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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1139-1144.)
© 1995 American Heart Association, Inc.

Articles

In Situ Localization and Quantification of mRNA for 92-kD Type IV Collagenase and Its Inhibitor in Aneurysmal, Occlusive, and Normal Aorta

William D. McMillan; Bruce K. Patterson; Richard R. Keen; Vera P. Shively; Maria Cipollone; William H. Pearce

From the Division of Vascular Surgery, Department of Surgery, Department of Pathology and Medicine (B.K.P.), and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Ill.

Correspondence to William D. McMillan, MD, 251 E Chicago Ave, Suite 628, Chicago, IL 60611.

	Abstract

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Abstract Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n=8), AOD (n=8), and NL (n=7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to -tubulin. Mean values±SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n=5), AOD (n=2), and NL (n=2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855±0.180) than NL (0.046±0.23) (P<.02), but differences between AOD (0.406±0.196) and AAA or AOD and NL were not significant. Differences in TIMP-1 mRNA levels between tissue types were not significant (AAA, 1.17±0.123; AOD, 1.79±0.351; NL, 0.652±0.378). Cultured AAA and NL aortic VSMCs constitutively expressed mRNA for TIMP-1 but not MMP-9. In situ hybridization of AAA and AOD tissue localized MMP-9 mRNA to adventitial macrophages in areas of neovascularization and TIMP-1 mRNA to adventitial VSMCs. MMP-9 mRNA levels are significantly greater in aneurysmal than normal aorta. Cultured VSMCs constitutively express TIMP-1 but not MMP-9. In the diseased aortic wall, MMP-9 mRNA is found in adventitial macrophages and TIMP-1 mRNA in adventitial VSMCs. Localization of MMP-9 mRNA expression to discrete areas surrounding vasa vasorum suggests that the enzyme is responsible for localized matrix alterations associated with neovascularization.

Key Words: aortic aneurysm • metalloproteinase • vasorum • TIMP

	Introduction

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The ultrastructure of the aortic wall is markedly altered in both aneurysmal and atherosclerotic occlusive disease.¹ Previous work from this laboratory has demonstrated the presence of inflammatory infiltrates within the wall of diseased aorta and noted increasing inflammation in aneurysms compared with atherosclerotic occlusive disease.² One theory of aneurysm formation maintains that the inflammatory response and associated cytokines modulate the balance between degradation and synthesis within the wall of the aorta and thereby the pathogenesis of aneurysmal and occlusive disease. Previous work from our laboratory and others has provided evidence for this theory by demonstrating increased levels of inflammatory mediators in diseased infrarenal aorta,^{3 4} increased collagen types I and III mRNA expression in aneurysmal aorta compared with normal aorta,⁵ and interleukin-1ß–mediated upregulation of human collagenase expression in aortic smooth muscle cells grown in culture.⁶

Matrix metalloproteinases (MMPs) represent an important class of aortic wall degradative enzymes whose expression is mediated via inflammatory cell cytokines.^{7 8} mRNA for human collagenase (MMP-1), 72-kD type IV collagenase (MMP-2), and stromelysin (MMP-3) have all been identified within the wall of diseased aorta, and cytokine stimulation upregulated their production by aortic smooth muscle cells.^{9 10} Ninety-two-kilodalton type IV collagenase (MMP-9) is especially intriguing because it is a potent elastase and collagenase that could alter multiple structural aspects of the aortic wall. In fact, alterations in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinases type 1 (TIMP-1), could lead to aneurysm formation.

MMP-9 and TIMP-1 are involved in a variety of diseases, including rheumatoid arthritis,¹¹ invasive carcinoma,¹² and matrix alterations associated with neovascularization.¹³ Several authors have identified MMP-9 in protein extracts of aneurysm tissue homogenates,^{14 15 16} although its production by aortic smooth muscle cells in culture has not been observed. Others have demonstrated increased enzymatic activity at 80 kD (corresponding to activated MMP-9) within protein extracts of aneurysmal tissue compared with normal aorta and that addition of TIMP-1 limits the observed in vitro elastase activity of aneurysmal supernatants on normal aorta.¹⁷ Given evidence for cytokine-mediated upregulation of metalloproteinases in vitro, we hypothesized that the observed increases in MMP-9 protein activity reflected localized changes in mRNA expression by subpopulations of inflammatory cells within the aneurysm wall. To date, no detailed study of MMP-9 and TIMP-1 mRNA expression within aneurysmal, occlusive, and normal aortic walls has been done.

The purpose of our study was to measure relative levels of MMP-9 and TIMP-1 mRNA expression in aneurysmal, occlusive, and normal aortic tissue specimens; determine the presence or absence of their expression by vascular smooth muscle cells cultured from aneurysms and normal aorta; locate the cell type responsible for mRNA production in situ; and thereby provide a clearer understanding of the role of MMP-9 in aneurysmal and occlusive aortic disease.

	Methods

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Materials
Infrarenal aortic specimens were obtained from patients who provided informed consent before either abdominal aortic aneurysm resection (15 patients; average age, 67 years; ratio of men to women, 14:1) or aortobifemoral bypass for occlusive disease (10 patients; average age, 60 years; ratio of men to women, 8:2) in accordance with the Institutional Review Board and Research Committee of Northwestern University/McGaw Medical Center. Specimens of normal aorta (9 patients; average age, 60 years; ratio of men to women, 6:3) were obtained at the time of organ procurement or autopsy. Specimens were placed immediately in either liquid nitrogen (tissue mRNA extraction), Streck Tissue Fixative (in situ hybridization, Streck Laboratory), or Dulbecco's modified Eagle's medium (DMEM, cell culture).

RNA Extraction and Northern Transfer Hybridization
Aortic tissue from patients undergoing aneurysm resection (n=8) or aortic bypass procedures for occlusive disease (n=8) and from healthy donors (n=7) was snap-frozen in liquid nitrogen and stored at -80°C until use. RNA extraction and Northern transfer took place as previously described,¹⁸ producing two membranes for hybridization. Probes were synthesized from previously characterized cDNA of MMP-9,¹⁹ TIMP-1,²⁰ or -tubulin,²¹ random primers, and [³²P]ATP by use of a Prime-It II Kit (Stratagene). Specific activity of each probe was determined with a scintillation counter and was >1x10⁶ cpm/ng. Hybridization proceeded at 68°C according to the protocol accompanying the Quik Hyb (Stratagene) hybridization solution. Probes for -tubulin and MMP-9 or TIMP-1 were hybridized to the same membrane. Autoradiographs were made by exposing the hybridized membranes to x-ray film (Hyperrad-TM, Amersham) at -80°C for 24 hours. Quantification of mRNA levels proceeded as described previously¹⁸ using laser densitometric assessment of autoradiographs. MMP-9 and TIMP-1 mRNA signals were normalized to the signal for -tubulin, a constitutively expressed protein in mammalian cells, to control for differences in intensity caused by variable amounts of RNA in each lane. Analysis of variance (ANOVA) and Fischer's protected least square of differences were used for statistical analysis. Differences were considered significant at 95% confidence (P<.05), were reported as mean±SEM, and were displayed graphically with standard deviation error bars.

Cell Culture
Tissue samples of aortic aneurysm and normal aorta were immediately processed after transport in sterile DMEM. Tissue was minced and placed in M-199 media with 15% fetal bovine serum (Sigma Chemical Co), 0.05 mg/mL endothelial cell growth supplement (Sigma), 120 U/mL penicillin G, 120 µg/mL streptomycin, and 2.5 µg/mL amphotericin B. Cells were grown to confluence in 5% CO₂, passaged with 0.25% trypsin, and harvested after passage 3. Cell type was confirmed by staining with monoclonal anti–-smooth muscle actin antibody (Sigma A-2547) counterstained with rhodamine-labeled goat anti-mouse immunoglobulin (Cappel Teknika). Cell medium was changed to serum-free ITS+ Premix (Collaborative Biomedical) in M-199 (1:100, vol/vol) for 48 hours before mRNA extraction. After RNA extraction and Northern transfer, membranes were hybridized with cDNA probes for TIMP-1¹⁹ and MMP-9.²⁰ Tissue RNA from a single aneurysmal specimen was added to the Northern gel as a positive control for MMP-9.

In Situ Hybridization
Bluescript plasmids containing inserts of probes for MMP-9¹⁹ and TIMP-1²⁰ were kindly provided by Drs Gregory Goldberg (Washington University) and Mark Johnson (Northwestern University). Each plasmid was linearized with restriction enzymes to create templates for unidirectional synthesis of digoxigenin-labeled RNA probes with specific promoters following the methods from a Genius IV Riboprobe Synthesis Kit (Boehringer Mannheim) (antisense MMP-9, Not I/T3; sense MMP-9, Xho I/T7; antisense TIMP-1, Xho I/T7; sense TIMP-1, Sac I [Klenow enzyme added to digest 3' sticky end]/T3). Transcripts were checked on 1% agarose gel, and concentrations were determined by serial dilution color reaction against known concentrations of control-labeled RNA. Hybridization to a Northern blot of tissue RNA from aneurysmal, occlusive, and normal tissue samples confirmed probe specificity.

Paraffinized 4-µm sections of aortic tissue (6 aneurysmal, 2 occlusive, and 2 normal) fixed in Streck Tissue Fixative were placed on Vectabond (Vector Laboratories)–coated Fischer Superfrost (Fisher Scientific) slides. Slides were deparaffinized and hydrated through descending ethanol concentrations into diethylpyrocarbonate (DEPC)-treated water. Pretreatment followed a modification of the methods of Hillian et al²² and included incubation with 7.5 µg/mL proteinase K, 4% paraformaldehyde fixation, and acetylation in 0.25% acetic anhydride. Slides were rinsed with 2x standard saline citrate (SSC), dehydrated through ascending ethanol concentrations, and prehybridized for 2 hours with 50% formamide in 2xSSC at 60°C (MMP-9) or 47°C (TIMP-1). A modification of the hybridization technique described by Young²³ was followed: Five microliters (500 ng) of newly synthesized probe was added to 3 µL of DEPC-treated water and 4 µL of ribonucleic acid mix (sheared salmon sperm DNA 2.5 mg/mL, yeast total RNA 6.3 mg/mL, yeast tRNA 6.2 mg/mL, and DEPC-treated water 187 µL/mL), heated to 65°C for 5 minutes, and cooled on ice for 1 minute. Hybridization buffer (88 µL) (1 mol/L Tris-HCl, pH 7.4, 24 µL/mL; 100 mmol/L EDTA, pH 8, 12 µL/mL; 3 mol/L NaCl, 125 µL/mL; formamide 100%, 595 µL/mL; dextran sulfate 10 mg/mL; Denhardt's solution 50x, 24 µL/mL; DEPC-treated water, 25 µL/mL) was added at room temperature. The resulting 100 µL of hybridization mix was added to each slide before coverslipping and placement in a humidified chamber at 60°C (MMP-9) or 47°C (TIMP-1) for 14 to 16 hours. Slides were washed four times in 4xSSC, treated with 300 µL RNase A (40 µg/mL) at 37°C for 30 minutes to remove unbound probe, washed for 10 minutes each in 2xSSC, 1xSSC, 0.5xSSC, and 0.1xSSC at room temperature, and washed twice in 0.1xSSC at 58°C for 30 minutes. Slides were then dehydrated quickly through ascending concentrations of ethanol and washed in digoxigenin buffer 1 (0.1 mol/L Tris-HCl, 0.15 mol/L NaCl, pH=7.5). Slides were blocked in a solution containing buffer 1, 3% Vector goat serum (Vector Laboratories), and 0.3% Triton X-100 for 30 minutes before incubation with 100 µL of diluted anti-digoxigenin antibody/alkaline phosphatase conjugate (antibody conjugate 1:3000 in buffer 1 with 3% goat serum and 0.3% Triton X-100) for 5 hours. Subsequent washes included buffer 1 for 10 minutes and buffer 3 (Tris-HCl 100 mmol/L, NaCl 100 mmol/L, MgCl₂ 50 mmol/L, pH=9.5) for 2 minutes. Color substrate solution (300 µL) (45 µL nitro blue tetrazolium salt, 35 µL X-phosphate, and 2.4 mg levamisole in 10 mL buffer 3) was added to each slide before placement in a humid dark chamber for 16 hours. Rinsing slides in buffer 4 (Tris-HCl 10 mmol/L, EDTA 1 mmol/L, pH=8) and washing in 500 mL of 0.1xSSC for 30 minutes completed the reaction. Slides were counterstained with hematoxylin and coverslipped with Crystalmount (Biomedia) aqueous-based mounting medium.

Alternating serial sections of aorta taken at operation were stained simultaneously with sense and antisense probes. All sections were examined under light microscopy in a blinded fashion by a single pathologist to determine positivity and the location and cell type producing the mRNA of interest. Immunohistochemical staining with antibody to -smooth muscle actin (Sigma) and macrophage/histiocyte antigen Mac 387 (Dakopatts) on unstained alternating sections aided cell type identification. More than 10 sections of each specimen were stained with each of the four RNA probes to limit the possibility of intrasample variability.

	Results

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Tissue mRNA
Signal for MMP-9 mRNA was present in all aneurysmal (n=5), all occlusive (n=3), and two of four normal aortic samples tested (Fig 1). Mean MMP-9/-tubulin mRNA signals in aortic aneurysm tissue were 18 times greater than in normal aorta. ANOVA demonstrated significant differences between groups (P<.04), and Fischer's protected least-squares difference testing identified the difference between aneurysmal and normal as solely responsible (0.855±0.180 versus 0.046±0.23, P<.02). Differences between occlusive and normal (0.406±0.196 versus 0.855±0.180, P=.22) or aneurysmal and occlusive (0.855±0.180 versus 0.406±0.196, P=.10) were not significant (Fig 2). Signal for TIMP-1 mRNA was present in all aneurysmal (n=3), all occlusive (n=5), and two of four normal samples (Fig 1). Differences in TIMP-1/-tubulin mRNA ratios between tissue types did not reach statistical significance (P=.096) (aneurysmal, 1.17±0.123; occlusive, 1.79±0.351; normal, 0.652±0.378) (Fig 2).

View larger version (46K): [in this window] [in a new window]   Figure 1. Northern blot analysis of mRNA from infrarenal aortic tissue of normal human aorta (NA), patients with aortoiliac occlusive disease (Occ), and patients with aneurysms (AAA). Radiolabeled cDNA probes for 92-kD type IV collagenase (MMP-9) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) hybridized to messages of lengths 2.8 and 0.9 kb. Probes for human -tubulin hybridized to 1.6-kb mRNA on both membranes in all lanes, indicating that adequate amounts of total RNA were loaded in each well. All aneurysmal and occlusive specimens tested demonstrated signal for MMP-9 and TIMP-1. A weak signal for MMP-9 was present in two of three normal aortas, whereas two of four normal aortas demonstrated signal for TIMP-1 mRNA.

View larger version (21K): [in this window] [in a new window]   Figure 2. Bar graphs showing MMP-9 tissue mRNA (left) and TIMP-1 tissue mRNA (right) in abdominal aortic aneurysms (AAA), occlusive aortoiliac tissue (AOD), and normal infrarenal aorta (NA). All signals were normalized to -tubulin to control for loading differences in total RNA and were expressed as mean with standard deviation error bars. MMP-9 mRNA levels were increased significantly in aneurysmal tissue compared with normal (P<.02). No significant differences between MMP-9 occlusive and normal or occlusive and aneurysmal tissue mRNA levels were identified. No significant differences in TIMP-1 mRNA levels were identified between any of the tissue types tested. Other abbreviations as in Fig 1.

Cultured Vascular Smooth Muscle Cell mRNA
MMP-9 mRNA levels were below limits of detection in vascular smooth muscle cells cultured from aneurysmal and normal aorta. Aneurysmal tissue mRNA served as a positive control during these hybridizations (Fig 3). Both normal and aneurysmal aortic cultured vascular smooth muscle cells expressed TIMP-1 mRNA (Fig 3).

View larger version (21K): [in this window] [in a new window]   Figure 3. Northern blot analysis of mRNA from cultured aneurysmal and normal vascular smooth muscle cells (VSMCs) probed with radiolabeled cDNA probes for MMP-9 (top) and TIMP-1 (bottom). Both normal and aneurysmal VSMCs contained TIMP-1 mRNA. Neither aneurysmal or normal VSMCs contained MMP-9 mRNA. Aneurysmal tissue RNA served as a positive control during MMP-9 cell culture hybridizations. Other abbreviations as in Fig 1.

In Situ Hybridization
In situ hybridization demonstrated MMP-9 and TIMP-1 in two atherosclerotic and six aneurysmal aortic tissue specimens. No binding of either probe was seen in two normal aortic specimens. No binding of sense probes was seen in any specimens.

In aneurysmal tissue, the probe for MMP-9 bound specifically to mRNA in adventitial macrophages adjacent to inflammatory infiltrates within the aortic wall (Fig 4). Macrophage/histiocyte cell type was confirmed by Mac 387 staining of adjacent sections (Fig 5). The binding was consistently in areas demonstrating vasa vasorum. In the occlusive specimens, binding was in macrophages near vasa vasorum but was not uniformly adjacent to inflammatory infiltrates (Fig 6).

View larger version (2K): [in this window] [in a new window]   Figure 4. In situ hybridization of abdominal aortic aneurysm tissue with digoxigenin-labeled RNA probes for MMP-9 counterstained with hematoxylin. Antisense probe bound to adventitial cells adjacent to inflammatory infiltrates (A, magnification x16, upper right of photograph), whereas no binding of sense probe was seen in adjacent sections (B, magnification x16). C, Higher-power view of antisense section demonstrates binding within adventitial macrophages (magnification x80).

View larger version (2K): [in this window] [in a new window]   Figure 5. Comparison of MMP-9 antisense in situ hybridization in aneurysmal tissue (B, magnification x80) and Mac 387 macrophage-specific immunohistochemical stain in an adjacent section (A, magnification x80). Note that both bind to similar rounded cells, indicating that MMP-9 mRNA is expressed by macrophages. Comparison of TIMP-1 antisense in situ hybridization in aneurysmal tissue (D, magnification x80) with smooth muscle cell -actin immunohistochemical staining in an adjacent lower-power section (C, magnification x40). Note arrow indicating the same vessel on both sections. Both techniques stain elongated thin cells and TIMP-1 in situ probes bound to the same cells that stain positively for smooth muscle actin.

View larger version (2K): [in this window] [in a new window]   Figure 6. In situ hybridization of abdominal aortic tissue from patients with occlusive aortoiliac disease by digoxigenin-labeled RNA probes for MMP-9 and TIMP-1. All samples were counterstained with hematoxylin. Antisense TIMP-1 probe bound to mRNA in adventitial smooth muscle cells (A, magnification x16), whereas antisense probe for MMP-9 bound to mRNA within adventitial macrophages (B, magnification x16; C, same section, magnification x80). Adv. indicates adventitia; Int., intima.

The probe for TIMP-1 bound to mRNA of adventitial smooth muscle cells in the aneurysmal (Fig 7) and occlusive (Fig 6) specimens. TIMP-1 probes bound diffusely in adventitial smooth muscle cells and were not localized exclusively to areas of vasa vasorum. Cell type was confirmed by positive staining of similar areas on alternating sections with -smooth muscle actin antibody.

View larger version (2K): [in this window] [in a new window]   Figure 7. In situ hybridization of abdominal aortic aneurysm tissue with digoxigenin-labeled RNA probes for tissue inhibitor of metalloproteinase type one (TIMP-1) counterstained with hematoxylin. Antisense probe bound to adventitial cells (A, magnification x40), whereas no binding of sense probe was seen in adjacent sections (B, magnification x40). Higher-power view (C, magnification x80) demonstrates binding within elongated adventitial smooth muscle cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Aortic wall degeneration and reparative synthesis are features of both aneurysmal and atherosclerotic disease. In 1980 Busuttil and coworkers²⁴ first described increases in aneurysmal collagenase activity, and their discovery stimulated intense research into degradative enzymes and aortic disease. Metalloproteinases and their inhibitors, such as MMP-9 and TIMP-1, have emerged as potentially important mediators of aortic wall elastin and collagen degradation.^{25 26 27} Our experiments examined MMP-9 and TIMP-1 mRNA expression in aortic disease.

Tissue extraction experiments demonstrated MMP-9 and TIMP-1 mRNA in all occlusive and aneurysmal specimens tested. Only half of the normal specimens tested demonstrated expression of TIMP-1 or MMP-9 mRNA. Attempts at quantification revealed an 18-fold increase of MMP-9 mRNA in aneurysmal compared with normal aorta (P<.02). Other comparisons failed to reach statistical significance, primarily because of small numbers of samples. This limits interpretation of the elevated MMP-9 message level. However, elevated expression of mRNA for the potent elastolytic and collagenolytic enzyme within aneurysmal tissue compared with normal aorta echoes previously reported protein data^{15 16 17} and suggests an ongoing destructive process within the aneurysm. The presence of MMP-9 and TIMP-1 mRNA in all diseased tissue tested suggests that they have a role in both aneurysmal and occlusive disease.

Results of the cell culture and in situ hybridization experiments addressed the origin and location of MMP-9 and TIMP-1 mRNA within the diseased aortic wall. MMP-9 mRNA was below limits of detection in extracts of aneurysmal and normal cultured aortic smooth muscle cells. Conversely, vascular smooth muscle cells cultured from normal and aneurysmal aortic specimens demonstrated expression of TIMP-1 mRNA. In situ hybridization confirmed tissue culture results by localizing expression of MMP-9 to macrophage/histiocyte cells and TIMP-1 to smooth muscle cells.

Probes for MMP-9 applied to aneurysmal tissue hybridized exclusively to macrophage mRNA in areas of adventitial neovascularization adjacent to, but distinct from, inflammatory infiltrates. Previous authors have observed increased adventitial neovascularity or vasa vasorum within both aneurysmal and occlusive aorta.¹⁶ Szekanecz et al²⁸ recently demonstrated increased aortic endothelial cell migration, a key component of neovascularization, in response to aneurysmal supernatants containing interleukin-8. MMP-9 has also been implicated in matrix remodeling associated with neovascularization in vitro.¹³ Given increased neovascularity in aneurysmal disease and in situ localization of MMP-9 mRNA to macrophages surrounding vasa vasorum, our finding of increased MMP-9 mRNA levels in aneurysmal tissue is not surprising. Our results strongly suggest that the enzyme is involved in matrix remodeling associated with increased adventitial neovascularity rather than diffuse structural changes characteristic of aneurysmal disease. The location of macrophages, or histiocytes, containing MMP-9 mRNA adjacent to inflammatory infiltrates suggests that inflammatory cytokines might mediate expression. Identification of expression by macrophages surrounding vasa vasorum in occlusive and aneurysmal specimens suggests that the enzyme has a similar role in both disease processes. Continued study to better quantify levels of expression in occlusive specimens and correlate the amount of neovascularity with amounts of MMP-9 mRNA would help to confirm these initial observations.

TIMP-1 probes hybridized to mRNA within aortic smooth muscle cells in both aneurysmal and occlusive specimens. TIMP was present both adjacent to and in areas distinct from MMP-9 mRNA and neovascularization. An obvious question concerns the role of TIMP-1 mRNA made by smooth muscle cells in areas distinct from MMP-9 expression by macrophages. As reported by Goldberg et al,²⁹ TIMP-1 inhibits MMP-9 via competitive binding and requires both enzymes to be present within the same area of extracellular matrix. The most likely explanation is that smooth muscle cells expressing TIMP-1 are involved in remodeling processes unassociated with MMP-9. Subpopulations of smooth muscle cells might have produced TIMP-1 mRNA to inhibit other metalloproteinases, such as human collagenase (MMP-1) or stromelysin (MMP-3), in matrix devoid of MMP-9.

Tissue extraction experiments demonstrated TIMP-1 and MMP-9 mRNA in normal tissue, but our in situ experiments failed to detect either in normal tissue. A likely explanation for the apparent inconsistency is that in normal specimens, local tissue concentrations of TIMP-1 and MMP-9 mRNA are below the sensitivity of in situ technique. Previous authors have noted that in situ probes hybridize effectively if concentrations of mRNA in localized subsets of cells are high but are less effective when large numbers of weakly expressive cells contain similar amounts of mRNA.³⁰ In tissue extraction experiments, local concentrations are irrelevant, and only total mRNA content matters.

A final question concerns the time course of the disease state and alterations in enzyme expression. All diseased aortic tissue specimens were obtained from patients with late-stage atherosclerotic or aneurysmal disease. MMP-9 or TIMP-1 could have been expressed in other areas of the wall earlier in the course of aneurysm formation. For instance, MMP-9 might have a role in medial elastin or collagen destruction early in the course of aneurysmal disease and only later be expressed exclusively by adventitial macrophages. Without the ability to obtain specimens early in the time course of disease, we can demonstrate only those findings specific to late-stage occlusive and aneurysmal aorta.

We conclude that both late-stage aneurysmal and occlusive atherosclerotic aortas contain mRNA for MMP-9 and TIMP-1, that MMP-9 mRNA levels are significantly greater in aneurysmal than normal aortic tissue, that MMP-9 mRNA is expressed by adventitial macrophages, and that TIMP-1 mRNA is expressed by adventitial smooth muscle cells. Localization of macrophages expressing MMP-9 to areas of adventitial vasa vasorum suggests that MMP-9 is involved in matrix alterations associated with neovascularization in both late-stage atherosclerotic and aneurysmal disease but not in the diffuse structural degradative changes characteristic of aneurysmal disease.

	Acknowledgments

 
This study was supported in part by the Alyce F. Salerno Foundation, the Gaylord T. Freeman Fund, and National Heart, Lung, and Blood Institute grant 1-K07 HL-02661-01A1.

Received December 22, 1994; accepted May 16, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Zarins CK, Glagov S. Aneurysms and obstructive plaques: differing local response to atherosclerosis. In: Bergan JJ, Yao JST, eds. Aneurysms: Diagnosis and Treatment. New York, NY: Grune & Stratton; 1982:61-82.

2. Koch AE, Haines GK, Rizzo RJ, Radosevich JA, Pope RM, Robinson PG, Pearce WH. Human aortic aneurysms: immunophenotypic analysis suggesting immune-mediated response. Am J Pathol. 1990;137:1199-1213. [Abstract]

3. Pearce WH, Sweis I, Yao JST, McCarthy WJ, Koch AE. Interleukin-1ß and tumor necrosis factor- release in normal and diseased human infrarenal aortas. J Vasc Surg. 1992;16:783-789.

4. Newman KM, Jean-Claude J, Li H, Ramey WG, Tilson MD. Cytokines that activate proteolysis are increased in abdominal aortic aneurysms. Circulation. 1994;90(suppl II):II-224-II-227.

5. McGee GS, Baxter BT, Shively VP, Chisholm R, McCarthy WJ, Flinn WR, Yao JST, Pearce WH. Aneurysm or occlusive disease-factors determining the clinical course of atherosclerosis in the infrarenal aorta. Surgery. 1991;110:370-376. [Medline] [Order article via Infotrieve]

6. Evans CH, Georgescu HI, Lin CW, Mendelow D, Steed DL, Webster MW. Inducible synthesis of collagenases and other neutral metalloproteinases by cells of aortic origin. J Surg Res. 1991;51:399-404. [Medline] [Order article via Infotrieve]

7. Duncan MR, Berman B. Differential regulation of glycosaminoglycans, fibronectin and collagenase production in cultured human dermal fibroblasts by interferon-alpha, -beta, and -gamma. Arch Dermatol Res. 1989;281:11-18. [Medline] [Order article via Infotrieve]

8. Dayer JM, de Rochemonteix B, Burrus B, Demczuk S, Dinarello CA. Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells. J Clin Invest. 1986;77:645-648.

9. Yangi H, Sasaguri Y, Sugama K, Morimatsu M, Nagase H. Production of tissue collagenase (matrix metalloproteinase 1) by human aortic smooth muscle cells in response to platelet derived growth factor. Atherosclerosis. 1992;91:207-216.

10. Keen RR, Murphy T, Cipollone M, Shively VP, Pearce WH. Upregulation of prostromelysin in aortic aneurysms is mediated by interleukin-1ß. In: Proceedings of the Association for Academic Surgery, 27th Annual Meeting, Hershey, Nov. 10-13, 1993.

11. Unemori EN, Hibbs MS, Amento EP. Constitutive expression of a 92 kD gelatinase (type V collagenase) by rheumatoid synovial fibroblasts and its induction in normal human fibroblasts by inflammatory cytokines. J Clin Invest. 1991;88:1656-1662.

12. Moscatelli D, Rifkin DB. Membrane and matrix location of proteinases, a common theme in tumor cell invasion and angiogenesis. Biochim Biophys Acta. 1988;948:67-85. [Medline] [Order article via Infotrieve]

13. Mignatti P, Tsuboi R, Robbins E, Rifkin DB. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor induced proteinases. J Cell Biol. 1989;108:671-682. [Abstract/Free Full Text]

14. Newman KM, Ogata Y, Malon AM, Irizarry E, Ghandi RH, Nagase H, Tilson MD. Identification of matrix metalloproteinases 3 (stromelysin-1) and 9 (gelatinase-B) in abdominal aortic aneurysms. Arterioscler Thromb. 1994;14:1315-1320. [Abstract/Free Full Text]

15. Vine N, Powell JT. Metalloproteinases in degenerative aortic disease. Clin Sci. 1991;81:233-239. [Medline] [Order article via Infotrieve]

16. Herron GS, Uremeric E, Wong M, Rapp JH, Hills MH, Stoney RJ. Connective tissue proteinases and inhibitor in abdominal aortic aneurysms. Arterioscler Thromb. 1991;11:1667-1677. [Abstract/Free Full Text]

17. Tilson MD, Newman KM. Proteolytic mechanisms in the pathogenesis of aortic aneurysms. In: Yao JST, Pearce WH, eds. Aneurysms: New Findings and Treatments. Norwalk, Conn: Appleton & Lange; 1994:5-6.

18. Mesh CL, Baxter BT, Pearce WH, Chisholm RL, McGee GS, Yao JST. Collagen and elastin gene expression in aortic aneurysms. Surgery. 1992;112:256-260. [Medline] [Order article via Infotrieve]

19. Wilhelm SM, Collier IE, Marmer BL, Eisen AZ, Grant GA, Goldberg GI. SV-40 transformed human lung fibroblasts secrete a 92 kDa type IV collagenase which is identical to that secreted by normal human macrophages. J Biol Chem. 1989;264:17213-17221. [Abstract/Free Full Text]

20. Gasson J, Golde D, Westbrook CA, Hewick RM, Kaufman RJ, Wang GG, Temple PA, Leary AC, Brown EL. Molecular characterization and expression of the gene encoding human erythroid-potentiating activity. Nature. 1985;315:768-770. [Medline] [Order article via Infotrieve]

21. Cowan NJ, Dobner PR, Fuchs EV, Cleveland DW. Expression of human alpha-tubulin genes: interspecies conservation of 3' untranslated regions. Mol Cell Biol. 1983;3:1738-1745. [Abstract/Free Full Text]

22. Hillian KJ, Farquharson M, Harvie R, McKee TA, MacSween RNM, McNicol AM. Detection of messenger RNA in formalin fixed rat tissues using RNA probes labeled with new nucleotide analogue digoxigenin-11-UTP. J Pathol. 1990;160:164A. Abstract.

23. Young WS III. In situ hybridization histochemistry. In: Bjorklund A, Hokfelt T, Wouterlood F, van den Pol A, eds. Handbook of Chemical Neuroanatomy, vol 8: Analysis of Neuronal Microcircuits and Synaptic Interactions. Philadelphia, Pa: Elsevier Science Publishers; 1990:481-512.

24. Busuttil RW, Abou-Zamzam AM, Machleder HI. Collagenase activity of the human aorta: a comparison of patients with and without aortic aneurysms. Arch Surg. 1980;115:1373-1378. [Abstract/Free Full Text]

25. Busuttil RW, Rinderbriecht H, Flesher A, Carmack C. Elastase activity: the role of elastase in aortic aneurysm formation. J Surg Res. 1982;32:214-221. [Medline] [Order article via Infotrieve]

26. Brophy CM, Marks WH, Reilly JM, Tilson MD. Decreased tissue inhibitor of metalloproteinases (TIMP) in abdominal aortic aneurysm tissue: a preliminary report. J Surg Res. 1991;50:653-657. [Medline] [Order article via Infotrieve]

27. Brown SL, Backstrom B, Busuttil RW. A new serum proteolytic enzyme in aneurysm pathogenesis. J Vasc Surg. 1988;7:210-214. [Medline] [Order article via Infotrieve]

28. Szekanecz Z, Shah MR, Harlow LA, Pearce WH, Koch AE. Interleukin-8 and tumor necrosis factor- are involved in human aortic endothelial cell migration. Pathobiology. 1994;62:134-139. [Medline] [Order article via Infotrieve]

29. Goldberg GI, Strangin A, Collier IE, Genrich LT, Marmer BL. Interaction of 92kDa type IV collagenase with tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase and activation of the proenzyme with stromelysin. J Biol Chem. 1992;267:4583-4591. [Abstract/Free Full Text]

30. Pringle JH, Primrose L, Kind CN, Talbot IC, Lauder I. In situ hybridization demonstration of polyadenylated RNA sequences in formalin fixed paraffin sections using a biotinylated oligonucleotide poly d(t) probe. J Pathol. 1989;158:279-286.[Medline] [Order article via Infotrieve]

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