HC-HA/PTX3 Purified From Human Amniotic Membrane Reverts Human Corneal Fibroblasts and Myofibroblasts to Keratocytes by Activating BMP Signaling

Abstract

Purpose Fibrosis or scarring is a pathological outcome of wound healing and is characterized by terminally differentiated myofibroblasts. Heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) is a unique matrix component purified from amniotic membrane that exerts an anti-inflammatory effect. Herein, we investigate whether HC-HA/PTX3 can also exert an antiscarring effect. Methods Human corneal fibroblasts and myofibroblasts were seeded on plastic, immobilized HA or HC-HA/PTX3 or on plastic with or without soluble HA and HC-HA/PTX3 in DMEM+10% FBS, with or without AMD3100 or SB431542 in DMEM+ITS with or without transforming growth factor–β1 (TGF-β1). Transcript expression of keratocyte and signaling markers was determined by RT-qPCR. Immunostaining was performed to monitor cytolocalization of signaling markers and α-SMA. Western blotting was used to measure relative protein level. Results Human corneal fibroblasts and myofibroblasts cultured in or on HC-HA/PTX3, but not HA, were refrained from cytoplasmic expression of αSMA and nuclear translocation of pSMAD2/3 when challenged with exogenous TGF-β1. Such an antiscarring action by suppressing canonical TGF-β1 signaling was surprisingly accompanied by phenotypic reversal to keratocan-expressing keratocytes through activation of BMP signaling. Further investigation disclosed that such phenotypic reversal was initiated by cell aggregation mediated by SDF1-CXCR4 signaling highlighted by nuclear translocation of CXCR4 and upregulation of CXCR4 transcript and protein followed by activation of canonical BMP signaling. Conclusions These findings collectively provide mechanistic understanding explaining how amniotic membrane transplantation exerts an antiscarring action. In addition, HC-HA/PTX3 and derivatives may be developed into a new biologic to treat corneal blindness caused by stromal scar or opacity in the future.

HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors

“…The rejuvenation of senescent LNC by HC‐HA/PTX3 involves a reprogramming (de‐differentiation) process, which is highlighted by early “morphological” cell aggregation that uniquely occurs in P4 LNC 38 and P10 LNC (Figure 2). Such aggregation also occurs as an early event in the reprogramming of human corneal fibroblasts and myofibroblasts into keratocytes, 37 yet it does not occur in human skin and tenon fibroblasts (Supplemental data Figure S5). Although cell aggregation is similarly promoted by HC‐HA/PTX3, restoration of nuclear Pax6 staining occurs in P10 LNC but not human corneal fibroblasts (Supplemental data Figure S5).…”

“…We, thus, envision a different mechanism by which HC‐HA/PTX3 activates CXCR4‐mediated signaling through the nuclear translocation of CXCR4. Because cell aggregation and nuclear translocation of CXCR4 also occur during the reprogramming of human corneal fibroblasts into keratocytes, 37 future studies are needed to delineate the mechanism of how HC‐HA/PTX3 promotes nuclear translocation of CXCR4 in LNC and human corneal fibroblasts.…”

“…Furthermore, CXCR4‐mediated signaling, but not BMP signaling, is pivotal in the reprogramming of P10 LNC. Although the reprogramming of human corneal fibroblasts into keratocytes by HC‐HA/PTX3 also involves cell aggregation mediated by CXCR4‐signaling followed by activation of BMP signaling, both CXCR4‐signaling and BMP signaling are required for reprogramming of human corneal fibroblasts into keratocytes 37 . Further studies are warranted to delineate the action mechanism of HC‐HA/PTX3 in reprogramming differentiated cells into progenitor cells and will shed light upon its potential role as a surrogate niche matrix in regenerative medicine, particularly for stem cell‐based therapies.…”

“…HC‐HA/PTX3 has been shown to exert anti‐inflammatory actions that extend from innate immune responses, through facilitating apoptosis of stimulated neutrophils and polarizing M2 macrophages, to adaptive immune responses, by suppressing the activation of Th1 and Th17 lymphocytes to downregulate alloreactive immune responses 35,36 . In addition, HC‐HA/PTX3 reverts human corneal fibroblasts and myofibroblasts to keratocytes by inducing cell aggregation mediated by CXCR4 signaling followed by activation of BMP signaling 37 . Different from 3D MG, in vitro reunion of LEPC and LNC on immobilized HC‐HA/PTX3 promotes cell aggregation and sphere growth highlighted by quiescence of LEPC by activating BMP and noncanonical Wnt (PCP) signaling in LNC 38 .…”

Visual Acuity and Number of Amniotic Membrane Layers as Indicators of Efficacy in Amniotic Membrane Transplantation for Corneal Ulcers: A Multicenter Study

“…This tissue has multiple biological properties, including the induction of cell proliferation, reduction of neovascularization, anti-scarring properties, increasing migration of cells such as keratinocytes, pool corneal regeneration, and little or no immunogenicity [ 7 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. Heavy chain hyaluronan/pentraxin 3 (HC-HA/PTX3), a matrix component of AM, is a key factor responsible for the aforementioned AM’s properties [ 16 , 17 , 21 ]. These properties favor its use in ophthalmological pathologies, such as persistent corneal ulcers (neurotrophic, post-herpetic) [ 7 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ], descemetoceles [ 29 ], perforations [ 7 , 14 , 26 , 30 , 31 , 32 ], and chemical burns [ 7 , 14 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ].…”