The in vivo bioactivity of vascular endothelial growth factor/vascular permeability factor is independent of N-linked glycosylation.
The carbohydrate moieties of glycoprotein hormones or growth factor molecules may have a variety of effects that impact biological potency. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a 45 kD heparin-binding, endothelial cell (EC) specific mitogen with a putative N-linked glycosylation site. Recent studies have shown that VEGF/VPF may successfully augment collateral development in animal models of myocardial and hindlimb ischemia. The extent to which glycosylation of the 75 asparagine site affects the angiogenic properties of VEGF/VPF has not been studied in vivo. Specifically unaddressed to date is the concern that nonglycosylated VEGF/VPF may be less stable, and therefore characterized by a shorter half-life, reducing its utility for therapeutic angiogenesis. Accordingly, the purpose of this study was to investigate the extent to which posttranslational modification, specifically glycosylation, mofies the angiogenic properties of VEGF/VPF in vivo. Glycosylated (g+) recombinant human VEGF165 was purified from media conditioned by Chinese hamster ovary (CHO) cells. Nonglycosylated (g-) VEGF165 was expressed, purified and refolded from E. coli. The purity of both materials was assessed by silver-stained SDS/PAGE and characterized by the presence of a single amino terminal sequence as indicated by Edman degradation. Tryptic mapping by reverse-phase HPLC confirmed that the potential glycosylation site at 75 asparagine was occupied by N-linked carbohydrate for the Chinese hamster ovary-derived VEGF/VPF, but not for E. coli-derived VEGF/VPF. The mitogenic effects of Chinese hamster ovary-derived (g+) VEGF165 and E. coli-derived (g-) VEGF165 wre studied in vitro using microvascular EC. At concentrations of VEGF/VPF ranging from 10(-4) to 10(2) nM, both produced similar concentration-dependent effects on EC proliferation. For in vivo studies, (g-) (n = 8) and (g+) (n = 8) formulations of VEGF/VPF were administered to New Zealand white rabbits with unilateral hindlimb ischemia. For (g-) versus (g+) VEGF/VPF-treated groups, respectively, calf blood pressure ratio was 0.40 +/- 0.04 versus 0.37 +/- 0.04; angiographic score (of collateral vessels) was 0.37 +/- 0.04 versus 0.35 +/- 0.04; capillary density (capillaries/mm2) at necropsy was 246.9 +/- 21.5 versus 253.9 +/- 18.8; and tissue perfusion (colored microspheres) was 92.8 +/- 5.5 versus 90.30 +/- 13.47 (all p = ns). Moreover, intravascular Doppler-based analyses of resting, maximum, and endothelium-dependent flow was similar for (g-) and (g+) VEGF/VPF. These in vitro and in vivo findings establish that the potential for VEGF/VPF to stimulate therapeutic angiogenesis persists unaltered in the nonglycosylated state.
Walter
D H
DH
Department of Medicine (Cardiology), St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.
Hink
U
U
Asahara
T
T
Van Belle
E
E
Horowitz
J
J
Tsurumi
Y
Y
Vandlen
R
R
Heinsohn
H
H
Keyt
B
B
Ferrara
N
N
Symes
J F
JF
Isner
J M
JM
D000792:Angiography; D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D016466:CHO Cells; D002196:Capillaries; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000737:chemistry / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D006031:Glycosylation; D007866:Leg / Q000098:blood supply; D008222:Lymphokines / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D010449:Peptide Mapping; D011817:Rabbits; D011994:Recombinant Proteins; D012039:Regional Blood Flow / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors