF induced valvular calcification, suggesting a GGPP dependent pathway of fluvastatin on VEGF signaling in valvular endothelial cells.