Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-β1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-β1) and collagen gel contraction (TGF-β1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (β-catenin, LEF1) and TGF-β (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3’s ability to inhibit PVR formation warrants evaluation in an animal model.
Chronic graft-versus-host disease (cGVHD) is a major complication of hematopoietic stem cell transplantation. Dry eye disease is the prominent ocular sequel of cGVHD and is caused by excessive inflammation and fibrosis in the lacrimal glands. Heavy chain-Hyaluronan/Pentraxin 3 (HC-HA/PTX3) is a complex purified from human amniotic membrane (AM) and known to exert anti-inflammatory and anti-scarring actions. In this study, we utilized a mouse model of cGVHD to examine whether HC-HA/PTX3 could attenuate dry eye disease elicited by cGVHD. Our results indicated that subconjunctival and subcutaneous injection of HC-HA/PTX3 preserved tear secretion and conjunctival goblet cell density and mitigated inflammation and scarring of the conjunctiva. Such therapeutic benefits were associated with suppression of scarring and infiltration of inflammatory/immune cells in the lacrimal glands. Furthermore, HC-HA/PTX3 significantly reduced the extent of infiltration of CD45+ CD4+ IL-17+ cells, CD45+ CD34+ collagen I+ CXCR4+ fibrocytes, and HSP47+ activated fibroblasts that were accompanied by upregulation of collagen type Iα1, collagen type IIIα1 and NF-kB in lacrimal glands. Collectively, these pre-clinical data help prove the concept that subcutaneous and subconjunctival injection of HC-HA/PTX3 is a novel approach to prevent dry eye disease caused by cGVHD and allow us to test its safety and efficacy in future human clinical trials.
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