Vascular endothelial growth factor regulates osteoblast survival – evidence for an autocrine feedback mechanismJohn Street1,2 and Brian Lenehan1,21Department of Orthopedic Surgery, National University of Ireland, Cork, Ireland2Combined Neurosurgical and Orthopedic Spine Program, University of British Columbia, Vancouver, BC, CanadaCorresponding author.John Street: [email protected] ; Brian Lenehan: [email protected] Received February 26, 2009; Accepted June 16, 2009.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.- Other Sections▼ o Abstract o Introduction o Methods o Results o Discussion o Competing interests o Authors' contributions o References AbstractBackgroundApoptosis of osteoblasts and osteoclasts regulates bone homeostasis. Skeletal injury in humans results in 'angiogenic' responses primarily mediated by vascular endothelial growth factor(VEGF), a protein essential for bone repair in animal models. Osteoblasts release VEGF in response to a number of stimuli and express receptors for VEGF in a differentiation dependent manner. This study investigates the putative role of VEGF in regulating the lifespan of primary human osteoblasts(PHOB) in vitro.MethodsPHOB were examined for VEGF receptors. Cultures were supplemented with VEGF(0–50 ng/mL), a neutralising antibody to VEGF, mAB VEGF(0.3 ug/mL) and Placental Growth Factor (PlGF), an Flt-1 receptor-specific VEGF ligand(0–100 ng/mL) to examinetheir effects on mineralised nodule assay, alkaline phosphatase assay and apoptosis.. The role of the VEGF specific antiapoptotic gene target BCl2 in apoptosis was determined.ResultsPHOB expressed functional VEGF receptors. VEGF 10 and 25 ng/mL increased nodule formation 2.3- and 3.16-fold and alkaline phosphatase release 2.6 and 4.1-fold respectively while 0.3 ug/mL of mAB VEGF resulted in approx 40% reductions in both.PlGF 50 ng/mL had greater effects on alkaline phosphatase release (103% increase) than on nodule formation (57% increase). 10 ng/mL of VEGF inhibited spontaneous and pathological apoptosis by 83.6% and 71% respectively, while PlGF had no significant effect. Pretreatment with mAB VEGF, in the absence of exogenous VEGF resulted in a significant increase in apoptosis (14 vs 3%). VEGF 10 ng/mL increased BCl2 expression 4 fold while mAB VEGF decreased it by over 50%.ConclusionVEGF is a potent regulator of osteoblast life-span in vitro. This autocrine feedback regulates survival of these cells, mediated via a non flt-1 receptor mechanism and expression of BCl2 antiapoptotic gene.- Other Sections▼ o Abstract o Introduction o Methods o Results o Discussion o Competing interests o Authors' contributions o References IntroductionBone is a complex, dynamic and highly specialized tissue that undergoes continuous regeneration and remodeling throughout life. Deposition and resorption of mineralized matrix occurs during development and growth, during physiological adult skeletal remodeling and during repair of surgically or traumatically injured bone. Appropriate blood supply, and intricate coupling of the vasculature with osteoblasts and osteoclasts is a prerequisite to regulation of this formation and removal of bone. Blood vessel formation, angiogenesis, and blood vessel removal, pruning, are strictly coordinated to facilitate the ever-changing demands of the skeleton. Within the temporary functioning structure of the basic multicellular unit (BMU), osteoblasts mediate bone formation, osteoclasts bone resorption, while both cells share intimate proximity with the vascular endothelium and haemopoietic and stromal cells of the bone marrow. These BMU's represent the spatial and temporal orchestration of the strictly controlled activities of osteoblasts, osteoclasts and cells of the vascular tree. The function of these cells is regulated by a number of systemic and local factors that modulate bone metabolism andvasclarization [1]. The systemic factors include parathyroid hormone, growth hormone, Vitamin D3, glucocorticoids, calcitonin and numerous vasoactive peptides. Local soluble factors known to enhance the formation of mineralized matrix include the insulin-like growth factors (IGF-I and -II), transforming growth factor beta (TGFβ), platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). Cytokines that enhance osteoclast function and bone resorption include interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) [2]. The principle 'angiogenic' cytokines that regulate blood vessel formation are vascular endothelial growth factor (VEGF), bFGF, PDGF, TGFβ, TNFα and angiopoietin-1 (Ang-I). Clearly the activities of many of these factors are common to the regulation of bone forming, bone resorbing and endothelial cells. Of these factors, vascular endothelial growth has been the focus of most recent interest [3]. This dimeric glycoprotein, with a molecular weight range from 17 to 22 kDa,has several isoforms with very similar biological activities. For a long time, VEGF was considered endothelial cell specific, however recent reports have confirmed the presence of VEGF receptors, flt-1 and/or KDR on numerous other cell types, including osteoblasts [4]. Placenta Growth Factor is another angiogenic protein specifically of the VEGF family. This protein is known to bind to Flt-1 receptor with high affinity but fails to bind the KDR VEGF receptor [5]. Recent studies have demonstrated that the mitogenic and antiapoptotic effects of the VEGF proteins on endothelial cells are mediated through specific receptors [5]. We have reported that isolated skeletal injury in humans results in local and systemic 'angiogenic' responses primarily mediated by VEGF [6,7]. VEGF has been identified as essential for bone repair in animal models [8], and is a prerequisite to hypertrophic cartilage removal and ossification during murine skeletal growth [3,5,9]. Osteoblasts may release VEGF in response to a number of stimuli, including myriad bonederived cytokines and hypoxia, simulating bone injury [[10-15]ejost]. Osteoblasts also express receptors for VEGF in a differentiation dependent manner [4]. Meanwhile osteoclasts express VEGF receptors and osteoclast differentiation and
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