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1 From the Department of Pathology, University of Geneva; and the 2 Department of Ophthalmology and Clinical Neurosciences, University Hospitals, Geneva Medical School, Switzerland.
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Abstract |
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METHODS. Samples of ERM were obtained from 23 patients during microsurgery for
PVR or PDR. Electron microscopy, immunohistochemistry, and confocal
microscopy with antibodies recognizing 
-smooth muscle (SM) actin,
desmin, TGF-ß1, TGF-ß receptors I and II, and ED-A FN were
performed.
RESULTS. -SM actin was detected in all ERMs, whereas desmin was present in
50% of the cases. ED-A FN was expressed in all ERMs in close relation
with -SM actin–positive myofibroblasts. In addition, TGF-ß1 and
TGF-ß R II were always present, TGF-ß RII being expressed in both
-SM actin–positive and negative fibroblastic cells.
CONCLUSIONS. Myofibroblast accumulation is a key event in ERM-associated traction
retinal detachment occurring during PVR and PDR. The current results
suggest that the presence of -SM actin–positive myofibroblasts is
probably dependent on the concomitant neoexpression of TGF-ß1,
TGF-ß RII, and ED-A FN. The results furnish new data on the mechanism
of -SM actin stimulation in fibroblasts in a human pathologic
setting.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The development of a scarlike ERM appears to be caused by a complex
sequence of cellular and biochemical events. After an initial phase,
characterized by a gliotic reaction,2
fibrotic changes,
similar to those characterizing the proliferative fibroblastic phase of
wound healing and subsequent scar formation,5
predominate.
During the process of granulation tissue formation and contraction,
fibroblasts modulate into myofibroblasts whose main feature is the
expression of the smooth muscle (SM) differentiation marker -SM
actin and, more rarely, desmin and SM myosin heavy
chains.6
Myofibroblasts have been shown to be responsible
for fibrocontractive situations such as hypertrophic scars,
fibromatoses and stromal reaction to epithelial tumors.7
8
The presence of cells showing myofibroblastic features in ERMs has been
previously reported in different ophthalmologic
diseases.9
10
11
A single study has also described the
presence of -SM actin expressing myofibroblasts in ERM after PVR or
PDR.12
In the present study, we systematically investigated the presence of
myofibroblasts in ERMs of different origins, using electron microscopy
and immunohistochemistry with antibodies against several cytoskeletal
markers. We extended our studies to the examination of transforming
growth factor (TGF)-ß1, observed in vitreous samples of
PVR,13
TGF-ß receptor II (RII), and the ED-A isoform of
cellular fibronectin (FN), the main inductors of -SM actin
expression.14
15
16
Our results show that ERMs in PVR and PDR are characterized by the
diffuse presence of -SM actin–positive myofibroblasts containing
TGF-ß1 and TGF-ßRII and expressing ED-A FN. In some fibroblastic
cells, TGF-ßRII expression allegedly precedes -SM actin’s
appearance. Confocal analysis demonstrates for the first time the close
connection of -SM actin–containing stress fibers and extracellular
ED-A FN. This reinforces the assumption that myofibroblast modulation
is responsible for retinal tractional detachment and furnishes some new
data on the mechanism of -SM actin stimulation in a human pathologic
setting.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Antibodies
We used the following primary antibodies: mouse monoclonal IgG2a
recognizing -SM actin,17
affinity-purified rabbit
polyclonal IgG against desmin,18
affinity-purified rabbit
polyclonal IgG recognizing SM myosin heavy chain types 1 and
2,19
mouse monoclonal IgG1 against vimentin, rabbit
polyclonal IgG specific for glial fibrillary-associated protein (GFAP),
mouse monoclonal IgG1 recognizing all types of cytokeratin
(anti-pankeratin, Lu-5, all from Dako, Glostrup, Denmark), mouse
monoclonal IgG1 specific for ED-A type III domain of cellular FN
(IST-9, gift from Luciano Zardi, National Institute for Cancer
Research, Genova, Italy)20
, affinity-purified chicken
polyclonal IgG against TGF-ß1 (R&D, Minneapolis, MN), and two
affinity-purified rabbit polyclonal IgGs against TGF-ß receptor I and
II (TGF-ßRI and II; Santa Cruz Biotechnology, Santa Cruz, CA).
Histology, Immunohistochemistry, and Immunofluorescence
Tissue samples (eight PVR and seven PDR membranes) were fixed in
4% buffered formaldehyde and embedded in paraffin. Sections
(5-µm-thick) were stained with hematoxylin and eosin. Immunostaining
was performed on sections adjacent to the sections stained
histologically. The presence of -SM actin, desmin, SM myosin,
TGF-ß1, and TGF-ßRI and II was determined by means of the
streptavidin-biotin complex peroxidase or alkalin-phosphatase method
(Dako), as previously described.21
Immunoreactivity of
desmin and SM myosin was intensified by one to three microwave
treatments for 5 minutes in 10 mM citrate buffer (pH 6.0) before the
first antibody was used. Sections were treated with one of the primary
antibodies 1 hour at room temperature, except for TGF-ßRI and II
antibodies which were applied overnight at 4°C. This was followed by
incubation with goat anti-mouse (for -SM actin and desmin),
anti-rabbit (for SM myosin and TGF-ßRI and II) or anti-chicken (for
TGF-ß1) biotinylated antibodies (Jackson ImmunoResearch, West Grove,
PA) and treatment with
streptavidin-biotin-peroxidase or
alkaline-phosphatase complex. The determination of peroxidase and
alkaline-phosphatase activities was performed with diaminobenzidine
(Serva, Heidelberg, Germany) and fast red (Dako), respectively. Slides
were counterstained with hemalun and mounted (Aquatex; Dako).
Double immunostaining with -SM actin and TGF-ßRII was performed as
previously described.21
Briefly, paraffin sections (from
three PVR and four PDR membranes), after treatment with
H2O2 to inhibit endogenous
peroxidase, were incubated with anti-TGF-ßRII antibody overnight.
This was followed by incubation with goat anti-rabbit biotinylated
antibody and treatment with streptavidin-peroxidase, which was revealed
with diaminobenzidine. After washing, anti--SM actin antibody was
applied for 1 hour, followed by incubation with goat
anti-mouse-alkaline-phosphatase–conjugated antibody, which was
revealed with fast red. Slides were counterstained with hemalun and
mounted.
Samples were observed with a photomicroscope (Axiophot; Carl Zeiss, Oberkochen, Germany) using an oil immersion plan-neofluar x40/1.3 objective. Images were acquired with a high-sensitivity camera (Photonic Science Coolview; Carl Zeiss) using an acquisition software (Image Access 2.04; Imagic, Zürich, Switzerland). Images were processed with analysis software (Adobe Photoshop 5.0; Adobe Systems, Mountain View, CA) and printed with a digital printer (Pictography 4000; Fujifilm, Tokyo, Japan).
Two researchers estimated independently the surface and the intensity of the staining for each antibody without taking into account the labeling into vessel walls. The evaluation of the staining was graded as follows: -, no labeling; +, focal labeling; ++, labeling in approximately 50% of the tissue; +++, extensive labeling. No differences were found in data obtained by the two observers.
For the study of FN distribution we used immunofluorescence, because
the antigenic properties of ED-A FN are destroyed by formol fixation.
Tissues (nine PVR and seven PDR membranes) were embedded in OCT 4583
(Miles, Naperville, IL) and snap-frozen in precooled liquid isopentane.
Three-micrometer-thick cryostat sections were fixed in acetone at
-20°C for 5 minutes and air dried for 2 hours at room temperature.
They were incubated with anti--SM actin and anti-vimentin,
anti-GFAP, anti-pankeratin, anti-ED-A FN antibodies for 30 minutes at
room temperature. Goat anti-mouse IgG2a conjugated with
tetramethylrhodamine isothiocyanate (TRITC; Southern Biotechnology,
Birmingham, AL), goat anti-mouse IgG1 conjugated with fluorescein
isothiocyanate (FITC; Southern Biotechnology), or goat anti-rabbit IgG
labeled with TRITC (Jackson ImmunoResearch) were used as secondary
antibodies. Nuclei were labeled with
4',6-diaminido-2-phenylindol-dihydroclorid (DAPI; Fluka, Buchs,
Switzerland). Preparations were mounted with polyvinyl alcohol. The
surface and intensity of staining for -SM actin and ED-A FN were
estimated as described for immunohistochemistry.
Confocal Laser Scanning Microscopy
Double immunofluorescence staining was performed on whole ERMs
(five PVR and one PDR) fixed in methanol at -20°C for 15 minutes.
Specimens were stained with anti--SM actin and anti-ED-A FN
antibodies as described, except that primary and secondary antibodies
were incubated overnight at 4°C.
Specimens were observed with a confocal laser scanning fluorescence
inverted microscope (LSM 410; Carl Zeiss) equipped with two lasers used
simultaneously: a helium-neon (He-Ne) laser (excitation wavelength at
543 nm) and an argon laser (excitation wavelength at 488 nm). The
excitation spectra were separated by a dichroic beam splitter of 488
and 543 nm and the emission spectra of the two fluorochromes were
separated by a 560-nm dichroic beam splitter. Two detectors were used
in parallel and were preceded with a 590- to 610-nm (rhodamine channel)
or a 510- to 525-nm (fluorescein channel) narrow-band barrier filter.
The partial superposition of the emission spectra of the two
fluorochromes was negligible.16
The specimen was observed
through an oil immersion plan-neofluar x63/1.4 objective, and the
visual field was enhanced by zooming in two times. Between 30 and 50
optical sections of 512 x 512 pixels separated by 0.2 µm were
performed in the z axis. A three-dimensional image was
reconstructed with Imaris software (Bitplane, Zürich,
Switzerland) running on computer workstation (Octane; Silicon Graphics,
Mountain View, CA). Images were printed with a digital printer
(Fujifilm). Colocalization of -SM actin (red) and ED-A FN
(green) was evaluated by counting the number of pixels containing both
stains (yellow) in all optical sections using the Colocalization
software (Bitplane).
Electron Microscopy
Membrane tissue samples (two PVR and one PDR) were fixed in 1.5%
glutaraldehyde in 0.1 M sodium cacodylate (Merck, Darmstadt, Germany)
containing 1% sucrose for 3 hours. This was followed by fixation in
1% osmium tetroxide for 1 hour and subsequent dehydration and
embedding in Epon. Semithin sections were stained with toluidine blue.
Thin sections were treated with uranyl acetate and lead citrate and
examined in an electron microscope (model 400; Philips, Eindhoven, The
Netherlands).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-SM actin was detected in all ERMs obtained from eyes affected by
PVR and PDR and was present in most of the fibroblastic cells (Fig. 2A
). Desmin was found in 50% of the specimens, either from PVR- or
PDR-affected eyes. It was expressed in a small proportion of
fibroblasts (approximately 10%) in five samples and in 50% of cells
in two samples (Fig. 2B) . SM myosin heavy chains were present in only
one sample of PDR and was graded as focal staining (data not shown).
Double immunofluorescence staining with anti--SM actin and
anti-vimentin or anti-GFAP or anti-keratin antibodies showed that
-SM actin–positive cells expressed vimentin as well, but not GFAP
or keratin. ED-A FN was strongly expressed in all ERMs, irrespective on
the diagnosis (Table 1)
, and localized around -SM actin–positive
cells. In specimens from a recurrence of the disease, cells expressed
features similar to those in primary ERMs (Table 1)
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-SM actin. TGF-ßRII was expressed constantly in myofibroblasts in
all specimens studied (Fig. 2D
and Table 1
), whereas TGF-ßRI was
negative. Double staining with -SM actin and TGF-ßRII (performed
on three PVR and four PDR membranes) showed that fibroblastic cells
expressed both; moreover, several cells positive for TGF-ßRII alone
were observed (Fig. 2E)
.
Three-Dimensional Reconstruction of Myofibroblasts
All PVR and PDR membranes used for confocal laser scanning
microscopic analysis were strongly positive for -SM actin and ED-A
FN. Three-dimensional reconstruction by shadow projection showed -SM
actin–positive myofibroblasts (red) in which stress fibers appeared
well defined (Fig. 4)
. ED-A FN (green) was clearly detected in the extracellular compartment
of the tissue and appeared as a network surrounding the myofibroblasts.
Continuity between -SM actin–positive stress fibers and ED-A FN
filaments was visible (Fig. 4
, insert). However, only 0.058% ±
0.006% of the total pixel number exhibited a colocalization of -SM
actin and ED-A FN demonstrating that these proteins were almost never
colocalized (yellow).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-SM actin and rarely or never desmin and SM myosin heavy
chains),7
are the main cellular components of ERMs in both
PVR and PDR. This suggests that myofibroblasts play a role in the
production of retractile phenomena causing retinal detachment in both
conditions, similar to other fibrocontractive
diseases.6
7
27
If untreated, ERMs tend to cause a
progressive retinal detachment that begins locally but eventually may
involve many retinal quadrants. It seems unlikely that the strong
tractional force necessary to induce such a detachment results merely
from extracellular matrix remodeling induced by isolated migrating
fibroblasts.
In parallel to myofibroblast accumulation, we have shown that
significant amounts of TGF-ß1, as well as one of its specific
receptors, TGF-ßRII, are present in ERMs. TGF-ß1 is mainly
localized within macrophages or in connection with extracellular
matrix. Our findings suggest that the initial stage of myofibroblast
differentiation coincides with the presence of TGF-ßRII followed by
-SM actin appearance. TGF-ß1 has been shown to be the most
important stimulus for -SM actin and collagen
synthesis.14
15
28
29
30
Recently, our laboratory has shown
that TGF-ß1 needs the presence of the cellular ED-A FN variant to
stimulate synthesis of both collagen and -SM actin.16
As described by Immonen et al.,31
we have observed that
ED-A FN was present in ERMs in all samples studied. Therefore, factors
known to be capable of producing contractile events resulting in
retinal detachment are present in ERMs. Further studies are needed to
establish the mediators of TGF-ß1 synthesis by macrophages and other
inflammatory cells. Presently, granulocyte macrophage
colony-stimulating factor is thought likely to be responsible for this
action.32
33
34
Cells with the ultrastructural characteristics of myofibroblasts have
been previously identified in contractile ERMs removed during
surgery,9
10
but their number was reported to be scarce,
probably because most of these reports concerned nonvascular ERMs
obtained during macular pucker surgery. More recently, -SM actin
expressing myofibroblasts were found in up to 90% of PVR and PDR
specimens,12
but they were not evaluated quantitatively,
nor was their organization within the membranes studied. In the present
study, -SM actin expressing myofibroblasts were found to be present
in 100% of the membranes. Immunostaining for -SM actin was
prominent in all membranes and dense bundles of actin microfilaments
forming stress fibers within the myofibroblasts were observed by
electron microscopy, indicating a high contractile potential.
Additionally, confocal microscopy clearly demonstrated that the
myofibroblasts formed a dense sheet of cells, similar to those
developing when fibroblasts are cultured in culture
dishes,35
connected to one another and associated with
ED-A FN. The continuity observed between -SM actin–positive stress
fibers and ED-A FN-positive extracellular bundles can be attributed to
the fibronexus, another structure typical of
myofibroblasts.36
It has been suggested that RPE cell migration into the vitreous cavity during various vitreoretinal diseases secondarily differentiates into fibroblasts and stimulates the production of collagen and FN at the retinal surface by releasing various cytokines, including TGF-ß1.37 However, transdifferentiation of RPE cells to fibroblasts is controversial.11 Indeed, RPE cells may adopt a fibroblastic appearance but remain keratin positive, thus retaining their epithelial nature.11 Hyalocytes have also been postulated as a possible source of fibroblasts, but they have macrophage rather than fibroblast characteristics.38
In the present study, myofibroblasts in ERM specimens were positive for
vimentin and -SM actin and negative for keratin and GFAP. The
absence of keratin staining excludes an RPE cell origin of the
myofibroblasts. That specimens were constantly negative for GFAP and
positive for vimentin suggests that myofibroblasts originate from
astrocytes. However, we cannot exclude an origin from GFAP-negative
nonactivated cells.39
40
Finally, because ERM contain
blood vessels, a pericytic origin of myofibroblasts is also
possible.41
42
In conclusion, we have shown that, similar to other fibrocontractive
settings, ERMs are characterized by the presence of typical -SM
actin–positive myofibroblasts accompanied by TGF-ß1, its specific
receptor TGF-ßRII, and ED-A FN. Further work examining the modulation
of TGF-ß1 and ED-A FN expression is needed to explore the mechanisms
of ERM development and possibly to intervene in their onset and
evolution.
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Acknowledgements |
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Footnotes |
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Supported by the Swiss National Science Foundation, Grants 32-49543.96 and 31.50568.97.
Submitted for publication February 3, 1999; revised November 30, 1999; accepted December 28, 1999.
Commercial relationships policy: N.
Corresponding author: Constantin J. Pournaras, Department of Ophthalmology, University Hospital of Geneva, Rue Alcide-Jentzer 22, 1211 Geneva 4, Switzerland. constantin.pournaras{at}hcuge.ch
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts J Cell Biol 122,103-111-smooth muscle actin by transforming growth factor-ß1 in quiescent human breast gland fibroblasts: implications for myofibroblast generation in breast neoplasia Lab Invest 68,696-707[Medline][Order article via Infotrieve]
-smooth muscle actin: a new probe for smooth muscle differentiation J Cell Biol 103,2787-2796-smooth muscle actin in normal and pathological soft tissues Cell Motil Cytoskeleton 11,260-274[Medline][Order article via Infotrieve]
-smooth muscle actin expression in fibroblasts by transforming growth factor-ß isoforms: an in vivo and in vitro study Wound Rep Reg 4,277-287
-smooth muscle actin containing myofibroblasts Virchows Arch B Cell Pathol Mol Pathol 60,73-82
-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes J Histochem Cytochem 37,315-321[Abstract]
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