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H. He, W. Li, D. Y. Tseng, A. J. Day, S. C. G. Tseng; Characterization of Hyaluronan and Its Binding With II and TSG-6 in AM Extracts. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5561.
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© ARVO (1962-2015); The Authors (2016-present)
The abundant hyaluronan (HA) is present in AM stroma, but it remains unknown whether HA is covalently linked with any proteins and contributes to AM’s activity against scarring.
Cryopreserved human AM was successively extracted with buffers containing increasing salt concentrations: 150 mM NaCl (A), 1 M NaCl (B), and 4 M guanidine-HCl (C). Additionally, AM was extracted by PBS (P) for biochemical and biological assays. The amount and sizes of HA in these AM extracts were determined by HA ELISA and agarose gel electrophoresis analyses, respectively. The binding of inter--trypsin inhibitor (II) or tumor necrosis factor-stimulated gene-6 (TSG-6) with HA in AM extracts was examined with hyaluronidase (HAase) digestion or NaOH treatment followed by Western blotting. The suppression of TGF-β1 gene expression by Extract P was measured with a luciferase reporter assay.
HA was present in all four AM extracts, but was mostly (70.1 ± 6.0 %) in Extract A, had an average molecular weight (MW) about 6x106 Daltons (Da), and was covalently linked with heavy chains (HCs) of II via a NaOH-sensitive bond. This complex of HC-HA was also more abundant in Extract A. Bikunin (or the light chain of II) and TSG-6 were mainly present in Extracts A and C, and not covalently linked with HA. A TSG-6 immunoreactive 50-kDa band was mostly present in Extract A. Extract P, similar to Extract A in containing HMW HA covalently linked with HCs of II, suppressed TGF-β1 promoter activation. This suppression was not mimicked by HMW HA alone and was abolished by digestion with HAase or heat treatment.
HMW HA in AM is predominantly extracted by a low salt buffer and presents as a complex covalently linked with II. This complex may contribute to the suppression of TGFβ1 promoter activation and the AM’s anti-scarring action.
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