Co-Expression of Podoplanin and CD44 in Proliferative Vitreoretinopathy Epiretinal Membranes

Abstract

Epiretinal membranes (ERMs) are sheets of tissue that pathologically develop in the vitreoretinal interface leading to progressive vision loss. They are formed by different cell types and by an exuberant deposition of extracellular matrix proteins. Recently, we reviewed ERMs' extracellular matrix components to better understand molecular dysfunctions that trigger and fuel the onset and development of this disease. The bioinformatics approach we applied delineated a comprehensive overview on this fibrocellular tissue and on critical proteins that could really impact ERM physiopathology. Our interactomic analysis proposed the hyaluronic-acid-receptor cluster of differentiation 44 (CD44) as a central regulator of ERM aberrant dynamics and progression. Interestingly, the interaction between CD44 and podoplanin (PDPN) was shown to promote directional migration in epithelial cells. PDPN is a glycoprotein overexpressed in various cancers and a growing body of evidence indicates its relevant function in several fibrotic and inflammatory pathologies. The binding of PDPN to partner proteins and/or its ligand results in the modulation of signaling pathways regulating proliferation, contractility, migration, epithelial-mesenchymal transition, and extracellular matrix remodeling, all processes that are vital in ERM formation. In this context, the understanding of the PDPN role can help to modulate signaling during fibrosis, hence opening a new line of therapy.

Keywords: cell migration; cluster of differentiation 44 (CD44); epiretinal membrane (ERM); fibrosis; interactomics; podoplanin (PDPN); proliferative vitreoretinopathy; transdifferentiation.

Figure 1

(a) Immunolocalization of PDPN in ERM cells showing expression of PDPN in different cells: VIM⁻/CK⁻, VIM⁻/CK⁺, VIM⁺/CK⁺ (upper row, arrows), VIM⁻/GFAP⁻, VIM⁺/GFAP⁻, and VIM⁻/GFAP⁺ (lower row, arrows). (b) Immunolocalization of CD44 in ERMs showing CD44 expression in both VIM⁺/CK⁺ and VIM⁺/CK⁻ cells (arrows). Scale bar 20 μm.

Figure 2

(a) Immunofluorescence of PDPN and CD44 showing co-expressing cells (arrows). Scale bar 20 μm. (b) Real-time qPCR on 9 patients (numbered from 1 to 9 on the x axis) confirmed PDPN and CD44 co-expression in all analyzed ERMs. Values are expressed as fold change with respect to one arbitrarily chosen ERM specimen that was used as reference sample for all the reactions (patient 1).

Figure 3

PDPN hub added to the [10]-DIN, previously obtained by processing 141 selected factors from ERM-identified proteins [26].

Figure 4

Enlargements of PDPN/[10]-network, in trace mode visualization, highlighting: (a) the 21 non-experimental factors added by the software to get PDPN into the net. These proteins form the FNI of PDPN and link it to at least one of the net experimental nodes; (b) the original net node HMGB1 that modulates the majority of the FNI proteins; (c) FNI proteins shared by EGFR and PDPN; and (d) CD44 and PDPN shared protein interactors from their FNIs. To visualize FNI proteins shared by PDPN and other net nodes, the trace mode visualization was centered on both PDPN and original net nodes that resulted as the principal interactors with the PDPN FNI (i.e., HMGB1, EGFR, and CD44). For this reason, some nodes also not belonging to the PDPN FNI (but to HMGB1, EGFR, and CD44 FNI) and not acting as a molecule bridge between PDPN and original nodes are visible in (b–d) panels.

Figure 5

Enlargements of YAP1/PDPN/[10]-network, in trace mode visualization, highlighting: (a) the YAP1 FNI, including HMGB1 and 13 factors necessary for YAP1 interaction with PDPN/[10]-network nodes; (b) all the 13 proteins from the YAP1 FNI are shared with the PDPN FNI; (c) YAP1 FNI proteins shared with CD44; and (d) YAP1 and EGFR FNI common proteins. To visualize FNI proteins shared by YAP1 and other net nodes, the trace mode visualization was centered on both YAP1 and PDPN/[10]-network nodes that resulted as the principal interactors with the YAP1 FNI (i.e., PDPN, CD44, and EGFR). For this reason, some nodes also not belonging to the YAP1 FNI (but to PDPN, CD44, and EGFR FNI) and not acting as a molecule bridge between YAP1 and original nodes are visible in (b–d) panels.

Figure 6

(a) Real-time qPCR on nine patients (x axis) revealed HMGB1 expression in all ERMs analyzed. Values are expressed as fold changes with respect to one arbitrarily chosen ERM specimen that was used as a reference sample for all the reactions (patient 1). (b) Immunofluorescence on ERMs with SOX2, STAT3, and YAP1 antibodies showing cytoplasmic and faint nuclear staining (arrows). Scale bar 10 μm.