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Regular Articles |
From the Departments of Pharmacology*
and
Cardiovascular Research,
Berlex
Biosciences, Richmond, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, Daugherty and colleagues7 reported that chronic infusion of angiotensin II (Ang II) induces AAA in 33% of female apoE-KO mice. The aneurysms were characterized by complex tissue remodeling in the adventitia similar to human disease. In addition, the aneurysmal tissue also showed exacerbated vascular inflammation, such as monocyte/macrophage infiltration in the vascular wall. Ang II has been shown to induce the expression of interleukin (IL)-6.8-10 IL-6 is a marker of vascular inflammation and it has been shown to increase in atherosclerotic lesions in apoE-KO mice in association with the infiltrating macrophages.11 In human AAAs, the circulating level of IL-6 has been reported to be elevated12 and positively correlated with aortic diameter expansion.13 Therefore, the aim of this study was to test the hypothesis that Ang II promotes vascular inflammation that in turn activates the uPA-plasmin-MMP system, thus causing aneurysm in apoE-KO mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osmotic minipumps (Alzet, model 2004, Palo Alto, CA) containing either phosphate-buffered saline (PBS) or Ang II (1.44 mg/kg/day) were implanted subcutaneously in 6-month-old male apoE-KO mice. After 30 days, blood pressure was measured noninvasively by the tail-cuff method. The diameter of the abdominal aorta was measured noninvasively by miniaturized ultrasound, and validated postmortem by direct measurement of cross sections of the suprarenal aorta. Serum cholesterol and triglycerides levels were measured by Consolidated Veterinary Diagnostics (West Sacramento, CA).
Noninvasive Measurement of Systolic Blood Pressure
Systolic blood pressure and heart rate were measured in conscious mice using a tail-cuff system (Kent Scientific, Litchfield, CT). Mice were trained to lie quietly in a restrainer placed on a warm pad for a period of at least 30 minutes for 1 to 4 days before the study. On the day of the study, the mice were placed in the temperature-controlled restrainer for 15 minutes. Blood pressure was then measured repeatedly and recorded on a data acquisition system (PowerLab, 16/s; ADInstruments, Australia). Systolic blood pressure and heart rate were averaged from consecutive five measurements.
Measurement of Aortic Diameter
Noninvasive Measurement by Ultrasound
A cardiovascular ultrasound system (GE Vingmed System FiVE, Fairfield, CT) and a linear transducer (10 MHz) were used to image the suprarenal aorta noninvasively in mice anesthetized with 2.5% isoflurane via an anesthesia machine (IMPAC6; VetEquip, Pleasanton, CA). A longitudinal view of the suprarenal aorta was obtained by sagittally scanning the abdomen with the transducer. Confirmation of the correct location of the aorta was achieved by pulsatile flow-velocity waveforms from the middle of the aortic chamber on the image. The segment with the maximum diameter and the adjacent segment of the suprarenal aorta were located on the longitudinal image. The two cross-sections were scanned transversely to obtain images for measurement of the luminal diameters of the aneurysmal and adjacent portions of the suprarenal aorta. The images were stored digitally and analyzed with an on board computer with a resolution of 0.01 mm.
Postmortem Direct Measurement
Direct measurements from the suprarenal aorta were made histologically (as described in the next section). The lumen and adventitial circumferences at the maximal expended portion of the suprarenal aorta were quantified by CSimple Imaging Systems (Compix, Mars, PA) that was then used to calculate the luminal and outer diameters of the vessel. The wall thickness was calculated from the difference between the luminal and outer diameters.
Histopathological Examination
Tissue Preparation
At the end of each experiment, the aortae were perfused at a constant pressure of 100 mmHg through the heart with PBS followed by warm (37°C) agarose (SeaPlaque GTG Agarose, low-melt; FMC BioProducts, Rockland, ME) diluted in saline (3% w/v) and colored with a green tissue dye. After the agarose had solidified, the abdominal aorta was dissected free from the surrounding connective tissue and pinned onto a wax block before fixation in 10% formalin. Cross-sections of aorta (2.5 mm in thickness) were made between the superior mesenteric and right renal arteries. A small portion of the right renal artery was left attached to the samples to facilitate orientation of the specimen. These tissues were dehydrated through a graded ethanol series, cleared with xylene, infiltrated with warm paraffin, embedded in paraffin blocks, cut at 5-µm thickness, and stained with hematoxylin and eosin. Elastin and collagen were stained by the elastin-van Gieson method.
Quantification of Atherosclerotic Plaques in the Carotid Arteries
The left and right carotid arteries were dissected, cut open longitudinally, and pinned down individually on silicon-coated Petri dishes. Atherosclerotic plaques are visible without staining. The images of the open luminal surface of the vessels were captured with a digital camera (Sony, Japan) mounted on a dissecting microscope. The plaque area was quantified using C-Simple system (Compix, Mars, PA) and expressed as a percentage of the total luminal surface area as described in detail previously.14
Recombinant Mouse uPA
Mouse uPA from amino acids 146 to 434 (with deletion of most of the A chain and cysteine 296 to serine mutation) was expressed in a BaculoGold Baculovirus system (Pharmingen, San Diego, CA). The expressed recombinant protein was purified by benzamidine-Sepharose affinity chromatography.
Determination of uPA, PAI-1, tPA, and MMP Expression
Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Tissue and Total RNA Preparation: Mice were anesthetized in CO2/O2 and the entire aorta rapidly dissected from fat and connective tissues. The arch and abdominal aorta were separated, transferred to vials, and snap-frozen in liquid nitrogen. Tissues were homogenized with an Omni TH homogenizer (Omni International, Warrenton, VA) at 4°C in denaturing solution (Ambion, Austin, TX). Total RNA was isolated using a RNAaqueous-4 PCR kit from Ambion.
Primers and Probe Design: Primers and probes for the uPA, tPA, and PAI-1 genes were designed to recognize the mouse uPA, tPA, and PAI-1 sequences by using the computer program Primer Express (Perkin-Elmer Applied Biosystems, Foster City, CA). The oligonucleotide primers and TaqMan probes were purchased from Synthetic Genetics (San Diego, CA). The primers and a probe for the housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were purchased from Perkin-Elmer as rodent GAPDH control reagent kits (vic-labeled probe). Specific uPA sequences for TaqMan primers and probe were forward primer: CGA TTC TGG AGG ACC GCT TA, reverse primer: CCA GCT CAC AAT CCC ACT CA, and probe CTG TAA CAT CGA AGG CCG CCC AAC T. Those for PAI-1 were forward primer: TGC ATC GCC TGC CAT T, reverse primer: CTT GAG ATA GGA CAG TGC TT, and probe: TGG CCC ATG GCA CCC TCC A. Those for tPA were forward primer: CAA CAG CGG CCT GGT ACA A, reverse primer: CCC CAT TGA AGC ATC TTG GTT, and probe: CTC AGT GCC TGT CCG AAG TTG CAG C.
Procedures: Reverse transcriptase (RT) generation of cDNA and PCR was performed by one-step RT-PCR using the TaqMan Gold RT-PCR kit (Perkin-Elmer Applied Biosystems, Foster City, CA). The thermal cycling parameters were: 30 minutes at 48°C for RT, AmpliTag Gold activation for 10 minutes at 95°C, and 40 cycles of PCR (denature for 15 seconds at 95°C and annealing/extension for 1 minute at 60°C). A standard curve was constructed with a 4-point dilution curve (0.3, 3, 30, 300 ng) of total RNA from mouse testis. A control without template was included with each PCR. Samples and standard curves were run in triplicate for each PCR experiment. The quality of the PCR data were determined from the correlation coefficient of the standard curve, and the data were then normalized to the mRNA level of the housekeeping gene, GAPDH.
Immunoblotting
Mouse AAA segments were pooled together (average of 10 mice) from each experimental group. Tissues were homogenized in PBS containing 0.1% Tween 20 (PBST) to extract proteins. Protein concentrations were determined by the method of Bradford using the Coomassie protein assay reagent from Pierce (Rockford, IL). Six hundred µg of protein sample was mixed with sample buffer (in a final concentration of 63 mmol/L Tris-HCl, 10% glycerol, and 2% sodium dodecyl sulfate) and separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane (BioRad, Hercules, CA) that was blocked with a 10% solution of nonfat dried milk in PBST for 1 hour at room temperature. The membrane was incubated with rabbit anti-rodent uPA antibody (1190, 5 µg/ml in PBST; American Diagnostica Inc., Greenwich, CT) overnight at 4°C, and washed three times with PBST. The membrane was subsequently incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After washing four times with PBST, the immunoreactive protein bands were detected by using the enhanced chemiluminescence kit (Amersham, Piscataway, NJ).
Zymography
Protein extracts were prepared from aortic tissues as described above. Samples were resolved under nonreducing, denaturing conditions, on a 12% acrylamide gel (cast with 1% nonfat dry milk and 5 µg/ml human plasminogen) for uPA activity and on a 10% gel containing 0.1% gelatin as the substrate for MMP activity. After electrophoresis proteins were renatured by washing in Triton X-100. The gels were then incubated overnight at 37°C in 50 mmol/L Tris-HCl, 200 mmol/L NaCl, 5 mmol/L CaCl2. Zones of lysis were visualized by staining with 0.5% Coomassie blue R-250.
Ex Vivo Aorta Organ Culture and IL-6 Secretion Assay
The aortas from apoE-KO mice treated with vehicle (n = 6) or Ang II (n = 5) were dissected aseptically and cut into three segments: aortic arch, suprarenal aorta, and the rest of the abdominal aorta. The fresh aortic segments were immediately placed in 1 ml of Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD) containing 1x ITS (insulin, transferrin, and selenium; Life Technologies, Inc.) and 0.1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO). The secretion of IL-6 was measured at different time points by using a commercially available murine IL-6 enzyme-linked immunosorbent assay (Biotrak IL-6 ELISA kit; Amersham Life Sciences, Arlington Heights, IL) as described.11 The IL-6 values were normalized to the weight of the aortic tissue segment and expressed as pg/ml per mg tissue.
Statistics
Results are presented as means ± SE for the number of animals (n) indicated. Comparison between two groups with different treatments was performed by Student’s t-test. The differences were considered statistical significant when the P value was less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, chronic
infusion of Ang II in apoE-KO mice for 1 month did not significantly
affect body weight, or cholesterol and triglyceride levels. However,
systolic blood pressure was higher and the atherosclerotic plaque area
in the carotid arteries was significantly greater in apoE-KO mice
treated with Ang II compared to vehicle treatment.
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Chronic infusion of Ang II for 1 month resulted in development of
AAA in all animals. Fifty-eight percent of the mice had large aneurysms
(>100% expansion) that were consistently localized to the left
lateral aspect of the suprarenal region of the aorta (Figure 1)
. None of the vehicle-treated apoE-KO
mice developed an aneurysm. Figure 2
shows an ultrasound image from a mouse treated with Ang II measured
noninvasively. The longitudinal view shows the characteristic
dilatation of the abdominal aorta in the suprarenal region (Figure 2
,
top). The inner diameter of the aneurysm (Figure 2
, bottom left) was
2.5-fold larger than the adjacent nonaneurysmal portion measured in
a cross-section (Figure 2
, bottom right). Direct measurements of
histological sections of the suprarenal aorta of the apoE-KO mice
treated with either Ang II or vehicle demonstrate that Ang II increased
the outer diameter, inner diameter, and vascular wall thickness by
75%, 60%, and 129%, respectively (Figure 3)
.
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Ang II treatment produced a distinct pathology in the supra renal
aorta. Pathological changes could be seen as early as 10 days of
treatment with four out of six mice showing inflammation in the
adventitia. In one case, there was focal, full-thickness necrosis of
the aortic wall characterized by an acellular, pale eosinophilic area
(Figure 4A)
. One of six aortae appeared
normal whereas another was characterized by mild atheroma
formation with adjacent focal elastin fragmentation. At day 20,
inflammatory cell infiltrates, both lymphocytic and monocytic cells,
were seen entering the lesion from the adventitial area. There were
also foci of acute hemorrhage present (Figure 4B)
.
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.
There was evidence of both acute and chronic hemorrhage in the
adventitia. In one mouse, a large neo-lumen formed within the
adventitia directly adjacent to a relatively undamaged segment of the
aorta. The wall of this neo-lumen was composed of a mature granulation
tissue containing histiocytes and a few lymphocytes embedded in a thin
collagen matrix. The neo-lumen appeared to be connected to the aorta as
shown by the presence of green-colored agarose in the space.
Elastin fragmentation and degeneration of the media sometimes occurred
in the outer lamellae close to areas of adventitial inflammation. In
other vessels, focal destruction of the inner elastic lamina was found
in association with focal atheroma formation (Figure 4C)
. In over half
the cases there was focal, full thickness destruction of the elastic
lamellae (Figure 4D)
. In addition, even in areas of intact media, the
smooth muscle cells appeared hypertrophied as compared to controls. The
aortae of the age-matched apoE-KO mice treated with vehicle appeared
completely normal.
Secretion of IL-6 Protein from the Isolated Aorta
To determine the IL-6 level in Ang II-induced aneurysm, we
measured the ex vivo secretion of IL-6 protein from the
freshly isolated aortae separated into the atherosclerotic aortic arch,
suprarenal, and infrarenal abdominal aorta. Ang II increased secretion
of IL-6 protein from the aneurysmal segment and aortic arch by 4.2-fold
and 2.4-fold, respectively, compared to the comparable aortic segments
from vehicle-treated mice (Figure 5)
. Ang
II had no effect on IL-6 production in the infrarenal nonaneurysmal
segments of the abdominal aorta.
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The expression of uPA, tPA, and their major inhibitor, PAI-1, was
studied with real-time quantitative RT-PCR. As shown in Figure 6A
, the expression of uPA mRNA was
increased significantly (13-fold) in the aneurysmal portion of the
abdominal aortae in apoE-KO mice treated with Ang II compared to
vehicle-treated controls. This compares with a twofold increase in uPA
mRNA in the atherosclerotic aortic arch from Ang II-treated animals. In
addition, the expression of PAI-1 mRNA was increased sixfold in the
aneurysmal tissue compared to that of vehicle-treated controls (Figure 6B)
. On the other hand, the expression of tPA was decreased 50% in
the aneurysmal tissue (Figure 6C)
.
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. The recombinant mouse uPA (rm-uPA)
was blotted with the same antibody showing that the antibody is
recognizing mouse uPA. Activated uPA was detected from the aneurysm
tissues on the zymographic gel comparable to a mouse kidney extract
containing high uPA activity (Figure 7B)
. MMP-2 and -9 activity was
also examined by zymography. Although the MMP-9 activity was increased
in the aorta of apoE-KO mice compared with that of wild type, there was
no significant difference in MMP-9 activity between the aneurysmal
section of the abdominal aorta and the nonaneurysmal aortic arch in
apoE-KO mice (Figure 7B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Daugherty and colleagues7
reported that approximately
one-third of the female apoE-KO mice developed bulbous aneurysm after
chronic infusion of Ang II for 1 month. By using the same method (1.44
mg/kg/day Ang II for 1 month) in male 6-month-old apoE-KO mice, we
observed that 100% of the mice developed aneurysms; 58% with large
aneurysms (>100% expansion of the aorta) and the rest with small
aneurysms (<100% expansion). The discrepancy between the two
laboratories may be the results of the sex difference and from the
subjectively different criteria in determination of the bulbous and
large aneurysm. To quantify the diameter of the vessel, we measured the
cross-sectional diameter directly in the aorta perfused with agarose
under normal pressure to prevent collapse of the vessel after animal
death. This measurement showed not only the expansion of the outer
diameter by 75%, but also an expansion by 60% of the lumen in apoE-KO
mice treated with Ang II compared to those with vehicle. Most
importantly, it revealed that the aortic wall thickness increased
125%. The thickening was because of a reactive fibrosis with a
predominantly chronic (mononuclear) inflammatory cell infiltrate. In
addition, we also developed an ultrasound method to measure the
diameters of the aorta with a resolution of 0.01 mm. This is the first
reported application of this technique in mice. The luminal expansion
was observed noninvasively under normal pressure. Despite increased
aortic wall thickness and localized intramural hemorrhage, the lumen
was found uniformly dilated in aneurysm (Figure 2)
. The measurement was
validated by postmortem direct measurement. Therefore, this technique
can be used for longitudinal monitoring of AAAs in small animals, such
as mice and rats, as well as following the progressive effects of
potential therapies.
Ang II is a potent vasocontractor and can cause hypertension. In the present study in conscious male apoE-KO mice Ang II increased blood pressure by 22 mmHg at 1 month. In a recent report, active uPA was also found to promote vascular smooth muscle contraction.16 It could be that the increased active uPA in aneurysm contributes to the elevation of blood pressure. It is also possible that the hemodynamic stress resulting from hypertension may contribute to aneurysm formation in our model.
Macrophages and foam cells are the major source of proteases including
uPA and pro-MMPs in the atherosclerotic plaque.1,2,4,17
Many pro-MMPs are activated by plasmin which, in turn, is activated by
uPA. uPA expression was increased 13-fold in the aneurysmal segment of
Ang II-treated apoE-KO mice compared to vehicle-treated controls. In
contrast, the expression of uPA was increased only twofold in the
macrophage-rich but not dilated atherosclerotic aortic arch of the Ang
II-treated mice. We also demonstrated the increased protein and
activity of uPA in aneurysm tissues, suggesting that the increased
PAI-1 (with a lesser increase in mRNA compared to that of uPA) was not
enough to inhibit uPA activity (Figure 7)
. Consistent with these
findings, increased expression of uPA,18,19
MMPs,20,21
as well as PAI-1,1
have been
reported in human aortic aneurysms.
We also found that the expression of tPA mRNA was reduced in the
aneurysm tissues. This is most likely caused by the depletion of smooth
muscle cells and endothelial dysfunction in the aneurysm as histology
showing the replacement of smooth muscle cell by noncellular structures
in the media (Figure 4)
. In the vehicle-treated wild-type mouse, we
found that the majority of plasminogen activities are from tPA but not
uPA on a zymographic gel (data not shown). These data are consistent
with previous reports showing that tPA is mainly produced by smooth
muscle cells and that uPA mainly by inflammatory cells.
Both MMP-2 and MMP-9 are expressed in human AAA tissue and are the
major elastolytic enzymes secreted by human AAA tissues in organ
culture.4,22
Recently, MMP-12 was reported to be
prominently expressed by aneurysm-infiltrating macrophages within the
media of human AAA.5
The roles of MMP-9 and MMP-12 in the
pathogenesis of AAA have been investigated in a mouse model of
elastase-induced aneurysm. In this model, Pyo and
colleagues23
found that MMP-9- but not
MMP-12-deficient mice were protected from aneurysm formation,
suggesting that MMP-9 plays a more critical role than MMP-12 in this
experimental model. In the present study, whereas MMP-9 was increased
in both the aneurysmal segment of the aorta and in the atherosclerotic
aortic arch compared to that of wild-type mice, MMP-9 activity
was not specifically increased in the aneurysm (Figure 7B)
.
Although the exact mechanism(s) by which Ang II induces aneurysm in
apoE-KO mice is not clearly understood, its proinflammatory actions may
be important. Chronic vascular wall inflammation has been proposed to
play an important role in the pathogenesis of AAA
formation.24-26
Ang II promotes recruitment of
inflammatory cells to the vessel wall by inducing the expression of
monocyte chemoattractant protein-1.27
Ang II increases
low-density lipoprotein oxidation28
and by interacting
with the Ang II subtype-1 (AT1) receptor on macrophage increases
12/15-lipoxygenase activity,29
suggesting that it may
exacerbate atherosclerosis. Indeed, the present study also demonstrated
that Ang II exacerbates atherosclerosis in apoE-KO mice.
Histologically, the chronic inflammation and reactive collagen
formation found in the adventitia suggests a chronic, resolving process
whose initial event is unknown. The presence of hemosiderin in some
adventitiae suggests that hemorrhage may have played a role in
eliciting the reactive adventitial changes. Moreover, Ang II
has also been shown to induce the expression of IL-6.8-10
IL-6 is increased in atherosclerotic lesions in apoE-KO mice in
association with the infiltrating macrophages.11
In human
AAAs, the circulating level of IL-6 has been reported to be
elevated12
and positively correlated with aortic diameter
expansion.13
Consistent with these observations, we showed
that ex vivo secretion of IL-6 from the aneurysmal segment
of the aorta was increased compared to the aortic arch in Ang
II-treated mice and to the aortic segments from the vehicle-treated
mice (Figure 5)
. These results suggest that the severity of vascular
inflammation measured as IL-6 production may be important in aneurysm
formation.
In summary, we demonstrated that chronic infusion of Ang II in male apoE-KO mouse led to induction of AAAs in 100% of the animals, accompanied by a significant increase in uPA in the aneurysmal segment of the aorta. Macrophage infiltration and focal elastin fragmentation were also observed in the aneurysm. These findings are in agreement with the observations in human AAA, and support a key role for uPA in the pathogenesis of AAA.
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Acknowledgements |
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Footnotes |
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Accepted for publication July 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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