The role of fibronectin, laminin, vitronectin and their receptors on

The Role of Fibronectin, Laminin, Vitronectinand Their Receptors on Cellular Adhesionin Proliferative Vitreoretinopathy

Ricardo P. Casaroli Marano*\ and Senen Vilaro*

Purpose. To examine the possible role of some adhesion multifunctional glycoproteins of theextracellular matrix, such as fibronectin (FN), laminin (LN). vitronectin (VN) and their recep-tors (/3|-subunit complex and avj0:, integrins) in events of cell migration and adhesion in prolifer-ative vitreoreiinopathy (PVR).

Methods. Optical and clectron-immunocytochcmical techniques were carried out on epiretinalmembranes. Electmphorelic immunoblotling methods and densitonietric analysis of normaland PVR vitreous were also undertaken. Chi-square (x'J) and unbalanced analysis of variancewere employed for statistical analysis.

Results. FN was detected as a major component in the extracellular matrix in both fibrillar andpericellular arrangement. A change in pericellular distribution to more fibrillous organizationwas related to the time of intraocular proliferative tissue development (P < 0.001). LN and VNwere observed as minor components in extracellular matrix. A colocali/ed pattern between VNand FN in collagenic bundles of the matrix was often observed. Beta-1 subunit and cvv/3;,receptors were usually localized in a position thai could mediate the interact ion of FN. VN,and/or I.N to the cell plasma membrane. Increased levels of FN concentration were observedin both subretinal lluid and pathologic vitreous; intravilreal FN concentration lends to in-crease with clinical stages of the evolution of PVR, whereas intravitreal VN levels tend todecrease.

Conclusions. Results suggest that FN could mediate the initial events involved in epiretinalmembrane formation, and VN could modulate the adhesion mechanisms in established mem-branes. Invest Ophlhalmol Vis Sci. 1004;:<f>:270l-2803.

X he biologic activities of the extracellular matrix(ECM) reside both in its special components and in itsstructural integrity.1 Most ECM contain a fibrous col-lagenic network or other fiber-forming proteins, suchas glycoproteins. Noncollagenous extracellular glyco-proteins have important roles in many cell-surface in-teractions, such as adhesion, migration, phenotypedifferentiation and polarization, wound healing, tu-mor-cell invasion, and metastasis.2 For example, fibro-nectin (FN; MVV 440 kD) is a multifunctional interac-tive glycoprotein found as a soluble plasma protein

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and an insoluble form organized into extensive ECM,produced and secreted by a wide variety of cell types.*"'Laminin (LN; MVV 850 kD) is a major component ofbasement membranes and possesses multiple func-tional sites that mediate its interactions with cells andother ECM elements.(1"s Finally, vitroneclin (VN; MVV(35 to 78 kD) is present in fibrillar pattern in the ECMof a variety of tissues"10 and also in circulation with apredominant role in cell adhesion and hemostasis.111^

The regulation of ECM assembly and cellular re-sponse often requires adhesion and specific interac-tions of a cell with its substrate. These interactionsinvolve specific cell-surface proteins that bind adhe-sive ligands of the ECM recognized by both Arg-Gly-Asp (RGD) peptide cell-binding sequences and non-RGD cell-binding sequences in adhesive ECM macro-molecules such as those described above.1'"1' Inparticular, integrins—heterodimeric molecules ofcell-adhesion receptors—have been implicated in a va-riety of cell-to-cell and cell-to-matrix interactions and

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2792 Investigative Ophthalmology & Visual Science, May 1994, Vol. 35, No. 6

are also active in transmitting signals from the extra tothe iniracellular compartment. IK~21 All integrins are asubunits, noncovalently associated with a /? subunit,and can be expressed in a wide variety of cell types.1"Many of the integrins thai share the fix subunit areknown to recognize FN,22-2:< LN,2/1-2fl and collagen.2"Cell attachment to VN occurs by any of several f3.6 sub-unit associations, including the classical VN-reccptor:the avj8s integrin.27 These receptors also bind to FN,fibrinogen, von Willebrand factor, and collagen.1'12*

Proliferative vitreoretinopathy (PVR) is a serioushuman intraocular disorder characterized by fibrocel-lular sheets of connective tissue proliferating on bothretinal surfaces, which causes structural and func-tional damage to the retina. PVR is most likely to de-velop after rhegmatogenous retinal detachment, per-forating trauma of the posterior segment of the eye,and after surgery for retinal reattachment.2'1 These fi-brocellular sheets (epiretinal membranes, ERM) arecomposed of different cell types, essentially retinalpigment epithelial cells, glial cells, fibroblasts, macro-phages, and myofibroblast-like cells*0"'2 surroundedby a fibrous matrix with an extensive amount of colla-gen.3ft:w Pathogenesis is poorly understood, especiallythe mechanisms of cellular migration, adhesion, andproliferation. Previous studies have suggested theparticipation of growth factors'4 sr' and serum pro-teins."1-*"

In the present work, we have studied the distribu-tion of extracellular matrix glycoproteins and their re-ceptors in PVR. The results suggest that KN couldhave an important role in the earliest pre-membrano-genic stages of this pathologic condition and later inthe mechanisms of cell-ECM adhesion.

MATERIAL AND METHODS

Human Tissue and SamplesHuman ERM (n = 54) were dissected and removed byappropriate intraocular vitreous forceps (Grieshaber,Switzerland; Cat. No. C-612.08, C-612.13, andC-612.98) from patients with retinal detachment com-plicated by PVR who were undergoing intraocularsurgery. Specimens were immediately fixed in 1% glu-taraldehyde and 4% paraformaldehyde in 0.1 M phos-phate-buffered saline (PBS), pH 7.4, for at least 1 2hours at 4°C. They were then rinsed in PBS 0.1 M pH7.4 and immersed in 2.1 M sucrose in PBS solution for2 to 4 hours. Part of them (n = 28) were mounted on ametal stub, rapidly frozen in liquid nitrogen and main-tained at —196°C before sectioning by cryoukrami-crotomy. The remainder (n = 26) were embedded inOCT (Miles, Elkhart, IN), frozen in isopentane andstored at —35°C for cryostat sections. Pathologic vitre-ous (n = 15) was obtained during surgery under visual

control by aspirating liquefied vitreous from thecenter of the vitreous cavity with a tuberculin syringebefore the vitrectomy infusion was opened. Subretinalfluid aspirates (n = 10) were obtained by externaldrainage. Samples (200 //I to 500 /il) were centrifuged(13,500g for 8 minutes at. room temperature), dividedinto aliquots, and then stored at —35°C. Control sam-ples (n = 9) were obtained from normal human eyesdonated for corneal transplant (Eye Bank, BarraquerOphthalmological Centre, Barcelona, Spain), as de-scribed above. ERM, subretinal fluid, and vitreous as-pirates were classed into A to D3 according to Hiltonet al classification.2'1 ERM were also classed arbitrarilyaccording to the time since ERM onset: up to 2months; 2 to (3 months; and more than 6 months. ERMonset was determined by vitreoretinal interface exami-nation, including fundus biomicroscopy and indirectophthalmoscopy.2'1

Antibodies

For immunochemical detection of ECM glycoproteinsa rabbit antiserum against human fibronectin (Dako-patts, Glostrup, Denmark) and a rabbit antiserumagainst murine LN (Sigma, St. Louis, MO) were used,both at a dilution of 1:200. A mouse mAb directedagainst human VN (mAb VN7) was a generous giftfrom Dr. KJaus Preissner and was used at a dilution of1:150. A polyclonal rabbit anti-murine/?, integrin sub-unit (anti-ASjSi) was provided by Dr. Carles Enrich andhis coworkers at a dilution of 1:50. The as.ft.K mAbLM609. prepared as described,M) was a kind gift fromDr. David Cheresh and was used at a dilution of 1:30.Rhodamine (TRITC) (Dakopatts) or FITC-conjugatedanti-rabbit or anti-mouse immunoglobulin antisera(Boehringer, Penzberg, Germany) were used for opti-cal immunodetection at a dilution of 1:25. For elec-tron microscopic immunocytochemistry, protein A/colloidal gold 16 nm (pA-Au 16 nm), produced in ourlaboratory, and rabbit anti-mouse immunoglobulin-gold (IgG-Au 10 nm) (Amersham, UK) were used at adilution of 1:50. In several electronic immunocyto-chemical experiments, we used rabbit anti-mouse sec-ondary immunoglobulin (Dakopatts) at a dilution of1:75 to amplify the mAb detection. Both primary andsecondary reagents were diluted in 1% ovalbumin inPBS-glycine 0.1 M (pH 7.4).

Indirect Immunofluorescence Procedure

For immunofluorescence techniques, ERM were ob-tained by both consecutive serial cryostat (Frigocut2800 E, Reichert-Jung, Wein, Austria) sections (6 /umto 10 fim) and semithin sections (0.3 ini to 0.4 fim) at-70°C (Ultracut FC4D, Reichert-Jung), prepared on0.5% gelatin-coated slides and placed in humidifiedchamber at 4°C before optical immunocytochemical


Glycoproteins and Receptors in PVR 2793

procedures. Indirect imniunolluorescence for cryo-stat sections was performed following Vilaro et al.4"Semithin frozen sections were air-dried at room tem-perature, washed (3 X 5 minutes) in PBS-glycine O.JM, and blocked (10 minutes) in 1% ovalbumin in PBS-glycine 0.1 M solution in a humidified chamber atroom temperature. Primary antibodies were incu-bated for 2 hours, slides were rinsed with PBS-glycine0.1 M, and the secondary conjugated antibodies (IgG-FITC or IgG-TRITC) were applied for \ hour at roomtemperature in darkness. After the last incubation,slides were washed and mounted with 70% glycerol in5% n-propyl galleate-buliercd mounting medium.Negative control sections were prepared by omissionof the primary antibodies. Fmmunostaining was visual-ized with an epilluorescence microscope (Polyvar II,Reichert-Jung).

For each specimen, 9 to 20 whole sections werestudied and interpreted at low magnification (X10 toX25) microscopy. Indirect immunolluorescence re-sults were always visualized in association with inter-ferential microscopy of the same field.

Electron Immunocytochemical Procedure

For electron immunocytochemical staining, consecu-tive serial ultrathin sections (00 inn to 0.1 um) al— 105°C were obtained by cryoultramicrotomy (Ultra-cut FC4D, Reichert-Jung), placed on gold grids (200mesh) formvar-coated lorTEM and maintained in PBS0.1 M pH 7.4 at 4°C before the electron immunocyto-chemical study. The grids were pretreated in ammo-nium chloride in PBS 0.1 M solution (10 minutes) toeliminate nonspecific: radicals, rinsed in PBS-glycine0.1 M solution ( 4 X 2 minutes), and then blocked in0.5%ovalbumin in PBS-glycine 0.1 M solution (10 min-utes) at room temperature. Primary antibodies wereincubated for 30 minutes at the dilutions mentionedabove and washed in PBS-glycine 0.1 M. Secondaryanti-mouse IgG-Au 10 inn or pA-Au 1(5 nm was thenapplied for 20 minutes. After successive washes, firstin PBS 0.1 M (4 X 2 minutes) and then in double dis-tilled water (6 X 2 minutes), grids were contrasted in0.03% uranyl acetate solution (10 minutes), and a thinsurface membrane of methyl-cellulose was applied.Double-staining procedures lor VN/FN was carriedout in a two-step method. First mAb VN7 was appliedas described, then grids were fixed in 2% paraformal-dehyde in PBS 10 mM pH 7.4 (20 minutes) and succes-sively rinsed in PBS 10 mM. The second step was thencarried out for antisera against FN. Negative controlsections were prepared by omission of the primary an-tibodies, to observe anti-mouse IgG-Au specificity andpA-Au affinity for human tissues. Results were ob-served in conventional TFM (Hitachi (500 AB, Hitachi,Japan).

Hemoglobin and Protein Sample MeasurementControl for blood plasma contamination in both sub-retinal fluid aspirates and normal vitreous sampleswere carried out by henioglobinometry.'11 On the basisof normal vitreous concentrations, we excluded allpathologic samples that showed hemoglobin levelshigher than 0.2 nig/ml (Coulter Counter Model Sr>,Beds, UK). Protein concentration of all samples wasdeterminated by protein-dye binding.'12

Electrophoresis and Western BlottingSodium dodecyl sulphate-polyacrylamide gel electro-phoresis (SDS-PAGE) was carried out as previously de-scribed."1 :< Briefly, normal and pathologic vitreous andsubretinal fluid samples were mixed in a 1:1 (vol/vol)electrophoresis sample buffer (1% SDS/10% 2-iner-captoethanol/10% [wt/vol] glycerol/0.001% bromo-phenol blue/0.125 M Tris-HCI, pH 6.8), kept at100°C for 5 minutes, and electrophoresed (Mini-Pro-tean II 200/2.0 Flectrophoresis Apparatus, Bio-Rad,Richmond, CA) for 2.5 to 4 hours at 50 to (50 V, on a7.5% or 10% polyacrylamide gel in SDS. Protein bandswere visualized by Coomassie blue or silver staining.Western blotting of proteins on nitrocellulose and de-tection using antibodies were performed as de-scribed." SDS-PAGE proteins were transferred(Trans-Blot 200/2.0 Transfer Apparatus, Bio-Rad) at20 V, overnight at 4°C onto a nitrocellulose sheet (Hy-Bond-c, Amersham). Primary antibodies were incu-bated overnight at 4°C in 1% nonfat dry milk in 50mM Tris-HCI (pH 7.4) at 1:1000 dilution. Secondaryanti-rabbit or anti-mouse peroxidase-conjugated im-munoglobulin (Dakopatts) diluted 1:2000 in 0.05%Tween 20 (Sigma) in 50 mM Tris-HCI (pH 7.4) wasapplied for 3 to 4 hours at room temperature. Thenitrocellulose strips were stained with diaminobenzi-dine (0.1% DAB in 50 mM Tris-HCI pH 7.4).

Slot Blotting and DensitometricalQuantificationTo quantify FN and VN, we used a slot blottingmethod (Bio-Dot SF Microfiltration Apparatus, Bio-Rad) on nitrocellulose sheets for each dilution bank ofnormal and pathologic vitreous samples.41 Nitrocellu-lose sheets were incubated with primary and second-ary antibodies and stained using the DAB techniquedescribed above. A direct densitometrical reading wasperformed by digitalized images of nitrocellulosesheets (IBAS II, Interactive Analysis System, Kontron,Munich, Germany). Standard concentrations of puri-fied VN and FN (0.2 ng/nl, Boehringer) were analyzedsimultaneously with each sample.

Statistical AnalysisChi-square analysis (x~) was used to estimate statisticaldifferences between the variabilities of frequency



among groups. To study the variability betweenmeans, we applied an unbalanced analysis of variance(ANOVA) by least significant difference (LSD).

RESULTS

FN Matrix Assembly in ERM, Glycoproteins,and Integrin ExpressionThe time course and pattern of distribution in vivo ofFN fibril formation in ERM matrix was established incryostat sections (n = 26 ERM) by indirect immunoflu-orescence. Large amounts of FN were evident in mostspecimens, in pericellular or fibrillar arrangement inmatrix, with short and thin or longer and thicker fibrils(Fig. 1). A pericellular pattern of distribution was

more frequently noted in the group of ERM with only2 months of evolution (Figs. 1A and IB) than in thegroup of ERM aged between 2 to 6 months (P < 0.005,by chi-square analysis test). Pericellular pattern wasrarely observed in the third group of ERM greaterthan 6 months of evolution, and differences were es-tablished with the first group (P < 0.05, by chi-squareanalysis). No significant difference was found betweenthe intermediate group and the last. Electron-immuno-cytochemistry with immunogold labeling techniquesconfirmed the FN in its pericellular pattern and incollagen fiber arrangement. Individual cells presentedlarge amounts of FN deposited along the plasma mem-branes and on cytoplasmic processes when present(Figs. 1C and ID); cells with a wide variety of morphol-ogy showed large amounts of this glycoprotein.

Dense fibrillar fluorescent labeling was the mostfrequent finding for FN in matrix material, sometimeslocalized (Figs. II and 1J), sometimes generalized dis-tributed across surface of the sample (Figs. 1E-H).The first group of ERM (<2 months of evolution)showed a clear localized distribution when comparedwith the group of intermediate time of evolution,which showed a generalized pattern in the extracellu-lar medium (P < 0.001, by chi-square analysis). FNappeared to decrease in matrix material with the timeof ERM evolution and showed a tendency to assume alocalized distribution in specimens with more than 6months of evolution when compared with the interme-diate group (P < 0.01, by chi-square analysis).

FIGURE 1. Fibronectin expression in ERM. (A) Semithin sec-tions prepared by indirect immunofluorescence stainshowed moderate amounts of pericellular FN (arrows)(X990, bar = 20 fim). (B) A wide variety of morphologic celltypes presented immunoreactive FN (arrows) (X800, bar =20 fitn). (C) Immunoelectron microscopy showed labeling(16 nm gold) (arrows) closely related with plasma membrane(XI 9,200, bar = 1 nm). (D) Higher magnification of brack-eted area in C; immunoreactive FN binds a peripheral elec-trodense material (arrows) related to cell membrane ob-served in some cell types. Collagenic matrix composed byfibers with clear periodicity present variable amounts of im-munogold label (X32.500, bar = 1 fim). (E) Cryostat-sec-tioned specimens analyzed by interference contrast micros-copy revealed abundant and dense fibrillar matrix in regulararrangement (X390). (F) By immunofluorescence, abundantamounts for this protein in generalized and regular thickerfibrillar pattern across the specimen surface were seen(X390). (G) Generalized and fine fibrillar FN irregular dis-tributed in matrix material were also common pattern oflabeling (X315). (H) The same section examined by interfer-ence contrast microscopy reveals areas of regular and ran-dom collagenous fiber arrangement (X390). (I) Localizedareas of regular pattern of immunofluorescence stain(X390) contrast with (J) areas of irregular fibrillar distribu-tion in the same specimen (X315, bar = 30 pim).


Glycoproteins and Receptors in PVR

A

2795

*te

FIGURE 2. Laminin expression in ERM specimens. (A) Cellsdistributed in matrix (arrows) visualized by interference con-trast microscopy (B) presented variable immunoreactivitystaining for LN (arrows) (X900, bar = 20 Mm). (C) Slightlabeling in a fine fibrillar pattern of distribution (D) in smalllocalized areas in the same sectioned specimen examined byinterference contrast microscopy (X360, bar = 40 #m). (E)By immunoelectron microscopy, pA-Au (16 nm) particleswere noted both related to the plasma membrane (arrow)and the extracellular material surrounding cells (X19.200,bar = 1 fixn). (F) Collagenic fibers with periodicity presentedimmunoreactivity for this glycoprotein (X39,500, bar = 0.5/an). (G) Scanty immunoreactivity for LN was common fea-ture in matrix (19,200, bar = 1 jim).

In all ERM examined (n = 54), LN was present in31 specimens (57%) but always as a minor componentin the tissue. Its pericellular distribution was also ob-served (Fig. 2B), but most frequently a fine fibrillarpattern was noted in localized areas of the specimen(Fig. 2C). Although the third group of ERM (>6months of evolution) tended to lack this glycoprotein,no significant difference was found among groups. Byelectron-immunocytochemistry, less extensive amountsof LN were occasionally noted around the plasmamembrane but were not directly related with it (Fig.2E). The distribution of LN in collagenic matrixshowed a similar pattern to that observed for FN, butwas restricted in confluent areas of immunogold label-ing, which in most cases occupied small areas of thespecimen (Fig. 2F and 2G).

In several consecutive serial cryostat sections, aclear pericellular preference for determinate celltypes was manifest for both proteins (FN and LN), but

FIGURE 3. Identification of pericellular immunoreactive FNand LN in ERM. Consecutive serial cryosections were incu-bated with specific antisera for FN and LN in indirect immu-nofluorescence experiments. (A) Specimens incubated withanti-FN antisera showed high cell population that presentstrong pericellular staining. (B) The same section examinedby interference contrast microscope revealed a group of pig-ment-laden cells localized at the edges of sample (betweenopen arrows). This area was negative for FN immunostaining;therefore, some pigmented cells at the inner aspect of thespecimen showed positive label {completearrows). (C) Consec-utive cryosection obtained for the same specimen and incu-bated with anti-LN antisera showed positive label for cells atthe periphery of sample corresponding the marginal areanonreactive for FN {between arrows) (X315, bar = 40 /tm).



FIGURE 4. Expression of /3, subunit complex and a\,/3a integrin in ERM. Semithin cryosec-tioned specimens were analyzed by indirect immunofluorescence after incubation with anti-AS/3, antisera and mAb LM609 directed against avj83 receptor. (A) Wide variety of cellspresented fluorescent staining for /?, complex receptor (arrows) (X730, bar = 20 j*m). (B) TheVN receptor (avfi.A) was occasionally found in isolated cells or organized in clusters or nest(arrows) (X360, bar = 40 /*m). (C) VN immunolocalization in the same sample shows a pericel-lular pattern of distribution (arrows) as its receptor (X450, bar = 30 jum). (D) High magnifica-tion showed immunofluorescence for /3,-subunit complex closely related to the plasma mem-brane (arrow) (XI,130, bar = 10 Mni)- (E) Beta-1 labeling for plasma membrane (pm) wascorroborated by immunoelectron microscopy (X39,500, bar = 0.5

mainly for LN (Fig. 3). Areas with pigment-laden cellsappeared to show a preference in labeling LN (Figs.3B and 3C), although there were regions in sectionsthat had significant labeling in the absence of pig-mented cells,

VN was demonstrated in 6 out of 17 (35%) speci-mens tested for this glycoprotein. Its pericellular dis-tribution (Fig. 4C) was more frequently seen than itsgeneralized fine fibrillar arrangement in the matrix.As expected, by electron-immunocytochemical proce-dures the labeling for VN was distributed around thecell membrane but was not restricted to it (not shown).A low level of immunogold reactivity was also presentin dense or loose bundles of collagen fibers in local-ized areas (not shown). Immunofluorescence labelingfor av/33 and /?] subunit complex integrins was occa-

sionally identified (Fig. 4). The latter was observedmore frequently, but it was observed less frequently inERM with more than 6 months' evolution. By electronmicroscopy methods, anti-jSj subunit complex labelingwas achieved infrequently but always correlated withcell cytoplasmic membrane (Fig. 4E); immunolabeledcells did not show any particular morphologic charac-teristic. Alpha-V Beta-3 integrin was infrequentlyidentified and was associated with slight immunogoldlabeling of plasma membrane (not shown).

In collagenous matrix, densely or looselyarranged, variable quantities of FN were also noted(Figs. 5A and 5B). Double-labeling experimentsshowed a clear colocalization of VN and FN over col-lagenous bundles (Figs. 5C and 5D) and occasionally ina pericellular pattern, with predominant immunoreac-



A B

tft**

FIGURE 5. Colocalization of FN and VN in collagenic matrixin ERM. Ultrathin sections were incubated with niAb VN7anti-VN and then with antl-FN antisera. (A) In single-labeling experiments, strong immunoreactivity for FN wasobserved in densely arranged collagenous material(X27,500). (B) High magnification shows labeling across col-lagen bundles (X39,500). (C) Double-label experiments al-ways showed more abundant FN immunoreactivity (pA-Au16 nm) {arrowhead) than VN (IgG-Au 10 nm) (arrows)(X30(000). (D) Colocalized label for FN and VN {arrows) wasusually seen (X27,500, bar = 0.5 Mm).

tivity for FN (not shown). Electron microscopy con-trols showed virtually no background of Au labeling.

Electrophoresis and Immunoblotting forNormal and Pathologic Vitreous, andSubretinal Fluid SamplesPathologic vitreous and subretinal fluid samples ofPVR patients were compared with pathologic vitreoussamples from other intraocular proliferative dis-orders. Controls were normal human serum and nor-mal vitreous samples obtained from human eyes afterdeath. Protein concentration of pathologic PVR vitre-ous for each stage of disease were substantially in-creased when compared with normal vitreous samples(0.506 ± 0.04 Mg//*1) (p < 0.001, by unbalanced AN-OVA test). Normal vitreous separated by 7.5% SDS-PAGE under reducing conditions and Coomassie blueor silver staining revealed a main band around 66 kD,corresponding to the albumin fraction when normal

2797

human serum was processed in the same experiment.In the pathologic vitreous and subretinal fluid sam-ples, main bands of low molecular weight (^66 kD)were noted, which coincided with the electrophoreticband pattern of the low-weight proteins observed inthe normal human serum samples analyzed. Proteinswith a molecular mass higher than 66 kD were lessevident, and similarities were found with the serumband profile (Fig. 6). By immunoblotting assays, FNwas detected in 210 to 230 kD bands in 7.5% SDS-PAGE gels, coincident with the electrophoretic mobil-ity of purified FN used as a mass marker. In normalvitreous samples, little FN immunoreactivity was de-tected (Fig. 7a). In 10% SDS-PAGE gels, VN immuno-reactivity was present in all the samples analyzed as adouble band (Fig. 7b) corresponding to the VN 75 kDand 65 kD bands.12 Several pathologic samples immu-noblotted with antibodies against FN or VN showedlow molecular mass bands that may represent de-graded fragments for intact FN or VN. LN immunore-activity was not detected in any subretinal fluid or vitre-ous sample.

FN and VN Concentration Measurement inNormal and Pathologic Samples

Densitometry by IBAS indicated significant differ-ences in the FN concentration among the stages ofPVR samples and other intraocular pathologic prolif-erative conditions. In the early stage of disease (stageA), low FN levels (0.188 ± 0.04 ng/)tg protein) wereobserved similar to normal samples (0.129 ± 0.03 ng/Mg protein). At the next stage of disorder (stage B), asubstantial raise in the intraocular FN levels (0.490 ±0.08 ng//xg protein) was detected, and this differencewas significant when compared with both the normaland the previous pathologic stage samples (P < 0.05,by unbalanced ANOVA test). Similar increased con-centrations of FN were noted in the later PVR stages.Decreased levels of intraocular VN concentrationwere observed in pathologic samples when comparedwith normal samples (0.195 ± 0.05 ng/^g protein).VN concentration appeared to decrease with develop-ment of intraocular proliferative disorder. The VN lev-els at stage A (0.144 ± 0.08 ng/jug protein) and B(0.163 ± 0.08 ng//itg protein) were significantly higherthan the later stage D (0.061 ± 0.03 ng//ig protein) (P= 0.05, by unbalanced ANOVA test). Similar VN levelswere noted in stage C (0.093 ± 0.04 ng/^ig protein),and when the later stages (C and D) were comparedwith the normal VN levels, statistical differences wereapparent (P < 0.05, by unbalanced ANOVA test). Ta-ble 1 provides quantitative measurements of total pro-teins, FN, and VN relative amounts in normal andpathologic conditions. The total variation of FN andVN concentrations at each stage of PVR are presentedin Figure 8.


2798

mw nl S

nl+S

Investigative Ophthalmology & Visual Science, May 1994, Vol. 35, No. 6

PVR (SRF) PVRA A B B A B C C D D

kDa

220

66

4536

24

FIGURE 6. Electrophoretic profile of normal (nl) and PVR vitreous proteins separated on7.5% SDS-PAGE under reducing conditions and Coomassie blue staining. Large amounts ofprotein of low molecular mass (^66 kD) characterize pathologic samples in both subretinalfluid (SRF) in early stages (A and B) and vitreous aspirates in later stages (C and D) of disease.Proteins of molecular mass higher than 66 kD observed in normal human serum (S) were lessevident in pathologic samples. Molecular weight (MW) standards were as follows: fibronectin(220 kD), bovine serum albumin (66 kD), ovaibumin (45 kD), glyceraldehyde-3-phosphatedehydrogenase (36 kD), and trypsinogen (24 kD).

DISCUSSION

Significant progress has been made in elucidating phy-siopathogenic mechanisms in PVR, although manyfundamental questions remain. One such questionconcerns the events of cell-matrix interaction inperiretinal proliferative fibrocellular tissue formation.

kDa

220

FN

1

FN

nl2

PVR A(SRF)

PVRB(SRF)

PVR PVRni C D7 8 9

Several studies have evaluated the involvement ofvarious cell types30"33 and the possible role of solubleand insoluble proteins35"38 in human ERM. Here wehave studied the cellular distribution of the main ex-tracellular glycoproteins that could mediate cell—ma-trix interaction in PVR. We have observed that FN isthe major protein involved in both extracellular andintravitreous environment, and we suggest it may havea leading role in membranogenesis. On the otherhand, the decreased levels of intraocular VN observedin the development of PVR could be involved in themechanisms of cell adhesion in the established ERM.

The regulation of extracellular matrix assemblyand cellular response to these matrices are importantfor the control of several cellular events. For instance,cell morphology, cell attachment and migration, tissue

PVR

PDR

1

A C(SRF)

2 3

IOFB

5

kDa

75 165 i

FIGURE 7. Immunoblot analysis of normal (nl) and PVR vitre-ous proteins separated by 7.5% and 10% SDS-PAGE underreducing conditions, (a) Western blot of subretinal fluid(SRF) in the early stages A (lanes 3 and 4) and B (lanes 5 and6) of PVR and later stages C (lane 8) and D (lane 9) werepositive for FN. Purified FN (lanes 1 and 2) was used aspositive control, (b) On 10% SDS-PAGE, PVR samples indifferent stages (lanes 2, 3, and 4) and from patients withintraocular foreign body (IOFB) and proliferative diabeticretinopathy (PDR) were incubated with mAb VN7 directedagainst VN. Double-band profile for VN (65 to 75 kD) wasobserved and low molecular mass fragments of degradedglycoprotein were also detected.



TABLE l. Concentrations of Total Proteins, FN, and VN in Normal (nVitreous (n = 25) Samples

Total Proteins Fibronectin(ng/fig Protein)

2799

9) and Pathologic

VitronectinProtein)

NormalPVR AfPVR BtPVR CPVR DIOFB

0.50013.31IT). 7015.1314.7020.80

± 0 .± 3.± 5.± 4.± 2.± 0.

0437§10§•r>0§

oo§oos

0.129 ± 0.030.188 ±0.040.400 ± 0.08+0.523 ± 0.07+0.454 ± 0.02+1.346 ± 0.f>0§

0.1 Of) ± 0.050.144 ±0.080.103 ± 0.080.093 ± 0.04+0.001 ± 0.03:0.154 ± 0.09

1'VR. prnlilcralivc vitrcorciiiif>p:ilhy and Mali's (A. B. ('.. D); IOFB. intraix.ular foreign body.* Mean ± SK.t Subrciinal lluid.: Slaiisikal .si^nilicancc from normal samples (I' < 0.0")).§ Siaiisikal si^niiic.incc from normal samples (/' < 0.001).

stability, cell polarity, and differentiation often re-quire adhesion and specific: interactions of a cell withits substrate. ERM formation can be understood as apathologic proliferative "model" in which FN matrixassembly shows interesting features. When intraocularproliferative tissue with less than 2 months of clinicalevolution was labeled for FN, pericellular distributionwas significantly more evident than in older speci-mens. In addition, a localized fibrillar FN pattern wasmore apparent in the first specimen group analyzedthan in others. On the other hand, fibrillar FN genera-lizably distributed in matrix was a common featurethat characterized tissue with longer evolution and wasless evident in specimens with more than 6 months ofclinical evolution; absence of FN immunoreactivitywas noted only in the latter group. Upon analyzing the

10

enc

oa.

5 -

i

1 ^

I •

|1 1

I 1NL

PVR stages

FIGURE 8. Profile of total concentration of FN and VN inclinical evolutive Mages (A, B, C, and D) of PVR. Normalvitreous samples (NL) were also analyzed.

role of FN in wound repair'1"10 some crucial chrono-logic events were observed: During the early stages ofwound healing, plasmatic-soluble FN stimulates di-rected cell migration toward the wound, making it actas a chemotactic element, and it also has a role in cell-to-cell, and cell-to-substrate adhesion; later, insolu-ble FN is secreted by cells to produce a fibrillar extra-cellular matrix that has important relationships withcellular anchorage and interaction with this matrix;finally, FN may enhance phagocytosis in wounds butgradually disappears with maturation of collagen. Theassembly of FN into fibrils was found as a cell surface-mediated process'21 ' ' that involves the binding of thisglycoprotein by a receptor-like system, whereas thea.r,/?,,4* and some other /3, integrins'1'1-'" seem to be re-quired as part of the adhesion mechanism. Thus, a cellsurface-correlated FN is observed first, followed by apartial assimilation into the matrix. For these reasons,we may consider, according to the behavior of FN ob-served in the intraocular fibrocellular tissue exam-ined, that PVR may represent modified intraocularwound repair because the assembly behavior of thisglycoprotein agrees with the classical model describedabove. Based on stereologic procedures, we observedthat the volume-density cell-matrix estimation inERM tends to decrease with the time of disease evolu-tion, which might be explained by a reduction of cellu-lar population and/or an increase in collagen deposi-tion in fibrocellular tissue (unpublished data, 1993).FN was abundant in most of the ERM studied, havingbeen reduced in amount and occasionally absent inspecimens with more than 6 months of evolution; atthis stage, similar to wound repair, FN tends to disap-pear gradually whereas stabilization of the collagenicframework is noted. Cells involved in ERM formationmay be considered the probable source of FN. Many-cell types identified as a constituent of these mem-branes (retinal glia, retina) pigment epithelium, andfibroblast-like cells were largely recognized as com-ponents in the intraocular fibrocellular tissue in hu-



man PVR.8") are known to be capable of FN synthesisin vitro,:>l:'~ and variable labeling of FN mRNA hasrecently been reported in cells in human detached ret-ina and in ERM."<8

LN and VN appear to be involved in fibrocellulartissue formation, but they only have a secondary role.Their identification was occasionally possible in peri-cellular and fibrillar patterns. Pericellular LN labelingwas noted in this study by optical and electron micros-copy immunochemistry procedures. Pigment-ladencells were frequently observed associated to this glyco-protein, but these were not the only characteristic celltype positive for immunoreactive LN. Piginented cells,as a component of ERM, may be represented by reti-nal pigment epithelial cells and macrophage-likecells.-H2 In addition, ultrastructural studies found thatretinal pigment epithelium and retinal glial cells areable to constitute basement membranes, and they actwith collagen type IV as structural proteins. Amongseveral functions, LN promotes epithelial cell adhe-sion to collagen"f>l and has the ability to convert em-bryonic mesenchymal cells into polarized-shapedform.r>> These data are sufficient evidence to justifyfurther double-labeling assays to elucidate two majorpoints: first, whether pigment-laden cells that label im-munoreactive LN represent retinal pigment epithelialcells, and second, whether cells positive for pericellu-lar LN have a glial origin. The significance of VN incell events involved in epiretinal formation could bemainly sought in its ability to promote cell adhe-sion141'" and its biochemical structure, which includesa domain capable of binding collagen.212-" The cellattachment activity of VN is based on the RGD se-quence,1'11" •which is recognized by a wide variety ofcell types.1" Properties of VN in hemostasis are exten-sively described,1112 but, in our view, it is difficult tocorrelate hemostatic phenomena with ERM formationin PVR.

A fine fibrillar pattern of VN arrangement in thematrix material was observed, and colocalization ofVN and FN was frequently noted in collagenic bundlesby elect ron-immunochemistry procedures. Several im-munolluorcscence studies suggest the deposition ofVN in a fibrillar pattern in loose connective tissue ofmany normal structures, including lung, kidney, skin,and smooth and skeletal muscle,*•'' where it is some-times colocalized with FN. These data provide evi-dence that appreciable amounts of VN may be depos-ited far from the liver where it is biosynthesized. Forthis reason, the mechanisms involved in VN and FNmatrix deposition during intraocular fibrocellular tis-sue formation in PVR could also involve the exposureof fibrillar collagenic framework and other proteinssynthesized by local cellular components to plasmaticVN and FN present in the altered vitreous. Our data(see below) showed an increasing intravitreal level of

FN and a slight alteration in VN concentration in sub-retinal fluid and vitreous aspirates, suggesting this pro-cess at least for FN.

Expression of 0,-subunit complex and av/33 inte-grins is corroborated by the pericellular pattern iden-tified by these glycoproteins. However, we do not havesufficient data to conclude that /3, complex was prefer-entially expressed in the group of ERM whose pericel-lular labeling for FN were most evident, or whetherthis integrin immunoreactivity decreased in specimenswith longer clinical evolution. In addition, in immu-noelectron microscopy labeling procedures, integrinimmunoreactivity seems to be scanty, which could bedue to normally diminished tissue antigenicity as aconsequence of the method. Another explanation forthe integrin activity in PVR involves its presence in theearliest stages of disease when constituted ERM arenot found, but the events correlated to membrano-genesis, such as cellular migration, adhesion, and pro-liferation, certainly do take place. Moreover, surgicallyexcised ERM represent a final product of a complexproliferative phenomenon and look like a stabilized"scar" in the wound repair process. The distributionand presumed functional interaction of receptors, FN,and LN are generally coincident in several struc-tures,4 ' ' but some peculiarities are remarkable. Forexample, responses to collagen and FN or LN arecommonly mediated by /i -class integrins, whereasnonplatelet receptors for VN and FN are composed ofav-subunit in association with Pu/3^, or jSr,.5""1'" In addi-tion, FN can bind with the classical VN-receptor («v/?:<)specifically and with high affinity, supporting cell ad-hesion to matrix proteins.2S Finally, VN-receptor(aj3;0 may act as an LN-receptor and can mediate celladhesion.S(jl All this evidence is indicative that inte-grin receptor complexes and specific sequence do-mains of glycoproteins may have a major role in intra-ocular fibrocellular tissue formation.

We present evidence that ERM formation can rep-resent a cell-mediated process. It is not clear how themechanisms involved in cell migration, adhesion, andgrowth observed in PVR are initiated, but we hypothe-size an important contribution of breakdown of theblood-retinal barrier, in this case reflected by a failureof the retinal pigment epithelium to function as abarrier."-' High concentrations of proteins in subreti-nal fluid (early stages) and in pathologic vitreous (laterstages) characterize the PVR intraocular environment.These levels tend to be constant at the different clini-cal stages of disease and were 25 to 30 times higherthan that of normal vitreous and about one-fourth ofprotein levels of normal serum. In fact, as previouslydescribed, the serum contains chemoattractant sub-stances that act by increasing migration for retinal-derived glial cells and retinal pigment epithelial cells,in vitro; cell migration appears to be mediated in a



dose-dependent manner, and, among these serum ele-ments, FN was highly active.:'"'':< In addition, impor-tant alterations in integrity and permeability of blood-retinal barriers have been found in eyes with diabeticretinopathy,'1'2 as well as in a transient form in eyes withretinal detachment.'1' Hemorrhage may also result inrelease of plasma or serum components into the vitre-ous cavity, and these are thought to be associated withthe increased incidence of PVR after penetrating ocu-lar injuries,b:i with or without intraocular foreign bod-ies.

Our findings showed increased levels of intravi-treal FN and protein in traumatic PVR with intraocu-lar foreign bodies, associated with accepted hemoglo-bin concentrations (<0.2 mg/ml) in samples. Break-down of blood-retinal barriers may explain thechanges in proteins and FN levels observed in patho-logic intraocular milieu. Intraocular FN levels vary sig-nificantly in the earliest stages of PVR (stages A and B)when we could only analyze subletinal iluid becauseintraocular surgery with vitreous manipulation wasrarely imperative to reattach the retina. At thesestages, we may observe clinical intraocular changes butconstituted ERM are not found, although mechanismsinvolved in membranogenesis are certainly present.'2"Similar relative levels of FN in normal vitreous areobserved in subretinal Iluid obtained at si age A ol dis-ease, although important changes in protein concen-trations were found. Associating experimental andclinical data, we suggest that FN may have a significantrole in initial events that characterize several clinicalintraocular aspects of F_RM formation in PVR.2" Anincrease in FN levels could not be exclusively ex-plained by plasmatic origin because the protein con-centration remains unchanged. An attractive hypothe-sis is that early cell production of FN at stage A isresponsible for the increased FN levels observed atstage B.

Unlike FN, intraocular VN levels decrease pro-gressively with the clinical evolution of disease, andslight differences in concentrations were noted whencomparing the earliest stages with normal vitreoussamples. These findings are difficult to explain be-cause plasmatic concentrations of FN (0.3 /ug//ul) andVN (0.2 to 0.4 /ug//xl) are similar.2"'1"-'1-' If permeabilityof ocular barriers reveals high concentrations of FN,which has a much higher molecular weight (450 kD)than VN (65 to 75 kD), it might be difficult to accept aselective mechanism that allows an exclusive passageof intraocular FN from circulation. For these reasons,we assume that VN could be adsorbed in some eventsof cellular adhesion and spreading, correlated to cell-substratum interaction. This fact might be acceptedand has been demonstrated in vitro/'-1"'1''7 It is likelythat, in vivo, FN and VN exert differential effects inmechanisms involving tissue growth and repair, and

the differential distribution of these glycoproteins invjvo'.i.ni.-i:i.i.H S U p p O I ( s t n j s hypothesis.

Finally, it has recently been reported that chronicwound Iluid samples, examined by immunoblottingand cell adhesion assays, showed a marked and some-times complete degradation of VN, as well as a lessevident degradation of FN that alters the cell adhesionnecessary for normal wound closure.'1'1 In our study,we noted the presence of some products of FN and VNdegradation in immunoblotting experiments in severalsamples. However, our data are insufficient to deter-mine whether degradation is time-dependent or is re-lated to clinical stages of disease. Despite this evi-dence, similarities between wound fluids and PVR in-traocular environment may be established, suggestingthat periretinal fibrocellular tissue formation ob-served in this disorder may represent a final productof a specific mechanism of intraocular wound repair.

Key Words

epirclinal membranes, retinal detachment, glycoproleins,integrins, iinmunocytochemistry

Acknowledgments

The authors thank the following people for their contribu-tions to this study: Dr. David Cheresh (Research Institute ofScripps Clinic:, La Jolla, CA) for niAb LM009; Dr. KlausPreissner (Kerckhoff-Klinik. Max-Planck-Gescllschaft, BadNaulieiin, Germany) for niAb VN7; Dr. Carles Enrich andcollaborators (University of Barcelona, Barcelona, Spain)for aiiii-AS|8|; Dr. Manuel Reina and Dr. Luis Lecea (Univer-sity of Barcelona. Barcelona, Spain) for his suggestionsabout the blotting techniques; Servians Cientifico-Tecnicosde la Universidad de Barcelona, especially to Francisca Peirofor her assistance in 1BAS analysis; Matilde Ruiz and Dra.Nora Mestrc (Ceniro de Oftalmologia Barraquer, Barce-lona, Spain) for hemoglubinometric methods; Mr. RobinRycroft for expert assistance in correcting (his manuscript;and Dr. Alfredo Muinos, Dr. Rafael Barraquer, Dr. Fran-cisco Matcus-Marquez, Dr. Carlos D. Heredia, and Dr. Dan-iel Vilaplana (Centro de Oftalmologia Barraqucr. Barce-lona. Spain), who kindly provided specimens for this work.

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The role of fibronectin, laminin, vitronectin and their receptors on