Abstract
Purpose: :
Amniotic membrane (AM) and its extract (AME) have anti-angiogenic and anti-inflammatory actions. We speculate that hyaluronan (HA) complex such as HC-HA (the heavy chain (HC) of inter--trypsin inhibitor (II) complexed with HA) contributes to these effects and sought to purify and characterize this complex from AME.
Methods: :
AME was obtained from AM extracted in PBS. Subsequently, H fraction, which contains HC-HA complex, was purified from AME by two rounds of ultracentrifugation with CsCl/4M guanidine. The purity of H fraction was examined by SDS-PAGE with Coomassie blue staining. The size and quantity of HA in H fraction was determined by agarose gel electrophoresis followed by staining with Stains-All dye.HC-HA complex in H fraction was demonstrated by treatment of H fraction with hyaluronidase (HAase) or 0.05 N NaOH followed by Western blotting with anti-II antibody. The effects of H fraction on cell morphology and proliferation of HUVEC and RAW264.7 were shown by phalloidin staining, MTT assay, and BrdU labeling/staining.
Results: :
H fraction contains very little protein based on Coomassie blue staining. HA in H fraction was high molecular weight (> 6000-kDa) and covalently linked with HC, which was released from HA after HAase digestion or NaOH treatment. Inclusion of H fraction in the medium significantly reduced filamentous actin, and significantly suppressed proliferation in HUVEC using MTT assay (from 0.28 ± 0.02 to 0.11 ± 0.02, p=0.0004) and in RAW264.7 cells (from 0.82 ± 0.13 to 0.43 ± 0.04, p=0.0002). BrdU labeling and staining further confirmed such suppression in HUVEC (from 0.32±0.04 to 0.02±0.03, p=0.00005) and in RAW264.7 cells (p=0.03).
Conclusions: :
HC-HA complex in H fraction was a potential factor contributing to suppressing activation and proliferation in HUVEC and macrophages. These effects explain, at least partially, AM’s activity against angiogenesis and inflammation.
Keywords: neovascularization • proliferation • phosphorylation