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Nitric Oxide Synthase-2 in Human Optic Nerve Head Astrocytes Induced by Elevated Pressure In Vitro
Bin Liu, PhD, MD;
Arthur H. Neufeld, PhD
Arch Ophthalmol. 2001;119:240-245.
ABSTRACT
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Objective To determine whether astrocytes of the human optic nerve head can induce
nitric oxide synthase-2 (NOS-2) in response to elevated hydrostatic pressure
as a mechanism for directly damaging the axons of the retinal ganglion cells
in glaucoma.
Methods Primary cultures of astrocytes from human optic nerve heads were placed
in chambers, either pressurized at elevated hydrostatic pressure (60 mm Hg)
or maintained at ambient pressure. The induction of NOS-2 was studied by immunocytochemistry,
immunoblot, and semiquantitative reverse transcription polymerase chain reaction.
Results In astrocyte cultures under ambient pressure, NOS-2 was almost undetectable.
In astrocyte cultures under elevated hydrostatic pressure for 24, 48, and
72 hours, intensive labeling of NOS-2 in the Golgi body and the cytoplasm
was observed by immunocytochemistry and intense bands of NOS-2 were detected
by immunoblotting. As detected by semiquantitative reverse transcription polymerase
chain reaction, the messenger RNA level of NOS-2 increased significantly in
the astrocytes under elevated hydrostatic pressure within 12 hours, peaking
earlier than the protein level of NOS-2.
Conclusion Elevated hydrostatic pressure induces the astrocytes of the human optic
nerve head to express NOS-2.
Clinical Relevance In glaucoma, the appearance of the neurodestructive NOS-2 in astrocytes
of the optic nerve head may be a primary response to elevated intraocular
pressure, in vivo, and therefore damaging to the axons of the retinal ganglion
cells.
INTRODUCTION
GLAUCOMA, a nerve degeneration that causes loss of retinal ganglion
cells and a characteristic visual field defect, is in many patients associated
with elevated intraocular pressure. In response to elevated intraocular pressure,
the optic disc is compressed and the cribriform plates of the lamina cribrosa
are stretched and become disorganized.1 The
initial site of neuronal degeneration in glaucomatous optic neuropathy is
believed to be the axons of the retinal ganglion cells at the level of the
lamina cribrosa of the optic nerve head.1-2
Within this region, the connective tissue undergoes extensive remodeling of
the extracellular matrix,3 and there are marked
changes in astrocytes4 and microglia5 in both morphologic characteristics and distribution.
These changes may result primarily in response to elevated intraocular pressure
or secondarily after tissue damage. Local cellular responses may alter the
microenviroment of the axons of the retinal ganglion cells and contribute
to axonal damage as the chronic glaucomatous process proceeds. From our past
work, we have demonstrated that one cellular response pathway that contributes
to local neurotoxic effects to cause degeneration of the axons of the retinal
ganglion cells in the glaucomatous optic nerve head is mediated by nitric
oxide (NO).6
Recently, NO has received much attention because of its wide range of
biological effects.6-8
Physiologically, NO serves both intracellularly, as a second messenger that
responds to activation of plasma membrane receptors, and extracellularly,
as a paracrine factor that carries information between cells.7-8
Pathologically, NO is cytodestructive and, in particular, can cause neuronal
degeneration.9-11
Nitric oxide is synthesized from the guanidino-nitrogen of L-arginine and
molecular oxygen by nitric oxide synthase (NOS). Three isoforms of NOS have
been cloned and demonstrated in many tissues. Constitutive NOS isoforms (NOS-1
and NOS-3) are activated by biological signals that transiently increase intracellular
Ca2+ and are identifiable in a variety of cells in normal tissues.
The inducible isoform (NOS-2) is usually not present under normal conditions,
is Ca2+ independent, and is induced by cytokines. The expression
of NOS-2 results in the sustained and unregulated release of excessive amounts
of NO that is cytotoxic to neighboring cells.12-13
Our laboratory has reported that NOS-2 is present in the optic nerve
heads of patients with primary open-angle glaucoma and that there is positive
staining for nitrotyrosine in the tissue, suggesting that NO through peroxynitrite
may contribute to the local damage of the axons of the retinal ganglion cells.6, 14 Support for the hypothesis of NO neurotoxic
effects in glaucoma is provided by pharmacologic experiments by Neufeld et
al15 that demonstrated that an NOS-2 inhibitor
can significantly protect against the loss of retinal ganglion cells in an
animal model of glaucoma. To understand the regulation of NOS-2 expression
in glaucomatous optic neuropathy, we demonstrated that the major cell type
that expresses NOS-2 in the glaucomatous optic nerve head is the reactive
astrocyte.16
In this study, we asked whether the expression of NOS-2 in reactive
astrocytes of the glaucomatous optic nerve head is a direct response to elevated
intraocular pressure or a secondary response to tissue destruction. In the
work presented herein, we have used cultured human optic nerve head astrocytes
to demonstrate that a mechanical stress in vitro, elevated hydrostatic pressure,
induces transcription of the NOS-2 gene and synthesis
of the NOS-2 protein. Our results suggest that, in glaucoma, elevated intraocular
pressure may directly induce astrocytes of the human optic nerve head to express
NOS-2, thus playing a primary role in neurotoxic effects by producing excessive
NO that can damage the axons of the retinal ganglion cells.
MATERIALS AND METHODS
Twelve human eyes from donors (aged 22-65 years) with no history of
eye disease were obtained within 24 hours after death from eye banks throughout
the United States. The eyes were stored at 4°C and processed within 8
hours of enucleation. Primary lamina cribrosa astrocyte cultures were derived
as described by Hernandez et al.17 The posterior
pole of the eyes was dissected and the optic nerve head was freed from sclera
and other neighboring tissues. The optic nerve head was sliced sagittally
and under a dissecting microscope the lamina cribrosa was identified. With
the use of a sharp blade, the lamina cribrosa was dissected from prelaminar
and postlaminar regions. Each half disk of tissue was then cut into 2 or 3
explants that were placed into culture. From every eye, 4 or 5 explants of
the lamina cribrosa were obtained.
The dissected samples were placed in 25-cm2 plastic tissue
culture flasks, which had been conditioned with Dulbecco modified Eagle medium
supplemented with 10% fetal bovine serum. The explants were incubated in 1.5
mL of medium at 37°C in a 5% oxygen humidified atmosphere; the medium
was changed twice a week. Two weeks after initial outgrowth, the cells were
treated with 0.05% trypsin-EDTA and split at a ratio of 1:2. The first-passage
cells were grown to confluency on glass coverslips for characterization by
immunofluorescent staining of glial fibrillary acidic protein (GFAP; 1:400)
(Sigma-Aldrich Corp, St Louis, Mo), HLA-DR (1:50; Accurate Chemical &
Scientific Corp, New York, NY), von Willebrand factor (1:300; Sigma-Aldrich
Corp), and  –smooth muscle actin (1A4; 1:100; Sigma-Aldrich Corp).
The primary cell cultures were purified for astrocytes by growing the cells
for 1 week in modified, astrocyte-defined, serum-free medium (Dulbecco modified
Eagle medium, 0.2% ITS+Premix [combination of insulin, transferrin, selenium,
linoleic acid, and bovine serum albumin]; Collaborative Biomedical Products,
Bedford, Mass; and 0.1% bovine serum albumin; Sigma-Aldrich Corp) containing
forskolin, which suppressed the growth of fibroblasts. The second-passage
cell cultures, which had more than 95% cells positive for GFAP, were grown
to preconfluency in serum-free medium and used for the following experiments.
At the preconfluent stage, 7 days after growing in serum-free medium,
cells, either in culture dishes or on coverslips, were placed in a closed
chamber equipped with a manometer that was designed and used previously by
Yang et al.18 Pressure was elevated in the
chamber to 60 mm Hg with the use of 92% air and 8% carbon dioxide. This pressure
was chosen because, at 60 mm Hg, marked alterations in collagen type I synthesis,18 increased synthesis of cyclic adenosine monophosphate,19 and selective expression of a specific isoform of
neural cell adhesion molecule synthesis20 have
been demonstrated in astrocytes of the human optic nerve head. As controls,
the same numbers of cells in culture dishes or on coverslips were placed in
a similar chamber at ambient atmospheric pressure in 95% air and 5% carbon
dioxide. The variation in carbon dioxide was necessary to maintain the pH
in both chambers at 7.4. Calculations with the Henry law showed that the amounts
of oxygen dissolved in the media and thus available to the cells did not differ
significantly under the pressurized and control conditions.18
Both pressurized and control chambers were placed in a tissue culture incubator
at 37°C and maintained for 12, 24, 48, and 72 hours. The pH of the culture
media of cells in both chambers was monitored daily and confirmed to remain
constant at 7.4. The numbers of cells were monitored by counting in an electronic
cell counter (Coulter Electronic Ltd, Luton, England).
Preconfluent cells grown on glass coverslips were fixed in 4% paraformaldehyde
at 4°C for 30 minutes, washed in phosphate-buffered saline (PBS), and
treated with 0.5% fetal bovine serum–0.2% Triton X-100–0.5% glycine
in PBS for 20 minutes. The coverslips were incubated with 50 µL of monoclonal
primary antibody against GFAP (Sigma-Aldrich Corp; working dilution, 1:50)
for 30 minutes. After washing several times with PBS, the coverslips were
incubated with goat anti–mouse Oregon green-X conjugated secondary antibody
(Molecular Probes Inc, Eugene, Ore; working dilution, 1:500). The coverslips
were then washed with PBS, incubated with a second, polyclonal primary antibody
against NOS-2 (Santa Cruz Biotechnology Inc, Santa Cruz, Calif; working dilution,
1:50), washed as above, and incubated with goat anti–rabbit rhodamine
red-X conjugated secondary antibody (Molecular Probes Inc; working dilution,
1:1000). After washing several times with PBS, the coverslips were mounted
in Vectashield with 4',6-diamidino-2-phenylindole (Vector Laboratories
Inc, Burlingame, Calif) and imaged by fluorescence microscopy (Olympus AX70;
Tokyo, Japan).
Immunoblot for NOS-2 was performed as described previously.16 Briefly, astrocyte monolayers were washed with PBS
and lysed in 8-mol/L urea solution containing protease inhibitor (cocktail
tablet from Boehringer Mannheim, Penzberg, Germany). Lysates were homogenized
and protein concentration was determined with the Bradford colorimetric assay.
Thirty micrograms of protein lysates for determination of NOS-2 was loaded
in each lane in a sample buffer (2% sodium dodecyl sulfate, 10% glycerol,
0.001% bromophenol blue, 1% dithiothreitol, and 0.05-mmol/L Tris hydrochloride,
pH 6.8), separated on 10% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis, and transferred to a nitrocellulose filter. The blots were
blocked with 5% nonfat milk in PBS, then incubated with anti–NOS-2 polyclonal
antibody (Santa Cruz Biotechnology Inc; working dilution, 1:100), followed
by peroxidase-conjugated goat anti–rabbit IgG2a, and imaged
by means of the enhanced chemiluminescence detection system (Amersham Life
Science Inc, Arlington Heights, Ill). Negative control was run in parallel
by preincubation at 4°C overnight with a specific blocking peptide (Santa
Cruz Biotechnology Inc; working dilution, 1:5) to neutralize the anti–NOS-2
antibody. Quantitation was performed by scanning the Hyperfilm radiographs
and measuring the band intensity by optical densitometry (OD) with an image
densitometer (Model GS670; Bio-Rad Laboratories, Hercules, Calif) and inage
quantitating software (Molecular Analysis 1.5; Bio-Rad Laboratories). The
comparative increase of the protein level from control to pressure-exposed
groups was calculated by comparing the OD values from 3 independent experiments
with cells from different donors.
Semiquantitative reverse transcription polymerase chain reaction (RT-PCR)
was performed as published previously.21 Briefly,
106 cells were homogenized in 1 mL of Trizol (Life Technologies,
Rockville, Md), and total RNA was extracted according to the manufacturer's
recommendations and quantified by measuring the absorbance at 260 nm of an
aliquot. The ratio of 260/280 nm was greater than 1.8. Complementary DNA (cDNA)
was synthesized by using 1 µg of RNA that was reverse transcribed by
reverse transcriptase (SuperScript II RT; Life Technologies) with oligo (dT)
primers according to the manufacturer's instructions (Life Technologies).
The primers used for NOS-2 detection were 112S (5'-CCAGTGACACAGGATGACCTTCAG-3'),
complementary to bases 112 through 135, and 715A (5'-TGCCATTGTTGGTGGAGTAACG-3'),
complementary to bases 715 through 694 of the human NOS-2 cDNA sequence. The
primers used for  -actin detection were 792S (5'-CTCTCTTCCAACCTTCCTTCCTG-3'),
complementary to bases 792 through 814, and 1110A (5'-CCAGACTCGTCATACTCCTGCTTG-3'),
complementary to bases 1110 through 1087 of human -actin cDNA sequence.
Polymerase chain reaction conditions were first optimized to ensure that the
PCR was in the linear range. As a control for calibrating an equivalent amount
of input cDNA, the messenger RNA (mRNA) level of constitutively expressed -actin
was determined in parallel aliquots of cDNA to control for differences in
cDNA synthesis efficiency. Polymerase chain reaction conditions were 25 cycles
of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute,
and extension at 72°C for 30 seconds for NOS-2 and 15 cycles for -actin.
After amplification, amplicons were electrophoresed on 2% agarose gel and
were blotted onto a positively charged nylon membrane (Hybond-N+;
Amersham Life Science Inc). Hybridization was performed at 60°C with specific
internal DNA probes corresponding to bases 142 to 552 of NOS-2 cDNA sequence
for the PCR production of NOS-2, or corresponding to bases 871 to 998 of -actin
cDNA sequence for PCR production of -actin. Samples were washed 2 times
at 60°C under increasingly stringent salt conditions. Chemiluminescent
detection of hybridized nucleotides was performed with enhanced chemiluminescent
(ECL) detection system (Amersham Life Science Inc) and autoradiography for
approximately 60 minutes on film (Hyperfilm-ECL; Amersham Life Science). Quantitation
was performed by scanning the Hyperfilm radiographs and measuring the band
intensity by OD with the use of an image densitometer (Model GS670; Bio-Rad
Laboratories) and ImageQuant software. The OD value of the band intensity
for NOS-2 was calibrated by dividing by that for -actin from the parallel
sample. The comparative increase of the RT-PCR production of NOS-2 mRNA from
control to pressure-exposed groups was calculated by comparing the OD values
from 3 independent experiments with cells from different donors.
RESULTS
Cultures of lamina cribrosa astrocytes from normal human optic nerve
heads have positive staining for intracellular filamentous GFAP, confirming
a distinguishing characteristic of astrocytes. When grown in a chamber at
ambient atmospheric pressure for 48 hours, the astrocytes have flat, star-shaped
cell bodies with a few short, thick processes. Immunocytochemical colocalization
for NOS-2 demonstrated that most of the GFAP-positive astrocytes grown under
ambient atmospheric pressure are negative for NOS-2, but a few have very faint
positive labeling for NOS-2 in the paranuclear area (Figure 1A). When exposed to elevated hydrostatic pressure (60 mm
Hg) for up to 48 hours, the shape and size of the astrocytes changed. These
cells have much larger cell bodies with extensive, thinner, and longer processes.
Immunocytochemical colocalization for NOS-2 demonstrated that most of the
astrocytes exposed to elevated hydrostatic pressure for 48 hours are intensely
positive for NOS-2. The presence of NOS-2 protein was apparent in the paranuclear
region, most likely in the rough endoplasmic reticulum–Golgi region,
as well as diffusely in the cell body and processes (Figure 1B).
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Figure 1. Immunofluorescent double-labeling
for nitric oxide synthase-2 (red), glial fibrillary acidic protein (green),
and the nucleus (blue-purple). Labeling for nitric oxide synthase-2 is not
present in control astrocytes (A) but is intensely present in the rough endoplasmic
reticulum–Golgi region and cytoplasm of astrocytes grown under elevated
hydrostatic pressure for 48 hours (B).
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The synthesis of NOS-2 protein in astrocytes under ambient pressure
and elevated hydrostatic pressure was also demonstrated by immunoblot. Using
a polyclonal antibody against NOS-2, a specific band, approximately 120 kd,
was detected by immunoblotting of the cell lysates. By pretreating the antibody
against NOS-2 with a specific blocking peptide, the band at 120 kd was not
apparent, confirming that this band is specific for NOS-2 (data not shown).
The cell lysates from astrocyte cultures exposed to elevated hydrostatic pressure
for 24, 48, and 72 hours contain much more intense bands for NOS-2 than the
control culture at ambient pressure (Figure
2A). Similar results were obtained from astrocyte cultures from
different human donors. Evaluation of the band intensity of NOS-2 at 24, 48,
and 72 hours of elevated hydrostatic pressure indicated that the level of
NOS-2 protein peaks at 48 hours of elevated hydrostatic pressure (Figure 2B).
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Figure 2. Immunoblot for nitric oxide synthase-2
(NOS-2) protein. A, Intense bands of protein for NOS-2 are detected in astrocytes
grown under elevated hydrostatic pressure for 24, 48, and 72 hours but not
in controls (C). B, Semiquantitation of the optical densitometry values (±SD)
for the immunoblot bands of the pressure-exposed cultures compared with controls
(n = 3).
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To investigate the effect of hydrostatic pressure on the gene transcription
of NOS-2, semiquantitative RT-PCR was performed on the NOS-2 mRNA of astrocytes
exposed to elevated hydrostatic pressure for 12, 24, and 48 hours and compared
with control cultures exposed to atmospheric pressure. Our results demonstrated
that the mRNA level for NOS-2 was significantly enhanced in the astrocytes
under elevated hydrostatic pressure. By RT-PCR, very intense bands indicating
mRNA for NOS-2 were detected by hybridizing with the specific NOS-2 DNA probe
from the samples of astrocytes exposed to elevated hydrostatic pressure for
12, 24, and 48 hours (Figure 3A),
but not from samples from the control culture. The RT-PCR for mRNA for -actin
from the same samples showed no significant difference (Figure 3B). The constancy of the -actin results confirms that
the amount of mRNA from the individual samples for the determination of NOS-2
was relatively similar and that the induction of gene transcription for NOS-2
was not a general phenomenon that up-regulates the transcription of all genes.
Similar results were obtained from astrocyte cultures from different human
donors. Evaluation of the band intensity for mRNA for NOS-2 at 12, 24, and
48 hours of elevated hydrostatic pressure indicated that the mRNA level for
NOS-2 in the astrocytes peaked markedly at 12 hours of exposure to elevated
hydrostatic pressure and remained at a high level through 24 hours. After
48 hours of exposure to elevated hydrostatic pressure, the mRNA level for
NOS-2 was still elevated compared with control but appeared to decrease from
the peak (Figure 3C).
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Figure 3. Reverse transcription polymerase
chain reaction for nitric oxide synthase-2 (NOS-2) messenger RNA. A, Intense
bands for NOS-2 messenger RNA are detected in astrocytes grown under elevated
pressure for 12, 24, and 48 hours but not in controls (C) (bp indicates base
pairs). B, Band intensity for -actin is similar under all conditions.
C, Semiquantitation of the optical densitometry values (± SD) for the
NOS-2 messenger RNA bands of the pressure-exposed cultures compared with controls
(n = 3).
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COMMENT
In this in vitro study, we investigated the effects of biomechanical
stress on the induction of NOS-2 in astrocytes of the human optic nerve head
by detecting the protein and mRNA levels for NOS-2 in cell cultures exposed
to elevated hydrostatic pressure. Our results show that, under elevated hydrostatic
pressure, the astrocytes of the human optic nerve head exhibit significantly
increased gene transcription and synthesis of NOS-2 in vitro. Although we
performed these initial experiments at 60 mm Hg, well above the intraocular
pressure normally observed in patients with glaucoma, we believe that further
investigations will demonstrate that the pressure-sensitive induction of NOS-2
will occur at lower hydrostatic pressures. The appearance of NOS-2 in the
optic nerve head in glaucoma could be a primary event in the disease or secondary
to cellular and tissue changes and/or the response to cytokines released in
the damaged tissue. Our studies suggest that, in glaucomatous optic neuropathy,
the astrocytes of the human optic nerve head may directly respond to elevated
intraocular pressure by expressing NOS-2 and thereby damage the axons of the
retinal ganglion cells by producing excessive NO.
Immunocytochemical analysis demonstrates an apparent increase in the
amount of NOS-2 protein in the astrocytes exposed to elevated hydrostatic
pressure when compared with controls. The immunolabeling of NOS-2 in the astrocytes
exposed to elevated hydrostatic pressure is most intense in the rough endoplasmic
reticulum–Golgi region, the site of new protein synthesis, as well as
in the cytoplasm. The appearance of the immunolabeling of NOS-2 in the astrocytes
induced by elevated hydrostatic pressure is the same as our previous observations
with astrocytes that were stimulated by cytokines. The astrocytes that are
exposed to elevated hydrostatic pressure exhibit larger cell bodies with extensive
processes, perhaps indicating that elevated hydrostatic pressure stimulates
the cells to become reactive astrocytes.
By immunoblot and semiquantitative RT-PCR, the protein and mRNA levels
for NOS-2 in the astrocytes were demonstrated and compared semiquantitatively
at sequential time points of exposure to elevated hydrostatic pressure and
to ambient pressure. The synthesis of NOS-2 protein induced by elevated hydrostatic
pressure reaches the peak level at 48 hours of exposure; however, the transcription
of mRNA for NOS-2 reaches the peak level earlier, at 12 hours of exposure.
The difference in the peak time between the mRNA level and the protein level
of NOS-2 is consistent with the process of protein synthesis.
In optic nerve head astrocytes in vitro, the mRNA for NOS-2 is relatively
short-lived. After stimulation with cytokines, the mRNA of NOS-2 is even more
transiently expressed, with a 6-hour half-life.22
At the 3' untranslated region of NOS-2 mRNA, there is a conserved adenine
and uracile–rich octanucleotide sequence that mediates mRNA instability.
Thus, the mRNA level of NOS-2 does not remain elevated for sustained periods,
but reaches a maximum, after which induction is terminated and the mRNA is
destroyed. It is therefore unlikely that any one astrocyte maintains induced
NOS-2 throughout the chronic glaucomatous process over many years. In vivo,
as glaucomatous optic neuropathy proceeds, individual astrocytes in local
areas of the optic nerve head may be serially induced to express NOS-2, leading
to focal areas of damage within the optic nerve head.
Induction of NOS-2 in astrocytes in response to a variety of stimuli
has been reported,23-25
but, to our knowledge, this is the first report that a mechanical stimulus,
elevated hydrostatic pressure, can induce astrocytes to express NOS-2. The
subcellular mechanisms of the responses of astrocytes to elevated hydrostatic
pressure are unknown. With the use of patch clamp techniques, membrane ion
channels have been shown to be pressure sensitive. In rat neonatal astrocytes,
one class of ion channels, called curvature-sensitive channels, is activated
when the cell membrane curves toward the soma under pressure, and another
class of ion channels, called stretch-activated channels, is activated by
suction.26-27 Using astrocytes
from the human optic nerve head in experiments similar to ours in vitro, Ricard
et al20 reported that hydrostatic pressure
causes changes in the cytoskeleton and an increase in expression and synthesis
of a specific isoform of neural cell adhesion molecule, which changes cell
adhesion properties. How an extracellular mechanical stress signal is transmitted
intracellularly and which signal cascades promote new gene expression in optic
nerve head astrocytes remain to be determined.
A variety of cell types, such as vascular endothelial cells,28-29 neuronal cells,30
bone cells,31 marrow cells,32
articular chondrocytes,33 vascular smooth muscle
cells,34 and periodontal ligament cells,35 have been reported to respond to hydrostatic pressure
or other biomechanical stresses. Biomechanical stress causes cellular responses,
including synthesis of cellular mediators such as growth factors and cytokines,
opening and closing of ion channels, and the synthesis and degradation of
extracellular matrix macromolecules. Agar et al30
reported that elevated hydrostatic pressure (100 mm Hg) causes 2 neuronal
cell line cultures to undergo apoptosis. In vascular endothelium, the gene
of another isoform of nitric oxide synthase, the endothelial nitric oxide
synthase (NOS-3), can be potently and rapidly up-regulated by certain kinds
of shear stress, particularly steady laminar shear stress, but not by turbulent
shear stress.36 The shear stress–mediated
stimulation of NO production through up-regulation of gene expression may
involve the activation of "shear stress receptors" that are present in the
endothelial cells, and the subsequent rapid production of an intracellular
second messenger.37 A variety of other proteins
are rapidly induced and activated by shear stress in endothelial cells, including
certain cell surface potassium channels, members of the mitogen-activated
and stress-activated protein kinase cascades, certain transcription factors
such as nuclear factor- B, and subsets of receptor-associated G proteins.38 Thus, for certain cell types, the biomechanical microenvironment
of the cells might be as important as the biochemical one.
Our studies in vitro indicate that the in vivo expression of NOS-2 in
the astrocytes of the human glaucomatous optic nerve head, which we have demonstrated
previously,6 may be a direct response to the
elevated intraocular pressure that is characteristic of glaucoma. The optic
nerve head fills the scleral canal and is, therefore, between 2 pressure compartments,
the intraocular compartment (intraocular pressure) and the central nervous
system (retrolaminar tissue pressure). The lamina cribrosa of the optic nerve
head is a connective tissue suspended perpendicularly to the pressure gradient
between the intraocular compartment and the central nervous system. In eyes
with normal intraocular pressure, this pressure gradient varies because of
the ocular pulse and the diurnal changes in intraocular pressure.39-40 In eyes with glaucoma, there are
elevated intraocular pressure, spikes of increased intraocular pressure, and
diurnal changes of intraocular pressure. Thus, significant deformation of
the lamina cribrosa occurs in glaucoma, which may generate biomechanical stress
on the astrocytes and other cell types that are embedded in the connective
tissue and attached to the extracellular matrix macromolecules.41
The heightened and changing hydrostatic pressure gradient within the optic
nerve head may directly induce astrocytes to express NOS-2 in vivo, as we
find in vitro. Further studies on the molecular regulation of intraocular
pressure–induced NOS-2 expression in astrocytes of the human optic nerve
head may lead to a new appreciation of the role of cellular responses in glaucomatous
optic neuropathy and new therapeutic approaches for accomplishing neuroprotection
in patients with glaucoma.
AUTHOR INFORMATION
Accepted for publication August 9, 2000.
This work was supported in part by grant EY12017 and core grant EY02687
from the National Eye Institute, Bethesda, Md, and by the Glaucoma Foundation,
New York, NY.
We acknowledge M. Rosario Hernandez, DDS, for developing the pressure
chamber used in these experiments and for assistance with the chamber and
the cell culture.
Corresponding author and reprints: Arthur H. Neufeld, PhD, Department
of Ophthalmology and Visual Sciences, Box 8096, Washington University School
of Medicine, 660 S Euclid Ave, St Louis, MO 63110 (e-mail: neufeld{at}vision.wustl.edu).
From the Department of Ophthalmology and Visual Sciences, Washington
University School of Medicine, St Louis, Mo.
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