The role of fibronectin, laminin, vitronectin and their receptors on cellular adhesion in proliferative vitreoretinopathy.

Abstract

To examine the possible role of some adhesion multifunctional glycoproteins of the extracellular matrix, such as fibronectin (FN), laminin (LN), vitronectin (VN) and their receptors (beta 1-subunit complex and alpha v beta 3 integrins) in events of cell migration and adhesion in proliferative vitreoretinopathy (PVR).
Optical and electron-immunocytochemical techniques were carried out on epiretinal membranes. Electrophoretic immunoblotting methods and densitometric analysis of normal and PVR vitreous were also undertaken. Chi-square (chi 2) and unbalanced analysis of variance were employed for statistical analysis.
FN was detected as a major component in the extracellular matrix in both fibrillar and pericellular arrangement. A change in pericellular distribution to more fibrillous organization was related to the time of intraocular proliferative tissue development (P < 0.001). LN and VN were observed as minor components in extracellular matrix. A colocalized pattern between VN and FN in collagenic bundles of the matrix was often observed. Beta-1 subunit and alpha v beta 3 receptors were usually localized in a position that could mediate the interaction of FN, VN, and/or LN to the cell plasma membrane. Increased levels of FN concentration were observed in both subretinal fluid and pathologic vitreous; intravitreal FN concentration tends to increase with clinical stages of the evolution of PVR, whereas intravitreal VN levels tend to decrease.
Results suggest that FN could mediate the initial events involved in epiretinal membrane formation, and VN could modulate the adhesion mechanisms in established membranes.

Full text

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The Role of Fibronectin, Laminin, Vitronectin
and Their Receptors on Cellular Adhesion
in Proliferative Vitreoretinopathy
Ricardo P. Casaroli Marano*\ and Senen Vilaro*
Purpose. To examine the possible role of some adhesion multifunctional glycoproteins of the
extracellular 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 epiretinal
membranes. Electmphorelic immunoblotling methods and densitonietric analysis of normal
and PVR vitreous were also undertaken. Chi-square (x'J) and unbalanced analysis of variance
were employed for statistical analysis.
Results. FN was detected as a major component in the extracellular matrix in both fibrillar and
pericellular arrangement. A change in pericellular distribution to more fibrillous organization
was related to the time of intraocular proliferative tissue development (P < 0.001). LN and VN
were observed as minor components in extracellular matrix. A colocali/ed pattern between VN
and 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 observed
in 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 to
decrease.
Conclusions. Results suggest that FN could mediate the initial events involved in epiretinal
membrane 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 its
structural integrity.1 Most ECM contain a fibrous col-
lagenic network or other fiber-forming proteins, such
as glycoproteins. Noncollagenous extracellular glyco-
proteins have important roles in many cell-surface in-
teractions, such as adhesion, migration, phenotype
differentiation 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 of
basement membranes and possesses multiple func-
tional sites that mediate its interactions with cells and
other ECM elements.(1"s Finally, vitroneclin (VN; MVV
(35 to 78 kD) is present in fibrillar pattern in the ECM
of a variety of tissues"10 and also in circulation with a
predominant 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 interactions
involve 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' In
particular, integrins—heterodimeric molecules of
cell-adhesion receptors—have been implicated in a va-
riety of cell-to-cell and cell-to-matrix interactions and
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2791

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Investigative Ophthalmology & Visual Science, May 1994, Vol. 35, No. 6
are also active in transmitting signals from the extra to
the iniracellular compartment. IK~21 All integrins are a
subunits, 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 are
known 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 serious
human intraocular disorder characterized by fibrocel-
lular sheets of connective tissue proliferating on both
retinal 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) are
composed of different cell types, essentially retinal
pigment epithelial cells, glial cells, fibroblasts, macro-
phages, and myofibroblast-like cells*0"'2 surrounded
by a fibrous matrix with an extensive amount of colla-
gen.3ft:w Pathogenesis is poorly understood, especially
the mechanisms of cellular migration, adhesion, and
proliferation. Previous studies have suggested the
participation 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 could
have an important role in the earliest pre-membrano-
genic stages of this pathologic condition and later in
the mechanisms of cell-ECM adhesion.
MATERIAL AND METHODS
Human Tissue and Samples
Human ERM (n = 54) were dissected and removed by
appropriate intraocular vitreous forceps (Grieshaber,
Switzerland; Cat. No. C-612.08, C-612.13, and
C-612.98) from patients with retinal detachment com-
plicated by PVR who were undergoing intraocular
surgery. 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 2
hours at 4°C. They were then rinsed in PBS 0.1 M pH
7.4 and immersed in 2.1 M sucrose in PBS solution for
2 to 4 hours. Part of them (n = 28) were mounted on a
metal stub, rapidly frozen in liquid nitrogen and main-
tained at —196°C before sectioning by cryoukrami-
crotomy. The remainder (n = 26) were embedded in
OCT (Miles, Elkhart, IN), frozen in isopentane and
stored at —35°C for cryostat sections. Pathologic vitre-
ous (n = 15) was obtained during surgery under visual
control by aspirating liquefied vitreous from the
center of the vitreous cavity with a tuberculin syringe
before the vitrectomy infusion was opened. Subretinal
fluid aspirates (n = 10) were obtained by external
drainage. Samples (200 //I to 500 /il) were centrifuged
(13,500g for 8 minutes at. room temperature), divided
into aliquots, and then stored at —35°C. Control sam-
ples (n = 9) were obtained from normal human eyes
donated for corneal transplant (Eye Bank, Barraquer
Ophthalmological Centre, Barcelona, Spain), as de-
scribed above. ERM, subretinal fluid, and vitreous as-
pirates were classed into A to D3 according to Hilton
et al classification.2'1 ERM were also classed arbitrarily
according to the time since ERM onset: up to 2
months; 2 to (3 months; and more than 6 months. ERM
onset was determined by vitreoretinal interface exami-
nation, including fundus biomicroscopy and indirect
ophthalmoscopy.2'1
Antibodies
For immunochemical detection of ECM glycoproteins
a rabbit antiserum against human fibronectin (Dako-
patts, Glostrup, Denmark) and a rabbit antiserum
against murine LN (Sigma, St. Louis, MO) were used,
both at a dilution of 1:200. A mouse mAb directed
against human VN (mAb VN7) was a generous gift
from Dr. KJaus Preissner and was used at a dilution of
1:150. A polyclonal rabbit anti-murine/?, integrin sub-
unit (anti-ASjSi) was provided by Dr. Carles Enrich and
his coworkers at a dilution of 1:50. The as.ft.K mAb
LM609. prepared as described,M) was a kind gift from
Dr. David Cheresh and was used at a dilution of 1:30.
Rhodamine (TRITC) (Dakopatts) or FITC-conjugated
anti-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 our
laboratory, and rabbit anti-mouse immunoglobulin-
gold (IgG-Au 10 nm) (Amersham, UK) were used at a
dilution of 1:50. In several electronic immunocyto-
chemical experiments, we used rabbit anti-mouse sec-
ondary immunoglobulin (Dakopatts) at a dilution of
1:75 to amplify the mAb detection. Both primary and
secondary reagents were diluted in 1% ovalbumin in
PBS-glycine 0.1 M (pH 7.4).
Indirect Immunofluorescence Procedure
For immunofluorescence techniques, ERM were ob-
tained by both consecutive serial cryostat (Frigocut
2800 E, Reichert-Jung, Wein, Austria) sections (6 /um
to 10 fim) and semithin sections (0.3 ^ini to 0.4 fim) at
-70°C (Ultracut FC4D, Reichert-Jung), prepared on
0.5% gelatin-coated slides and placed in humidified
chamber at 4°C before optical immunocytochemical

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Glycoproteins and Receptors in PVR2793
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.J
M, and blocked (10 minutes) in 1% ovalbumin in PBS-
glycine 0.1 M solution in a humidified chamber at
room temperature. Primary antibodies were incu-
bated for 2 hours, slides were rinsed with PBS-glycine
0.1 M, and the secondary conjugated antibodies (IgG-
FITC or IgG-TRITC) were applied for \ hour at room
temperature in darkness. After the last incubation,
slides were washed and mounted with 70% glycerol in
5% n-propyl galleate-buliercd mounting medium.
Negative control sections were prepared by omission
of the primary antibodies. Fmmunostaining was visual-
ized with an epilluorescence microscope (Polyvar II,
Reichert-Jung).
For each specimen, 9 to 20 whole sections were
studied and interpreted at low magnification (X10 to
X25) 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 (200
mesh) formvar-coated lorTEM and maintained in PBS
0.1 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) to
eliminate nonspecific: radicals, rinsed in PBS-glycine
0.1 M solution (4X2 minutes), and then blocked in
0.5%ovalbumin in PBS-glycine 0.1 M solution (10 min-
utes) at room temperature. Primary antibodies were
incubated for 30 minutes at the dilutions mentioned
above and washed in PBS-glycine 0.1 M. Secondary
anti-mouse IgG-Au 10 inn or pA-Au 1(5 nm was then
applied for 20 minutes. After successive washes, first
in PBS 0.1 M (4 X 2 minutes) and then in double dis-
tilled water (6 X 2 minutes), grids were contrasted in
0.03% uranyl acetate solution (10 minutes), and a thin
surface membrane of methyl-cellulose was applied.
Double-staining procedures lor VN/FN was carried
out in a two-step method. First mAb VN7 was applied
as 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 then
carried out for antisera against FN. Negative control
sections were prepared by omission of the primary an-
tibodies, to observe anti-mouse IgG-Au specificity and
pA-Au affinity for human tissues. Results were ob-
served in conventional TFM (Hitachi (500 AB, Hitachi,
Japan).
Hemoglobin and Protein Sample Measurement
Control for blood plasma contamination in both sub-
retinal fluid aspirates and normal vitreous samples
were carried out by henioglobinometry.'11 On the basis
of normal vitreous concentrations, we excluded all
pathologic samples that showed hemoglobin levels
higher than 0.2 nig/ml (Coulter Counter Model Sr>,
Beds, UK). Protein concentration of all samples was
determinated by protein-dye binding.'12
Electrophoresis and Western Blotting
Sodium dodecyl sulphate-polyacrylamide gel electro-
phoresis (SDS-PAGE) was carried out as previously de-
scribed."1 :< Briefly, normal and pathologic vitreous and
subretinal 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 at
100°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 a
7.5% or 10% polyacrylamide gel in SDS. Protein bands
were 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
(Trans-Blot 200/2.0 Transfer Apparatus, Bio-Rad) at
20 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 50
mM Tris-HCI (pH 7.4) at 1:1000 dilution. Secondary
anti-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) was
applied for 3 to 4 hours at room temperature. The
nitrocellulose strips were stained with diaminobenzi-
dine (0.1% DAB in 50 mM Tris-HCI pH 7.4).
transferred
Slot Blotting and Densitometrical
Quantification
To quantify FN and VN, we used a slot blotting
method (Bio-Dot SF Microfiltration Apparatus, Bio-
Rad) on nitrocellulose sheets for each dilution bank of
normal and pathologic vitreous samples.41 Nitrocellu-
lose sheets were incubated with primary and second-
ary antibodies and stained using the DAB technique
described above. A direct densitometrical reading was
performed by digitalized images of nitrocellulose
sheets (IBAS II, Interactive Analysis System, Kontron,
Munich, Germany). Standard concentrations of puri-
fied VN and FN (0.2 ng/nl, Boehringer) were analyzed
simultaneously with each sample.
Statistical Analysis
Chi-square analysis (x~) was used to estimate statistical
differences between the variabilities of frequency

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2794
Investigative Ophthalmology & Visual Science, May 1994, Vol. 35, No. 6
among groups. To study the variability between
means, we applied an unbalanced analysis of variance
(ANOVA) by least significant difference (LSD).
RESULTS
FN Matrix Assembly in ERM, Glycoproteins,
and Integrin Expression
The time course and pattern of distribution in vivo of
FN fibril formation in ERM matrix was established in
cryostat sections (n = 26 ERM) by indirect immunoflu-
orescence. Large amounts of FN were evident in most
specimens, in pericellular or fibrillar arrangement in
matrix, 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 only
2 months of evolution (Figs. 1A and IB) than in the
group of ERM aged between 2 to 6 months (P < 0.005,
by chi-square analysis test). Pericellular pattern was
rarely observed in the third group of ERM greater
than 6 months of evolution, and differences were es-
tablished with the first group (P < 0.05, by chi-square
analysis). No significant difference was found between
the intermediate group and the last. Electron-immuno-
cytochemistry with immunogold labeling techniques
confirmed the FN in its pericellular pattern and in
collagen fiber arrangement. Individual cells presented
large 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 most
frequent finding for FN in matrix material, sometimes
localized (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 compared
with the group of intermediate time of evolution,
which showed a generalized pattern in the extracellu-
lar medium (P < 0.001, by chi-square analysis). FN
appeared to decrease in matrix material with the time
of ERM evolution and showed a tendency to assume a
localized distribution in specimens with more than 6
months 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 stain
showed moderate amounts of pericellular FN (arrows)
(X990, bar = 20 fim). (B) A wide variety of morphologic cell
types 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 by
fibers 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 regular
arrangement (X390). (F) By immunofluorescence, abundant
amounts for this protein in generalized and regular thicker
fibrillar pattern across the specimen surface were seen
(X390). (G) Generalized and fine fibrillar FN irregular dis-
tributed in matrix material were also common pattern of
labeling (X315). (H) The same section examined by interfer-
ence contrast microscopy reveals areas of regular and ran-
dom collagenous fiber arrangement (X390). (I) Localized
areas of regular pattern of immunofluorescence stain
(X390) contrast with (J) areas of irregular fibrillar distribu-
tion in the same specimen (X315, bar = 30 pim).

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Glycoproteins and Receptors in PVR
A
2795
*te
FIGURE 2. Laminin expression in ERM specimens. (A) Cells
distributed in matrix (arrows) visualized by interference con-
trast microscopy (B) presented variable immunoreactivity
staining for LN (arrows) (X900, bar = 20 Mm). (C) Slight
labeling in a fine fibrillar pattern of distribution (D) in small
localized areas in the same sectioned specimen examined by
interference contrast microscopy (X360, bar = 40 #m). (E)
By immunoelectron microscopy, pA-Au (16 nm) particles
were noted both related to the plasma membrane (arrow)
and the extracellular material surrounding cells (X19.200,
bar = 1 fixn). (F) Collagenic fibers with periodicity presented
immunoreactivity 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 in
31 specimens (57%) but always as a minor component
in the tissue. Its pericellular distribution was also ob-
served (Fig. 2B), but most frequently a fine fibrillar
pattern was noted in localized areas of the specimen
(Fig. 2C). Although the third group of ERM (>6
months of evolution) tended to lack this glycoprotein,
no significant difference was found among groups. By
electron-immunocytochemistry, less extensive amounts
of LN were occasionally noted around the plasma
membrane but were not directly related with it (Fig.
2E). The distribution of LN in collagenic matrix
showed a similar pattern to that observed for FN, but
was restricted in confluent areas of immunogold label-
ing, which in most cases occupied small areas of the
specimen (Fig. 2F and 2G).
In several consecutive serial cryostat sections, a
clear pericellular preference for determinate cell
types was manifest for both proteins (FN and LN), but
FIGURE 3. Identification of pericellular immunoreactive FN
and LN in ERM. Consecutive serial cryosections were incu-
bated with specific antisera for FN and LN in indirect immu-
nofluorescence experiments. (A) Specimens incubated with
anti-FN antisera showed high cell population that present
strong pericellular staining. (B) The same section examined
by interference contrast microscope revealed a group of pig-
ment-laden cells localized at the edges of sample (between
open arrows). This area was negative for FN immunostaining;
therefore, some pigmented cells at the inner aspect of the
specimen 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 at
the periphery of sample corresponding the marginal area
nonreactive for FN {between arrows) (X315, bar = 40 /tm).