Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-α-inhibitor (HC·HA) purified from extracts of human amniotic membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic ExtractA(70.1±6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMWHA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (1α1) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein TSG-6). This HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced macrophage apoptosis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.