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Regular Article |
From the Ophthalmology Research Laboratories*
and
Neurosurgical Institute,
Burns and Allen
Research Institute, Cedars-Sinai Medical Center, University of
California Los Angeles Medical School Affiliate, Los Angeles,
California; the Department of Ophthalmology,
Kyoto Prefecture University of Medicine, Kyoto, Japan; the School of
Biological Sciences,§
University of East
Anglia, Norwich, England; and the School of
Dentistry,
Indiana University,
Indianapolis, Indiana
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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1, 5,
{beta}1,
1; and epithelial integrin
3{beta}1 in human diabetic retinopathy (DR)
corneas. Here, using 142 human corneas, we tested
whether these alterations might be caused by decreased gene expression
levels or increased degradation. By semiquantitative reverse
transcription-polymerase chain reaction, gene expression levels
of the 1, 5, and {beta}1 laminin chains;
nidogen-1/entactin; integrin 3 and {beta}1
chains in diabetic and DR corneal epithelium were similar to normal.
Thus, the observed basement membrane and integrin changes were
unlikely to occur because of a decreased synthesis. mRNA levels of
matrix metalloproteinase-10 (MMP-10/stromelysin-2) were significantly
elevated in DR corneal epithelium and stroma, and of
MMP-3/stromelysin-1, in DR corneal stroma. No such elevation
was seen in keratoconus corneas. These data were confirmed by
immunostaining, zymography, and Western blotting. mRNA
levels of five other proteinases and of three tissue inhibitors of MMPs
were similar to normal in diabetic and DR corneal epithelium and
stroma. The data suggest that alterations of laminins,
nidogen-1/entactin, and epithelial integrin in DR corneas may
occur because of an increased proteolytic degradation. MMP-10
overexpressed in the diabetic corneal epithelium seems to be the major
contributor to the observed changes in DR corneas. Such alterations may
bring about epithelial adhesive abnormalities clinically seen in
diabetic corneas.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Because many of the diabetic corneal abnormalities are apparently
related to changes in cell adhesion and tissue repair, they might be
due to alterations of adhesive molecules of the extracellular matrix
(ECM) and BM. We have recently conducted a systematic study of >30
various ECM and BM components and their integrin receptors in corneas
from patients with diabetes and DR.15
Our data showed that
DR corneas had a significant decrease in immunostaining for major
epithelial BM components, nidogen-1/entactin, laminin-1 (1{beta}11),
laminin-10 (5{beta}11), and of their binding integrin,
3{beta}1. These
alterations, especially of
3{beta}1 integrin, seemed
to be specific for diabetic corneas because they were not pronounced in
corneas from patients with a common edematous corneal disease, bullous
keratopathy.15
We tested here whether BM and integrin alterations in human diabetic
and DR corneas could occur because of decreased synthesis or increased
degradation. Because gene expression of the affected components did not
change with diabetes and DR, decreased synthesis could not easily
explain our previous data. On the other hand, gene and protein
expression of two matrix metalloproteinases (MMP), MMP-3 and MMP-10
(stromelysin-1 and stromelysin-2, respectively) was increased in
diabetic corneas without DR and especially, in diabetic corneas with
DR. These data give reason to suggest that overexpression of MMP-10,
found both in corneal epithelium and stroma (MMP-3 was expressed only
in stroma), may lead to the diabetes-related alterations of corneal
epithelial BM and laminin-binding integrin,
3{beta}1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Normal and diabetic autopsy corneas were obtained from the National Disease Research Interchange (Philadelphia, PA) within 40 hours after death. Keratoconus corneas removed during penetrating keratoplasty were received from collaborating surgeons within 30 hours after surgery. They were used as a control group to assess specificity of changes observed in diabetic corneas. For reverse transcription-polymerase chain reaction (RT-PCR) only corneas received within 30 hours after death or surgery were used. The diabetic group consisted of 33 age-matched normal corneas (from 30 individuals; mean age, 70.2 years; 31 corneas without ocular history, and two corneas after uneventful radial keratotomy that were used only for immunostaining), 45 diabetic corneas without DR (from 34 individuals; 23 with insulin-dependent diabetes mellitus and 22 with noninsulin-dependent diabetes mellitus; mean age, 67.5 years), and 37 diabetic corneas with DR (from 26 individuals; 22 with proliferative DR, 27 with insulin-dependent diabetes mellitus, and 10 with noninsulin-dependent diabetes mellitus; mean age, 65.6 years). The keratoconus group, in which corneas came from significantly younger people than diabetics, consisted of eight age-matched normal corneas (from eight individuals; mean age, 27.9 years) and 11 keratoconus corneas (from 11 individuals; mean age, 33.7 years). Eight diseased corneas were used as positive controls to test antibodies to various proteinases in immunohistochemistry. They included one neovascularized cornea with an ulcer, one neovascularized cornea with an acid burn, one cornea with herpetic scar, three failed corneal transplants, and two corneas after failed laser in situ keratomileusis. To confirm staining specificity, antibodies to proteinases were additionally used to stain sections of one normal brain, one meningioma, three glioblastomas multiforme, and one astrocytoma.
In diabetic corneas, all ECM and integrin abnormalities were previously documented in the central part only where Bowman’s layer lies underneath the epithelium.15 Also, in keratoconus corneas, only a central part removed during transplantation was available. Therefore, for gene expression analysis by RT-PCR, normal and diabetic corneas were trephined before freezing and central parts only analyzed to ensure adequate comparison between groups. For some RT-PCR experiments, epithelium, stroma, and endothelium of trephined corneas were mechanically separated and then frozen. The vast majority of corneas were analyzed individually, without being pooled. Only in six diabetic and DR cases, trephined corneas were cut in half and then one half was embedded in OCT for immunofluorescence and the other one used for RT-PCR or Western blotting with pooling halves from fellow eyes. Because of this fact and the small amount of tissue available from a trephined cornea, a different set of normal and diabetic or keratoconus corneas was analyzed by semiquantitative RT-PCR, Western blotting/zymography, and immunofluorescence.
Semiquantitative RT-PCR
Immediately on arrival, corneas were trephined, frozen in liquid
nitrogen, and stored at -80°C. The RT-PCR procedure has been
described in detail previously.16
Briefly, total RNA was
isolated from whole corneas or from mechanically separated epithelium,
stroma, and endothelium with TRIZOL reagent (Life Technologies Inc.,
Gaithersburg, MD) as per the manufacturer’s instructions. cDNA was
synthesized from 2.0 µg of total RNA using SuperScript II reverse
transcriptase (Life Technologies, Inc.). cDNA samples were subjected to
PCR using specific primers for different ECM proteins, proteinases, and
for {beta}2-microglobulin
({beta}2-MG) that served as an internal standard for
sample normalization. Most primers were designed using the Primer3
Internet software program (The Whitehead Institute, Boston, MA) and
their specificity was confirmed by BLAST (National Library of Medicine,
Bethesda, MD) Internet software-assisted search of nonredundant
nucleotide sequence database.16-18
The sequences of
primers for RT-PCR are shown in Table 1
.
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For Southern blot analysis, PCR products were transferred to positively
charged Hybond N+ (Amersham Inc., Arlington
Heights, IL) nylon membranes using an alkaline blotting procedure.
Filters were hybridized with oligonucleotide probes (Table 1)
5'
end-labeled with [-32P]ATP (222 TBq/mmol;
Dupont-New England Nuclear, Boston, MA), washed at high stringency, and
exposed to Kodak X-OMAT AR (Eastman Kodak, Rochester, NY) X-ray film.
To confirm sequence identity, PCR fragments were excised from the gels,
purified using Wizard PCR Prep (Promega Biotech), reamplified, cloned
into Plasmid PCR II using TA cloning kit (Invitrogen, Carlsbad, CA),
and sequenced in an automated sequencer at the Cedars-Sinai DNA
Sequencing Core Facility.
Indirect Immunofluorescence
Corneas were embedded in OCT and OCT blocks and sections were
stored at -80°C. General procedure, secondary antibodies, and
routine controls were as described previously.15,16
Primary antibodies are listed in Table 2
.
Because there are controversial data in the literature about tissue
localization of various proteinases and little work has been done on
cryostat sections, we have compared many antibodies using various
pathological cases as positive controls and several fixation protocols.
The necessity for this strategy has been recently
emphasized.19
The recommendations for
immunohistochemical use of particular antibodies are also described in
Table 2
.
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Routine controls without primary antibodies were included with each
experiment and were negative (see Figure 8
). Each antibody was analyzed
at least twice on most cases, with identical results. Since this work
was being done over a considerable amount of time, different number of
cases was analyzed for different proteinases. Purified monoclonal
antibodies were used at 20 to 30 µg/ml, and polyclonal antibodies
were diluted according to suppliers’ recommendations.
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Gelatin zymography was done as described.20 Briefly, frozen whole corneas were pulverized under liquid nitrogen, and extracted with 50 mmol/L Tris-HCl, pH 7.4, with 10 mmol/L CaCl2 and 0.25% Triton X-100, for 30 minutes at 4°C. The cleared solution is referred to as S1 fraction. The pellet was heat-extracted again in 50 mmol/L Tris-HCl, pH 7.4, with 10 mmol/L CaCl2 and 0.15 mol/L NaCl at 60°C for 6 minutes. The extract was cleared again and the supernate is referred to as S2 fraction. Samples were normalized for protein using the bicinchoninic acid protein assay as per the manufacturer’s recommendations (Pierce Chemical Co., Rockford, IL). Equal amounts of protein per sample were electrophoresed in 10% nonreducing Laemmli sodium dodecyl sulfate gels with 1% gelatin. Gels were soaked in 1% Triton X-100 for 30 minutes, rinsed and incubated overnight in the assay buffer containing 50 mmol/L Tris-HCl, pH 7.4, with 5 mmol/L CaCl2 and 0.02% NaN3. The gels were then stained with Coomassie Brilliant Blue and destained in acetic acid/methanol (10%/10% v/v). Gelatinase bands appeared clear against the blue background. Casein zymography was done in the same way as gelatin zymography, using precast 12.5% casein-containing gels (Bio-Rad, Hercules, CA). Separate control gels were incubated in the assay buffer supplemented with 2 mmol/L phenylmethyl sulfonyl fluoride to block serine proteinases or with 10 mmol/L ethylenediaminetetraacetic acid to inhibit MMPs. Addition of phenylmethyl sulfonyl fluoride did not alter the band pattern, and ethylenediaminetetraacetic acid eliminated gelatinolytic and caseinolytic bands.
Western Blot Analysis
Frozen and powdered whole corneas were extracted as above and the protein was determined by the bicinchoninic acid method (Pierce Chemical Co.). The extracts were heated at 95°C for 10 minutes in 4x Laemmli’s sample buffer with proteinase inhibitors and 2-mercaptoethanol (Boehringer Mannheim, Indianapolis, IN). Equal amounts of protein per sample were electrophoresed in 4 to 20% gradient sodium dodecyl sulfate gels (Bio-Rad). Western blotting with goat anti-mouse alkaline phosphatase conjugate as a secondary antibody was as described.16 Primary mouse monoclonal antibodies 10D6 to MMP-3 (R&D Systems, Minneapolis, MN) and IVC5 to MMP-10 (NeoMarkers, Fremont, CA) were used at 3 to 5 µg/ml.
Statistical Analysis
Immunostaining results were analyzed using a double-sided Fisher’s exact test, and RT-PCR results, by nonparametric Mann-Whitney test (InStat software program; GraphPad Software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Our previous finding of decreased immunostaining of non-DR
diabetic and especially DR corneas for laminins, nidogen-1/entactin,
and epithelial integrin
3{beta}1 could be due
either to decreased synthesis or increased degradation. To assess
changes in synthesis, gene expression levels were analyzed by
semiquantitative RT-PCR. When whole corneal RNA was used, gene
expression of 1 and {beta}1 laminin chains, nidogen-1/entactin, and
integrin subunits 3 and
{beta}1 was increased approximately twofold in
non-DR diabetic or DR corneas (Figure 1
and Table 3
). Because the
diabetes-related BM and integrin alterations15
likely
concerned epithelial cell products, gene expression of the affected
components was evaluated in separated corneal epithelium. Gene
expression of 1, 5, {beta}1 laminin chains; nidogen-1/entactin; and
integrin subunits 3 and
{beta}1 did not significantly change in non-DR
diabetic or DR corneal epithelium (Figure 2
and Table 3
).
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The results presented above suggested that the observed alterations in diabetic and DR corneas were unlikely to be caused by a decreased synthesis of affected components. Another possibility was that there was increased degradation of these components in diabetic corneas. Therefore, we have next analyzed gene and protein expression of a number of extracellular or surface proteinases in corneal epithelium and stroma including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-14, two plasminogen activators (urokinase and tissue plasminogen activator), and a recently described cathepsin V/L2 localized at the level of corneal epithelial BM.21
Gene expression of MMP-1, MMP-7, and MMP-9 in corneal epithelium or
stroma could not be revealed by semiquantitative RT-PCR in the
conditions used, with several sets of primers for each enzyme (not
shown here). In the epithelium, MMP-2 and MMP-3 were not expressed and
gene expression of cathepsin V/L2, urokinase, or tissue plasminogen
activator did not differ in normal, non-DR diabetic, or DR corneas
(Figure 3
and Table 3
). In contrast,
MMP-10 showed significantly increased gene expression in non-DR
diabetic and DR corneal epithelium compared to normal (Figure 3
and
Table 3
). In the stroma, gene expression of MMP-14, cathepsin V/L2,
urokinase, or tissue plasminogen activator did not differ significantly
in diabetic versus normal corneas (Figures 4 and 5
and
Table 3
). However, significantly increased gene expression of MMP-2,
MMP-3, and MMP-10 was noted in diabetic and DR stromal cells (Figure 4
and Table 3
).
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.
Therefore, MMP-3 and MMP-10 gene expression seemed to be selectively
elevated in diabetic corneas.
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and Table 3
).
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Carefully pretested antibodies to various proteinases (Table 2)
have been used in immunofluorescence studies of normal, diabetic, and
keratoconus corneas. In agreement with our inability to detect MMP-1,
MMP-7, and MMP-9 gene expression by RT-PCR, these enzymes were only
seen in occasional keratocytes of 1 to 2 cases per each group of
corneas (not shown here). The same was true for MMP-2 and MMP-14
despite the presence of their mRNA in the corneas. Possibly, their
amounts were too low to be detected by the immunostaining method used.
Procathepsin V/L2 was revealed in the epithelial BM and limbal blood
vessels. The staining intensity was similar among different groups of
corneas (Table 4)
. Urokinase antibodies
stained the epithelium and some keratocytes. Tissue plasminogen
activator was revealed in some keratocytes (often also positive for
urokinase) and weakly in the epithelium. The epithelial staining for
urokinase and tissue plasminogen activator remained similar in normal
and diabetic corneas, but some diabetic and DR cases had more positive
keratocytes than normal (Table 4)
.
|
. However,
in non-DR diabetic corneas, specific punctate staining for MMP-3 was
revealed in some keratocytes and the number of positive cells
significantly increased in DR corneas (Figure 8
and Table 4
). The
epithelium was negative in accordance with RT-PCR data. The intensity
of diffuse epithelial staining for MMP-10 was at the background level
in normal corneas. The staining significantly increased in a minority
of non-DR diabetic and in most of DR corneas (Figure 8
and Table 4
). In
normal corneas, keratocytes were mostly negative. The number of cases
with positive keratocyte staining increased in non-DR diabetic and
especially in DR corneas (Figure 8
and Table 4
). These findings were in
complete accordance with RT-PCR data. Stromelysin Activity Is Increased and MMP-2 Activity Is Not Changed in Diabetic Corneas
There was a discrepancy between our RT-PCR data showing increased
MMP-2 expression in diabetic corneas and our inability to detect it by
immunostaining in most cases. To resolve this controversy, gelatinase
activity (mostly due to MMP-2 and/or MMP-9) was assayed by zymography
in detergent corneal extracts. We have used our previous extraction
protocol and obtained a more soluble (detergent extract, S1) and a more
insoluble (heat extract, S2) fractions. Gelatin zymography revealed one
major band of lysis at 
65 kd, consistent with the proform of MMP-2
(migration is increased in nonreducing gels20
) that is the
major corneal MMP (Figure 9)
. This band
was present in all corneas and its intensity did not differ
significantly between groups in either S1 or S2 fraction. In normal
corneas, very weak bands in the MMP-9 size region were seen, and they
were absent from diabetic corneas.
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50 kd was observed, corresponding in size to the slower moving
band of the doublet of the MMP-3 standard. Some activity was seen in
normal corneas and it appeared to be increased in diabetic and DR
corneas (Figure 10)
. As with
gelatinolytic activity, heterogeneity in the band intensity from cornea
to cornea within the same group was evident.
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Increased expression of MMP-10 and MMP-3 mostly in DR corneal S2 fractions was confirmed by Western blotting although the bands were rather weak (not shown here).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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3{beta}1.15
These results agreed well with clinical observations of corneal
epithelial abnormalities in diabetic corneas.6
In this
study, the mechanism of BM and epithelial integrin alterations in
diabetic human corneas was explored. Two major possibilities were
tested: 1) decreased synthesis of BM components and
3{beta}1 integrin, and 2)
increased degradation of these components by extracellular proteinases.
Laminin gene expression was previously found to be increased in
diabetic mouse kidneys.25
We also found increased gene
expression of laminin chains 1, 5, {beta}1; nidogen-1/entactin; and
integrin chains in diabetic and DR human corneas. This was probably due
to stromal and/or endothelial cell activity because the increase was
not seen in the epithelium (Figures 1 and 2)
. Although
posttranscriptional changes could not be ruled out by these
experiments, the data presented here suggested that the observed
alterations in diabetic corneas were unlikely to be occuring because of
decreased synthesis of the affected components.
To explore a possible role of proteolytic degradation in the observed BM abnormalities, gene and protein expression of various proteinases was studied. Seven MMPs known to cleave select or many BM components24,26-29 were chosen including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, and MMP-14 as a membrane-type MMP. We also looked at the expression of the plasminogen activator system including urokinase and tissue plasminogen activator, which is important for both BM degradation and activation of pro-MMPs.30,31 Finally, a newly discovered cathepsin V/L2 was studied because it can degrade BM components and is localized at the corneal epithelial BM.18,21,32
Gene expression of MMP-1, MMP-7, and MMP-9 was not revealed in any corneal group. Gene expression of MMP-14, urokinase, tissue plasminogen activator, and cathepsin V/L2 did not change in diabetic corneal epithelium and stroma. These data were essentially corroborated at the protein level. Despite increased gene expression of MMP-2 in diabetic and DR corneal stroma, immunostaining and gelatin zymography did not reveal any diabetes-associated changes in its localization or activity.
In the epithelium of diabetic and DR corneas, an increased gene expression of MMP-10 was observed. In the stroma of diabetic and DR corneas, an increased gene expression of MMP-10 and especially MMP-3, was found. Elevated gene expression of MMP-3 or MMP-10 in diabetic corneas seemed specific because it was not observed in keratoconus corneas.
Differences in gene expression of stromelysins in diabetic corneas paralleled the increase in respective protein expression in diabetic and especially DR corneas. Casein zymography of corneal extracts also revealed a band similar in size to stromelysins that appeared to be more expressed in diabetic and DR corneas. Similar results were obtained for both MMP-3 and MMP-10 by Western blotting (not shown here). Noteworthy, elevated levels of MMP-3 and MMP-10 were not accompanied by significantly increased TIMP levels, which would potentially result in net increase of proteolytic activity.
These data support the idea that BM and integrin alterations in diabetic and DR corneas may result from degradation by select proteinases activated in diabetic conditions. MMP-10 might well be one such proteinase because it was the only one that was overexpressed in diabetic corneal epithelium. The presence of collagenous Bowman’s layer in human corneas between the epithelium and the stroma makes it less likely that a stromal proteinase may be involved in epithelial BM alterations. However, it cannot be excluded completely.
MMP-3 and MMP-10 are closely related proteins and belong to a group of broad spectrum MMPs capable of degrading various ECM and BM components and activating other pro-MMPs.33-35 Their expression is important for tissue development and remodeling, immune response, as well as for wound healing.36-42 MMP-3 and/or MMP-10 expression is also induced or increased in various pathological conditions including cancer, osteoarthritis, rheumatoid arthritis, and chronic gastrointestinal and leg ulcers.24,43-50 MMP-3 may play a direct role in mammary tumor development and may be considered as a natural mammary tumor promoter.50,51
MMP-3 seems to be involved in the regulation of glomerular ECM turnover. Its expression is decreased in diabetic nephropathy and this may contribute to mesangial ECM expansion.52 To the best of our knowledge, no studies have been conducted on MMP-10 in diabetes. This report thus provides the first demonstration of MMP-10 mRNA and protein in the cornea. It also documents MMP-10 increase in diabetic and DR corneas, which may contribute to BM alterations and subsequent epithelial abnormalities typical for these corneas.
What may be the mechanisms of increased expression of MMP-3 and MMP-10 in diabetic corneas? The first possibility concerns an abnormal activity of certain corneal growth factors and cytokines.53 Our preliminary data show that in diabetic and DR corneas, gene expression of one such growth factor, insulin-like growth factor-I (IGF-I), is significantly increased (M. Saghizadeh and A. V. Ljubimov, unpublished data). IGF-I has been implicated in DR development both directly54 and indirectly, by up-regulating a potent angiogenic factor, vascular endothelial growth factor.55,56 It was recently shown that IGF-I can increase MMP-3 expression in the trabecular meshwork cells.57 Moreover, at least MMP-3 can degrade IGF-binding proteins and in turn, modulate IGF-I activity.58,59 However, other growth factors and cytokines known to modulate the expression of stromelysins33,37,39 might also influence their activity in diabetic corneas.
The second possibility concerns nonenzymatic glycosylation (glycation)
of ECM and BM proteins as a result of long-term diabetic hyperglycemia.
Advanced glycation end products have been found in the diabetic corneas
primarily in the epithelial BM.60
Interestingly, cultured
corneal epithelium showed reduced adhesion and spreading on glycated
laminin-1 (1{beta}11) that could lead to abnormal adhesion of
diabetic corneal epithelium. Glycated BM type IV collagen may be turned
over more slowly than normal.61
This may result in turn,
in an increase of MMP expression, which would lead to enhanced
proteolysis of other BM components, eg, laminins and
nidogen-1/entactin, and exacerbate diabetic epitheliopathy.
In summary, alterations of BM and epithelial integrin in diabetic human corneas could occur because of proteolytic degradation by specific proteinases, notably by MMP-10. Increased proteolysis in the corneal epithelial layer may be the molecular mechanism leading to abnormalities of epithelial cell adhesion and wound healing typical for diabetic corneas. It should be noted, however, that more direct experiments using, for example, treatment of normal, diabetic, and DR organ-cultured corneas with purified MMPs and their inhibitors, are needed to demonstrate the actual involvement of select MMPs in diabetic corneal alterations. Alternative mechanisms, such as abnormal posttranscriptional and/or posttranslational modifications of BM proteins and integrins leading to their decreased expression in diabetic corneas, should also be explored.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgements |
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Footnotes |
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Supported by Cedars-Sinai Medical Center Young Investigator Award (to A. V. L.); the Iris and B. Gerald Cantor Foundation (to M. C. K. and A. V. L.); the National Institutes of Health grant no. EY06807 (to M. C. K.), and The Discovery Fund for Eye Research.
Accepted for publication October 4, 2000.
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