BritishJ7ournalofOphthalmology 1993; 77: 238-242

Origin of fibronectin in epiretinal membranes ofproliferative vitreoretinopathy and proliferativediabetic retinopathy

Salvatore Grisanti, Klaus Heimann, Peter Wiedemann

AbstractFibronectins, high molecular multifunctionalglycoproteins of the extracellular matrix andplasma, have been a popular area of researchin the pathogenesis of proliferative disordersof the retina. Several immunohistochemicalstudies have revealed that fibronectin is amajor constituent ofepiretinal membranes andthat the cell types involved in proliferativeintraocular disorders may synthesise it. How-ever, owing to the fact that plasma and cellularfibronectin are similar in their overall structure,the origin of fibronectin in epiretinal mem-branes has not yet been clearly defined. In thisstudy, we used two monoclonal antibodies:FN-3, which recognises an extra domainpresent in the cellular but not plasma form offibronectin; and FN-4, which reacts with anantigenic site on both plasma and cellularfibronectin. In 37 epiretinal membranesobtained from eyes with proliferative vitreo-retinopathy and proliferative diabetic retino-pathy, we demonstrated the presence ofcellular fibronectin, thus indicating localproduction. The significantly stronger andpositive immunostain with FN-4 in the samespecimens suggests the colocalisation ofplasma fibronectin, that may be derived fromthe breakdown of the blood-retinal barrier andtrapped in membranes during their formation.In pathological vitreous we demonstrated bothtypes of fibronectin by western blot analysis.(BrJ Ophthalmol 1993; 77: 238-242)

Department ofVitreoretinal Surgery,University Eye HospitalCologne, Cologne,GermanyS GrisantiK HeimannP WiedemannCorrespondence to:Dr P Wiedemann, UniversityEye Hospital Cologne, Joseph-Stelzmann-Strasse 9, D-5000Cologne, Germany.Accepted for publication9 December 1992

The uncontrolled growth and proliferation ofseveral types ofnon-neoplastic cells at the vitreo-retinal interface is the common final pathway ofthe eye's reaction towards vitreoretinal alteration.In proliferative vitreoretinopathy (PVR) thedevelopment of tractional membranes leading toa significant and definitive visual loss seems to becaused by a complex series of cellular andbiochemical interactions similar to the pro-liferative fibroblastic phase of physiologicalwound healing. Tractional epiretinal membranesalso develop during the course of proliferativediabetic retinopathy (PDR).Fibronectins constitute a class of high

molecular weight proteins (440 kD) which arewidely present on the cell surface of several typesof cells, in basement membranes, in stromalmatrices, and in plasma.' The multiple biologicalfunctions of these glycoproteins result from theirability to promote cell migration adhesion anddifferentiation, to enhance the synthesis of cyto-skeletal proteins and stabilisation of the extra-cellular matrix, and finally to participate in the

process of phagocytosis, wound healing, 'andblood coagulation.23 It is also well known thatfibronectins have a domain with special affinityto collagen, heparin, fibrin, fibronogen,hyaluronic acid, and complement factor Clq.'These interactions may be of particularimportance in the development of contractilemembranes at the vitreoretinal interface in PVRand PDR as they allow the cells to attach to thematrix.

Research has focused on the involvement offibronectins in proliferative intraocular disordersas they were found to be chemotactic for theretinal pigment epithelial (RPE) cells,4 present invitreous aspirates from patients with PVR andPDR obtained during vitrectomy,56 andidentified as the major constituent of youngepiretinal membranes.7 8 9 However, neitherthese findings, which introduced fibronectin asan important pathogenetic factor into the diseaseconcept, nor the fact that the major cell typesinvolved in PVR and PDR may synthesisefibronectin,3l01' could clearly define the origin offibronectin in epiretinal membranes.

Recently, the first approach ofrecognition of alocal production of fibronectin in epiretinalmembranes was made by Immonen et al, 12 whodemonstrated the occurrence of cellular fibro-nectin in a small number of epiretinalmembranes, and by Hiscott et al, who showedfibronectin related mRNA by in situ hybridis-ation. In this study we directly identified fibro-nectin located in contractile membranes usingtwo monoclonal antibodies (FN-3 and FN-4)that recognise two different antigenic sites on thecell-associated fibronectin; one of them (theFN-3 determinant) is not found in humanplasma fibronectin.'4

Materials and methods

SAMPLES AND CELL CULTURESVitreoretinal traction membranes were obtainedfrom patients undergoing surgical treatment for

Table I Expression ofcellularfibronectin (FN-3) andcellular/plasmafibronectin (FN4) in surgically excisedhuman epiretinal membranes (The staining intensityforfibronectins was graded as strong (+ +), weak (+),absent (-))

FN-3 FN-4Diagnosis No -/+/++ -/+/++

Idiopathic PVR 7 2/5/0 0/3/4Traumatic PVR 18 8/9/1 0/11/7Macular pucker 7 3/4/0 0/5/2PDR 5 2/2/1 0/3/2

PVR=proliferative vitreoretinopathy; PDR=proliferativediabetic retinopathy

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Origin offibronectin in epiretinal membranes ofproliferative vitreoretinopathy andproliferative diabetic retinopathy

traction retinal detachment due to PVR andPDR and processed as described elsewhere.9 Weincluded 18 membranes from eyes with traumaticPVR, seven with idiopathic PVR, seven withmacular pucker, and five specimens from eyeswith PDR. Central vitreous from donor eyes forkeratoplasty was aspirated within 6 hours afterdeath and immediately stored at -20°C. Vitreousaspirates were obtained from patients withidiopathic (n=3) and traumatic PVR (n=3), andPDR (n=3) by needle aspiration from the centreof the vitreal cavity before vitrectomy. Pooledhuman serum (Sigma S-2257) and purifiedfibronectin from human plasma (Sigma F-2006)were also examined.

BUFFERS AND REAGENTSThe following buffers and solutions wereemployed: phosphate buffered saline (PBS) (pH7 3). PBS-Tween containing 0 05% (blotstaining) Tween 20 (Serva 37470); fast red TR(Sigma F-2768), naphthol AS-MX phosphate(Sigma N-4875) stain buffer (pH 8-2).

IMMUNOHISTOCHEMICAL ANALYSIS OFEPIRETINAL MEMBRANESThe preparation of slides for immunohisto-chemistry and the general immunostaiming

technique of epiretinal membranes wereperformed as previously described.9 '5 For assay,all antibodies were diluted in PBS containing0-5% bovine serum albumin. The followingprimary antibodies were used with the indicateddilutions: anti-human (cellular) fibronectin FN-3, 1:10 (Cymbus Bioscience CBL 181); anti-human (cellular and plasma) fibronectin FN-4,1:10 (Cymbus Bioscience CBL 182); anti-humanfibronectin, 1:100 (Calbiochem 341643).

Primary antibody was labelled with a biotin-streptavidin system consisting of antimouseimmunoglobin (1:500 Dakopatts E 418 01), andstreptavidin conjugated with alkalinephosphatase (1:1000, Dakopatts ) 396 21).

This immunohistochemical staining wasanalysed and documented with a photomicro-graphic camera (MC 100, Zeiss, Germany).

Fig IC

..

.....

u..

Fig 1B Fig IDFigure I Alkaline phosphatase staining ofserialfrozen sections ofsurgically excised human epiretinal membranes with FN-4(A, C), andFN-3 (B, D). Phosphatase convertsfast red substrate to a redproduct. Counterstaining with haematoxylin and eosinshows cell nuclei (some marked by arrows). Magnification AIB: x 70; CID x 280.

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Grisanti, Heimann, Wiedemann

Qualitative differences were concluded fromserial sections.

SDS-PAGE AND WESTERN BLOTTINGThe specificity of the primary antibodies againstfibronectin was confirmed by sodium dodecyl-sulphate-polyacrylamide gel electrophoresis(SDS-PAGE)16 of human plasma fibronectin andsubsequent western blot analysis." Thesetechniques were also used for the examination ofvitreous samples.

CONTROLSAs a means of negative control, parallel slideswere stained with the first antibody substitutedby non-immune IgG from mouse (Sigma 1-538 1)or rabbit (Sigma I-5006), both in 50% higherconcentrations. Positive controls of plasmafibronectin (Sigma S-2257) were achieved withSDS-PAGE and western blotting.:

Results

Figure 2 Detection ofcellular (FN-3, *) andcellularlplasma (FN4, -)

fibronectins by western blotstain. Note the occurrence(arrow) offibronectin-relatedbands revealing a molecularweight over 180 kD. Nofibronectins could be detectedin physiological vitreous.Bothforms offibronectincould be shown in samplesfrom pathological vitreous.id (idiopathic) PVR; tr(traumatic) PVR; PDR(proliferative diabeticretinopathy).

MWin kDa

27

.49 =

.58

180

:...

IMMUNOHISTOCHEMICAL MICROSCOPY OFEPIRETINAL MEMBRANESWe examined 37 epiretinal membranes frompatients with PVR (n=32) or PDR (n=5) for thepresence of two different antigenic determinantsof fibronectins. The fibronectins stain labelintensity was graded as follows: absent (-),weak (+), or strong (++) (see Table 1). Apositive signal for FN-4 - that is, staining an

epitope on cellular and plasma fibronectin, was

seen in all 37 specimens. The antibody FN-3,which reacts selectively with the cell-associatedform of fibronectins,'4 did not yield a positivesignal in 15 of 37 examined epiretinal mem-

branes. Usually the staining was more intense forFN-4 than for FN-3. Negative staining forcellular fibronectin, but also a weaker signal forFN-4, was found in older membranes. Thesemembranes are characterised by a diminishednumber of cells and fibrotic transformation.Staining with the antibody FN-4 showed, incontrast to staining with FN-3, mainly a homo-geneous distribution of the glycoprotein. One

can see areas which stained with FN-4, but didnot stain with FN-3 (Fig 1).

WESTERN BLOTAntibody FN-4 clearly precipitated fibronectinin pooled human plasma and purified fibronectinwhile antibody FN-3 did not. '4 No fibronectinscould be detected by western blot in normalhuman postmortem vitreous (Fig 2).We analysed vitreous samples from patients

with idiopathic (n=3) and traumatic PVR (n=3)and PDR (n=3). Although cellular or cell-associated fibronectin, respectively, forms higheroligomers than plasma fibronectin and is there-fore much less soluble,'8 9 its presence could bedemonstrated in the vitreous of the pathologicaleyes by the selective FN-3 reactivity and differentstain pattern with regard to FN-4 reactivity(Fig 2). While cellular fibronectin can be recog-nised as a narrow band positioned in a highermolecular weight area than the 180 kD markerprotein, FN-4 (cellular and plasma) stainingrevealed a broader band on the same molecularweight position. One vitreous sample in thetraumatic PVR and one in the PDR group didnot contain enough cellular fibronectin to bevisualised by the western blotting technique.

DiscussionThe pathogenesis of PVR is characterised by adispersion of cells into the vitreal cavity, wherethey proliferate, form conglomerates, and finallydetach the retina. The disease progression hasmany features of a maladapted wound healingprocess, and there is extensive experimentalhistological and clinical evidence which supportsthis hypothesis.20

Although different interactions are importantin initiating and controlling PVR and PDR, thephase of cell adhesion and interaction with theextracellular matrix and matrix proteins seems tobe based on similar mechanisms in both diseases.Cell adhesion is preconditional for the growthand therefore for all following steps in theprogress ofthe disease. In this stage ofmembraneformation and organisation, fibronectin mayplay a major role as a chemoattractant4 as well asan adhesion-promoting factor providing atemporary scaffold for cell migration.Two principal forms of fibronectins that are

similar in their overall structure but not identicalin their chemical composition2' and biologicalactivities2223 were described. One has identifiedplasma fibronectin produced by liver cells24 andcellular fibronectin produced by fibroblasts andother cell types.'8 The differences betweencellular and plasma fibronectins, which areencoded by a single gene,2'25 seem to arise frompost-transcriptional events via alternativesplicing of fibronectin mRNA.2128 Fibronectinsform dimers of two subunit polypeptides (200-250 kD) connected near one end by disulphidebonds, but cellular fibronectin can form higheroligomers'829 and is therefore less soluble thanthe plasma form.

Several studies have demonstrated that themajor cell types involved in PVR and PDR - forexample, RPE cells and fibroblasts, maysynthesise fibronectin.3'I " Since it has been

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Origin offibronectin in epiretinal membranes ofproliferative vitreoretinopathy andproliferative diabetic retinopathy

demonstrated that plasma fibronectin can bedeposited in tissues and incorporated into. thecellular matrix,n0 it has not been possible toidentify the definitive source of fibronectin intissue sections3' and therefore in epiretinalmembranes, by means of polyclonal antisera.

Recently, the first approach of recognition of a

local production in fibronectin in epiretinalmembranes was made by Hiscott et al,'3 whoshowed variable labelling of retinal pigmentepithelium, retinal glia, and fibroblast-like cellsin membranes at the vitreoretinal junction with a

fibronectin mRNA probe. This observationcorrelates with the fact that RPE cells in vitroproduce and deposit fibronectin in the extra-cellular matrix,'" and supports our findings of a

direct identification of cellular fibronectin inepiretinal membranes that was also confirmed byImmonen et al. 12

In situ hybridisation can show 'up-regulation'or activation of fibronectin production, respect-ively, and therefore demonstrates a productivedisease stage. However, it will fail during'down-regulation' or stagnation of productionrespectively, although a previous glycoproteinproduction may have induced a participation oflocally synthesised cellular fibronectin inmembrane formation. Owing to the fact that thedirect identification of cellular, locally producedfibronectin can fill this information gap, thecombination with the fibronectin mRNA probewould optimise the observation of stage andprogress of the disease.The significantly stronger positive immuno-

stain with FN-4 and the occasionally negativeimmunostain with FN-3 and positivity with FN-4 in the same specimens suggests the colocalisa-tion of plasma fibronectins, that enters the eye

after breakdown of the blood-retinal barrier andis trapped in membranes during their formation.The great difference between FN-4 and FN-3staining in all membranes (for example, as in Fig1) suggests that fibronectin in epiretinalmembranes is predominantly plasma derived.Additionally, we observed the presence of bothplasma and cellular fibronectin mostly in younger(only a few months), cell-rich membranes than inolder fibrotic membranes.

Although several studies suggested that theparticularly high vitreous fibronectin levels seen

in patients with proliferative vitreoretinal diseaseare derived from plasma fibronectin followingbreakdown of the blood-retinal barrier," a con-

tributing local production could not be excluded.Despite the small number of the analysed vitrealspecimens in our study, we demonstrated thepresence of cellular fibronectin by western blot.Owing to the fact that the western blot techniqueis qualitative rather than quantitative, we cannotassess the level of contribution by intravitrealproduction. However, lower levels of cellularfibronectin than of plasma fibronectin inepiretinal membranes, and the fact that cellularfibronectin is much less soluble than the plasma

form, suggest a smaller contribution of locallyproduced glycoprotein.The pathophysiological role of plasma fibro-

nectin may be based on its quality as a structuralprotein. Circulating plasma fibronectin mayserve as a building block for the early assembly of

extracellular matrix by providing the possibilityof attachment to vitreous collagen. In compari-son with it cellular fibronectin seems to play itspivotal part in the regulation of cellular events22enhancing the autonomity in membrane growthand transformation.

This study was supported by the Retinovit Foundation and theDeutsche Forschungsgemeinschaft (Wi880/3-2), and waspresented in part at the ARVO meeting.The authors would like to thank Mrs B Martiny for skilful

technical assistance.

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doi: 10.1136/bjo.77.4.238 1993 77: 238-242Br J Ophthalmol

 S Grisanti, K Heimann and P Wiedemann retinopathy.vitreoretinopathy and proliferative diabeticmembranes of proliferative Origin of fibronectin in epiretinal

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