Heparin octasaccharides inhibit angiogenesis in vivo.
BACKGROUND
In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2.


METHODS
Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity.


RESULTS
Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing > or =16 saccharide residues were investigated.


CONCLUSIONS
Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.
Hasan
Jurjees
J
Cancer Research UK, Department of Medical Oncology, Manchester, UK.
Shnyder
Steven D
SD
Clamp
Andrew R
AR
McGown
Alan T
AT
Bicknell
Roy
R
Presta
Marco
M
Bibby
Michael
M
Double
John
J
Craig
Steven
S
Leeming
David
D
Stevenson
Kenneth
K
Gallagher
John T
JT
Jayson
Gordon C
GC
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D045744:Cell Line, Tumor; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006493:Heparin / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000098:blood supply / Q000473:pathology / Q000517:prevention & control*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009844:Oligosaccharides / Q000494:pharmacology*; D010314:Partial Thromboplastin Time; D023041:Xenograft Model Antitumor Assays / Q000295:instrumentation / Q000379:methods
D020533:Angiogenesis Inhibitors; D000925:Anticoagulants; D009844:Oligosaccharides; D016222:Fibroblast Growth Factor 2; D006493:Heparin