title: Factors in the fracture microenvironment induce primary osteoblast angiogenic cytokine production.
abstract: Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (<1% oxygen) conditions for 0, 3, 6, 12, and 24 hours, and VEGF gene expression was analyzed by Northern blot analysis. To determine whether rhPDGF-BB-induced VEGF messenger RNA (mRNA) expression was transcriptionally mediated or required de novo protein synthesis, transcription, and translation, studies were performed using actinomycin D and cycloheximide, respectively. Treatment with 50 ng/ml rhPDGF-BB resulted in a 2.4-fold increase in VEGF mRNA expression after 3 hours. Interestingly, rhPDGF-BB and hypoxia seemed to have an additive effect, resulting in a 3.7-fold increase in VEGF mRNA expression after 6 hours in primary neonatal rat calvarial osteoblasts. Furthermore, by using actinomycin D and cycloheximide, the authors demonstrated that the rhPDGF-BB-induced VEGF mRNA expression was transcriptionally mediate and not dependent on de novo protein synthesis. These data demonstrate that rhPDGF-BB transcriptionally increases osteoblasts VEGF mRNA expression in vitro. Furthermore, the semiquantitative results suggest that rhPDGF-BB and hypoxia act additively to increase VEGF mRNA expression. It is postulated that similar mechanisms may occur in vivo, at a site of skeletal injury, to induce neoangiogenesis and promote fracture repair.
authors.affiliation: Department of Surgery, Stanford University School of Medicine, 257 Campus Drive, Stanford, CA 94305-5148, USA.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Bouletreau
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Warren
authors.forename: Jason A
authors.initials: JA
authors.lastname: Spector
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Steinbrech
authors.forename: Babak J
authors.initials: BJ
authors.lastname: Mehrara
authors.forename: Michael T
authors.initials: MT
authors.lastname: Longaker
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000077214:Becaplermin; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000235:genetics*; D005260:Female; D017102:Fracture Healing / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D008222:Lymphokines / Q000235:genetics*; D008297:Male; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010006:Osteoblasts / Q000187:drug effects*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D012886:Skull / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D016228:Endothelial Growth Factors; D008222:Lymphokines; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000077214:Becaplermin