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Shunsuke R. Sakurai, Hua He, Lorraine Siok May Chua, Scheffer C. Tseng; Characterization Of The Role Of PTX3 In Enhancing The Anti-angiogenic Action Of HC·HA Purified From The Chorion. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4881.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous work has shown that HC·HA, the complex formed from the heavy chains (HC) of inter-α inhibitor covalently linked to hyaluronan (HA), has anti-angiogenic effects in vitro when purified from amniotic membrane (AM) extract. The same purification method used on chorion extract yielded an HC·HA sample that was 25-fold more potent in HUVEC proliferation assays, and had a higher amount of total protein, including the known anti-angiogenic molecule PTX3. The purpose of this work is to complete the characterization of the protein content of the two formulations of HC·HA, and to identify the component responsible for the enhanced potency of chorion HC·HA.
HC·HA samples were purified from amniotic membrane and chorion extracts by two rounds of density-gradient ultracentrifugation with CsCl/4M guanidine HCl. Both extracts were subjected to a semi-quantitative multiplex array to identify angiogenic or angiostatic proteins. IαI was purified from pooled human plasma using a previously reported method. HC·HA was reconstituted in vitro from HMW Healon HA, purified IαI, and TSG-6, in the presence or absence of PTX3. The reconstituted HC-HA was characterized by Western Blot with HAase and NaOH digestion, and its anti-angiogenic action on HUVEC was measured by BrdU ELISA.
The proteomic profile array identified several key proteins in both amnion and chorion HC·HA, including PF4, IGFBP1, and PTX3. Of these, PTX3 was further investigated due to its known association with HC·HA in the cumulus-oocyte complex. Amnion and chorion HC·HA significantly inhibited HUVEC proliferation at concentrations of 25 µg/ml and 1 µg/ml, respectively (p<0.0001), while reconstituted HC·HA increased HUVEC proliferation (p<0.05). The addition of PTX3 during reconstitution at concentrations from 1-25 µg/ml negated the increase caused by reconstituted HC·HA, but did not cause any further inhibition. As expected, HMW HA showed no effect at this concentration (25 µg/ml).
These results collectively suggest that the HC·HA complex alone is not responsible for the anti-angiogenic effects of amnion and chorion HC·HA, but rather acts as a platform to deliver or enhance the action of other anti-angiogenic proteins. Because PTX3 was shown not to be effective in restoring full anti-angiogenic activity to reconstituted HC·HA, further characterization is needed to identify the protein responsible for the anti-angiogenic effects of amnion and chorion HC·HA.
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