title: Expression of VEGF isoforms by epiphyseal chondrocytes during low-oxygen tension is HIF-1 alpha dependent.
abstract: OBJECTIVE
  To establish the role of hypoxia and HIF-1 alpha for VEGF expression of murine epiphyseal chondrocytes. To analyze the effect of hypoxia on VEGF isoform expression.
  
  
  METHODS
  VEGF mRNA and VEGF isoform expression was investigated in epiphyses of murine newborns by in situ hybridization and real-time PCR. Further, epiphyseal chondrocytes were isolated from newborn mice with homozygous flanking of the HIF-1 alpha gene with lox-P sites. HIF-1 alpha was deleted by infection with adenovirus containing cre-recombinase. After chondrocytes reached confluency they were exposed to 0.5% or 20% oxygen, respectively. Total VEGF and VEGF isoform mRNA expression levels were measured by real-time PCR. Secreted VEGF protein was determined by ELISA.
  
  
  RESULTS
  VEGF mRNA signals were detected in the hypertrophic zone and in the center of the proliferative zone of the murine epiphysis, which is considered to be hypoxic. Real-time PCR revealed that VEGF(120)is the dominant isoform in vivo. In cultured epiphyseal chondrocytes strongly increased VEGF gene expression levels were detected after exposure to hypoxia. Furthermore, secretion of VEGF protein was significantly enhanced under 0.5% oxygen. Remarkably, functional inactivation of HIF-1 alpha abolished the hypoxic increase of VEGF expression in chondrocytes completely. Furthermore, the soluble isoforms VEGF(120)and VEGF(164)are the most abundantly expressed splice variants in chondrocytes exposed to low oxygen levels.
  
  
  CONCLUSIONS
  The data presented here clearly indicate that hypoxia is able to induce the synthesis of soluble VEGF isoforms by epiphyseal chondrocytes, most likely through stabilization of HIF-1 alpha. Thus it can be speculated that HIF-1 alpha is an essential prerequisite for hypoxic VEGF synthesis in the epiphysis, thereby contributing to the formation and invasion of blood vessels in long bone development.
authors.affiliation: Molecular Biology Section, Division of Biology, University of San Diego, CA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Cramer
authors.forename: E
authors.initials: E
authors.lastname: Schipani
authors.forename: R S
authors.initials: RS
authors.lastname: Johnson
authors.forename: B
authors.initials: B
authors.lastname: Swoboda
authors.forename: D
authors.initials: D
authors.lastname: Pfander
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D004838:Epiphyses / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017403:In Situ Hybridization; D051379:Mice; D020033:Protein Isoforms / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D042442:Vascular Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; D042442:Vascular Endothelial Growth Factors