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H. He, E. Shay, S. C. G. Tseng; Inhibition of Spreading and Activation in Macrophages by HC•HA Complex Purified From Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1986.
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Human amniotic membrane extract (AME) suppresses the inflammatory responses in macrophages and one active component has recently been identified as HC•HA, a complex formed by a covalent linkage between heavy chains (HC) of inter-α-inhibitor and hyaluronic acid (HA). Herein, we investigated the effect of HC•HA on adhesion, spreading, and activation of macrophages.
Macrophage RAW264.7 cells were treated with HMW HA, AME or HC•HA (1-50 µg/ml HA) for 24 h, along with 1 µg/ml LPS. During 24 h treatment, the cell morphology was monitored, cell attachment was analyzed after 1-3 h, cell viability, spreading, and proliferation [bromodeoxyuridine (BrdU) was added 2 h before the cell cultivation was terminated] were measured. In a separate experiment, 2 µg of HMW HA or HC•HA was covalently coupled to each Covalink-NH 96 well, onto which cell adhesion and TNF-α levels secreted in the medium were measured 24 h after LPS treatment.
Treatment with 0.2 - 50 µg/ml HMW HA for 24 h did not cause a noticeable morphological change. In contrast, AME and HC•HA (1-50 µg/ml HA) dose-dependently caused macrophages to shrink and become round and smaller, with HC•HA being more potent than AME. Cell adhesion was affected by HC•HA, but not by HMW HA at 25 µg/ml. Cell viability and spreading were significantly inhibited by 5-50 µg/ml HC•HA (p < 0.01), but not by HMW HA. However, cell adhesion on to immobilized HC•HA was much higher than that on to immobilized HMW HA on Covalink-NH wells. TNF-α production was significantly downregulated by HC•HA (Ctrl: 7947 ± 135; HMW HA: 7723 ± 261; HC•HA: 5154 ± 227 pg/mg protein, p = 0.0002 for HC•HA vs Ctrl or HMW HA). Interestingly, 25 µg/ml HC•HA did not inhibit cell proliferation measured by BrdU ELISA and immunostaining.
The HC•HA complex purified from AME promotes adhesion, inhibits spreading and viability, down-regulates the inflammatory responses, but does not inhibit cell proliferation in macrophages. The data suggest that HC•HA might selectively eliminate the non-proliferating, activated, and differentiated macrophages that are responsible for promoting inflammatory responses.
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