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E. Shay, H. He, S. C. G. Tseng; In vitro Anti-Angiogenic Effect of the Covalent Complex of Hyaluronan and the Heavy Chain of Inter--Inhibitor (HC·HA) Purified From the Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2814.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic membrane (AM) has potent anti-inflammatory, anti-scarring and anti-angiogenic actions. Our previous work has identified and purified a covalent complex formed between the heavy chain (HC) of inter-α-trypsin inhibitor covalent-complexed with hyaluronan (HA) termed HC·HA as one active component that exerts AM’s anti-inflammatory and anti-scarring actions. Thus we speculate that HC·HA may also exert a potent antiangiogenic action.
Amniotic membrane extract (AME) obtained from AM extracted in PBS was submitted to two rounds of ultracentrifugation with CsCl/4M guanidine HCl to HC·HA. The effects of HC·HA (from 0.2-50 µg/ml) on morphology, viability, proliferation, attachment, and morphogenesis of human umbilical vein endothelial cells (HUVEC) were examined by light microscopy and phalloidin staining, MTT assay and Live/Dead assay, BrdU staining and ELISA, fibronectin/collagen attachment assay, and Matrigel tube formation assay, respectively. These effects of HC·HA on angiogenesis were compared to HMW HA at the same HA concentration.
Inclusion of HC·HA at 5 µg/ml in the medium significantly altered cell morphology, evident in reduced filamentous actin, and suppressed cell viability by MTT assay (from 0.28 ± 0.02 to 0.11 ± 0.02, p=0.0004) and Live/Dead staining, while HMW HA had no effect. HC·HA significantly and dose-dependently inhibited HUVEC proliferation from 1 to 50 µg/ml (P<0.01), while HMW HA only significantly inhibited proliferation at 100 µg/ml (P<0.01). Cell attachment was significantly decreased only by 50 µg/ml HC·HA. 25 µg/ml HC·HA, but not HMW HA, significantly suppressed HUVEC tube formation.
These results collectively indicate that HC·HA complex suppresses in vitro angiogenesis of HUVEC through suppression of VEGF signaling and explains partially why AM is developmentally avascular and how AM clinically exerts anti-angiogenic action.
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