title: Overexpression of VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis.
abstract: BACKGROUND
  Systemic sclerosis (SSc) is characterized by widespread microangiopathy, fibrosis, and autoimmunity. Despite the lack of angiogenesis, the expression of vascular endothelial growth factor A (VEGF) was shown to be upregulated in SSc skin and circulation; however, previous studies did not distinguish between proangiogenic VEGF(165) and antiangiogenic VEGF(165)b isoforms, which are generated by alternative splicing in the terminal exon of VEGF pre-RNA.
  
  
  OBJECTIVE
  We investigated whether VEGF isoform expression could be altered in skin and circulation of patients with SSc.
  
  
  RESULTS
  Here, we show that the endogenous antiangiogenic VEGF(165)b splice variant is selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF(165)b expression correlated with increased expression of profibrotic transforming growth factor-β1 and serine/arginine protein 55 splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. Circulating levels of VEGF(165)b were significantly higher in patients with SSc than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF(165)b than healthy MVECs. Transforming growth factor-β1 upregulated the expression of VEGF(165)b and serine/arginine protein 55 in both SSc and healthy MVECs. In SSc MVECs, VEGF receptor-2 was overexpressed, but its phosphorylation was impaired. Recombinant VEGF(165)b and SSc-MVEC-conditioned medium inhibited VEGF(165)-mediated VEGF receptor-2 phosphorylation and capillary morphogenesis in healthy MVECs. The addition of anti-VEGF(165)b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC-conditioned medium. Capillary morphogenesis was severely impaired in SSc MVECs and could be ameliorated by treatment with recombinant VEGF(165) and anti-VEGF(165)b blocking antibodies.
  
  
  CONCLUSIONS
  In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia.
authors.affiliation: Department of Anatomy, Histology and Forensic Medicine, University of Florence, Viale G.B. Morgagni 85, Florence, Italy. mirkomanetti@yahoo.it
authors.forename: Mirko
authors.initials: M
authors.lastname: Manetti
authors.forename: Serena
authors.initials: S
authors.lastname: Guiducci
authors.forename: Eloisa
authors.initials: E
authors.lastname: Romano
authors.forename: Claudia
authors.initials: C
authors.lastname: Ceccarelli
authors.forename: Silvia
authors.initials: S
authors.lastname: Bellando-Randone
authors.forename: Maria Letizia
authors.initials: ML
authors.lastname: Conforti
authors.forename: Lidia
authors.initials: L
authors.lastname: Ibba-Manneschi
authors.forename: Marco
authors.initials: M
authors.lastname: Matucci-Cerinic
mesh_terms: D017398:Alternative Splicing / Q000502:physiology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D020405:Dermis / Q000098:blood supply; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007511:Ischemia / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D016601:RNA-Binding Proteins; D012595:Scleroderma, Systemic / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D042561:Vascular Endothelial Growth Factor B / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C489470:SRSF6 protein, human; D053773:Transforming Growth Factor beta1; C467484:VEGFA protein, human; C467485:VEGFB protein, human; D042461:Vascular Endothelial Growth Factor A; D042561:Vascular Endothelial Growth Factor B; D000068103:Serine-Arginine Splicing Factors; D040301:Vascular Endothelial Growth Factor Receptor-2