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H. He, W. Li, S.J. Zhang, D. Tseng, A.J. Day, S.C. G. Tseng; Role of Hyaluronan, Inter–Alpha–Trypsin Inhibitor, and TSG–6 Complex in Amniotic Membrane in Inhibiting TGF–Beta Transcription . Invest. Ophthalmol. Vis. Sci. 2006;47(13):599.
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© ARVO (1962-2015); The Authors (2016-present)
Clinically, amniotic membrane (AM) transplantation has consistently been shown to suppress inflammation and scarring on the ocular surface. Hyaluronan (HA), a known component of AM stroma, is covalently linked with heavy chains (HC) of inter–α–trypsin inhibitor (IαI), which occurs via a TSG–6–mediated mechanism, in the cumulus matrix of ovulated oocytes and in inflamed synovial fluids. Both HC–HA and TSG–6 have been implicated in suppressing inflammation. We thus sought to determine whether AM stroma matrix might contain HC–HA complexes and TSG–6 and whether such a HA–containing complex may suppress TGF–ß signaling.
Human AM was subjected to HABP–immunohistochemistry (for detecting HA) and immunostaining with an antibody to IαI, and sequentially extracted with 150 mM NaCl, 1 M NaCl, and 4 M guanidine HCl buffers. These three protein extracts with or without 50 U/ml hyaluronidase digestion were subjected to agarose gel electrophoresis and to Western blot analyses using antibodies to IαI and TSG–6. Water–soluble AM stromal extracts with or without hyaluronidase digestion, heat denaturation, and different MW cut offs, were assayed for their abilities of suppressing TGF–ß1 promoter activity in cultured human corneal fibroblasts.
Both HA and IαI were immunolocalized in the AM stroma. Western blot showed free light and heavy IaI chains in 150 mM NaCl extracts, and HC–HA complex in all three extracts. In contrast, TSG–6 was present only in 150 mM NaCl extracts and its amount was not changed by hyaluronidase digestion, indicating that TSG–6 was not covalently bound to HA. TGF–ß promoter activity was dose–dependently suppressed by water–soluble AM stromal extracts, but not by HA alone. Such suppression was lost when the extract was heated at 90 °C for 10 min or treated with hyaluronidase at 37 °C for 1 h, but preserved in the retentate through 30–, 50–, and 100–kDa cutoff membrane filtration.
AM stroma contains HC–HA and TSG–6, which can be extracted by a low salt buffer, and the HC–HA complex may be responsible for suppressing TGF–ß1 transcription and inflammation.
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