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Fu Li, Ying-Ting Zhu, Hua He, Su-Zhen Zhang, Scheffer Tseng; HC-HA Complex Purified from Amniotic Membrane Exerts Anti-scarring Effect by Suppressing TGFβ1 but Activating TGFβ3 Signaling in Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2068.
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© ARVO (1962-2015); The Authors (2016-present)
Experimental and clinical studies support an anti-scarring therapeutic action by cryopreserved amniotic membrane (AM). Our recent studies demonstrated that heavy chain-hyaluronan complex (HC-HA) is uniquely produced by and can be purified from AM and suppresses the TGF-β1 promoter activity in human corneal fibroblasts. Herein, we further characterize this HC-HA’s anti-scarring effect.
Human corneal fibroblasts were seeded on plastic with or without immobilized HC-HA in DMEM or DMEM+10% FBS for 48 h, or DMEM for 24 h and then treated with 10 ng/ml TGFβ1 for 24 h. Real-time qPCR was used to measure mRNA levels of TGFβ family members. Immunostaining was performed to monitor α-smooth muscle actin ( α-SMA) expression and SMAD2/3 signaling.
On plastic as reported, TGFβ1 mRNA was increased 2-fold by addition of FBS and 4-fold by addition of TGFβ1. In contrast, on HC-HA, TGFβ1 mRNA was not increased by FBS but dramatically decreased 8-fold by addition of TGFβ1. In parallel, on plastic, TGFβ3 mRNA was not increased by FBS, but increased 2-fold by TGFβ1. On HC-HA, TGFβ3 mRNA was increased 3-fold than plastic, and 2-fold and 5-fold after addition of FBS and TGFβ1, respectively. TGFβ2 mRNA was not affected by any treatments when cells were seeded on plastic or HC-HA. In addition, HC-HA inhibited SMAD2/3 nuclear translocation and substantially reduced cytoplasmic α-SMA normally upregulated by TGFβ1.
Collectively, these results indicate that HC-HA purified from AM is responsible for AM’s anti-scarring action by marked downregulation of TGFβ1 but upregulation of TGFβ3, which is known to counteract TGF-β1 signaling. Furthermore, such an anti-scarring effect is more apparent under the challenge of TGF-β1 or serum, during which time HC-HA also suppresses Smad2/3 signaling, and expression of α-SMA.
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