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Investigative Ophthalmology & Visual Science, Vol. 33, No. 2, February 1992
Copyright © Association for Research in Vision and Ophthalmology
Localization of Smooth Muscle and Nonmuscle Actin
Isoforms in the Human Aqueous Outflow Pathway
Annelies W. de Kater, Aliakbar Shahsafaei, and David L. Epstein
a-Smooth muscle actin is the isoform of actin restricted to vascular smooth muscle, pericytes, myofibroblasts and, certain other cells that are of myoid origin. We investigated the distribution of a-smooth
muscle actin and nonmuscle specific filamentous actin in the human aqueous outflow system by immunohistochemical methods. Filamentous actin was observed in all cellular constituents of the outflow
pathway, while distribution of a-smooth muscle actin was restricted to the ciliary muscle, to specific
cells throughout the trabecular meshwork, and to cells adjacent to the outer wall and the collector
channels. The ciliary muscle extended deep into the corneoscleral meshwork, far anterior to the scleral
spur. These findings agree with our previous study localizing the distribution of smooth muscle myosin
in the human aqueous outflow pathway. Although functionality of the immunoreactive cells needs to be
demonstrated, our data show that a potentially contractile apparatus exists in a subpopulation of
trabecular meshwork cells and in certain cells of the more distal components of the outflow system.
Invest Ophthalmol Vis Sci 33:424-429,1992
Previous studies have demonstrated the presence of
cells rich in actin in the human aqueous humor outflow pathway, specifically in the trabecular meshwork
(TM) and adjacent to the outer wall of Schlemm's
canal.12 In addition, smooth muscle-like cells have
been reported in the aqueous outflow system of the rat
and rabbit.3"6 We recently showed that cells containing smooth muscle-specific myosin are found within
the TM, adjacent to the outer wall of Schlemm's canal
and the collector channels.7 Such structural and cytoplasmic contractile proteins are involved in many biological functions, and the presence of these proteins in
cells of the outflow pathway could be important in
aqueous humor outflow dynamics.
The objectives of the present study were to determine, by immunohistochemical methods, whether
smooth muscle-specific actin is present in the human
outflow pathway and to compare the distribution of
this protein to the distribution of cytoskeletal filamentous actin (F-actin). We used a monoclonal antibody
specific to the a-smooth muscle actin (a-sm actin)
isoform to identify smooth muscle actin in cells of the
outflow system and compared its distribution to that
of F-actin as visualized by rhodamine-conjugated
phalloidin.
Materials and Methods
Ostensibly normal human autopsy eyes (n = 16;
ages 23-87 yr) were obtained from the New England
Eye Bank or from the National Disease Research Interchange. Anterior segments were immersed in a fixative solution of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3 (freshly prepared), within 24
hours after death. The anterior segments were divided
in quadrants, cut in meridional wedges, and frozen
and stored at -75 °C. At least two quadrants of each
eye were examined.
From the Howe Laboratory of Ophthalmology, Massachusetts
Eye and Ear Infirmary, and Harvard Medical School, Boston, Massachusetts.
This study was supported in part by NEI EYO1894, and by a
grant from National Glaucoma Research, a program from the
American Health Assistance Foundation, Rockville, MD.
Presented in part at the Annual Meeting of the Association for
Research in Vision and Ophthalmology, Sarasota, Florida, May
1990.
Submitted for publication: February 15, 1991; accepted October
9, 1991.
Reprint requests: Annelies W. de Kater, University of Texas
Southwestern Medical Center, Dept. of Ophthalmology, 5323
Harry Hines Blvd., Dallas, TX 75235.
Immunofluorescence
Four-micron sections were cut, placed on gelatin
coated slides, and air dried for several hours at room
temperature. All steps were carried out at room temperature. Sections were subsequently washed in phosphate buffered saline (PBS), incubated in 0.15 M glycine in PBS to quench residual free aldehydes, and
permeabilized by incubation in PBS supplemented
with 0.1% Triton-X, 0.015 M glycine, and 0.1% bo-
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MUSCLE AND NONMUSCLE ACTIN ISOFORMS IN THE AQUEOUS OUTFLOW PATHWAY / de Korer er ol
vine serum albumin.8 The same buffer was used
throughout the experiment as an antibody diluent
and washing medium. Steps were performed in a rotating apparatus to improve access of the reagents to
the tissue. To identify a-sm actin, a primary antibody
(monoclonal anti-a-sm actin; Sigma, St. Louis, MO;
working dilution 1/400) was applied for 1 hr in a
moist chamber. The specificity of the antibody to the
single isoform of a-sm actin has been characterized by
immunoblotting assays and was described previously9. The slides were rinsed with buffer and incubated with a secondary antibody (rhodamine-conjugated goat anti-mouse IgG; Cappel, West Chester, PA;
working dilution 1/75). To visualize F-actin, tissue
sections were incubated with rhodamine-conjugated
phalloidin for 1 hr (Sigma; working dilution 1/20).
After a PBS wash, coverslips were mounted with a
medium of PBS, glycerol, and paraphenylenediamine.10 Negative control tissue sections (primary antibody omitted) were run routinely with each antibody-binding study. A positive control was binding of
the antibody to the ciliary muscle, which was present
in each section. The slides were viewed and photographed with a Zeiss Photomicroscope III (Oberkochen, West Germany), equipped for epiilumination.
Results
F-actin was observed in all cellular constituents of
the aqueous outflow pathway (Figs, la, 2a), while distribution of a-sm actin was restricted to the ciliary
muscle, to specific cells throughout the TM, and to
cells adjacent to the outer wall and the collector channels (Figs, lb, 2b). The pattern of distribution of a-sm
actin varied within the TM of different sections. However, positive antibody binding was seen in all eyes
examined. The pattern of binding did not correlate
with individual quadrants, nor was there apparent
correlation with the donor's age. Fluorescence was absent in negative controls.
F-Actin
The ciliary muscle and its anterior extensions into
the TM exhibited intensefluorescence.In the trabecular cells lining the beams, brightfluorescencewas distributed in a continuous band along the long axis of
the cells. In addition, foci of brighter fluorescence
were seen throughout the TM. F-actin was noted in
the elongated juxtacanalicular cells underlying the inner wall of Schlemm's canal, and bright fluorescence
was associated with the endothelial lining of
Schlemm's canal. The area adjacent to the outer wall
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of Schlemm's canal was consistently rich in F-actin
(Fig. 2a).
a-SM Actin
Ciliary muscle: Brightfluorescencewas seen in the
cells of the ciliary muscle, which appeared to insert
directly into the corneoscleral meshwork anterior to
the scleral spur (Fig. 2b). At the insertion of the ciliary
muscle (ciliary muscle tips) into the TM, a-sm actin
was present in ciliary muscle cells as well as in contiguous trabecular cells that lined trabecular beams.
(Fig. 3).
Trabecular meshwork: In all eyes examined, a-sm
actin was identified in focal cells of the uveal and corneoscleral TM (Fig. 2b) and was distributed in continuous bands along the longitudinal axis of the cells.
The pattern of binding in the different specimens
ranged from the presence of a-sm actin in a few uveal
and corneoscleral TM cells (Fig. lb) to binding in a
majority of these cells (Fig. 2b). Frequently, individual juxtacanalicular cells contained a-sm actin (Fig.
4). We did not observe immunoreactive cells in the
inner wall endothelial lining of Schlemm's canal.
Outer wall: Positive antibody binding was seen in
cells adjacent to the outer wall of Schlemm's canal in
a majority of eyes. Binding patterns varied from single
discontinuous cells to large groups of cells exhibiting
intense fluorescence (Figs, lb, 2b). The long axis of
the a-sm actin-containing cells appeared positioned
parallel to the longitudinal aspect of Schlemm's canal.
No immunoreactivity was observed in the outer wall
endothelial cells.
Collector channels: Discontinuous immunofluorescent cells were seen adjoining the endothelial cells lining the collector channels (Fig. 5). The a-sm actincontaining cells appeared fusiform and in an orientation parallel to the lumen of the collector channels. In
tissue sections where an ostia of a collector channel
could be observed, fluorescence was associated with
cells close to the collector channel opening (Fig. 6). In
addition, a-sm actin was noted along septae in
Schlemm's canal (Fig. 6).
Discussion
This study shows that nonmuscle, as well as muscle-specific actins, are present in the human aqueous
outflow system. F-actin was identified in all of the
cellular components, with fluorescence appearing
most intense in the ciliary muscle and in other identifiable muscular elements of the outflow pathway.
Bright fluorescence also was associated with cells lining trabecular beams immediately anterior to the cili-
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Fig. 1. Overviewfluorescencemicrographs of the anterior chamber angle, (a) F-actin. Nonmuscle F-actin is seen in all cellular components
of the outflow pathway. Intensefluorescenceis associated with the ciliary muscle and other muscular structures. Brightfluorescenceis seen in
the endothelial lining of Schlemm's canal (X100). (b) a-sm actin. The distribution of a-sm actin is restricted to the ciliary muscle, to focal cells
within the TM and to cells adjacent to the outer wall and collector channels (X100).
Fig. 2. Higher-magnificationfluorescencemicrographs of the outflow system, (a) F-actin. Microfilaments can be visualized in the trabecular
cell lining the beams. Foci of brighterfluorescenceare seen within the TM, adjacent to the outer wall and the ostia of the collector channels
(X750). (b) a-sm actin. Smooth muscle-specific actin is seen in the ciliary muscle and in a large number of uveal and corneoscleral TM cells.
Focal immunoreactive cells are observed associated with the outer wall and collector channel (X250).
ary muscle tips. In contrast, a-sm actin was restricted
in its distribution to the ciliary muscle and other focal
cells of the outflow pathway. Anterior to the ciliary
muscle tips, a-sm actin was noted in cells lining the
corneoscleral trabecular beams far anterior to the
scleral spur. Frequently, focal cells in the juxtacanalicular tissue (JCT) contained a-sm actin. In all eyes,
this smooth muscle-specific actin isoform was found
in cells adjacent to the outer wall of Schlemm's canal
and to the collector channels. This distribution of asm actin was similar to that of smooth muscle myosin, which we have described previously.7 In the
current study, we made no attempt to quantify labeling found per total number of eyes as we did in the
smooth muscle myosin manuscript. Although it
might appear there was a difference in the frequency
with which positive cells were found in a given eye,
the pattern of antibody labeling was similar or identical. That fewer eyes appeared to show immunoreactivity for smooth muscle myosin in the trabecular meshwork and JCT could be a result of sampling size, because frequently only one or a few cells were positive
for this antigen. We examined at least two segments
per eye. However, if only very few cells show immunoreactivity, the sample size may need to be much larger
to accurately quantify labeling per total number of
eyes.
a-sm actin, one of the 6 major isoforms of actin, is
limited in distribution to vascular smooth muscle,9
pericytes,11 myofibroblasts,12 and certain other specific cells that are of myoid origin. We used a monoclonal antibody to the single isoform for visualizing a-sm
actin in the human outflow pathway.9 Although further studies are needed to determine the nature of the
subpopulation of cells in the outflow pathway containing smooth muscle-specific proteins, our data
suggest that the cell population of the human TM is
heterogeneous and that at least two distinct cell types
are present. Of note is that a recent study showed electrical and morphological heterogeneity of bovine TM
cells, with a subpopulation of TM cells demonstrating
smooth muscle-like characteristics.13 Although functional contractility of these smooth muscle proteincontaining cells in human and bovine still needs to be
determined in vivo, our data further confirm that a
potentially contractile apparatus is present in a subpopulation of human TM cells.
Evidence for a role of cytoskeletal proteins in
aqueous outflow comes from perfusion studies.14"15
Several pharmacologic agents, which decrease resistance to outflow in perfusion studies in vivo, have a
demonstrable effect on cytoskeletal proteins of TM
cells in vitro. Specifically, cytochalasin B and D have
an effect on outflow facility when perfused in the intact eye and on the cytoskeleton in tissue culture. Recently, studies from our laboratory have shown that
the sulfhydryl agent ethacrynic acid (ECA) increases
outflow facility in enucleated bovine eyes, in the in
vivo monkey eye, and in the organ cultured human
anterior segment.16-17 These effects were correlated
with induced changes in cell shape and cytoskeletal
proteins of cultured human and bovine TM cells.18'19
Pharmacological agents that are used clinically to increase outflow facility also have been shown to cause
changes in cytoskeletal proteins in TM cells in vitro.
Epinephrine and pilocarpine produced alteration in
cell shape associated with changes in actin and vimentin20>21. The combined results from these studies
strongly suggest that cytoskeletal proteins may be directly involved in aqueous outflow mechanisms.
Agreeing with our previous study that localized
smooth muscle myosin to the outflow pathway, in the
present study a-sm actin was consistently found in
cells adjacent to the outer wall of Schlemm's canal
and the collector channels. If these potentially contractile cells are actually functional, they may influence the distal resistance to outflow. Furthermore,
these current data confirm results from our previous
study, in which we demonstrated that the longitudinal
portion of the ciliary muscle inserts directly into the
corneoscleral TM far anterior to the scleral spur.
In summary, we have shown that smooth musclespecific contractile proteins are present in cells of the
TM as well as in cells of the more distal components
of the outflow system in human eyes. The presence of
contractile elements in these cells suggests they may
participate in the modulation of aqueous humor outflow resistance.
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Fig. 3. Fluorescence micrograph of a-sm actin at the anterior insertion of the ciliary muscle. Both ciliary muscle cells (black arrows) and
trabecular cells (white arrows) lining the adjacent corneoscleral beams contain a-sm actin (X650).
Fig. 4. High-magnification fluorescence micrograph of a-sm actin. Focal corneoscleral TM cells, as well as juxtacanalicular cells contain
a-sm actin. a-sm actin is also visualized in cells close to the outer wall of Schlemm's canal (X1000).
Fig. 5. Fluorescence micrograph of a-sm actin in cells adjacent to a collector channel, a-sm actin appears to be distributed in a perinuclear
fashion (X675).
Fig. 6. Fluorescence micrograph, a-sm actin is associated with the ostia of a collector channel, with a septum in Schlemm's canal and with
cells adjacent to the outer wall. In addition, it is seen in focal TM cells (X250).
Key words: aqueous humor outflow pathway, trabecular
meshwork, immunohistochemistry, a-smooth muscle actin, smooth muscle
Acknowledgements
The authors thank Drs. Nancy C. Joyce and Ilene K. Gipson for advice and help in the initial stages of this project.
We also are grateful to Dr. R. Rand Allingham for valuable
comments and criticism.
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