title: Slit2 modifies VEGF-induced angiogenic responses in rabbit skeletal muscle via reduced eNOS activity.
abstract: OBJECTIVE
  Slit2 is a possible modulator of VEGF-induced angiogenesis, but its effects have not been tested on large animal models. We studied the effect of Slit2 on therapeutic angiogenesis induced by VEGF receptor 2 (VEGFR2) ligands Vammin and VEGF-D(ΔNΔC) in vivo in rabbit skeletal muscles. The Slit2 target genes were also studied by RNA sequencing in endothelial cells.
  
  
  RESULTS
  Adenoviral intramuscular gene transfers were performed into New Zealand White rabbit hindlimbs. Confocal and multiphoton microscopes were used for blood vessel imaging. Signaling experiments and gene expression analyses were performed to study mechanisms of Slit2 action. Slit2 decreased VEGFR2-mediated vascular permeability. Slit2 also reduced VEGFR2-mediated increase in blood perfusion and capillary enlargement, whereas sprouting of the capillaries was increased. Slit2 gene transfer alone did not have any effects on vascular functions or morphology. VEGFR2 activation was not affected by Slit2, but eNOS phosphorylation was diminished. The transcriptome profiling showed Slit2 down-regulating angiogenesis-related genes such as Nuclear receptor subfamily 4 group A member 1 (NR4A1) and Stanniocalcin-1 (STC-1) as well as genes related to endothelial cell migration and vascular permeability.
  
  
  CONCLUSIONS
  Combining Slit2 with VEGFs adjusts VEGFR2-mediated angiogenic effects into a more physiological direction. This possibly allows the use of higher VEGF vector doses to achieve a more widespread vector and VEGF distribution in the target tissues leading to a better therapeutic outcome while reducing excess vascular permeability.
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Tiina
authors.initials: T
authors.lastname: Nieminen
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Pyry I
authors.initials: PI
authors.lastname: Toivanen
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Johanna P
authors.initials: JP
authors.lastname: Laakkonen
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Tommi
authors.initials: T
authors.lastname: Heikura
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Minna U
authors.initials: MU
authors.lastname: Kaikkonen
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Kari J
authors.initials: KJ
authors.lastname: Airenne
authors.affiliation: Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland Science Service Center and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland seppo.ylaherttuala@uef.fi.
authors.forename: Seppo
authors.initials: S
authors.lastname: Ylä-Herttuala
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D000818:Animals; D002465:Cell Movement / Q000235:genetics; D002478:Cells, Cultured; D005260:Female; D015316:Genetic Therapy / Q000379:methods; D006614:Hindlimb / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011817:Rabbits; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D000098552:Slit Homolog 2 Protein
chemical_list: D043925:Angiogenesis Inducing Agents; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D000098552:Slit Homolog 2 Protein
keywords: Angiogenesis; Slit2; Vascular endothelial growth factor; eNOS