The extracellular matrix complexity of idiopathic epiretinal membranes and the bilaminar arrangement of the associated internal limiting membrane in the posterior retina

Abstract

Purpose To study the composition of the internal limiting membrane (ILM) of the retina, the extracellular matrix (ECM) of idiopathic epiretinal membranes (iERMs), and the relationships occurring between the two membranes. Methods Forty-six iERMs, 24 of them associated with the ILM, were collected and included in this study. The investigation has been carried out by immunofluorescence and confocal microscopy on glutaraldehyde- and osmium-fixed epon-embedded samples and on frozen samples. Sections were double or triple labelled with antibodies against vimentin; collagens I, III, IV, α5(IV), and VI; laminin 1 + 2; laminin α2-, α4-, α5-, β1-, β2-, β3-, γ1-, and γ2-chains; entactin; and fibronectin. Results iERM thickness was not uniform. Almost 14% of iERMs showed thickenings due to folding of their ECM component under the cell layer. The vitreal side of iERMs was often shorter than the attached ILM. In this case, the ILM resulted folded under the iERM. ILMs contained laminin 111; laminin α2-, α5-, β1-, β2-, and γ1-chains; entactin; collagens I; α5(IV); [α1(IV)]2α2(IV); and VI. Laminins, entactin, and α5(IV) were gathered on the retinal half of the ILM, whereas collagens [α1(IV)]2α2(IV) and I were restricted to the vitreal side. Collagen VI was detected on both sides of the ILM. iERMs expressed laminin 111, collagens III, [α1(IV)]2α2(IV) and VI, entactin, and fibronectin. Entactin co-localized with laminins and collagen IV. Conclusions Analysis of laminins and collagen chain expression indicates that ILM contains laminin 111 (former laminin 1), laminin 521 (former laminin 11), laminin 211 (former laminin 2), collagen [α1(IV)]2α2(IV), and collagen α3(IV)α4(IV)α5. In contrast, iERMs express only collagen [α1(IV)]2α2(IV) and laminin 111. In addition, both iERMs and ILMs contain entactin. The presence of three major constituents of the basement membranes co-localized together in iERMs is suggestive for a deranged process of basement membrane formation which fails to assemble properly. In view of the many interactions occurring among its proteins, the ECM of either the iERMs or the ILMs can account for their reciprocal adhesiveness. In addition, the peculiar deposition of the ECM observed in some samples of iERM is suggestive for its involvement in the formation of macular puckers.

Untangling the Extracellular Matrix of Idiopathic Epiretinal Membrane: A Path Winding among Structure, Interactomics and Translational Medicine

“…Collagen IV is a constant constituent of iERMs [ 12 , 18 , 19 , 20 , 27 ], as well as of ERMs from PVR eyes [ 22 , 23 , 35 ]. A diffuse and considerable intense staining, which does not fit with a mere basement membrane-related immunoreactivity, has been reported by many investigators [ 12 , 19 , 20 , 21 , 27 ]. A study of collagen IV distribution in iERMs has been recently carried out by confocal microscopy showing that this collagen is deposited interstitially, intermingled with collagen I, with a staining intensity that progressively increases towards the vitreous side of the membrane.…”

“…Collagen I has been repetitively reported in iERMs [ 12 , 18 , 19 , 20 , 21 ], and it is certainly shared with other types of ERMs [ 22 , 23 , 24 ]. As collagen I is a hallmark of all fibrotic responses, one might expect to find it in all ERMs, and this was the case in many studies [ 12 , 20 , 21 ]. However, some investigators also observed a certain number of iERMs apparently lacking collagen I [ 18 , 19 ].…”

“…When the ratio of the two collagens is in favour of collagen III, collagen fibrils assume the aspect of reticular fibres [ 29 ], but when the ratio is in favour of collagen I, fibrils are plainly referred to as collagen I fibrils. Although collagen III in iERMs has received little attention, when searched, it was found with the same frequency as collagen I [ 18 , 21 ]. Similar results have been observed in ERMs from PVR eyes [ 22 , 23 ], where the areas occupied by collagen III and collagen I on histologic sections matched [ 23 ].…”

“…Transmission electron microscopy confirms interstitial condensations of collagen IV-immunoreactive material that are frequently infiltrated by collagen I fibrils ( Figure 1 ). In this respect, collagen IV-immunoreactive areas mostly also co-localize with collagen III and collagen VI [ 12 , 20 , 21 ]. Anti-collagen IV antibody-labelled material can even be observed within intracytoplasmic vesicles or lysosome-like bodies, suggesting active remodelling of the ECM by ERM cells.…”

“…Collagen IV α-chains occur in six different variants (α1–α6) which assemble to give rise to three collagen IV isoforms: [α1(IV)] 2 α2(IV), α3(IV)α4(IV)α5(IV) and [α5(IV)] 2 α6(IV). So far, the only isoform detected in iERMs by immunohistochemistry is the [α1(IV)] 2 α2(IV) [ 21 ]. Proteomic analysis not only corroborates the presence of the α1(IV) and α2(IV) collagen chains but also allowed the detection of α3(IV), α4(IV) and α5(IV) collagen chains [ 13 ].…”