Factor Xa and thrombin induce epithelial to mesenchymal transition by retinal pigment epithelial cells via PDGF-signaling

Abstract

Purpose: Proliferative vitreoretinopathy (PVR) is a difficult to treat inflammatory fibrotic disorder complicating retinal detachment. Understanding of the processes involved in PVR is incomplete. However, proliferation, epithelial to mesenchymal transition (EMT) and secretion of cytokines and collagen by RPE cells are considered to be central in PVR development. Because breakdown of the outer blood-retinal barrier is one of the first events in PVR development we hypothesize that coagulation proteins induce pro-inflammatory and fibrotic changes in RPE and as such play an important role in PVR development.

Methods: RPE cells were stimulated with factor Xa (1 U/ml) or thrombin (5 U/ml). Changes in cytokine, growth factor, α-smooth muscle actin (α-SMA), ZO-1 and collagen expression levels were determined by RQ-PCR, ELISA and/or fluorescence microscopy. Involvement of protease-activated receptor (PAR) subtypes as well as NF-κB and PDGF signaling was examined using specific antagonists and inhibitors.

Results: Factor Xa and thrombin both induced pro-inflammatory and pro-fibrotic mediator expression levels like GM-CSF, IL-6, IL-8, MCP-3, PDGF-AA and PDGF-BB by RPE cells. Blocking studies revealed that these effects were mediated via PAR1 induced NF-κB activation. Additional experiments showed that factor Xa and thrombin downregulate ZO-1 and upregulate α-SMA and collagen expression. Blocking experiments showed that induced α-SMA and collagen expression by factor Xa and thrombin is inhibited when PDGF-signaling is blocked.

Conclusions: The coagulation cascade proteins factor Xa and thrombin stimulate the production of pro-inflammatory and pro-fibrotic mediators by RPE cells via PAR1 induced NF-κB activation. Factor Xa and thrombin induce the expression of mesenchymal markers like α-SMA and collagen in a PDGF dependent manner. These data demonstrate that factor Xa and thrombin may contribute to PVR-associated changes in RPE cell behaviour.

Keywords: 512 EMT (epithelial mesenchymal transition) • 490 cytokines/chemokines • 701 retinal pigment epithelium