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(Circulation. 1997;96:2228-2232.)
© 1997 American Heart Association, Inc.
Articles |
Size Matters
The Relationship Between MMP-9 Expression and Aortic Diameter
From the Feinberg Cardiovascular Research Institute (D.A.J., M.A.P.) and Division of Vascular Surgery, Department of Surgery (W.D.M., N.A.T., M.C., W.H.P.), Northwestern University Medical School, Chicago, Ill.
Correspondence to William H. Pearce, MD, 251 E Chicago Ave, No. 626, Chicago, IL 60611.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background Despite a wealth of data detailing increased metalloproteinase (MMP)-9 expression and activity in abdominal aortic aneurysms (AAAs), no studies examine the relationship between aortic size and MMP-9 expression. Because elastolysis occurs early in AAA formation, we hypothesized that MMP-9 expression would vary with aortic diameter. The purpose of this study was to measure MMP-9 mRNA levels in AAAs of various diameters and define the relationship between AAA size and MMP-9 expression.
Methods and Results MMP-9 mRNA levels were measured by competitive polymerase chain reaction (PCR) using gene-specific external standards with cDNA from AAAs (n=19) and normal aortas (n=4). Levels were normalized to GAPDH mRNA, determined separately via competitive PCR, to control for efficiency of reverse transcription. AAA size was measured on CT scans obtained within 6 weeks of surgery. MMP-9/GAPDH mRNA transcript levels in AAAs were expressed as mean±SEM and analyzed by ANOVA with a Tukey adjustment. There was a fourfold elevation in MMP-9/GAPDH mRNA transcript levels in 5.0- to 6.9-cm AAAs (98.06±15.19) compared with small (3.0- to 4.9-cm) AAAs (20.87±5.15, P<.03), large (>7-cm) AAAs (27.16±14.56, P<.01), or normal aortas (3.57±1.13, P<.003). The results did not change when they were normalized to patient height, nor were there significant differences in risk factors, age, or sex in each AAA group.
Conclusions MMP-9 mRNA expression is significantly higher in moderate-diameter (5- to 6.9-cm) AAAs than either small (<4.0-cm) or large (>7.0-cm) AAAs. Increased MMP-9 expression may account for the propensity of AAAs >5 cm to continue to expand, in contrast to smaller aneurysms. Lower levels in AAAs >7 cm suggest that increases in other enzymes or in diameter-dependent mechanical stress on the aortic wall are responsible for their characteristic rapid expansion and high rupture rates.
Key Words: aorta • aneurysm • metalloproteinases
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Aortic aneurysms are characterized by the degradation of structural proteins, including both collagen and elastin.1 2 3 MMPs are responsible for collagen and elastin degradation within the aortic wall, and many authors have demonstrated increases in MMP protein expression and activity within aneurysms.4 5 6 7 8 9 10 11 Type IV collagenase (MMP-9; 92 kD) is the primary elastolytic enzyme within the aneurysm wall.4 5 8 9 Work from our laboratory demonstrates that the relative increase in MMP-9 mRNA expression in aneurysms far exceeds increases in the expression of other MMPs, including interstitial collagenase (MMP-1) and 72-kD type VI collagenase (MMP-2).12
Hypothetical relationships between MMP expression and aneurysm size may have potential clinical significance. Aortic elastolysis occurs early within aneurysm formation, and collagen fatigue is thought to be responsible for the rapid expansion of large aneurysms antecedent to eventual rupture.13 14 15 16 17 Historical data indicate that aneurysms >5 cm in diameter are prone to undergo continued expansion but that the fate of smaller aneurysms is less predictable.18 19 20 21 Taken together, these data suggest that particular MMPs with specific substrate specificities (ie, for elastin or collagen) may be expressed at various times during aneurysm formation and expansion. If present, relationships between aneurysm size and expression of particular MMPs could be exploited by therapies designed to slow aneurysm expansion and prevent late rupture.
Given the observed increase in MMP-9 expression in aneurysms and its role in elastinolysis, we hypothesized that MMP-9 expression would vary with aortic diameter. The purpose of this study was to measure MMP-9 mRNA levels, determined via competitive PCRs, in aneurysms of various diameters to define the relationship between aneurysm size and MMP-9 expression.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Tissue Specimens
Infrarenal aortic specimens were obtained from patients who provided informed consent before abdominal aortic aneurysm repair (19 patients) in accordance with the Institutional Review Board and Research Committee of Northwestern University/McGaw Medical Center. Specimens of normal aortas (4 patients) were obtained at the time of organ procurement from the Regional Organ Bank of Illinois. All specimens, obtained 3 to 6 cm below the renal arteries, were immediately snap-frozen in liquid nitrogen and stored at -80°C until use.
All aneurysm specimens analyzed had corresponding CT scans of the abdomen and pelvis within 6 weeks of surgery. The largest single diameter of the infrarenal aorta was measured and taken to be the size of the aneurysm. Patients without recent CT scans or for whom scans were not available were excluded from this study before analysis of tissue specimens. All measurements of aneurysm diameter were made by the same observer using loop magnification before tissue analysis. All normal aortic specimens had diameters of <3 cm at the time of organ harvest.
RNA Extraction and Preparation of SCDNA Templates
Total RNA was extracted as previously described.22
RNA quality was confirmed via agarose checking gel (tight ribosomal
bands) and spectrophotometric analysis (260/280 >1.5). Total
RNA (5 µg) was treated with 0.5 U RQ1 DNAase (Promega) according to
the instructions provided by the manufacturer. After incubation, 50
µL of Tris-EDTA buffer and 10 µL of 2 mol/L sodium acetate
(pH 4) were added, followed by phenol/chloroform extraction and
ethanol precipitation. Reverse transcription of RNA was performed with
a SuperScript preamplification system for first-strand cDNA synthesis
(Life Technologies) with oligo d(T)18 primers according to
the instructions provided by the manufacturer. Control reactions
omitting reverse transcriptase (RT-) were set up for each RNA sample.
After incubation, each sample was heated to 70°C to
inactivate the enzyme and was diluted 10-fold with
distilled water.
Primer Design
Published cDNA sequences for human GAPDH23 and
MMP-924 were used for primer construction. PCR primers
were chosen by use of the oligonucleotide
analysis program OLIGO 4.0 (National Biosciences). The primer
sequences and lengths of the expected PCR products are listed in
Table 1
.
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ESDNA Synthesis
External standard fragments of cDNA were designed to compete
with SCDNA for primers in each PCR reaction. ESDNA was prepared as
described previously25 with the external standard primer
listed in Table 1
and with an additional reamplification step using
primers 3 and 5. After synthesis, the ESDNA was purified with a QIAEX
purification kit (Qiagen Inc) as described in the instructions provided
by the manufacturer. ESDNA concentrations were determined as previously
described, with ethidium bromide fluorescence26
and low DNA mass ladder (Life Technologies) as a control.
Competitive PCRs
Each competitive PCR contained a known concentration of ESDNA
and a fixed amount of SCDNA. Serial reactions that differed only in the
amount of ESDNA used were run for each specimen. In addition, every PCR
reaction was run with a control consisting of specimen RNA only (RT-
mixture, see "Preparation of SCDNA Templates") to control for
contamination with genomic DNA. If any product was produced in a
control reaction, the entire cDNA sample was discarded, the RNA was
retreated with DNAase, the reverse transcription step was repeated, and
another control reaction was run. A typical 25-µL PCR reaction
contained a fixed amount of SCDNA (1 to 10 µL, depending on optimal
concentration), 20 mmol/L Tris-HCl (pH 8.4), 50
mmol/L KCl, 2 mmol/L MgCl2, 0.2
mmol/L of each dNTP, 200 nmol of each primer (3' and 5'), 0.5 U
Taq polymerase (Roche Molecular Systems), and a variable
but known amount of ESDNA. Separate reactions were run for MMP-9 and
GAPDH. Amplification was performed with a PowerBlock system (Ericomp)
for 35 cycles (denaturation at 94°C for 30 seconds, annealing for 30
seconds [MMP-9, 56°C; GAPDH, 60°C], extension at 72°C for 30
seconds).
Analysis
After competitive PCR, the products corresponding to
SCDNA and to ESDNA were separated by gel electrophoresis in 1.35% or
1.8% agarose with 0.5 mg/mL ethidium bromide and photographed
on Polaroid 667 film. The film negatives were scanned with the model
GS-670 imaging densitometer (BioRad). Peak areas for both amplified
products (SCDNA area and ESDNA area) were calculated by Molecular
Analyst software. The ratio of ESDNA area to SCDNA area was calculated
for each PCR reaction and corrected for the lower ethidium bromide
incorporation of ESDNA as follows (in number of copies): SCDNA area
(corrected)=ESDNAx(ESDNA length/SCDNA length). The correction
coefficient was 0.654 for GAPDH and 0.744 for MMP-9.
Corrected ratios of ESDNA area/SCDNA area were plotted on the y axis against the number of ESDNA copies in each corresponding PCR reaction. A regression curve with correlation coefficient >0.9 was fitted for PCR reactions performed on each cDNA sample by Mathematica software (Wolfram Research). The specific SCDNA template in each starting cDNA sample was equimolar to ESDNA when that ESDNA area/SCDNA area (corrected) was equal to 1. ESDNA concentration and SCDNA concentration in number of copies per microliter (corresponding to ESDNA area/SCDNA area=1) were obtained graphically from the regression curve.
To normalize for variations in the amount of starting specimen RNA and in the efficiency of the reverse transcription reactions for each specimen tested, the mRNA transcript number was expressed as a ratio of GAPDH mRNA transcripts determined via separate competitive PCR reactions on the same SCDNA.
ANOVA statistics were used to test for significant differences in MMP-9 levels between specimens grouped by aortic size. A Tukey adjustment was used to control for multiple comparisons. Statistical significance was set at 95% (P=.05). All results were reported as mean±SEM.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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There was a statistically significant elevation in MMP-9/GAPDH (No. of copies MMP-9/No. of copies GAPDH) levels in aneurysms ranging in diameter from 5 to 6.9 cm (98.06±15.19 MMP-9/GAPDH) compared with either small aneurysms ranging in size from 3 to 4.9 cm (20.87±5.15 MMP-9/GAPDH, P<.03), large aneurysms with diameters >7 cm (27.16±14.56 MMP-9/GAPDH, P<.01), or normal aortic specimens (3.57±1.13 MMP-9/GAPDH, P<.003). The Figure
demonstrates the fivefold elevation of MMP-9 levels in moderate-size
aneurysms compared with small aneurysms and the roughly
fourfold elevation compared with large aneurysms. The results
did not change significantly when the MMP-9/GADPH ratios were
normalized to patient height (Table 2),
nor was there any significant difference in the atherosclerotic risk
factors, age, or sex of patients in each aneurysm group (Table 3).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Three decades ago, identification of the relationship between aortic diameter and risk of aneurysm rupture established a rationale for operative repair and presumably prolonged the lives of countless subsequent patients. Although most now accept the concept that aneurysms >5 cm merit repair, few mechanistic data are available to explain why or how larger aneurysms continue to expand or rupture. Although biomechanical forces and Laplace's law certainly have a role, these mechanical arguments do not fully explain the observed exponential relationship between diameter and risk of rupture that begins abruptly at 6 cm, nor do they account for the significant risk of rupture observed even in small aneurysms.27
Recently, the inflammatory model of aneurysm formation has gained increasing acceptance. In this model, an aortic wall inflammatory response to an as yet undetermined antigen results in ongoing matrix destruction and eventual aortic expansion. Despite the ever-increasing volume of literature detailing elevated expression and protein levels of MMPs in aortic aneurysms, few reports in the literature address the relationship between aortic aneurysm diameter and MMP expression. A single study using immunohistochemistry suggests a differential pattern of metalloproteinase production in large and small aneurysms.10 The authors identified qualitative increases in MMP-9 in larger aneurysms, whereas MMP-2 appeared to predominate in smaller aneurysms. Our findings detailing the relationship between MMP-9 mRNA expression and aortic size represent the first quantitative study.
Our results demonstrate a fourfold to fivefold elevation in MMP-9 mRNA expression by aneurysms with diameters between 5 and 6.9 cm compared with either small (3 to 4.9 cm) or large (>7 cm) aneurysms. This suggests that elastolytic activity may be maximal in moderate-size aneurysms, although no data relating enzymatic activity to aneurysm diameter are available as yet. The peak of MMP-9 mRNA expression in moderate-size aneurysms may be related to the degree of inflammation. Previous studies from our own laboratory using in situ hybridization have localized MMP-9 mRNA expression to macrophages within adventitial inflammatory infiltrates, whereas other studies have demonstrated increasing numbers of inflammatory cells in aneurysms of increasing size (up to 7 cm).9 10 28 However, this does not explain why larger aneurysms produce less MMP-9 mRNA. Although there is no clear explanation, it is possible that an absence of substrate elastin in large aneurysms resulted in decreased expression via feedback mechanisms. Alternatively, because little is known about the relative amount of inflammation in aneurysms >7 cm, very large aneurysms may have few inflammatory cells and consequently low MMP activity. The latter hypothesis would suggest that mechanical forces have more to do with the observed rapid expansion of very large aneurysms than do inflammation-mediated biological processes. It is also possible that in large aneurysms, MMPs other than MMP-9 are responsible for ongoing aortic expansion and that diameter-dependent alterations in mechanical forces, such as shear stress, result in alterations in the patterns of MMP expression.
It is interesting to note that the greatest variability in MMP-9 mRNA levels also occurred in moderate-size aneurysms. This corresponds to the clinical observation that not all aneurysms expand at the same rate. It is tantalizing to speculate that aneurysms with increased MMP-9 mRNA expression might expand at a faster rate than would those displaying lower levels of expression. Unfortunately, in our own study, serial scans were unavailable for most patients (because patients are generally referred to our specialty center specifically for repair), and no data regarding the rate of expansion were available. Studies designed to correlate that rate of expansion with MMP levels are needed. If present, such relationships could be exploited; tests designed to measure MMP activity might allow one to determine the propensity for expansion of small to moderate-size aneurysms and consequently the need for early repair.
One important question relating to this study and many others concerns the role of translational and posttranslational modification on MMP-9 protein levels. The efficiency of translation of increased MMP-9 mRNA levels observed in moderate-diameter aneurysms is unknown. However, previous authors have identified increased MMP-9 protein levels in aneurysms as opposed to normal aortas,4 5 8 9 29 and the present work supports our previous findings demonstrating increased expression of MMP-9 mRNA in aneurysms.9 12 Thus, identification of elevated MMP-9 expression in moderate-diameter aneurysms, in combination with previous studies demonstrating corresponding increases in MMP-9 activity in aneurysms, suggests that the increases in MMP-9 mRNA levels are in fact reflected in the elastolytic activity of the aneurysm.
Another important question concerns whether factors other than
aneurysm size, such as variability in atherosclerotic risk
factors or in the mean diameter of the unaffected aorta, could account
for the observed differences in MMP-9 expression between groups. After
the results were normalized to patient height to control for
differences in mean aortic diameter, the same pattern of MMP-9 mRNA
expression was observed, including the statistically significant
elevation in moderate-size aneurysms. In addition, no
significant differences between groups were observed in relation to
age, sex, or atherosclerotic risk factors (Table 2).
As with any study using reverse transcription–based competitive PCR for mRNA quantification, adequate controls are extremely important and limitations in the accuracy of the technique must be acknowledged. The most important controls test for contamination of cDNA samples by genomic DNA. This study used DNAase to completely digest all genomic DNA after tissue mRNA extraction but before reverse transcription. As mentioned in the "Methods" section, for every DNAase-treated tissue mRNA sample reverse transcribed to SCDNA, there was a corresponding control reaction in which reverse transcriptase was intentionally omitted (RT-). Before cDNA was used for quantification, the RT mixture was run as a template in PCR reactions. If any product was seen (corresponding to genomic contamination), the cDNA was discarded, the tissue RNA sample retreated with DNAase, and the reverse transcription reaction repeated. Our study also used a second form of internal control: namely, each specimen was run multiple times with serial dilutions of external standard before copy numbers were determined. It is unlikely that a cross-contamination involving a single PCR reaction could affect the results. In fact, regression analysis of band densities confirmed correlation between the many separate reactions run for each particular enzyme on each individual sample. Finally, competitive reverse transcription PCR has recognized limitations.30 31 Specifically, slight differences in size and guanine-cytosine content between specimen and external standard products limit the absolute accuracy of the technique. Within these limitations, competitive PCR remains an established method for quantification of mRNA levels for multiple enzymes in small tissue samples.30 31
We conclude that MMP-9 mRNA expression is significantly increased in moderate-diameter (5- to 6.9-cm) aneurysms compared with either small (<4.0-cm) or large (>7.0-cm) aneurysms. This increase in MMP-9 expression may account for the observed propensity of aneurysms >5 cm to continue to expand, in contrast to smaller aneurysms. However, lower levels of expression in aneurysms >7 cm suggest that increases in the expression of other enzymes or in diameter-dependent mechanical stress on the aortic wall are responsible for the more rapid expansion and high rupture rates that characterize very large aneurysms. Further work relating MMP-9 expression to aneurysm growth rates is needed.
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Selected Abbreviations and Acronyms |
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Received March 17, 1997; revision received May 12, 1997; accepted May 20, 1997.
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References |
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H. Nakashima, M. Aoki, T. Miyake, T. Kawasaki, M. Iwai, N. Jo, M. Oishi, K. Kataoka, S. Ohgi, T. Ogihara, et al. Inhibition of Experimental Abdominal Aortic Aneurysm in the Rat by Use of Decoy Oligodeoxynucleotides Suppressing Activity of Nuclear Factor {kappa}B and ets Transcription Factors Circulation, January 6, 2004; 109(1): 132 - 138. [Abstract] [Full Text] [PDF]
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P. W.M. Fedak, M. P.L. de Sa, S. Verma, N. Nili, P. Kazemian, J. Butany, B. H. Strauss, R. D. Weisel, and T. E. David Vascular matrix remodeling in patients with bicuspid aortic valve malformations: implications for aortic dilatation J. Thorac. Cardiovasc. Surg., September 1, 2003; 126(3): 797 - 805. [Abstract] [Full Text] [PDF]
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E. F. Steinmetz, C. Buckley, and R. W. Thompson Prospects for the Medical Management of Abdominal Aortic Aneurysms Vascular and Endovascular Surgery, May 1, 2003; 37(3): 151 - 163. [Abstract] [PDF]
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C. A. Warnes and J. S. Child Aortic Root Dilatation After Repair of Tetralogy of Fallot: Pathology From the Past? Circulation, September 10, 2002; 106(11): 1310 - 1311. [Full Text] [PDF]
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Z. S. Galis and J. J. Khatri Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: The Good, the Bad, and the Ugly Circ. Res., February 22, 2002; 90(3): 251 - 262. [Abstract] [Full Text] [PDF]
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S. Saito, N. Zempo, A. Yamashita, H. Takenaka, K. Fujioka, and K. Esato Matrix Metalloproteinase Expressions in Arteriosclerotic Aneurysmal Disease Vascular and Endovascular Surgery, January 1, 2002; 36(1): 1 - 7. [Abstract] [PDF]
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G. Sangiorgi, R. D'Averio, A. Mauriello, M. Bondio, M. Pontillo, S. Castelvecchio, S. Trimarchi, V. Tolva, G. Nano, V. Rampoldi, et al. Plasma Levels of Metalloproteinases-3 and -9 as Markers of Successful Abdominal Aortic Aneurysm Exclusion After Endovascular Graft Treatment Circulation, September 18, 2001; 104 (2009): I-288 - I-295. [Abstract] [Full Text] [PDF]
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M. Lindsey, K. Wedin, M. D. Brown, C. Keller, A. J. Evans, J. Smolen, A. R. Burns, R. D. Rossen, L. Michael, and M. Entman Matrix-Dependent Mechanism of Neutrophil-Mediated Release and Activation of Matrix Metalloproteinase 9 in Myocardial Ischemia/Reperfusion Circulation, May 1, 2001; 103(17): 2181 - 2187. [Abstract] [Full Text] [PDF]
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K. Niwa, J. K. Perloff, S. M. Bhuta, H. Laks, D. C. Drinkwater, J. S. Child, and P. D. Miner Structural Abnormalities of Great Arterial Walls in Congenital Heart Disease : Light and Electron Microscopic Analyses Circulation, January 23, 2001; 103(3): 393 - 400. [Abstract] [Full Text] [PDF]
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K Kozuma, M.A Costa, M Sabate, C.J Slager, E Boersma, I.P Kay, J.P.A Marijnissen, S.G Carlier, J.J Wentzel, A Thury, et al. Relationship between tensile stress and plaque growth after balloon angioplasty treated with and without intracoronary beta-brachytherapy Eur. Heart J., December 2, 2000; 21(24): 2063 - 2070. [Abstract] [PDF]
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D. P. Mason, R. D. Kenagy, D. Hasenstab, D. F. Bowen-Pope, R. A. Seifert, S. Coats, S. M. Hawkins, and A. W. Clowes Matrix Metalloproteinase-9 Overexpression Enhances Vascular Smooth Muscle Cell Migration and Alters Remodeling in the Injured Rat Carotid Artery Circ. Res., December 3, 1999; 85(12): 1179 - 1185. [Abstract] [Full Text] [PDF]
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L. J. Walton, I. J. Franklin, T. Bayston, L. C. Brown, R. M. Greenhalgh, G. W. Taylor, and J. T. Powell Inhibition of Prostaglandin E2 Synthesis in Abdominal Aortic Aneurysms : Implications for Smooth Muscle Cell Viability, Inflammatory Processes, and the Expansion of Abdominal Aortic Aneurysms Circulation, July 6, 1999; 100(1): 48 - 54. [Abstract] [Full Text] [PDF]
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L. E. P. Rohde, L. H. Arroyo, N. Rifai, M. A. Creager, P. Libby, P. M. Ridker, and R. T. Lee Plasma Concentrations of Interleukin-6 and Abdominal Aortic Diameter Among Subjects Without Aortic Dilatation Arterioscler Thromb Vasc Biol, July 1, 1999; 19(7): 1695 - 1699. [Abstract] [Full Text] [PDF]
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J. R. Parra, R. A. Cambria, J. A. Freischlag, G. R. Seabrook, and J. B. Towne Smoking Increases Proteolytic Activity in the Human Abdominal Aorta Vascular and Endovascular Surgery, November 1, 1998; 32(6): 617 - 622. [Abstract] [PDF]
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P. K. Shah Inflammation, Metalloproteinases, and Increased Proteolysis : An Emerging Pathophysiological Paradigm in Aortic Aneurysm Circulation, October 7, 1997; 96(7): 2115 - 2117. [Full Text]
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S. Uemura, H. Matsushita, W. Li, A. J. Glassford, T. Asagami, K.-H. Lee, D. G. Harrison, and P. S. Tsao Diabetes Mellitus Enhances Vascular Matrix Metalloproteinase Activity : Role of Oxidative Stress Circ. Res., June 22, 2001; 88(12): 1291 - 1298. [Abstract] [Full Text] [PDF]
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