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SuZhen Zhang, Hua He, Scheffer C. Tseng; Expression of IαI Family Proteins and Production of HC·HA by Human Amniotic Membrane Epithelial and Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4321.
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Recently we reported HC•HA, a covalent complex formed between heavy chains (HCs) of inter-α-inhibitor (IαI) and hyaluronan (HA) by catalytic action of TSG-6, is responsible for human amniotic membrane (AM)’s anti-inflammatory and anti-scarring actions. At the present time, the only known source of IαI is in the serum produced by the liver. We wonder if AM epithelial and stromal cells can also synthesize HCs, IαI, and HC•HA complex.
RT-PCR and Western blot were used to determine HC types in mRNAs and lysates of primary AM epithelial and stromal cells cultured in serum-free conditions and treated with or without siRNA to HC1 or TSG-6 siRNA. HC•HA complex was isolated from lysates of serum-free AM epithelial and stromal cells by two successive ultracentrifugations in the presence of CsCl and 4M guanidine HCl, and similarly characterized by Western blot with or without hyaluronidase (HAase) digestion.
Western blot analysis indicated that HC1, but not HC2, HC3, bikunin and TSG-6, was present in HC·HA complex purified from AM. AM epithelial and stromal cells expressed mRNAs and proteins of individual HC1, HC2, and HC3, bikunin, and TSG-6, of which the level was promoted by TNFα. As a result, these cells also produced IαI, which contained HC1, HC2 and bikunin linked by chondrointin sulfate. Knockdown of HC1 or TSG-6 by respective siRNA downregulated production of IαI expression in both AM epithelial and stromal cells. Western blot analysis confirmed the synthesis of HC•HA complex by AM epithelial and stromal cells as a high molecular weight band in the bottom of the loading well, which was eliminated after HAase digestion, resulting in a marked increase of the HC1 band.
HCs and bikunin are locally expressed by both AM epithelial and stromal cells to generate IαI. Through TSG-6, a HC•HA complex containing only HC1 is also produced. Thus, AM epithelial and stromal cells are likely a new source other than the liver to provide IαI and anti-inflammatory and anti-scaring HC•HA complex.
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