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Reduced Choroidal Neovascular Membrane Formation in Matrix Metalloproteinase-2–Deficient Mice

¹From the Departments of Ophthalmology and ²Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden; and the ³Riken Brain Science Institute, Saitama, Japan.

	Abstract

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PURPOSE. Findings in studies have suggested a role for matrix metalloproteinase (MMP)-2 in angiogenesis, including choroidal neovascularization (CNV). To investigate further, the current study was conducted to observe the formation of experimental CNV in MMP-2–deficient mice.

METHODS. CNV was induced in wild-type and MMP-2–deficient mice by krypton laser photocoagulation of the fundus. The time-course of expression of MMP-2 mRNA after laser treatment was determined by in situ hybridization with anti-sense and sense cRNA probes. MMP-2 protein distribution was determined by immunohistochemistry. Ten days after treatment, the extent of CNV was evaluated on hematoxylin-eosin stained serial sections. The maximum height of the CNV lesions was calculated by image analysis of digitized histologic images.

RESULTS. Expression of MMP-2 mRNA was detected in the CNV lesions at day 3 after laser treatment and peaked at day 5, after which it slowly declined. MMP-2 mRNA expression appeared to be highest at the margins of the membrane. Immunostaining for MMP-2 confirmed the presence of MMP-2 protein in the CNV lesions. The CNV lesions of MMP-2–deficient mice showed that relative thickness was reduced by 31% compared with wild-type mice (P = 0.006).

CONCLUSIONS. The present study demonstrated that MMP-2 mRNA and protein are upregulated during experimental CNV in the mouse. The marked difference in thickness of the CNV membrane between wild-type and MMP-2–deficient mice shows that MMP-2 is involved in the formation of experimental CNV in the mouse. These results suggest that pharmacologic targeting of MMPs, including MMP-2, may reduce formation of CNV in conditions such as age-related macular degeneration.

Angiogenesis, the formation of new vessels from preexisting ones, is a complex multistep process that involves the stimulation of angiogenic growth factor receptors on vascular endothelial cells, proteolytic breakdown of the endothelial basal membrane, endothelial cell proliferation and migration, degradation of the surrounding extracellular matrix (ECM), vessel stabilization, recruitment of supporting cells (e.g., pericytes), and closure of the newly formed arteriovenous loops.³

Matrix metalloproteinases (MMPs) are a family of more than 20 enzymes capable of degrading most, if not all, of the components of the vascular basal membrane and ECM. MMPs have been implicated in wound healing, arthritis, tumor infiltration and metastasis, and angiogenesis.^{4 5 6 7} Most MMPs are secreted as inactive proenzymes (zymogens) that are activated by proteolysis in the ECM or on the plasma membrane. Belonging to this group are collagenases, stromelysins, and gelatinases (i.e., MMP-2 and -9).

MMP-2 (gelatinase A) and -9 (gelatinase B) have similar substrate specificity, preferentially cleaving gelatin, elastin, and collagens type I, IV, and V, and their involvement in angiogenesis has been studied extensively. Whereas both MMP-2 and -9 appear to be important during tumor angiogenesis,^{8 9 10} their roles in the formation of CNV are not well understood. We have previously demonstrated expression of both MMP-2 and -9 mRNAs in CNV membranes surgically excised from patients with AMD, with MMP-2 mRNA being expressed in the vascularized core of the membranes.¹¹ The result was confirmed in a rat CNV model in which MMP-2 was expressed in the growing CNV membranes.¹² Taken together, the results in these studies suggest that MMP-2 may be involved in CNV. By contrast, the expression of MMP-9 mRNA is concentrated in the modified Bruch’s membrane and retinal pigment epithelial (RPE) layer of surgically excised CNV membranes, suggesting a role in the modulation of these structures, rather than in formation.¹¹ Accordingly, we were unable to detect MMP-9 mRNA in the growing CNV membranes in the rat model.¹²

To further investigate the possible role of MMP-2 in CNV we studied the expression of MMP-2 mRNA in a well-characterized model of laser-induced CNV in the mouse and compared the formation of CNV in wild-type and MMP-2–deficient mice.

	Materials and Methods

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Genetically Modified Mice
The generation and genotyping of MMP-2–deficient mice has been described.¹³ Briefly, homozygous wild-type and MMP-2–deficient animals of either sex, with a mixed genetic background of 97% C57BL/6 and 3% 129 strain, were used throughout the study. The genotype of the animals was determined with Southern blot analysis. The animals were maintained in a 12-hour light–dark cycle and had free access to food and water. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Generation of CNV in Mice
CNV was generated by krypton laser–induced rupture of Bruch’s membrane, essentially as previously described.¹⁴ Briefly, 16- to 18-week-old animals were anesthetized and the pupils were dilated. One krypton laser burn (50-µm spot size, 0.1-second duration, 120 mW) was delivered to the 9 and 3 o’clock positions of the posterior pole of the retina in each eye by using a slit lamp delivery system and a handheld contact lens. The formation of a subretinal bubble, indicating a rupture of Bruch’s membrane, appears to correlate with the formation of CNV in this model.¹⁵ Only eyes in which a bubble was produced by both burns were included in the study. The eyes were enucleated at the indicated time points and either fixed in formaldehyde overnight and embedded in paraffin or snap frozen and stored at -80°C for further processing. The specimens were serially sectioned and either stained with hematoxylin-eosin for light microscopy or prepared for in situ hybridization or immunohistochemistry.

In Situ Hybridization
In situ hybridization was performed on formaldehyde-fixed serial sections by using T3 and T7 RNA polymerase-derived sense and antisense cRNA probes (generated from a rat cDNA probe to MMP-2 [1825 bp]; kindly provided by Paul Basset, University of Louis Pasteur, Strasbourg, France) labeled with ³⁵S-uridine triphosphate (UTP), as previously described.^{12 16} Sections from all membranes were analyzed in at least two independent experiments. After a final wash at 20°C in 0.1x SSC plus 1 mM dithiothreitol (DTT), the sections were dehydrated in graded ethanol, air dried, dipped in photograph emulsion, and exposed for 6 weeks at 4°C. After development, the sections were counterstained with Mayer hematoxylin and studied by light microscopy.

Immunohistochemistry
Cryosections (7 µm), fixed in acetone and blocked with 1% normal rabbit serum in phosphate, were incubated for 1 hour with primary antibody (rat monoclonal antibody against CD31 (platelet-endothelial cell adhesion molecule [PECAM]-1; BD Biosciences Pharmingen, San Diego, CA) or sheep polyclonal antibody against human MMP-2 (a kind gift from Rosalind M. Hembry, University of East Anglia, Norwich, UK).¹⁷ The sections were subsequently incubated with biotinylated secondary antibody (rabbit anti-rat or rabbit anti-sheep IgG; Vector Laboratories, Burlingame, CA) for 30 minutes, followed by an incubation with avidin-horseradish peroxidase complex (Vectastain kit; Vector Laboratories) for 30 minutes. Peroxidase activity was visualized using 3-amino-9 ethylcarbazole (AEC, Vectastain; Vector Laboratories) as a red chromogen, after which the sections were counterstained with hematoxylin and mounted with glycerol-gelatin. All procedures were performed at room temperature.

Quantitative Analysis of Formation of CNV
To evaluate the extent of CNV in wild-type and MMP-2–deficient mice, 20 animals in each group were laser treated. Two animals in the MMP-2–deficient group were excluded: one that had prelaser cataract and one that died shortly after the anesthesia. The remaining animals (20 wild-type and 18 MMP-2–deficient mice) were killed at day 10 after laser treatment, and the eyes were processed for quantitative analysis. Serial sections, 4 µm thick, were cut throughout the entire extent of each burn. Hematoxylin-eosin–stained sections of all treated eyes in each group were examined at 200x magnification with a bright-field microscope and a digital color camera (respectively, Axioscope and Axiomat; Carl Zeiss, Jena, Germany). Digitized images were analyzed and measured with the accompanying image-analysis software (Axiovision; Carl Zeiss). All sections were masked to the observer. An exact delineation of the CNV membrane was often difficult, due to heavy pigmentation of the CNV membrane and the underlying choroid. The actual thickness of the CNV membrane was therefore estimated indirectly by measuring the difference between the thickness from the bottom of the pigmented choroidal layer to the top of the neovascular membrane (T) and the thickness of the intact, pigmented choroid adjacent to the lesion (C). The data from each animal were pooled, and a mean T and C were calculated (C was calculated as a mean between the choroidal thickness on each side of the CNV lesion). The relative thickness of the CNV membranes was then calculated using the formula: T - C/C. Two animals (MMP-2–deficient) were excluded, because the observer failed to detect any measurable membrane in either eye.

Statistical Analysis
Data were analyzed by computer (Statistica 5.5; StatSoft Inc, Tulsa, OK; and MedCalc; MedCalc Software, Mariakerke, Belgium). After confirming that data were normally distributed, we used Student’s two-tailed t-test for unpaired data to determine whether there were significant (P < 0.05) differences between the height of CNV in wild-type and MMP-2–deficient mice. The observer later repeated calculations of CNV height in 41 randomly selected sections, masked to the initially obtained value. Interobserver reproducibility was then assessed by calculating the 95% limits of agreement and using a Bland-Altman plot.

	Results

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Expression of MMP-2 mRNA and Protein in Experimental CNV in the Mouse
Normal C57BL/6J mice were krypton-laser–treated and observed for up to 20 days. Animals were killed at days 1, 3, 4, 5, 7, 10, 15, and 20. One day after laser treatment, histologic analysis of hematoxylin-eosin–stained sections showed disruption of the RPE layer with subretinal exudation and occasional hemorrhage and cellular infiltration into the subretinal space. Subretinal vessel ingrowth was first detected at day 3 after treatment, and this neovascularization increased, together with membrane size, until 10 days after treatment. After this time point the membranes gradually became smaller and more fibrotic. The membranes, often large dome-shaped subretinal protrusions at the site of the laser burns, consisted of fibrovascular tissue with single or multilayered RPE cells that occasionally formed a continuous layer facing the neurosensory retina but usually were scattered within the fibrous stroma.

The distribution and time-course of expression of MMP-2 mRNA was examined on serially sectioned laser-treated eyes from normal mice using in situ hybridization. The expression of MMP-2 mRNA in the untreated retina and choroid was detectable but low. After laser treatment, the expression of MMP-2 mRNA increased in the treated areas where individual MMP-2 mRNA–positive cells could be detected. This increase in expression of MMP-2 mRNA was detected at day 3 after laser treatment (not shown) and peaked at day 5 after treatment (Figs. 1B 1D) , after which the expression of MMP-2 mRNA gradually declined (not shown). The expression of MMP-2 mRNA appeared to be highest at the emerging margins of the growing CNV membrane (Fig. 1B 1D) . The presence of MMP-2 in the CNV lesions was further confirmed by immunohistochemical assay in which MMP-2 protein was mainly detected at the margins of the membrane (Fig. 2A) .

View larger version (103K): [in this window] [in a new window]   FIGURE 1. Expression of MMP-2 mRNA after laser-induced CNV. Expression of MMP-2 mRNA in CNV membranes of mice was analyzed by in situ hybridization at day 5 after laser treatment (B, C, D, E). (A) Control section stained with hematoxylin-eosin. Note red blood cells in newly formed vessel lumina within the membrane (oblique arrow). (B, D) Hybridization with antisense 35S-labeled MMP-2 riboprobes demonstrated a strong signal at the margins of the membrane (vertical arrows). (C, E) Control hybridization with sense MMP-2 riboprobes. (B, C) Bright-field and (D, E) dark-field images of the same sections are shown for comparison. In the dark-field images, the pigmented cells of the retinochoroid gave a strong background signal that was also present in the sense control (D, E). Scale bar, 50 µm.

View larger version (64K): [in this window] [in a new window]   FIGURE 2. Expression of MMP-2 protein after laser-induced CNV. (A) Immunostaining of a CNV lesion at day 10 after laser treatment demonstrated MMP-2 protein expression that was most intense at the margins of the membrane (arrows). (B) Control staining of a CNV membrane from an MMP-2–deficient mouse demonstrated weak background staining. Scale bar, 50 µm.

View larger version (86K): [in this window] [in a new window]   FIGURE 3. CNV in wild-type and MMP-2–deficient mice. Hematoxylin-eosin staining of CNV membranes from laser-treated wild-type (A) and MMP-2–deficient (B) mice. Arrows: measured thickness of the CNV membrane and underlying choroid (T) and the underlying choroid (C) alone. Immunostaining for PECAM showed new vessels within the CNV lesions in wild-type (C) and MMP-2–deficient (D) animals. Scale bar, 50 µm. (E) The relative thickness of the CNV membranes from wild-type and MMP-2–deficient mice was calculated using the formula: T - C/C. Absolute values: wild-type: n = 20; mean ± SEM = 4.24 ± 0.37; MMP-2–deficient: n = 16; mean ± SEM = 2.93 ± 0.25.

	Discussion

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The finding that MMP-2 is involved in development of CNV adds MMP-2 to a growing list of molecules that are probable participants in the pathogenesis of CNV. For instance, the presence and importance of both vascular endothelial growth factor (VEGF), a hypoxia-induced protein that stimulates vascular endothelial proliferation and migration, and the angiopoietin/Tie2 receptor system implicated in vascular stabilization, sprouting, and network formation have been demonstrated in surgically excised neovascular membranes from patients with AMD^{18 19} and in experimental models.^{15 20 21}

In the present study we found a 31% reduction in CNV in MMP-2–deficient mice. The formation of CNV principally involves the progressive subretinal ingrowth of new vessels paralleled by the development of a fibrovascular stroma. MMP-2 may promote growth of CNV in two principle ways: by breakdown of the vascular endothelial cell basal membrane or by degradation of the surrounding ECM. Although the mechanism by which MMP-2 is involved in CNV in the mouse model was not directly addressed in the present study, it is interesting to note that both MMP-2 mRNA and protein expression tended to be highest at the margins of the expanding membrane. Breakdown of the ECM is probably most severe in this area of the membrane, whereas vascular endothelial cell proliferation (initialized by degradation of the basal membrane) has been suggested to occur farther down the vascular stalk.^{22 23} This suggests that MMP-2 mainly acts by breaking down the ECM surrounding the growing CNV membrane. In support for this hypothesis there was no apparent difference in the vascularity between the CNV membranes from wild-type and MMP-2–deficient mice.

Our finding that MMP-2 is involved in formation of CNV is in line with recent findings that MMP-2 is necessary for optimal FGF-2 stimulated corneal angiogenesis.²⁴ The fact that we could observe only a partial decrease in CNV in the MMP-2–deficient animals may have several explanations. First, the proteolytic specificity of the MMPs show a substantial redundancy, suggesting that MMPs other than MMP-2 may also be involved in CNV. Examples of other MMPs with angiogenic properties are MMP-9 and membrane-type matrix metalloproteinase (MT-MMP)-1.^{7 9} Second, the action of endogenous MMP inhibitors, such as tissue inhibitors of matrix metalloproteinases (TIMPs) and PEX (an MMP-2 fragment with MMP-2 inhibitory properties), may reduce the proteolytic activity of synthesized MMP-2.^{4 5 25} Third, there is a chance that we may have underestimated the difference in the thickness of CNV between wild-type and MMP-2–deficient animals, because it was often difficult to detect and delineate the smaller lesions that more frequently were found in the MMP-2–deficient mice. In fact, in two of the animals (both MMP-2–deficient) we were unable to detect CNV lesions in any of the eyes, and they were consequently excluded from statistical analysis.

In addition to the MMP system the plasminogen activator (PA)-plasmin system of serine proteases has been implicated in angiogenesis.²⁶ These two protease systems do not function independently, but instead there appears to be synergistic interaction between them, as described in tumor models.²⁷ Accordingly, many of the MMPs are secreted as inactive zymogens that are activated by limited proteolysis by plasmin that in turn is proteolytically activated by its proenzyme plasminogen. A recent study demonstrated that plasminogen activator inhibitor (PAI)-1 knockout mice show a near 50% reduction in laser-induced CNV.²⁸ Together with the present study this suggests that MMP-2 and PAI-1 may act together in regulating the breakdown of matrix molecules during formation of CNV in the mouse model.

The results of the present study support the idea that pharmacologic inhibition of MMPs may be a novel way to treat patients with CNV. In fact, there have already been experimental and clinical trials of different MMP inhibitors in different neovascular conditions including CNV in AMD.²⁹ Regarding CNV, most studies have involved the broad-spectrum MMP inhibitor prinomastat, which potently inhibits CNV in experimental models.³⁰ However, in a recent clinical trial, prinomastat failed to induce regression of CNV in patients with AMD.³¹ This discrepancy probably reflects the fact that MMP inhibitors, similar to other anti-angiogenic agents, are more effective in preventing progressive growth of CNV (e.g., CNV in experimental models) than in causing regression of the manifest CNV membranes present in clinical CNV.

In conclusion, the present study shows that MMP-2 is involved in the progression of experimental CNV in the mouse and suggests that pharmacologic targeting of MMPs including MMP-2 may be a means of treating conditions involving CNV, such as neovascular AMD.

	Acknowledgements

 
The authors thank Berit Spångberg, Margareta Oskarsson, and Monica Aronsson for excellent technical support.

	Footnotes

 
Supported by grants from the Crown Princess Margaretas Foundation (KMA), the Edwin Jordans Foundation, and the Swedish Diabetic Society.

Submitted for publication February 21, 2002; revised July 2, 2002; accepted August 9, 2002.

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: Anders Kvanta, St. Erik’s Eye Hospital, Polhemsgatan 50, SE-112 82 Stockholm, Sweden; anders.kvanta{at}sankterik.se.

	References

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