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From the Casey Eye Institute, Oregon Health Sciences University, Portland, Oregon.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Flow rates were increased in perfused human anterior segment organ cultures and the matrix metalloproteinase (MMP) levels and IOP were evaluated. Human TMs in stationary anterior segment organ culture were mechanically stretched, and MMP levels were analyzed. TM cells were grown on membranes, which were then stretched, and MMP levels were evaluated. Western immunoblots, zymography, and confocal immunohistochemistry were used to evaluate changes in MMPs and their tissue inhibitors, the TIMPs.
RESULTS. Doubling the flow rate in perfused human organ cultures increased gelatinase A levels in the perfusate by 30% to 50% without affecting gelatinase B or stromelysin levels. Immediately after doubling the flow rate, the measured IOP doubled. However, over the next few days the IOP gradually returned to the initial level, although the flow rate was maintained at double the initial value. Stretching stationary organ cultures or stretching TM cells grown on membranes resulted in similar increases in gelatinase A without changes in gelatinase B or stromelysin levels. The gelatinase A increases occurred between 24 and 72 hours and were approximately proportional to the degree of stretching. Although coating the membranes with different ECM molecule affected the gelatinase A response, the optimum response occurred when the cells had been grown long enough to produce their own ECM. By Western immunoblot and confocal immunohistochemistry, the stretch-induced increases in gelatinase A were accompanied by strong decreases in TIMP-2 levels and moderate increases in one membrane type MMP, MT1-MMP. After mechanical stretching of the membrane, gelatinase A, MT1-MMP and TIMP-2 all exhibited a similar punctate immunostaining pattern over the TM cell surface.
CONCLUSIONS. These results are compatible with the hypothesis that elevations in IOP are sensed by TM cells as ECM stretch/distortion. TM cells respond by increasing gelatinase A and MT1-MMP, while decreasing TIMP-2 levels. This will increase ECM turnover rates, reduce the trabecular resistance to aqueous humor outflow, and restore normal IOP levels. This hypothesis provides a regulatory feedback mechanism for IOP homeostasis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We hypothesized earlier that TM cells could modulate aqueous outflow facility by changing ECM turnover and subsequent extracellular matrix (ECM) replacement rates.1 2 15 16 17 18 We recently reported two observations that support this hypothesis: (1) manipulation of TM matrix metalloproteinase (MMP) activity reversibly modulates outflow facility in perfused human anterior segment organ culture19 ; and (2) laser trabeculoplasty, a common treatment for glaucomatous IOP elevations, induces sustained MMP expression specifically within the trabecular juxtacanalicular region.20 21 Thus, a plausible molecular mechanism for trabecular modulation of aqueous outflow facility is present. Interference with endogenous MMP activity causes dramatic reduction in outflow facility; thus, maintenance of the appropriate outflow resistance requires ongoing ECM turnover.19
However, some mechanism of TM cell sensing of IOP or outflow facility would be required for this mechanism to effectively maintain IOP homeostasis.1 2 16 17 A significant portion of the resistance to outflow is thought to reside within the juxtacanalicular ECM.1 2 16 22 23 24 25 Because of the structure of the TM, which might be thought of as a "semi-porous diaphragm" stretched across the outflow pathway and Schlemm’s canal, increases in IOP will tend to selectively stretch or distort the TM.22 26 27 This should be particularly acute in the juxtacanalicular ECM, because a large portion of the resistance to outflow appears to reside there. Thus one possible mechanism that would allow TM cells to sense IOP would be for these cells to be able to recognize mechanical stretch or distortion.1 2 16 17 By now, several reports have appeared showing that TM cells can sense such mechanical stretching forces.28 29 30 31 32 33
A variety of cell types have been shown to use integrins, cellular receptors for ECM macromolecules, to detect stretching or distortions of these ECMs and transduce regulatory responses.34 35 36 37 TM cells express a variety of integrins,38 and may also detect stretching by similar mechanisms. Thus, to test the hypothesis that TM cells can sense ECM stretching or distortion and will respond by adjusting ECM turnover, we used three separate methods of creating mechanical stretching stresses on TM cells and evaluated the effects this had on MMP levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Models to Exert Mechanical Stretch on TM Cells: Perfused Organ
Culture
Stationary human anterior segment organ culture was conducted
serum-free as previously described.39
Perfused human
anterior segment organ culture was conducted as previously
described,19
except constant flow rate perfusion was used
and IOP was measured.40
Anterior segments were maintained
in stationary organ culture for 1 week before being placed in perfusion
culture and were perfused for 3 to 5 days until the IOPs had
stabilized, before conducting experiments. Flow rates were maintained
at 2.5 µl/min, and measured IOPs averaged about 7 to 8 mm Hg. To
produce stretch/distortion stresses on TM cells in perfused organ
cultures, flow rates were doubled (to 5 µl/min) and maintained for
several days, while measuring IOP at various time intervals as
indicated. In one set of experiments designed to verify the effects of
elevated IOP on gelatinase A levels, perfusion rates were doubled for
24 hours and then returned to 2.5 µl/min. The perfusate was collected
in a groove on the rim of the clamping ring at various times as
indicated and stored at -20°C for analysis. Probable resultant
forces on the TM cells of the juxtacanalicular region of the meshwork
are shown in Figure 1A
(small arrows). Presumably, the primary impact of doubling the flow
rate will be largest at the site of the outflow resistance, putatively
within the juxtacanalicular region of the TM (JCT in Fig. 1A
). This
flow pressure, the normal IOP of the eye (large arrows, Fig. 1A
),
should tend to bow the juxtacanalicular region into Schlemm’s canal.
The resultant forces (smaller arrows, not shown to scale) that a cell
within or on either side of the juxtacanalicular ECM (gray region in
Fig. 1A
) of the TM should experience include the following: (1) a
circumferential stretching force running parallel to Schlemm’s canal;
(2) a radial stretching force perpendicular to Schlemm’s canal with
vectors running toward the scleral spur and toward the cornea; and (3)
a force opposite to the IOP due to the venous pressure (not present in
the organ culture model).
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TM Cell Culture and Mechanical Stretch/Distortion
Porcine and human TM cells were cultured as previously
described.18
By passages 3 to 5, cells were plated at a
density of approximately 90% confluence onto cell culture insert
membranes in 6-well culture plates. After 3 to 5 days, serum-free
medium was added to the cells for 24 or 48 hours before and
during stretching experiments. To apply mechanical stretch/distortion,
a glass bead of precise diameter was placed beneath the insert in the
center of the membrane, and weight was applied to the lid of the plate
to force the lip of the insert down onto the upper lip of plate’s well
(Fig. 1C)
. This produced a defined upward bowing of the membrane, which
increased the surface area by an estimated 10%. The cells should
experience radial and circumferential stretch forces, the sum of which
are proportional to the reciprocal of the cell’s distance from the
center of the membrane. As indicated in one study, a smaller diameter
bead was also used to produce a 5% increase.
Western Immunoblots and Zymograms
Culture medium, perfusate or cellular extracts were analyzed by
zymography, using either gelatin or ß-casein as substrates for
gelatinases A and B (MMP-2 and MMP-9) or stromelysin (MMP-3),
respectively, or by Western immunoblot to determine the activity or
protein levels of the various MMPs or TIMPs.41
For
immunoblots, second antibody with conjugated horseradish peroxidase and
chemiluminescence detection was used, following the manufacturers’
directions. When cellular/ECM extracts were to be analyzed, proteins
were extracted using a modified RIPA buffer with a variety of
proteinase inhibitors as previously described.42
Confocal Immunohistochemistry
For localization studies, inserts were rinsed with
phosphate-buffered saline (PBS), and TM cells and proteins were fixed
on the membranes in 4% paraformaldehyde in PBS for 10 minutes and then
rinsed twice for 5 minutes each in PBS. Membranes were then washed in
room temperature TBS (50 mM Tris, 150 mM NaCl, pH 7.4) and incubated
with blocking buffer (TBS with 0.05% Triton X-100, 2% goat serum, and
an additional 150 mM NaCl) for 30 minutes. Membranes were then
incubated with primary antibody (5 µg/ml) in blocking buffer for 1
hour in a humidified chamber, rinsed in TBS three times more for 3
minutes each time, and incubated with fluorescein-conjugated secondary
antibody (17.2 µg/ml) in blocking buffer for 45 minutes in the dark
at 100% humidity. Membranes were then rinsed twice for 5 minutes each
in TBS, removed from the inserts, and placed on microscope slides.
After preequilibration with antifade buffer for at least 5 minutes,
antifade reagent was added, and slides were coverslipped, sealed, and
stored in the dark at 4°C until analyzed.
For analysis of the effects of 1x or 2x flow rate on TM cells in perfused anterior segments, 6-µm cryosections were analyzed after 24 hours at the designated flow rate. Explants were rinsed in PBS, and two wedges from opposite sides of the explants were removed and embedded in OCT containing 2.5% glycerol, quick-frozen, and stored at -80°C. Sections (6 µm) were cut with a Leitz Digital 1720 Cryostat (Leica, Germany), using standard methods. Briefly, sections were thaw-mounted onto Superfrost Plus slides (Fischer Scientific, Pittsburgh, PA), immersed in cold acetone for 2 seconds, and stored at -80°C. Slides were warmed to room temperature, fixed in 4% paraformaldehyde in PBS for 10 minutes, and then rinsed briefly in PBS. After pre-equilibration for 5 minutes at 37°C in buffer, 0.1 U of proteinase-free chondroitinase A was added per milliliter, and incubated for 8 minutes at 37°C. Subsequent steps were similar to those detailed above for the membranes.
Confocal microscopy was conducted as detailed earlier.43 In addition to nonstretched paired controls, stretched or nonstretched membranes without primary antibody were evaluated. Confocal instrument gain and zero settings were optimized below saturation on an intensely staining sample, and then no setting changes were made for the complete set with that antibody; images were processed together to avoid introducing artificial differences.
Live–dead staining (Molecular Probes) to evaluate the condition of TM cells was conducted as previously detailed.19 DNA analysis using a PicoGreen fluorescence assay (Molecular Probes) following the manufacturer’s instructions was used to correct for differences in cell density on membranes. When perfusion flow rates were doubled, twice the volume of perfusate was applied to gel lanes to correct for the dilution. Gels or autoradiographs were scanned, and relative band density was analyzed44 using a densitometry program (BioImage, Ann Arbor, MI). Student’s t-test or Mann–Whitney ranked sum analysis was used to determine significance when comparing treatment results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the effect of changing the degree of stretch, 24-hour stretch was conducted with beads of two different sizes. The smaller sized bead, which produces only a 5% surface area increase, results in a gelatinase A increases that is roughly half as large (i.e., in the range of 15%–20%). This is compared with the 30% to 50% increases seen with 10% surface area increases conducted in parallel (data not shown).
We also compared the efficacy of membranes coated with laminin, Matrigel, fibronectin, type IV or type I collagen, or gelatin with the noncoated membranes. Laminin and Matrigel were the least effective, dramatically attenuating the gelatinase A increases. Fibronectin and type IV collagen were of intermediate efficacy, producing less response than type I collagen or gelatin coating. The time after plating the cells on these membranes, that is, the time the cells were allowed to lay down their own ECM components, appears to be of primary importance. When the cells were allowed several days to establish an ECM before initiating the experiment, the gelatinase A responses were maximized, whether on coated or on uncoated membranes. Thus, all the studies presented herein were conducted on membranes without coatings.
Possible Involvement of TIMPs and MT-MMPs
Gelatinase A is unique among the MMPs in that its apparent
physiologic activation mechanism requires TIMP-2 and the
MT-MMPs.45
46
Thus, we investigated the effects of TM
cellular stretch/distortion on these proteins. TM cells grown on insert
membranes were subjected to 10% stretch for 24, 48, or 72 hours, and
media and cellular extracts were analyzed for changes in gelatinase A,
TIMP-2, and MT1-MMP levels. Typical zymograms and Western immunoblots
are shown (Fig. 5A)
. Media gelatinase A is seen as a latent 72-kDa pro-form and activated
68-kDa form; media TIMP-2 is a single band at 20 kDa, and cellular
extract MT1-MMP is seen predominantly as a very light latent pro-form
at 72 kDa, a dominant activated form at 63 kDa and a smaller breakdown
form at 45 kDa (Fig. 5A) . These band sizes are as predicted based on
literature values.45
46
47
48
Densitometric scans were analyzed
for each (Figs. 5B
5C
5D)
after 24, 48, or 72 hours of stretch.
Two separate experiments with each time point in triplicate resulted in
the indicated significance values, when comparing control versus
stretch pairs. Media gelatinase A and cellular MT1-MMP were increased
moderately, whereas media TIMP-2 was dramatically reduced by mechanical
stretch. The media levels of MT1-MMP were very low with or without
treatment (not shown). The cellular extract contained both TIMP-2 and
gelatinase A, although at much lower levels than the media. Gelatinase
A increased, and TIMP-2 decreased with stretch as observed in the
media.
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TIMP-2 immunostaining is also shown for cryosections through the outflow pathway of perfusion-cultured explants (Fig. 7) , which had been flowed at 1x (2.5 µl/min) for several days until the measured IOP had stabilized at about 7 to 8 mm Hg. They were then exposed for 24 hours at either 1x or 2x flow (2.5 or 5 µl/min, respectively) before termination of the experiment. The "-AB control" with no primary antibody added is also shown. Reduced TIMP-2 in response to doubling the flow rate is quite apparent. Gelatinase A and MT1-MMP immunostaining in these explants also change with stretch, reflecting the patterns detailed earlier for the membranes (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gelatinase A, TIMP-2, and MT1-MMP are normally expressed at moderate levels by TM cells in all three of these model systems. Evidence that has been presented for a complex pattern of gelatinase A regulation includes the following: transcriptional regulation, translational regulation, post-translational processing, secretion, ECM sequestration, recruitment by TIMP-2 to the membrane for activation by MT-MMPs, additional auto-activation steps, proteolytic processing to smaller active and then inactive forms, inhibition by TIMPs particularly TIMP-2 and possibly eventual degradation by TM cells. None of these processes have been unraveled in detail. Considerable evidence exists for a specialized relationship between TIMP-2 and pro-gelatinase A, involving heterodimerization via binding domains on each molecule’s carboxyl terminus.49 TIMP-2 has also been shown to recruit pro-gelatinase A to the cell surface, where TIMP-2 also binds to MT1-MMP and facilitates pro-gelatinase A’s proteolytic activation by MT1-MMP.45 46 The precise details and regulatory nuances of this activation process remain to be resolved. The punctate, apparent cell surface localization pattern of these three macromolecules after stretch is suggestive of colocalization, although we have not yet conducted detailed colocalization studies. In addition, pro-gelatinase A activation at the cell surface exhibits a bell-shaped TIMP-2 concentration dependence.45 When TIMP-2 is at low levels, well below 1:1:1 for these three molecules, the rate of MMP-2 activation is slow; when large excesses of TIMP-2 are present, the rate of MMP-2 activation is also slow, because both gelatinase A and MT1-MMP are also inhibited by TIMP-2. Our data can speculatively be interpreted by this model. Before stretch, TIMP-2 may be in excess thus restricting pro-gelatinase A activation rates; stretch reduces TIMP-2 approaching the optimum 1:1:1 ratio and triggering increases in pro-gelatinase A activation rates. Independently from this event, the observed increases in pro-MMP-2 and MT1-MMP levels will likely also increase active MMP-2 levels. In a number of other tissues, somewhat similar MMP responses to mechanical stretch have been reported.50 51
The inability of short-duration stretch to trigger gelatinase A increases in this system is intriguing. One interpretation of this data is that this trabecular homeostatic mechanism would need to discriminate against acute fluctuations in IOP. This would avoid overadjusting in response to transient changes in IOP because of physical activity. It seems reasonable that there would be two separate IOP regulatory mechanisms, one acute and one chronic, necessary to deal with the different types of pressure fluctuations that could be anticipated.
Although it seems clear that TM cells in the three different model systems that we used will experience mechanical stretching forces, the magnitude of these forces is difficult to estimate. In the perfusion system, which is the closest to the normal physiologic situation, the radial and circumferential forces on the juxtacanalicular TM cells (Fig. 1) seem likely, although their actual magnitudes are unknown. Contributions of other forces on these cells and forces on the remainder of the meshwork and on the remainder of the tissues are difficult to access. When we subjected scleral fibroblasts to mechanical stretching on insert membranes in cell culture, they did not respond with similar MMP/TIMP changes (data not shown). However, this model contains several other cell types. Another possible consideration is fluid shear forces in the perfused explant model. However, the aqueous humor flow rate is quite low. Estimations of the potential shear stress that uveal, corneo-scleral, or juxtacanalicular TM cells should experience at normal flow rates give values one to two orders of magnitude below those normally observed in blood vessels (C. Ross Ethier, University of Toronto, personnel communication). Although not absolutely ruling out any shear stress contributions, it seems unlikely that this would be a major contributor to our studies.
The stretched stationary culture model is the least well defined of the three that we used. The cornea and sclera will undoubtedly experience some stretch/distortion, and the magnitude of the trabecular stretch is very difficult to access. Although the resultant stretch forces shown in Figure 1A are likely to be important and acting on the juxtacanalicular TM cells, their relative contribution has not been ascertained. In spite of this limitation, the data reinforce that obtained with the other two models.
Although the most clearly defined of the three models, the membrane insert cell culture model suffers theoretically from the nonlinearity of the stretching forces. The forces on the cells in this model should vary as a function of 1/radius. Our confocal studies, however, argue that a threshold stretching force is achieved throughout the membrane and that the peripheral decline is not a major factor. When the MT1-MMP changes with stretch are evaluated on different regions of the membrane (as in Fig. 6 ), the observed changes in MT1-MMP are uniform across the membrane (data not shown). If a minimal stretch threshold were not reached throughout the membrane, cells on the more peripheral portions of the membrane should show considerably less increase in MT1-MMP than those on the central region.
Thus, our working hypothesis is supported, although certainly not unequivocally established, by these and earlier data.19 20 21 (1) Elevated IOP appears to be sensed as stretch by TM cells; (2) TM cell stretch triggers gelatinase A and MT1-MMP increases and TIMP-2 decreases; (3) this will increase ECM turnover and reduce the outflow resistance19 ; (4) putatively, this results in a return to physiologic IOP levels. This working hypothesis thus provides a plausible self-contained homeostatic mechanism that could explain chronic IOP regulation.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 17, 2000; revised January 3, 2001; accepted February 6, 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: Ted S. Acott, Casey Eye Institute (CERES), Oregon Health Sciences University, 3375 SW Terwilliger, Portland, OR 97201. acott{at}ohsu.edu
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-crystallin: an early cellular response to mechanical stretch Biochem Biophys Res Commun 235,69-73[Medline][Order article via Infotrieve]
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) as relative band density from zymograms.
(B) The active gelatinase A levels (•) and pro-gelatinase
A (
) and pro-gelatinase B (
92 kDa) and the
pro- and activated forms of gelatinase A (Gel A at 
,
control; 
, stretch; statistical significance is shown above each
pair of bars using Student’s t-test or Mann–Whitney ranked
sum analysis.
