Different collagen types define two types of idiopathic epiretinal membranes

Different collagen types define two types of idiopathicepiretinal membranes

Michaela Kritzenberger, Benjamin Junglas, Carsten Framme,1* Horst Helbig,1 Veit-Peter Gabel,1

Rudolf Fuchshofer, Ernst R Tamm & Jost Hillenkamp2

Institute of Human Anatomy and Embryology and 1Department of Ophthalmology, University of Regensburg, Regensburg,

and 2Department of Ophthalmology, University Medical Center Schleswig-Holstein, Campus Kiel, Germany

Date of submission 11 February 2010Accepted for publication 10 August 2010

Kritzenberger M, Junglas B, Framme C, Helbig H, Gabel V-P, Fuchshofer R, Tamm E R & Hillenkamp J

(2011) Histopathology 58, 953–965

Different collagen types define two types of idiopathic epiretinal membranes

Aims: To identify differences in extracellular matrixcontents between idiopathic epiretinal membranes(IEM) of cellophane macular reflex (CMRM) or pre-retinal macular fibrosis (PMFM) type.Methods and results: Idiopathic epiretinal membraneswere analysed by light and quantitative transmissionelectron microscopy, immunohistochemistry andWestern blotting. Substantial differences betweenCMRM and PMFM were observed regarding the natureof extracellular fibrils. In CMRM the fibrils were thin,with diameters between 6 and 15 nm. Between thefibrils, aggregates of long-spacing collagen were ob-served. In PMFM the diameters of fibrils measured either18–26 or 36–56 nm. Using immunogold electron

microscopy, 6–15 nm fibrils in CMRM were labelledfor collagen type VI, while the fibrils in PMFM remainedunstained. Using Western blotting and immunohisto-chemistry, a strong signal for collagen type VI wasobserved in all CMRM, while immunoreactivity wasweak or absent in PMFM. In contrast, PMFM showedimmunoreactivity for collagen types I and II, which wasweak or absent in CMRM. Both types of membranesshowed immunoreactivity for collagen types III and IV,laminin and fibronectin with similar intensity.Conclusion: The presence of high amounts of collagentype VI in CMRM and the relative absence of collagentypes I and II is the major structural difference toPMFM.

Keywords: collagen type III, collagen type VI, epiretinal membrane, immunogold techniques, immunohistochem-istry, transmission electron microscopy, Western blotting

Abbreviations: CMRM, cellophane macular reflex membrane; ECM, extracellular matrix; IEM, idiopathic epiretinalmembranes; ILM, inner limiting membrane; PMFM, preretinal macular fibrosis membrane

Introduction

Idiopathic epiretinal membranes (IEM) are caused bythe migration of cells to the vitreoretinal junction andthe formation of epimacular membranous tissue in theabsence of any known underlying retinal disease. Theprocesses that characterize IEM formation, such as cell

migration and deposition and contraction of extracel-lular matrix, show marked similarities to those whichcharacterize wound healing at other sites in the body.1

Accordingly, IEM formation can be interpreted as anaberrant wound healing process at the vitreoretinaljunction. The prevalence of the entity is high in theelderly population.2–4 IEM are associated with adysfunction of the macula, the area of high acuityvision, which is probably related to an impairment ofthe inner retinal layers.5 For some time, no generallyaccepted nomenclature or classification existed, andIEM were termed macular pucker,6,7 preretinalmacular fibrosis,8 epiretinal fibrosis,9 premaculargliosis,10 surface wrinkling retinopathy,11 cellophane

Address for correspondence: E R Tamm, Institute of Human Anatomy

and Embryology, University of Regensburg, Universitatsstr.

31, D-93053 Regensburg, Germany.

e-mail: [email protected]

*Present address: University Eye Hospital Bern, Inselspital,

Switzerland.

� 2011 Blackwell Publishing Limited.

Histopathology 2011, 58, 953–965. DOI: 10.1111/j.1365-2559.2011.03820.x

maculopathy12 or fibrocellular epiretinal mem-branes.13 More recently, several epidemiological studiessuch the Beaver Dam Eye Study,3 the Blue MountainsEye Study2,14 and the Los Angeles Latino Eye Study4

defined two major types of IEM distinguishing a lesssevere form, termed ‘cellophane macular reflex’, from amore severe form termed ‘preretinal macular fibrosis’.The distinction is based on the funduscopic appearancewhere membranes of the cellophane macular reflextype (CMRM) present as transparent patch or patchesof irregular increased reflection from the inner surfaceof the retina, while membranes of the preretinalmacular fibrosis type (PMFM) have a more opaque,greyish or whitish appearance on the retinal sur-face.15,16 The reasons for the different fundoscopicappearances are not entirely clear, as some histopath-ological analyses have reported a correlation betweenthe biomicroscopic opacification of IEM and theircollagen content,7 while others have found essentiallyno correlation between membrane opacification andcellularity or collagen content of the membranes.13

Besides their variation in fundoscopic appearance, IEMshow a quite substantial variation in their biomechan-ical properties which, in our hands, becomes evidentduring the delicate surgical peeling manoeuvre that isrequired for their removal from the retinal surface.17

CMRM that appear somewhat transparent and cello-phane-like by fundoscopy are usually ‘easy to peel’ andcan be removed in one stable piece once an edge of themembrane has been delaminated successfully fromthe retinal surface. By contrast, PMFM that aresomewhat opaque and whitish are usually relatively‘difficult to peel’. These membranes are comparativelyunstable, can be removed only by repeatedly graspingsmall fragments that consist of soft material, andprovide a greater risk of retinal damage during surgery.The aim of the present study was to identify distinctmolecular differences that could account for thedifferent fundoscopic and biomechanical properties ofCMRM or PMFM.

Material and methods

Idiopathic epiretinal membranes were obtained duringstandard three-port vitrectomy performed by experi-enced vitreoretinal surgeons (C.F., H.H., V.P.G., J.H.).IEM were classified by the operating surgeon as eitherCMRM or PMFM according to the following criteria:CMRM appeared thin, transparent and cellophane-likeupon funduscopy. During surgery CMRM were ‘easy topeel’, mechanically stable and were removed in onepiece. PMFM appeared thick, opaque, greyish orwhitish upon funduscopy. During surgery, PMFM were

‘difficult to peel’, mechanically unstable, removed inseveral fragments and required repeated graspingduring removal. For each IEM, the funduscopic criteriawere assessed and documented before surgery. OnlyIEM which clearly fulfilled both the funduscopic andsurgical criteria were included. Membranes wereremoved completely and processed for light and elec-tron microscopy. A total number of 60 IEM wereprocessed. Thirty-two IEM (53%) were classified asCMRM and 28 as PMFM (47%).

l ight and electron microscopy

Eighteen membranes (nine PMFM and nine CMRM)were processed for light and transmission electronmicroscopy. Membranes were fixed for several hours inone half-strength Karnovsky’s fixative (2% paraformal-dehyde ⁄ 2.5% glutaraldehyde).18 After rinsing in 0.1 m

cacodylate buffer, postfixation was accomplished in amixture of 1% OsO4 and 0.8% potassium ferrocyanidein 0.1 m cacodylate buffer for 2 h at 4�C. Specimenswere then dehydrated in a graded series of ethanol andembedded in Epon (Serva, Heidelberg, Germany).Semithin sections (1 lm) were collected on uncoatedglass slides and stained with methylene blue ⁄ azure IIafter Richardson et al.19 for light microscopy. Ultrathinsections were mounted on uncoated copper grids,stained with uranyl acetate and lead citrate20 andexamined on a Zeiss EM 10A electron microscope.(Zeiss, Oberkochen, Germany)

immunohistochemistry

Twenty (10 PMFM and 10 CMRM) membranes werefixed in 4% paraformaldehyde for several hours andthen rinsed carefully in 0.1 m phosphate-buffered saline(PBS), pH 7.4. After processing through a graded seriesof ethanol, specimens were immersed in xylene andembedded in Paraplast (Sherwood, St Louis, MO, USA).Sections were mounted on uncoated glass slides anddewaxed in a graded series of ethanol and xylene.Immunohistochemistry was carried out in a moistchamber on sections surrounded by water-repellentPAP-PEN (Science Service, Munich, Germany) accord-ing to the following protocol: (i) preincubation(45 min) with blocking buffer containing 10% fetalcalf serum (Seromed, Munich, Germany), 10% normalgoat serum (Sigma-Aldrich, Munich, Germany), 0.8%TritonX-100, 0.8% NaCl, 0.15% Timerosal in 0.08 m

Tris-buffer, pH 7.4, (ii) overnight incubation at 4�Cwith 1:100 diluted primary antibody [collagen I(Calbiochem, Merck, Darmstadt, Germany), collagen II(Chemicon via Millipore, Schwalbach, Germany),

954 M Kritzenberger et al.

� 2011 Blackwell Publishing Ltd, Histopathology, 58, 953–965.

collagen III (BioGenex, San Ramon, CA, USA), collagenIV (Rockland via Biomol, Hamburg, Germany), colla-gen VI (Rockland), fibronectin (Chemicon), laminin(Chemicon)] in blocking buffer, (iii) rinsing with Tris-buffered saline (TBS), (iv) incubation (60 min) in theappropriate secondary antibody [1:100 goat antimouseAlexa 546 or goat antirabbit biotinylated immuno-globulin (IgG) followed by streptavidin coupled withAlexa 555 (all from Invitrogen, Karlsruhe, Germany)]in blocking buffer, and (v) rinsing as in step 3 andmounting in fluorescent mounting medium (DakoCytomation, Hamburg, Germany) supplemented with10% Vector shield mounting medium containing 4¢-6-diamidino-2-phenylindole (DAPI) (Vector via Linaris,Wertheim-Bettingen, Germany). Sections were analy-sed on a Zeiss Axio Imager light microscope. Controlsections were incubated with PBS instead of theprimary antibody.

ultra- immunohistochemistry

Eight membranes (four PMFM and four CMRM) werefixed in 4% paraformaldehyde overnight and rinsedthoroughly in cacodylate buffer. After a graded series ofethanol, specimens were embedded in LR-white (Sci-ence Service). Ultrathin sections were mounted onnickel grids. Immunocytochemistry was performed in amoist chamber according to the following protocol:(i) preincubation with blocking buffer containing 10%normal goat serum and 0.005% fresh water fish gelatin(Biotrend, Cologne, Germany) in 0.1 m TBS, pH 7.6,(ii) overnight incubation with primary antibodies(collagen IV: 1:50, collagen VI: 1:25, both fromRockland), (iii) carefully rinsing with washing buffer(TBS supplemented with 0.2% acetylated bovine serumalbumin (BSA-c; Biotrend) and 0.8% NaCl), (iv) incu-bation with 10 nm gold conjugated goat-antirabbitantibody (Aurion via Biotrend), (v) rinsing with wash-ing buffer followed by TBS, (vi) postfixation with 2.5%glutaraldehyde in TBS for 5 min, (vii) rinsing with TBSand H2O, and (viii) staining with uranyl acetate.Sections were analysed on a Zeiss EM10A electronmicroscope. Control sections were incubated with PBSinstead of the primary antibody.

western blot analysis

A total of 14 IEM (six PMFM and eight CMRM) wereprocessed for Western blotting. To obtain proteinextracts, 200 ll of TRIzol (Invitrogen) was added toIEM followed by homogenization with a Power Gen 125(Fisher Scientific, Schwerte, Germany). Proteins wereisolated according to the manufacturer’s instructions

and protein content was measured by comparing a dotblot stained with Sypro Ruby (BioRad, Munich, Ger-many) to a bovine serum albumin standard. Proteinyield in four CMRM was too low to allow furtherprocessing. Proteins were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF)membranes (Millipore, Billerica, MA, USA) by semidryblotting. Western blot analyses were performed withspecific antibodies as described previously.21 Antibodieswere used as follows: rabbit anticollagen type VI (1:500;Rockland), rabbit anticollagen type III (1:500; SantaCruz Biotechnology, Santa Cruz, CA, USA) and chickenantirabbit IgG, coupled to horseradish peroxidase (HRP)(1:2000; Santa Cruz). Collagen type VI Western blottingwas conducted first followed by stripping each mem-brane with Roti�-free stripping buffer (Carl Roth,Karlsruhe, Germany), according to the manufacturer’sinstructions. Stripped membranes were blocked againand collagen type III labelling was performed. Chemi-luminescence was detected with the help of Immobi-lon� Western HRP substrate (Millipore) on a LAS 3000imaging workstation (Fujifilm, Dusseldorf, Germany).The molecular mass of the detected bands wasdetermined using appropriate software (AIDA imageanalyser; Raytest, Straubenhardt, Germany).

Results

While PMFM were separated from the retina in severalfragments during surgery CMRM were removed in onepiece, which formed numerous folds after histologicalprocessing (Figure 1A). By light microscopy, CMRMconsisted largely of three ordered layers: an incompleteinner cellular layer which was attached to an inter-mediate extracellular layer that stained only faintlywith methylene blue ⁄ azur II (Figure 1B) and an outerextracellular layer that stained more intensely. Byelectron microscopy, the outer extracellular layerconsisted of both fine filamentous and amorphousmaterial with the typical ultrastructural characteristicsof a basal lamina. The intermediate extracellular layercontained bundles of extracellular fibrils (Figure 1C,D).We concluded that the outer extracellular layer iscomprised of parts of the inner limiting membrane(ILM), while the intermediate extracellular layer andthe inner cellular layer had been formed duringpathogenesis of the CMRM. The structural elementswhich formed the three ordered layers in CMRM werealso found in specimens classified clinically as PMFM(Figure 1E,F). However, in contrast to CMRM, cells andthe two kinds of extracellular matrices did not formdistinct ordered layers in PMFM. Fragments containing

Collagen in epiretinal membranes 955


A B

C D

E F



ILM material were usually found to be separated fromfragments containing larger amounts of extracellularfibrils and associated cells (Figure 1E,F).

diameters of extracellular fibres differ

between membranes of cellophane macular

reflex or preretinal macular fibrosis type

In order to find structural differences that couldaccount for the different biomechanical properties ofboth CMRM and PMFM, we investigated in more detail

the ultrastructure of the extracellular matrix in theintermediate extracellular layer of both kinds of IEM.The intermediate extracellular layer of CMRM consistedof fibrils that formed a dense irregular network withoutany preferential spatial orientation (Figure 2A). Uponhigher magnification, the fibrils showed no obviousperiodicity (Figure 2B). Quantitative evaluationshowed that the extracellular fibrils of the CMRMintermediate layer had diameters between 6 and15 nm, with a mean diameter of 9.2 ± 1.9 nm(Figure 3). In some samples, aggregates of fibrous

Figure 1. Light microscopy (A, B, E) and low-power electron microscopy (C, D, F) of cellophane macular reflex membrane (CMRM) (A–D) and

preretinal macular fibrosis membrane (PMFM) (E, F). Light micrographs of semithin sections stained with methylene blue ⁄ azur II. A, B,

CMRM largely consist of three layers: a cellular layer (arrow) and two layers of extracellular matrix (asterisk and arrowheads). B is a

magnification of A. Scale bars: A, 10 lm; B, 5 lm. C, D, Using electron microscopy, the intermediate extracellular layer contains bundles of

extracellular fibrils (asterisks, higher magnification in 2A and B), while the outer extracellular layer (arrowheads) consists of fine filamentous

material with the structural characteristics of the inner limiting membrane (ILM). D is a magnification of C. Scale bars: C, 2 lm; D, 1 lm.

E, In PMFM, fragments of ILM (arrowheads) are typically separated from cells (arrow) and intermediate extracellular layer (asterisk). Scale bar:

10 lm. F, Using electron microscopy, dense bundles of collagen fibrils (asterisks) are found in the intermediate layer. Scale bar: 2 lm.

A B C D E

F G H

Figure 2. Electron microscopy of 6–15 nm extracellular fibrils in cellophane macular reflex membrane (CMRM) (A, B), and of 18–26 nm

fibrils (E, C) and 36–56 nm fibrils (G, D) in samples of preretinal macular fibrosis membrane (PMFM). A, E, G or B, C, D show the same

magnification, respectively. Scale bars: 0.5 lm (A, E, G) and 100 nm (B, C, D); 36–56 nm fibrils show the typical periodicity of collagen

fibrils (arrowheads in D). F, H, Aggregates of fibrous long spacing collagen are found between the 6–15 nm fibrils of the intermediate

extracellular layer in CMRM. Scale bars: 1 lm (C), 250 nm (D).



long-spacing ‘zebra’ collagen were observed among the6–15 nm fibrils (Figure 2F). Long-spacing collagenconsisted of broad bands of electron-dense material,which were interrupted by electron-lucent areascontaining fine fibrillar material at a periodicity of80–100 nm (Figure 2H). The intermediate layer ofPMFM also contained numerous extracellular fibrilswhich appeared to be orientated at random. Quantita-tive measurements showed that the fibrils in PMFMwere thicker than those observed in CMRM, with amean diameter of 30.8 ± 10.2 nm (Figure 3). In five ofthe seven samples, the fibrils showed diameters between18 and 26 nm, with the majority of the fibres having adiameter of 21 nm (Figure 2E,C). In the remaining twospecimens of PMFM, the extracellular fibrils in theintermediate layer were considerably thicker withdiameters between 36 and 56 nm (Figure 2G).On higher magnification, the 36–56 nm fibres showedthe typical periodicity of collagen fibres (Figure 2D).

high amounts of collagen type vi

characterize cellophane macular reflex

type membranes

Because electron microscopy had shown a distinctdifference in diameters of extracellular fibrils betweenCMRM and PMFM, we hypothesized that different sizefibrils might consist of different types of collagen. As inmultiple tissues in- and outside the eye, aggregates oflong-spacing collagen surrounded by small-diameterextracellular microfibrils have been observed in extra-cellular matrices that are rich in collagen type VI,22–30

we hypothesized that the intermediate layer of CMRMmight contain high amounts of collagen type VI. Toclarify this hypothesis, we performed immunohisto-chemistry for collagen type VI both in CMRM andPMFM samples. Indeed, a strong homogeneous immu-noreactivity for collagen type VI was observed in all 11samples of CMRM that were investigated (Figure 4A).In contrast, immunoreactivity for collagen type VI wasweak, patchy and barely detectable in all 10 samples of

CMRM

% o

f fib

ers

40

30

20

10

01 10 19 28

nm37 46 55

CMRM PMFM

PMFM

PMFM

nm

0.0

10.0

20.0

30.0

40.0

50.0A

B

Figure 3. Morphometric analysis of intermediate layer fibril dia-

meter. A, Mean ± standard deviation (SD) of diameter of fibrils in

cellophane macular reflex membrane (CMRM) (n = 5) and preretinal

macular fibrosis membrane (PMFM) (n = 7). B, distribution of fibril

diameter. Measurements were taken from high (·75 000) magnifi-

cation transmission electron microscopy (TEM) micrographs and a

total of 150 fibrils were selected randomly and measured in each

specimen.

A

B

C

Col VI CMRM

Col VI PMFM

Col VI

Col III

PMFM

CMRM

Figure 4. Immunohistochemistry for collagen type VI (A, B) and

Western blotting (C) in proteins from cellophane macular reflex

membrane (CMRM) and preretinal macular fibrosis membrane

(PMFM). Collagen type VI shows intense immunoreactivity in

CMRM (A), but only moderate staining in PMFM (B).



PMFM that were investigated by immunohistochemis-try (Figure 4B). The results obtained by immunohisto-chemistry correlated with those obtained by Westernblotting. A strong signal for collagen type VI wasobserved in all four CMRM that were investigated,while the signal was absent or barely detectable in thesix PMFM that were processed for Western blotting(Figure 4C). In contrast, a signal for collagen type IIIwas obtained in both types of membranes with equalintensity (Figure 4C). Differences between CMRM andPMFM were also observed regarding the distribution ofcollagen types I and II, which form striated collagenfibrils in situ. Of 11 CMRM samples that were investi-gated, only three showed faint immunoreactivity forcollagen type I or type II, respectively (Table 1), whilethe rest remained unstained. In contrast, staining forcollagen types I and II was more common in PMFM.

Six of 10 samples showed moderate to intense immu-noreactivity for collagen type I (Figure 5A, Table 1).Those samples that did not react with antibodiesagainst collagen type I stained with antibodies againstcollagen type II (Figure 5B) and vice versa (Table 1).Only one sample showed intense immunoreactivity forboth collagen types I and II. Extracellular moleculesthat are distinct components of the ILM such ascollagen type IV and laminin could be detected in allsamples of CMRM and PMFM that were investigated(Figure 5E,F, Table 1). The same was true forfibronectin (Figure 5G, Table 1), which is supposed toconnect the ILM with the collagen fibrils of thevitreous.31 Control sections in which the primary anti-body had been omitted did not show immunoreactivity(Figure 5C,D), and the same was true when thesections were labelled with an unrelated rabbit anti-

Table 1. Immunohisto-chemistry, semiquantitativeevaluation

Collagen I Collagen II Collagen IV Fibronectin Laminin

CMRM (+) ++ + ++

(+) ++ ++ ++

(+) (+) + + +

++ ++ +

+ + ++

++ + ++

++ + ++

++ + ++

+ + ++

+ + +

PMFM ++ ++ + ++ ++++ + ++ +

+ + + ++

+ + + ++

(+) + ++ (+)

(+) + + +

+ + ++ ++

+ ++ ++ ++

+ ++ + +

+ + + +

CMRM, Cellophane macular reflex membrane; PMFM, preretinal macular fibrosis membrane.(+), Patchy staining in some areas of the sample; +, homogeneous staining throughout theentire sample; ++, intense staining.



body (against Ki67) at the same concentration (notshown). In summary, the data obtained by immuno-histochemistry and Western blotting correlated mark-edly with those obtained by transmission electronmicroscopy. CMRM that contained small diameter fibrilsand long-spacing collagen showed strong immunoreac-tivity for collagen type VI that was weak or absent inPMFM. In contrast, PMFM that contained thicker sizefibrils with typical periodicity stained for collagens typesI and II while immunoreactivity for these collagen typeswas almost absent in CMRM. Labelling for collagen IV,laminin and fibronectin in both CMRM and PMFMcorrelated with the finding of fine filamentous basallamina material presumably of ILM origin. In order toconfirm that the 6–15 nm extracellular fibrils in theintermediate layer of CMRM are composed of collagentype VI, ultra-immunohistochemisty with immunogold-labelling was performed. In CMRM, the fibrils of theintermediate layer were labelled distinctly with anti-bodies directed against collagen type VI (Figure 6A,B).Gold particles were aligned along and in the vicinity of6–15 nm extracellular fibrils, but were essentiallyabsent intracellularly (Figure 6A) or in the adjacentparts of the ILM (Figure 6C). In contrast, immunogold-labelling for collagen type VI was considerably weakerin the intermediate layer of PMFM, and only very fewgold particles were observed in this area (Figure 6D).Immunogold-labelling for collagen type IV, which was

used as a positive control, was observed in the ILM areasof both CMRM and PMFM (Figure 6E,F). Negativecontrols in which the primary antibody had beenomitted remained unstained (Figure 6G).

cells present in membranes of cellophane

macular reflex or preretinal macular

fibrosis type are not obviously different

As the different types of collagens that were observed inthe intermediate layers of CMRM and PMFM mightderive from different types of cells in the adjacent cellularlayers, their ultrastructural details were investigated. Inboth types of membranes, cells were either arranged as asingle layer or multilayers consisting of two to threecellular sheets. Two types of cells predominated: oval-shaped cells which contained dense bundles of 8–10 nmintermediate filaments, showed numerous cellular pro-cesses and resembled fibrous astrocytes (Figure 7A,B)and flat myofibroblast-like cells (Figure 7C) with bundlesof 6-nm actin filaments and dense bodies in theircytoplasm (Figure 7D). In CMRM, seven of nine mem-branes contained fibrous astrocytes, while PMFMshowed this cell type in five of seven membranes.Myofibroblast-like cells were found in six of the nineCMRM, and in four of the seven PMFM. In places, themyofibroblast-like cells were separated from the inter-mediate extracellular layer by an incomplete basal

A B C

D

E F G

Figure 5. Immunohistochemistry in cellophane macular reflex membrane (CMRM) and preretinal macular fibrosis membrane (PMFM).

A, collagen I; B, collagen II; E, collagen IV; F, laminin; G, fibronectin (all in PMFM). C, D, Negative controls of secondary antibodies (C, goat

antimouse; D, goat antirabbit) with omission of primary antibodies show only 4¢-6-diamidino-2-phenylindole (DAPI) staining of nuclei. Scale

bar: 100 lm.



lamina (Figure 7E). In two cases (one PMFM and oneCMRM), macrophage-like cells with an indented nucleusand secondary lysosomes in the cytoplasm were observed(Figure 7F). None of the samples contained pigmentedcells or cells with an epithelial phenotype. Overall, nodistinct differences regarding the phenotype of cells in thecellular layer was found between CMRM and PMFM.

Discussion

Based on the results of our study, we conclude that thetwo major types of IEM differ substantially regardingtheir collagenous fibrillar extracellular matrix. Thepresence of high amounts of 6–15 nm fibrils consistingof collagen type VI characterizes CMRM. In contrast,fibres of collagen types I and II are rare or absent inCMRM, but common in PMFM. This conclusion isbased on data obtained by electron microscopy inconjunction with morphometry, by immunohisto-

chemistry at the light- and electron microscopicallevels and by Western blotting.

Collagen type VI is an ubiquitous extracellularmatrix protein which typically forms networks of6–15 nm microfibrils.23,29,32,33 Consistent with thepresence of collagen type VI in CMRM is the observa-tion of aggregates of fibrous long-spacing collagenbetween the 6–15 nm microfibrils. Aggregates offibrous long-spacing collagen have been found invarious tissues in- and outside the eye and have beenshown to consist of collagen type VI fibrils.23,34

Collagen type VI fibrils form cross-banded fibrillaraggregates through end-to-end longitudinal assemblyand overlapping.35 Alignment of the aggregatesgenerates fibrous long-spacing collagen.25,34 It is ofinterest to note that in early studies on the ultrastruc-ture of eyes with subtle IEM, aggregates of long-spacingcollagen were already observed in the ECM of themembranes.36 In the absence of tools to identify the

A B C

D E F

G

Figure 6. Immunogold electron microscopy. A–D, Collagen VI. A, Strong expression for collagen VI is seen in the intermediate layer of

cellophane macular reflex membrane (CMRM), while cells remain unstained (right margin). B, Immunogold particles specific for collagen type VI

are associated with the 6–15 nm microfibrils in the CMRM. C, Immunoreactivity for collagen type VI is absent in the inner limiting membrane

(ILM) of CMRM. D, In the intermediate layer of preretinal macular fibrosis membrane (PMFM), only few immunogold particles specific for

collagen type VI are observed. E, F, Immunogold labelling for collagen type IV in the ILM of PMFM. G, Negative control of secondary antibody

with omission of primary antibody. Scale bars 0.5 lm (A, E), 1 lm (E) and 200 nm (B, C, D, F, G).



true nature of collagen fibrils, the surrounding fibrilswere regarded at that time as ‘vitreous fibrils’.

In addition to collagen type VI, CMRM showedstrong immunoreactivity for collagen type IV, lamininand fibronectin, three proteins that are found consti-tutively in the ILM37 which had been invariablyremoved together with the membranous tissue in allCMRM that were investigated. Collagen fibrils thatwere considerably thicker than the 6–15 nm collagentype VI microfibrils were not observed in CMRM, butwere seen commonly in PMFM. The thicker collagenfibrils in PMFM consisted of two groups of fibrils withdiameters ranging from 19–28 to 37–55 nm. It isreasonable to assume that both groups of fibrils wereidentical to those that labelled strongly for collagentypes I or II in the PMFM samples that were investi-gated by immunohistochemistry, although we did notperform immunogold labelling to confirm this. Onlythree of 11 CMRM samples that were investigatedshowed weak immunoreactivity for collagen types I

and II, while the others were essentially unlabelled,indicating strongly that the relative amounts of colla-gen types I and II are very low in CMRM compared toPMFM. In addition to collagen types I and III, PMFMalso labelled with antibodies against collagen type IV,laminin and fibronectin corroborating findings ofprevious studies.38–45 PMFM also contained somecollagen type VI microfibrils that were labelled withimmunogold between the thicker collagen fibrils ofdifferent nature. However, the combined results ofimmunohistochemistry, immunogold labelling andWestern blotting indicate clearly that collagen type VImicrofibrils are less numerous in PMFM than in CMRM.

Networks of type VI collagen fibrils are present inbasically all connective tissues and are found typicallyin close association with basement membranes. Thereis considerable evidence that type VI collagen functionsto anchor basement membranes through strong inter-action with type IV collagen.24,46–48 Collagen type VIalso interacts with several other extracellular matrix

A B C

D E F

Figure 7. Electron micrographs of cells in the cellular layer of idiopathic epiretinal membranes (IEM). A, B, Fibrous astrocytes (A, scale bar:

2 lm; B, scale bar 0.5 lm) with numerous cellular processes (asterisk in B) and tightly packed intermediate filaments (arrowheads in B).

C–E, Myofibroblast-like cells with elongated cells body and oval-shaped nucleus (C, scale bar: 2 lm), and 5–6 nm actin filaments (arrows)

with dense bodies (arrowheads, scale bar 0.5 lm). Myofibroblast-like cells are surrounded by basal lamina (E, arrowheads, scale bar 1 lm).

F, Macrophage-like cells in the intermediate layer with indented nuclei and secondary lysosomes, scale bar: 2 lm.



constituents in vitro, including the fibrillar collagentypes I and II.49,50 By light microscopy, immunoreac-tivity for collagen type VI has been observed at thevitreoretinal junction of normal eyes,37 and it appearsto be reasonable to assume that collagen type VIcontributes to anchor vitreous collagen type II fibrilsto the ILM.37,38 To our knowledge, an age-relatedincrease in the amounts of collagen type VI in thevitreoretinal junction of normal eyes has not beenstudied so far, either by transmission electron micros-copy or by biochemical methods. However, data fromstudies which compared the ultrastructure of theextracellular matrix at the vitreoretinal junctionbetween normal eyes and those with IEM indicatestrongly that the increase in collagen in the interme-diate layer of CMRM is not an age-related phenome-non, but part of the pathological processes duringCMRM formation. We hypothesize that excess collagentype VI in the intermediate layer of CMRM is themolecular reason for the high biomechanical stabilityof the membranes during surgical removal. Collagentype VI fibrils in the intermediate layer of CMRM shouldattach firmly to collagen type IV in the ILM (Figure 8),which is peeled from the retinal surface along with theCMRM during surgery. In contrast, the ECM in theintermediate layer of PMFM, which contains lesscollagen type VI, should be attached less firmly to theILM and may easily break during surgery.

It seems reasonable to assume that the ECM in theintermediate layer of both CMRM and PMFM is secreted

and synthesized by cells in the immediate adjacentcellular layer. In our material, cells with a glia- orastrocyte-like, or a myofibroblast-like ultrastructurewere predominant in both types of membranes, afinding that correlates with those of previous investi-gations on the ultrastructure of epiretinal mem-branes.7,51–56 Based on ultrastructural criteria alone,we did not find evidence for the presence of retinalpigmented epithelial cells in our material. Myofibro-blast-like cells comprise most probably the cell type inepiretinal membranes which is responsible for theircontraction.57 Both astrocytes and Muller cells havebeen shown to be capable of dedifferentiation to amesenchymal myofibroblast-like phenotype under cer-tain in vivo and in vitro conditions,58–64 and may welldo so in epiretinal membranes, as cells with a mixedastrocyte ⁄ myofibroblast-like phenotype have beenobserved in vitreous membranes.56,65,66 Both astro-cytes and Muller cells are capable of secreting collagentype VI and the other ECM molecules that wereobserved in both CMRM and PMFM.67–69

During a 5-year period, 9.3% of patients enrolled inthe Blue Mountains Eye Study progressed from CMRMto PMFM.14 In CMRM, IEM cells appear to secreteprimarily collagen type VI, a molecule that is alsonormally secreted by retinal glial cells. In contrast, inPMFM, IEM cells appear to secrete primarily collagentypes I, II and III, which are normally not producedby retinal glial cells, but by cells of a myofibroblast-like phenotype. During the progress from CMRM toPMFM, IEM cells may change their differentiation, andthis change may account for the secretion of differenttypes of collagens. Transforming growth factor (TGF)-b signalling might be involved in this process, as TGF-b is potent in inducing myofibroblast-like phenotypesin glial cells,57,61 but also in a broad variety of othercell types.70–75 While such a change in differentiationis a possible scenario, it needs to occur without majorchanges in the ultrastructural phenotypes of IEM cells,as we observed essentially the same types of cells inboth CMRM and PMFM by transmission electronmicroscopy. A quantitative immunohistochemicalcharacterization of the different types of IEM cellsappears to be a requirement to support the concept ofa change in differentiation of those cells that secretethe different collagen types in CMRM or PMFM.

Acknowledgements

This work was supported by a DFG grant (FOR 1075).The authors thank Margit Schimmel for excellenttechnical assistance and Antje Zenker for doing theschematic drawing.

CL

Collagen VIIL

ILM

Basal lamina of cellular layer

Figure 8. Schematic drawing of the three-layered structure in

cellophane macular reflex membrane (CMRM). Collagen type VI

fibrils in the intermediate layer connect with collagen type IV in the

inner limiting membrane (ILM) and with cells and their basal lamina

in the cellular layer (CL).



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Different collagen types define two types of idiopathic epiretinal membranes