title: Perlecan heparan sulfate proteoglycan is a critical determinant of angiogenesis in response to mouse hind-limb ischemia.
abstract: BACKGROUND
  Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown.
  
  
  METHODS
  Heparan sulfate (HS)-deficient perlecan (Hspg2(Δ3/Δ3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF)2 protein expression at days 2 and 28.
  
  
  RESULTS
  Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Δ3/Δ3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Δ3/Δ3) mice at day 28 (0.67 ± 0.12 vs 0.26 ± 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Δ3/Δ3) mice on day 28 (122 ± 30 cells vs 84 ± 34 cells per high-power field [HPF]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) were significantly decreased in the ischemic limbs of Hspg2(Δ3/Δ3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences.
  
  
  CONCLUSIONS
  These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.
authors.affiliation: Schulich Heart Center, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Beiping
authors.initials: B
authors.lastname: Qiang
authors.affiliation: Schulich Heart Center, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; Korea University Ansan Hospital, Ansan, Korea.
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lim
authors.affiliation: Schulich Heart Center, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Lekas
authors.affiliation: Keenan Research Centre in The Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Kuliszewski
authors.affiliation: Schulich Heart Center, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Rafael
authors.initials: R
authors.lastname: Wolff
authors.affiliation: Schulich Heart Center, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Azriel B
authors.initials: AB
authors.lastname: Osherov
authors.affiliation: Keenan Research Centre in The Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Rudenko
authors.affiliation: Keenan Research Centre in The Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada.
authors.forename: Howard
authors.initials: H
authors.lastname: Leong-Poi
authors.affiliation: University of Toronto, Toronto, Ontario, Canada; Toronto General Research Institute, Toronto, Ontario, Canada.
authors.forename: Hossein
authors.initials: H
authors.lastname: Noyan
authors.affiliation: University of Toronto, Toronto, Ontario, Canada.
authors.forename: Mansoor
authors.initials: M
authors.lastname: Husain
authors.affiliation: Karolinska Institute, Stockholm, Sweden.
authors.forename: Kiet
authors.initials: K
authors.lastname: Tran
authors.affiliation: Karolinska Institute, Stockholm, Sweden.
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
authors.affiliation: Karolinska Institute, Stockholm, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Hedin
authors.affiliation: Karolinska Institute, Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Tran-Lundmark
authors.affiliation: Schulich Heart Center, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada. Electronic address: bradley.strauss@sunnybrook.ca.
authors.forename: Bradley H
authors.initials: BH
authors.lastname: Strauss
mesh_terms: D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D004195:Disease Models, Animal; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D019812:Heparan Sulfate Proteoglycans / Q000502:physiology*; D006614:Hindlimb / Q000098:blood supply*; D007150:Immunohistochemistry; D007511:Ischemia / Q000150:complications / Q000378:metabolism* / Q000473:pathology; D017078:Laser-Doppler Flowmetry; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: D019812:Heparan Sulfate Proteoglycans; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D016222:Fibroblast Growth Factor 2; C071927:perlecan