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1 From the Division of Ophthalmology and the 2 Department of Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, New Mexico; and 3 Veterans Administration Medical Center, Albuquerque, New Mexico.
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Abstract |
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as a regulator of MMPs in the retinal neovascularization
process.
METHODS. C57/Bl6 mice were treated with 75% oxygen (experimental) or room air
(control) from postnatal days (P)7 through P12, followed by room air
until P17. Retinas were collected at P13, P15, or P17 and total RNA
analyzed for the relative level of TNF, TNF receptor (p55), and
TNF-converting enzyme (TACE). Immunostaining was used to identify
changes in TNF protein expression as well as to localize TNF within
specific retinal cell types. The role of TNF in stimulating retinal
microvascular endothelial cell (RMVEC) proteinase production was
evaluated using isolated murine RMVECs grown in normoxic or hypoxic
conditions. Message expression was analyzed by RT-PCR and protein
expression by zymographic analysis.
RESULTS. TNF mRNA was increased in the retinas of experimental animals on P13
and P15, during the early stages of retinal neovascularization. In
addition to being expressed by Müller glial cells and the inner
nuclear layer, additional expression was noted in the outer nuclear
layer of experimental animals. No significant level of apoptosis was
detected in the retina of experimental animals with retinal
neovascularization. Isolated RMVECs did not significantly increase MMP
production directly in response to a hypoxic stimulus, but required the
presence of exogenous TNF. TNF increased the expression of
MT1-MMP, MMP-3, and MMP-9 in these cells. The levels of TACE and p55,
proteins important in mediating the response of cells to TNF, were
found to be increased by the angiogenic protein, vascular endothelial
growth factor (VEGF), which was also elevated in the experimental
retinas.
CONCLUSIONS. TNF levels increase in experimental mouse retinas exposed to hypoxic
stimuli. Increased production of MMPs by RMVECs does not occur directly
in response to a hypoxic stimulus. These cells are responsive, however,
to stimulation by TNF, which enhances the production of specific
members of the MMP family. VEGF also plays a role in this process
through its regulation of TACE and p55 mRNA in the vascular endothelial
cells. These findings support the hypothesis that these two growth
factors have a role in the regulation of extracellular proteinase
expression during retinal neovascularization.
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Introduction |
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In addition to VEGF, other proteins, including tumor necrosis factor
(TNF)-, have been found to be expressed in the retinas of humans
with proliferative eye diseases9
10
11
and in animal models
of retinal neovascularization.12
TNF is a 26-kDa
transmembrane protein that is processed by TNF-converting enzyme
(TACE), to yield a 17-kDa soluble protein.13
14
TNF
functions through its binding to two receptors: p55, implicated in
apoptosis and NF
B activation, and p75, involved with lymphocyte
proliferation.15
16
The objective of the present study was to determine the role of TNF
in the neovascular response of the mouse retina to a hypoxic stimulus
and the role of this factor in the regulation of RMVEC behavior.
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Methods |
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In Vitro Cell Culture System
Mouse RMVECs were obtained from the laboratory of Jeff Gidday
(Washington University, St. Louis, MO). Briefly, retinal tissue was
collected and homogenized, followed by digestion with a solution of
collagenase, dispase, DNAse I, and
N()-p-tosyl-L-lysin
chloromethyl ketone (TLCK). The microvessels were separated
from other cells by density-dependent centrifugation in a 50% density
gradient (Percoll, Pharmacia & UpJohn, Uppsala, Sweden). The RMVEC band
was collected, washed, and cultured on collagen-coated plates
in DMEM containing 10% fetal calf serum and 30 µg/mL endothelial
cell growth supplement (Sigma Chemical Co., St. Louis, MO). Endothelial
cells were characterized by immunostaining with factor VIII and were
incubated in culture medium containing either TNF (1 ng/mL) or
VEGF165 (10 ng/mL; R & D Systems, Minneapolis, MN) for 24 hours at
37°C, and the RNA was extracted as described in the following
section. Three cultures were used for each treatment.
RNA Isolation RT-PCR Analysis
RNA was isolated from murine retinas (n = 3 at
each time point) or RMVECs (n = 6–8) using extraction
reagent (Trizol; Gibco, Rockville, MD). First-strand cDNA was prepared
from 0.5 µg total RNA using an oligo-dT primer and reverse
transcriptase (Superscript; GibcoBRL, Gaithersburg, MD). For
semiquantitative PCR, 1 µL of each first-strand reaction was then
amplified using MMP-, TACE-, p55-, and 18S-specific oligonucleotide
primers. Standard PCR amplification was performed at 94°C, 1 minute;
60°C, 1 minute; and 72°C, 1 minute for 27 cycles, which was
determined to be within the linear range of product amplification.
After completion, 10 µL of the reactions were analyzed by agarose gel
electrophoresis and ethidium bromide staining to determine the presence
or absence of specific transcripts in the cells or tissue, as well as
the levels of transcript relative to the control transcript 18S RNA.
Quantitation of band density was performed with image analysis software
(Alpha Imager; Alpha Innotech, San Leandro, CA). The following specific
primer pairs were used for these analyses: MMP-2:
5'-CTATTCTGTCAGCACTTTGG-3' and 5'-CAGACTTTGGTTCTCCAACTT-3'; MMP-9:
5'-AAATGTGGGTGTACACAGGC-3' and 5'-TTCACCCGGTTGTGGAAACT-3'; MT1-MMP:
5'-TGGGTAGCGATGAAGTCTTC-3' and 5'-AGTAAAGCAGTCGCTTGGGT-3'; TACE:
5'-CCTTTAAACCCTGCATTATCC-3' and 5'-GGGCAGAATATAACATAGAG-3'; p55:
5'-CACTGGAAGTGTGTCTCACTC-3' and 5'-ATCTGCTGCACCAAGTGCC-3'; and 18S:
5'-GAGCTCACCGGGTTGGTTTTG-3' and 5'-TACCTGGTTGATCCTGCCAG-3'.
Analysis of Activity Levels of MMP-2 and -9
Zymographic analysis was performed using RMVECs solubilized in
0.2% Triton X-100, 0.1 M phosphate (pH 7.3). Samples were equalized to
total DNA content and electrophoresed in 10% polyacrylamide minigels,
into which gelatin was cross-linked. After electrophoresis, the enzymes
were renatured by soaking the gels in a solution of 2.5% Triton X-100
followed by incubation for 24 to 48 hours in LSCB buffer (50 mM Tris,
0.2 M sodium chloride, 5 mM calcium chloride, 0.02%
polyoxyethyleneglycol dodecylether (Brij 35; Sigma), and 0.02% sodium
azide [pH 7.6]). The zones of proteolysis corresponding to the
presence of MMP-2 and -9 were visualized by staining the gel with
0.125% Coomassie blue and comparing the position of the bands with a
molecular weight standard and with a standard of HT1080
cell–conditioned medium that contains ample quantities of both
enzymes. The degree of digestion related to the amount of enzyme
present in the various samples was quantified using the image analysis
software (Alpha Imager; Alpha Innotech).
TNF Protein Detection and Localization
Western blot analysis was performed with equal amounts (10 µg)
of murine retinal tissue or RMVEC extract. The extracts were boiled in
SDS sample buffer, fractionated on a 15% SDS polyacrylamide gel, and
electrophoretically transferred to a nitrocellulose filter. The filters
were blocked overnight at 4°C (TBS, 0.1% Tween, 1% milk), rinsed
with TBS-0.1%Tween (TBST), and incubated with rat anti-mouse antibody
against TNF (Pharmingen, San Diego, CA) at 1 µg/mL in TBST for 1
hour at 25°C. Membranes were rinsed for 1 hour in TBST and incubated
with a secondary antibody coupled to biotin. Detection was accomplished
by the avidin biotin system (Vector Laboratories, Burlingame, CA).
For immunohistochemical localization of TNF, murine eyes from
experimental and control animals were collected at P15 or P17 and fixed
in 2% paraformaldehyde in 0.1 M phosphate buffer. Eyes were embedded
in optimal cutting temperature (OCT) compound (Tissue Tek; Miles
Laboratories, Elkhart, IN) and frozen. Tissue sections were incubated
in 10% goat serum for 1 hour at room temperature followed by 1 µg/mL
TNF antibody. The sections were washed with TBST and incubated with
a secondary biotinylated antibody (Vector Laboratories) for 1 hour.
Detection was achieved using the avidin biotin system (Vector
Laboratories). Slides were coverslipped (Permount; Fisher Scientific,
Fairlawn, NJ) and examined by light microscopy.
Detection of Apoptosis
Apoptosis detection was performed with a kit, according to
instructions (In Situ Death Detection kit; Roche Molecular
Biochemicals, Indianapolis, IN). Briefly, paraffin-embedded tissue
sections were processed for TUNEL staining. The TUNEL reaction was
prepared using the recommended 9:1 buffer-to-enzyme ratio. The sections
were placed in a dark, humidified chamber at 37°C for 1 hour.
Sections were rinsed once for 5 minutes with PBS and mounted using
diamidinophenylindole (DAPI) mounting medium (Vector Laboratories).
Apoptotic nuclei were visualized by FITC and counted (n = 25
sections).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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was significantly increased in experimental animals during the early
stages of retinal neovascularization at P13 and P15 compared with the
levels seen in control animals (Fig. 1)
. The TNF processing enzyme, TACE was also significantly elevated early
in the process and returned to control levels as the neovascularization
developed (Fig. 1)
. Increased expression of the TNF receptor I (p55)
was also seen in the experimental animals, but this occurred after the
increased expression of the growth factor and TACE (Fig. 1) . In
addition to the mRNA levels, the levels of both the soluble and
membrane-bound forms of TNF protein were significantly increased in the
retina of experimental animals at both P15 and P17 (Fig. 2)
. No difference was seen in the levels of soluble and membrane-bound
TNF proteins on P13 (data not shown). TNF was localized by
immunostaining to the inner nuclear layer in both control and
experimental retinas at P17. Because the stained cell processes were
seen to extend from the inner nuclear layer toward the ganglion cell
layer similar to the foot processes of the Müller cells, we
speculate that these cells are Müller cells. Additional staining
was detected in the outer nuclear layer of experimental animals only
(possibly photoreceptors; Fig. 3A
3B
). Sections incubated without the primary antibody showed no
significant background staining (Fig. 3C)
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protein is the
induction of this process. Examination of TUNEL-stained retinas
revealed a small number of apoptotic cells throughout the retina at
P13, P15, and P17 and no significant difference between experimental
and control animals (Fig. 4)
. This result suggests that TNF may have other roles in the retina
during neovascularization, one of which may be in the regulation of
extracellular proteinase production.
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on the regulation of these
specific MMPs was subsequently examined in cultures of isolated RMVECs.
Cells were treated with TNF for 24 hours followed by analysis of
mRNA or protein levels. In some cases, cells were treated with VEGF as
a positive angiogenic control. The effects of VEGF on endothelial cell
behavior and MMP expression have been well
characterized.19
20
21
Treatment of cells with TNF significantly and selectively altered
the expression of MMPs at the levels of both mRNA and protein. RT-PCR
analysis demonstrated that TNF increased the expression of MT1-MMP
and MMP-9 in the cultured cells, but had little effect on the
expression of MMP-2 (Fig. 5)
. In comparison, these cells responded to VEGF treatment with a
significant increase in the expression of MT1-MMP and smaller increases
in MMP-2 mRNA levels. VEGF did not appear to effect the expression of
MMP-9 (Fig. 5)
. Results from the zymographic analysis paralleled that
of the message levels of MMP-2 and -9 in cells treated with TNF
(Fig. 6) . There were increases in both the pro and active forms of MMP-9, with
no change in the levels of MMP-2 when compared with untreated control
cells. The effect of VEGF was seen primarily in the expression of the
pro form of MMP-2. In addition, cells treated with VEGF showed
increased expression of TACE and a slight although nonsignificant
increase in the expression of p55 (Fig. 7)
.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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TNF binds to the p55 receptor on responsive cell types and
elicits apoptosis or other events that require the activity of the
NFB transcription factor.22
23
24
25
The level of TNF
message was increased significantly at P13 and less dramatically at P15
in the model of retinal neovascularization. TNF expression was found
to be significantly increased, along with its processing enzyme TACE
during the early stages of angiogenesis after a hypoxic stimulus.
Immunohistochemical analysis localized the TNF protein to
Müller glial cells and their processes in the inner nuclear
layer. Additional staining was evident in a population of cells
(possibly photoreceptors) present in the basal portion of the outer
nuclear layer of experimental animals. The staining pattern suggests
that either these cells are being stimulated themselves by TNF or
that they are the source of the growth factor for action on other cell
types, including the capillary endothelial cells. It is possible that
the Müller cells secrete TNF that may be released through the
foot processes onto the capillary endothelial cells located in the
nerve fiber layer of the retina. Further studies using in situ
hybridization may resolve this question to some extent.
The absence of significant apoptosis in the experimental retina
suggests an alternative function for TNF in this system. The
cytoplasmic domain of the TNF receptor, p55, has an 80-amino-acid
residue "death domain" that can regulate the apoptotic
pathway.26
This is not the only outcome of p55 ligation,
however. NF-B is also activated by p55 stimulation, although the
mechanism that determines the choice of pathways is not completely
clear.27
Stimulation of NF-B activity by the
alternative pathway may result in a variety of cellular responses,
including the transcriptional regulation of expression of select
members of the MMP family of extracellular
proteinases.28
29
30
Previous studies have reported an early and rapid increase in
VEGF expression in the model used for these
experiments.31
32
33
The VEGF expression precedes the
formation of new vessels that becomes maximal on P17. The overlapping
temporal expression of VEGF and TNF may suggest an interactive role for
these two proteins during the angiogenic period. Indeed, the expression
of the MMPs detected in the retinal tissues of this model appears to
require both TNF and VEGF activity for increased expression, as
shown in the isolated RMVECs. The TNF increased the expression of
MT1-MMP and MMP-9, but had no effect on MMP-2. VEGF, in contrast, was
seen to increase the expression of MT-MMP-1 and -2, but had little
effect on MMP-9 mRNA levels. It was interesting to note that VEGF also
significantly affected the expression of TACE in the cultured cells.
This early change in TACE production, in response to VEGF stimulation,
may result in the release of membrane-bound TNF from cells, such as
Müller glial cells, yielding a soluble form of the protein, which
has been shown to be a more potent stimulator and may have more distant
effects on other cell types.34
We have previously shown that a synthetic MMP inhibitor, BB-94,
can significantly inhibit the development of retinal neovascularization
in the mouse model.35
In addition to inhibiting the active
forms of MT1-MMP and MMP-2 and -9, BB-94 also appeared to significantly
reduce the levels of the pro form of MMP-9 in the retinal tissues of
animals treated with this compound. This can now be explained by
results from the present study, in which TNF and TACE were expressed
in the mouse retina and TNF had a major effect on the expression of
MMP-9 in isolated microvascular endothelial cells. The solubilization
and activity of TNF is dependent on the activity of TACE, an
integral membrane metalloproteinase that causes the secretion of the
active form of TNF from its plasma membrane
precursor.13
14
In addition to inhibiting the members of
the MMP family, BB-94 has been shown to inhibit the activity of TACE
and other -disintegrin and metalloproteinase domains
(ADAMs).36
37
Inhibition of TACE activity and therefore
TNF activity would thus be expected to lower the expression of MMP-9
in the retinal tissues of BB-94–treated animals.
In conclusion, the results of this study suggest a major role for
TNF in the initiation of retinal neovascularization and provide a
more complete understanding of how retinal neovascularization in
general may be regulated. This information may be critical to the
identification of potentially new targets for therapeutic intervention
in the treatment of this disease process, thereby alleviating some of
the negative side effects of the current laser treatment.
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Acknowledgements |
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Footnotes |
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Submitted for publication February 6, 2001; revised September 12, 2001; accepted October 2, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Arup Das, Division of Ophthalmology, Department of Surgery, 2ACC, University of New Mexico Hospital, Albuquerque, NM 87131; adas{at}unm.edu.
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), membrane bound (26
kDa); (
), soluble (17 kDa). (n = 2 mice, four retinas for
each point). Values are mean ± SEM. *Significantly different from
control (P < 0.01). (B) Representative
Western blot. Lane 1: P15 experimental;
lane 2: P15 control; lane
3: P17 experimental; lane 4: P17
control. Top arrow: 26-kDa membrane bound TNF
