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(Hypertension. 2002;39:865.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Altered Regulation of Matrix Metalloproteinase-2 in Aortic Remodeling During Aging

Mingyi Wang; Edward G. Lakatta

From the Laboratory of Cardiovascular Sciences, Gerontology Research Center, National Institute on Aging/National Institutes of Health, Baltimore, Md.

Correspondence to Edward G. Lakatta, MD, Laboratory of Cardiovascular Sciences, Gerontology Research Center, National Institute on Aging/National Institutes of Health, 5600 Nathan Shock Drive, 3-B-03, Baltimore, MD 21224-6825. E-mail lakattae{at}grc.nia.nih.gov

	Abstract

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To elucidate potential mechanisms of enhanced type 2 matrix metalloprotease levels and activity within the thickened aged rat aorta, the present study measured its mRNA and protein levels and those of its membrane bound activator, MT1-MMP, its endogenous tissue inhibitor, TIMP-2, tissue type, and urokinase plasminogen activators and their receptors, and an inhibitor of plasminogen activation in aortae from Fisher 344X Brown Norway rats, 2 to 30 months of age. Semiquantitative immunohistochemistry, in situ hybridization, and in situ zymography of aortae detected a marked age-associated increase in gelatinolytic activity of type 2 metalloprotease within the thickened intima, internal elastic lamina, and elastic fibers in the inner part of the thickened tunica media, whereas the intimal tissue inhibitor of metalloprotease-2 mRNA and protein levels were not age related. Both activators of plasminogen and their receptors increased approximately 2-fold within the intima between 2 to 30 months. Similar, but not identical, age-associated changes in factors that regulate protease activity within the aortic media were also observed. We conclude that discordant regulation of factors that determine the activation status of type 2 matrix metalloprotease, coupled with an increase in the expression of its zymogen, occur with aging, which lead to an increase in the amount of activated protease. These factors are candidate mechanisms for age-associated vascular remodeling, a potent risk factor for vascular diseases with advancing age.

Key Words: aging • aorta • extracellular matrix • gene expression • immunohistochemistry • metalloprotease

	Introduction

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Age-associated macroscopic changes within large blood vessels in rodents are similar to those that occur in humans and include luminal dilation, medial thickening, elastic membrane fragmentation, and formation and thickening of an intima.^1–3 The thickened intima contains matrix molecules, including fibrinogen, collagen, and proteoglycan, and vascular smooth muscle cells (SMC), and also exhibits markedly higher levels of transforming growth factor ß (TGF-ß), the intercellular adhesion molecule (ICAM), and type-2 matrix metalloprotease (MMP-2), a 72 kDa type IV collagenase, than do younger vessels.⁴

Recent evidence suggests that MMP-2, a zinc dependent endopeptidase,⁵ promotes matrix protein degradation in vascular disease remodeling,⁶ similar to its role in tumor invasion,⁷ and facilitates SMC migration.⁸ In the aged aortae, MMP-2 accumulates in the area surrounding SMCs that are located near breaks in the internal elastic lamina and along elastic laminae throughout the media,⁴ suggesting that MMP-2 may also have a role in fragmentation of the elastic laminae with aging.

The MMP zymogen is regulated by gene transcription, and a delicate balance among its activators and inhibitors controls its activation status⁹ and its potential impact on arterial remodeling. In fibroblasts,¹⁰ endothelial cells,^11,12 and tumor cells,^13–15 cleavage and activation of MMP-2 is achieved by a novel membrane-type matrix metalloproteinase (MT1-MMP), synthesized as a pro-form and cleaved by both the intracellular protease furin¹⁶ and extracellular plasmin.¹⁷ An increased level of MT1-MMP and increased MMP-2 activation have been detected in the neointima following carotid injury.¹⁸

TIMP-2, an endogenous tissue inhibitor of MMP-2, acts directly to inhibit MMP-2 activation by binding to it and also binds to MT1-MMP, blocking its activation.^19,20 Overexpression of TIMP-2 reduces neointimal hyperplasia following carotid arterial injury.²¹ The serine protease, plasmin, inactivates TIMP-2 and can also induce a complete conversion of pro–MMP-2 form to the active form.²² Tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), through their binding to the endogenous uPA receptor (uPAR), facilitate the conversion of plasminogen to plasmin. Pro–MMP-2 activation is inhibited by plasmin inhibitors-1 (PAI-1) or anti-uPA antibodies.^22,23

The present study provides evidence for an age-associated increase in the amount and activity of pro–MMP-2 in the aortic wall of aged rats, occurring in the context of an imbalance among MT1-MMP, TIMP-2, and factors within the plasminogen activation pathway.

	Materials and Methods

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Animals and Aortic Histology
Two-, eight-, and thirty-month-old male F344XBN rats (Harlan Sprague Dawley, Indianapolis, Ind) (n=8 each) were obtained, housed, acclimated, and studied with appropriate approvals as indicated previously.⁴ Following measurement of systolic blood pressure (SBP) by the tail-cuff technique,⁴ animals were killed by an overdose of sodium pentobarbital, and aortae were immediately removed. Segments of the thoracic aorta were harvested, and morphometric evaluation of intimal and medial thickness was implemented as described previously.⁴ The ratios of the areas of elastin and total collagen type I fibers to that of media or adventia were measured from elastin-specific elastin van Gieson (EVG) or picricsirius red-stained slides at x200 magnification microscopy²⁴ (Zeiss) and expressed as a percentage of the medial or adventitial area. The relative amounts of collagen types I and III were assessed via polarization microscopy, the former emitting red-yellow and the latter emitting green fluorescence.²⁴

Immunohistochemistry
Immunohistochemistry was performed according to the biotin-streptavidin kit’s instruction (Zymed Laboratories, Inc). Antigen was recovered in citrate buffer at pH 6.0 (Zymed Laboratories, Inc) for 1 to 3 minutes in a microwave oven. The slides were incubated overnight at 4°C with primary antibodies (Table 1).

View this table: [in this window] [in a new window]   Table 1. Primary Antibodies and Their Concentrations

In Situ Hybridization
Digoxigenin (DIG)-labeled MMP-2 (330 bp), TIMP-2 (361 bp), and MT1-MMP(2338 bp) riboprobes were obtained from plasmid cDNA (kindly provided by Michael Crow, Gerontology Research Center, Baltimore, Md) by standard RNA synthesis, using SP6, T7, or T3 RNA polymerase. Incorporation of DIG-conjugated uridine-5’-triphosphate (UTP) was determined using a commercial kit (Boehringer Mannheim Corp) following the manufacturer’s instruction. In situ hybridization was performed by modification of the methods previously described.²⁵ Corresponding sense probes, hybridization buffer without probes, and RNase digestion of tissue sections for 30 minutes before hybridization were used as controls to determine specific hybridization.

In Vitro and In Situ Zymography
Frozen aortae were extracted and assayed via electrophoretic gelatin zymography as previously described.⁴ Recombinant human active and inactive MMP-2 mixtures (Oncogene Research Products) were used as positive controls. Zymography was performed in the presence of an inhibitory antibody against MMP-2 to determine the enzymatic specificity of gel digestion.

Statistical Analyses
All results are expressed as the mean±SEM. Statistical comparisons for age differences were made via an ANOVA followed by Bonferoni post hoc tests. A P value of <0.05 was taken as statistically significant.

	Results

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Aortic Structure
Figure 1 presents representative aortic sections of each age group, and Table 2 lists the average arterial pressure and structural features of the rat aortae by age. Both the intimal and medial thickness increased with age, and the elastic lamellae became thinned, fragmented, and distended with advancing age (Figure 1). The elastin fraction decreased and total medial collagen increased because of an increase in type III collagen. As previously reported in this hybrid rat strain,⁴ systolic pressure increases moderately with age (Table 2).

View larger version (88K): [in this window] [in a new window]   Figure 1. Representative aortic sections from 2 months, 8 months, and 30 months. The left panels were stained with picric sirius red and visualized under light microscopy. Collagen stains with a red color. Middle panels were stained with picric sirius red and visualized under polarized light microscopy. Collagen type I gives off a red-yellow color, and collagen type III, a green color. Right panels were stained with EVG. Elastin fibers stain with dark blue color. L indicates lumen; M, media. Average values for intimal and medial thickness and matrix components are listed in Table 2.

View this table: [in this window] [in a new window]   Table 2. Aortic SMC and Matrix Structural Characteristics and Blood Pressure

Immunostaining for MMP-2, TIMP-2, and MT1-MMP
Figure 2 depicts representative aortic sections of each age group stained with antibodies against MMP-2, TIMP-2, or MT1-MMP. At 2 months of age, MMP-2 was rarely visualized within the intima. Between 2 and 8 months, both the intimal and medial MMP-2 staining increased, and further increased between 8;T3 and 30 months (Table 3). In contrast, TIMP-2 was abundantly expressed in the intima at all ages (Figure 2, middle panels). However, with increasing age, particularly between 8 and 30 months, the medial content of TIMP-2 became dramatically reduced (Figure 2 and Table 3). Thus, the relative intensities of staining pattern for MMP-2/TIMP-2 in both the intima and the media increased monotonically with age (Figure 3). Intimal MT1-MMP staining increased with age (Figure 2, right panels), and this increase was statistically significant between 8 and 30 months (Table 3). Medial MT1-MMP staining did not significantly change with age. Thus, the MT1-MMP/TIMP increased with age in both the intima and media (Figure 3).

View larger version (122K): [in this window] [in a new window]   Figure 2. Representative aortic sections from 2 months, 8 months, and 30 months rats stained with an antibody against MMP-2, TIMP-2, and MT1-MMP. The antibodies for all three stain brown. L indicates lumen; M, media. Average intima and media semiquantitative staining scores are listed in Table 3.

View this table: [in this window] [in a new window]   Table 3. Aortic Intimal and Medial Immunohistochemistry

View larger version (35K): [in this window] [in a new window]   Figure 3. Top, The MMP-2:TIMP-2:M or MT1-MMP:TIMP-2 protein staining as a function of age in the aortic intima (A) and media (B). A ratio was constructed for each individual preparation and the mean ratio ±SEM for n=4 of each group is depicted. Middle, Ratios of mRNA for proteins listed in top panels. Bottom, tPA:PAI-1 or uPA:PAI-1 protein staining as a function of age in the intima (A) and media (B). *Overall age effect via ANOVA; significant difference versus 2 months; significant difference versus 8 months.

Immunostaining for tPA, uPA, uPAR, and PAI-1
tPA was expressed mainly in the intima, and its staining became more intense with advancing age (Figure 4, left panels, and Table 3). PAI-1 was abundantly expressed in the intima and media; intimal PAI-1 did not change with age, but within the media decreased significantly between 2 and 8 and 8 and 30 months of age (Figure 4, right panels, and Table 3). There were no significant differences among 3 age groups in the tPA:PAI-1 within the intima (Figure 3), but this ratio increased with aging within the media. With advancing age, staining for uPA tended to increase in both the intima and media, but this trend was statistically significant only within the intima at 30 months. The medial, but not the intimal, uPA:PAI-1 increased significantly with age between 8 and 30 months, (Figure 3). In contrast, uPAR staining markedly increased with age in both compartments (Figure 4, Table 3).

View larger version (103K): [in this window] [in a new window]   Figure 4. Immunohistostaining (brown color) of representative aortae from 2 months, 8 months, and 30 months rats for tPA, uPA, uPAR, and PAI-1. L indicates lumen; M, media. Average semiquantitative staining scores are listed in Table 3.

In Situ Hybridization for MMP-2, TIMP-2, and MT1-MMP mRNA
MMP-2 mRNA was detected within intimal endothelial cells and medial SMC (Figure 5). The average staining for intimal MMP-2 mRNA was low at 2 months and increased with age (Table 4). TIMP-2 mRNA was detected in some intimal endothelial cells and medial SMC, with the staining localized within or around nuclei. Medial TIMP-2 mRNA decreased with age, with significant reductions occurring between 8 and 30 months (Figure 5, Table 4). MT1-MMP mRNA staining increased with age within both compartments, and this increase reached statistical significance between 2 and 30 months.

View larger version (100K): [in this window] [in a new window]   Figure 5. In situ hybridization with an antisense MMP-2 mRNA probe, a TIMP-2 mRNA probe, or a MT1-MMP mRNA probe (dark purple) in representative aortae from 2 months, 8 months, and 30 months rats. In situ hybridization with the corresponding sense mRNA probes in 30 months rats as control (right panels). Sections were counterstained with nuclear fast red (red color). L indicates lumen; M, media. Average semiquantitative staining scores are listed in Table 4.

View this table: [in this window] [in a new window]   Table 4. Aortic Intimal and Medial In Situ Hybridization

Measurement of Gelatinolytic Activity by In Vitro and In Situ Zymography
Figure 6 shows representative in vitro zymograms from each age group. A clear gelatinolytic 72 kDa band was present at 2 months. Clear and weak 72 kDa and 62 kDa bands, corresponding to pro– and active forms of MMP-2, respectively, were detected at 8 months, and predominant 72 kDa and 62 kDa bands were detected at 30 months (Figure 6A). Densitometric quantification indicated no significant differences in pro–MMP-2 among 3 groups (Figure 6B); active MMP-2, however, increased 1.5-fold at 8 months compared with 2 months (P<0.05) and increased an additional 1.7-fold at 30 months (P<0.01).

View larger version (36K): [in this window] [in a new window]   Figure 6. A, Representative zymograms for latent and active MMP-2 from 2 months (lanes 2 to 4) 8 months (lanes 5 to 7), and 30 months (lanes 8 to 10) aortae. B, Average densitometry of the activated form of MMP-2 by age. Significant difference versus 2 months; significant difference versus 8 months.

Gelatinolysis during in situ zymography is indicated by green fluorescence against a dark background (Figure 7). The in situ gelatinolytic activity increased with age (Figure 7, middle panels) and was mainly localized to intima, internal elastic lamina, and elastin fibers in the inner tunica media. The in situ gelatinolytic activity was almost completely inhibited by an antibody against MMP-2 (Figure 7, right panels) demonstrating that it mainly resulted from MMP-2 activity. Aortic elastin also exhibits green autofluorescence, and control aortic sections clearly demonstrate the decrement of elastin density in the aged aorta (Figure 7, left panels).

View larger version (85K): [in this window] [in a new window]   Figure 7. Representative examples of in vivo zymography in aortae of 2 months, 8 months, and 30 months rats. Control (left), zymograms (middle), and zymograms after coincubation with reactive buffer including the MMP-2 antibody (right). Inset in middle lower panel is the area within rectangle at x1000 magnification. Control sections show that green-autofluorescence of elastin fibers decreases with aging (left). Experimental sections without antibody against MMP-2 show a strong increase in age-associated strong gelatinolytic activity (bright green fluorescence) localized to intima, internal elastic lamina, and elastin fibers in the inner tunica media (middle). Experimental sections with antibody against MMP-2 show gelatinolytic activity was almost completely inhibited (right).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present results, demonstrating age-associated changes in factors that regulate MMP-2 activity, extend our prior observation of increased MMP-2 levels and activity within the aortic wall with aging.⁴ Figure 8 integrates and summarizes the main quantitative findings. Within both the aortic intima and media, staining of MMP-2 increases progressively with age from 2 to 30 months (Figure 8, upper graphs), and MMP-2:TIMP-2 protein staining in both compartments increases progressively with age (Figure 8 lower graphs). An age-associated increase in intimal MT1-MMP protein staining parallels that of MMP-2 (Figure 8A, upper graph), and the MT1-MMP:TIMP-2 protein staining also increases with age in both compartments (Figure 8A, lower graph). This imbalance of the ratios of MMP-2 activators to inhibitors is accompanied by an increase of MMP-2 activity in situ (Figure 7). Age-associated differences were also observed in the activation cascade of the serine protease plasmin, also a potent activator of pro–MMP-2.²⁶ Intimal tPA, uPA, and PAI-1 showed appreciable changes with aging (Figure 8), and uPAR staining increased 2- to 3-fold. An increase in uPAR density might be expected to facilitate uPA/tPA conversion of plasminogen to plasmin, contributing to the age-associated increased activation of MMP-2. In contrast to the intima, medial uPA or tPA staining did not increase with age, but the medial PAI decreased, whereas both the uPA/PAI-1 and the uPAR increased with age.

View larger version (30K): [in this window] [in a new window]   Figure 8. Combined summary of the age-associated changes in immunostaining of MMP-2 and that of potential activators or inhibitors of MMP-2 in the intima (A) or media (B). The figure combines data from Table 3 and Figure 3.

The observed imbalances in MMP-2:TIMP-2 and MT1-MMP: TIMP-2 and tPA, uPA:PAI-1, and the increased uPAR with aging, coupled to the increased level of total MMP-2 zymogen and increased MMP-2 activity, are likely candidate mechanisms for the observed age-associated increase in MMP-2 activity within the vessel wall. The age-associated increases in intimal or medial MMP-2 and MT1-MMP and the reduction in TIMP-2 are, in part, transcriptionally regulated, as suggested by an increase in the steady levels of intimal or medial mRNA for both MMP-2 and MT1-MMP and a reduction for that of TIMP-2. The basal production of MMP-2, which is typically low, is increased by factors such as cytokines, phorbol esters, or contact with components of the extracellular matrix. MMPs are synthesized in a latent form as pro-enzymes (zymogens) that are activated by proteolytic cleavage of an amino-terminal domain²⁸ or independently of proteolytic processing by conformational changes such as those induced by oxidative stress.²⁹ Factors relevant to the activation of pro–MMP-2 are produced by both endothelial cells and vascular smooth muscle cells.^11,27,30,31 It is noteworthy that, in early passage, SMC from aortic explants, at MMP-2 production in response to stimulation by cytokines IL-1a, TNF-, or TGF-ß, increases with age.³²

Increased MMP activity may affect the vascular remodeling that accompanies aging in multiple ways. MMP-2 accumulates in SMC and elastic lamina (present study) and in the area surrounding smooth muscle cells located in the vicinity of breaks in the internal elastic lamina and along elastic laminae throughout the media of the aged aorta.⁴ Migration from the media is one plausible mechanism to account for the presence of SMC within the thickened intima of the aged aorta. Activated MMP-2, required for migrating cells to invade basement membrane, also requires dedifferentiation and lysis of their own basement membrane type IV collagen and invasion of the elastic lamellae and internal elastic membrane. The development of a synthetic, migratory phenotype of SMC in vitro occurs with successive culture passage and is accompanied by a high constitutive production of MMP-2;⁸ serum withdrawal reduces MMP production and prevents their invasion of basement membranes.⁸ Thus, increased MMP activity in situ may reflect an age-associated shift in some cells within the aortic media from the contractile to the synthetic-migratory phenotype. Additional evidence for in situ dedifferentiation of some aortic SMC with aging includes the observation that, unlike aortic medial SMC from young rats which in early passage in culture fail to migrate in response to a platelet-derived growth factor gradient, early-passaged aortic medial cells from old rats readily migrate in response to this stimulus.³²

Protease dysregulation is a feature of in vitro cell senescence.³³ In addition to increased protease activity within the aorta wall with aging, other markers of in vitro senescence in other cell types, including TGF-ß and ICAM, increase. Increased MMP activation induces the expression of the matrix glycoprotein, tenacin (TN),³⁴ which has been linked to integrin ligation and induction of mitogen-activated protein kinase activity,³⁵ SMC proliferation, and anti-apoptotic effect,³⁴ each of which has been implicated in vascular diseases.^36,37 Thus, the aging aortic wall is a heretofore relatively undiscovered in situ "petri dish," suitable for investigation of vascular smooth muscle dedifferentiation, proliferation, senescence, and apoptosis.

Perspectives
Age-associated remodeling of large vessels, manifest by intimal-medial thickening, dilation, stiffening, and endothelial dysfunction, occurs not only in rodents, but also in nonhuman primates, and in otherwise healthy humans as well. There is a growing body of evidence that increased arterial intimal-medial thickening, increased stiffness, and endothelial dysfunction in apparently otherwise healthy older persons, formerly thought to be part of "normal" aging, predict a higher risk for developing atherosclerosis, hypertension, stroke, and heart failure. In other words, aging blood vessels provide fertile soil in which vascular diseases can flourish, and this may explain, in part, why aging, per se, is so "risky" with respect to these diseases. The marked age-associated increase in arterial MMP-2 activity in situ, demonstrated here in rodents, which also occurs in nonhuman primates (Wang and Lakatta, unpublished observations, 2002), and other markers of vascular senescence observed in rodents, eg, increased TGF-ß and ICAM, are apparent mechanisms of the vascular remodeling that accompanies aging. Should similar mechanisms underlie age-associated arterial remodeling in humans, these would become potential targets, not only with respect to retarding "vascular aging," but also to reducing the marked risk that vascular aging, per se, confers for the development of human hypertension and atherosclerosis.

	Acknowledgments

 
We appreciate Drs Michael Crow and Linda Cheng for a thoughtful reading of the manuscript and Christina R. Link and Denise L. Dunaway for secretarial assistance.

Received November 27, 2001; accepted February 21, 2002.

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