Cigarette smoke enhances abdominal aortic aneurysm formation in angiotensin II-treated apolipoprotein E-deficient mice
Katrin Stollea*, An Bergesb, Michael Lietza, Stefan Lebrunc, Thomas Walleratha
Author affiliations:
a Philip Morris International R&D, Philip Morris Research Laboratories GmbH, Fuggerstr. 3, 51149 Cologne, Germany
b Philip Morris International R&D, Philip Morris Research Laboratories bvba, Grauwmeer 14, B-3001 Leuven, Belgium
c Philip Morris International R&D, Philip Morris Products S. A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland
*Corresponding author
Address: Philip Morris Research Laboratories GmbH, Fuggerstrasse 3, 51149 Cologne, Germany. Phone: ++49-2203-303-554; Fax: ++49-2203-303-362.
E-mail address: Katrin.Stolle@pmintl.com.
Funding: This work was supported in part by Philip Morris USA, Inc. prior to the spin-off of Philip Morris International, Inc. by Altria Group, Inc. on March 28, 2008.
Word count: 5981
ABSTRACT
Cigarette smoke, hyperlipidemia, and hypertension with the risk of development and progression of atherosclerosis and associated pathologies such as abdominal aortic
aneurysm (AAA) are correlated. We examined the interaction of cigarette mainstream smoke (MS) and angiotensin-II (Ang II)-induced hypertension in the atherosclerotic process using hyperlipidemic apolipoprotein E-knockout (ApoE-/-) mice. ApoE-/- mice were treated with Ang II for 4 weeks and then further exposed to MS or to fresh air for 4 weeks. AAA formation was observed in all mice treated with Ang II, regardless of smoke exposure; however, smoke exposure increased the incidence of AAA in these mice. Ang II treatment resulted in higher gene expression of matrix metalloproteinases (MMP)-2, -3, -8, -9, and -12 in the abdominal aortas, which was further increased by MS exposure. The proteolytic activity of MMP-2 and MMP-9 was also enhanced in Ang II-treated mice exposed to MS, but only minor changes were seen with either smoke exposure or Ang II treatment alone. This study shows for the first time that both formation and severity of AAA in hypertensive ApoE-/- mice are
accelerated by exposure to MS and that the proteolytic activity of MMPs is enhanced by the combination of Ang II and MS.
Keywords: Abdominal aortic aneurysm, Angiotensin II, Apo E knockout mice, Cigarette mainstream smoke, Metalloproteinases
1. Introduction
Cigarette smoking is one of the most important and modifiable risk factors associated with the development and progression of cardiovascular disease (CVD) (Schroeter et al., 2008; The United States Surgeon General’s Report, 2004). In the United States, over 830,000 deaths were attributed to CVD in the year 2006, and CVD is the number one cause of death globally. (Lloyd-Jones et al., 2010; WHO, 2009). Cigarette smoking also increases the risk of death from cardiovascular heart disease by two- to three-fold (The United States Surgeon General’s Report, 2004). Epidemiologic studies using carotid intimal-medial thickness as a marker for subclinical disease have demonstrated associations between cigarette smoking and the remodeling of the vasculature, which results in atherosclerosis and CVD (The United States Surgeon General’s Report, 2004). Atherosclerosis is a hardening of the arteries that is often progressive through lipid deposition, fibrosis, and thickening of the arterial wall. Atherosclerotic plaques develop over time, decreasing the space within the lumen of the artery. Eventually, the plaques become destabilized, leading to clinical events such as myocardial infarction or stroke. In addition, atherosclerosis in the abdominal aorta can lead to abdominal aortic aneurysm (AAA) (The United States Surgeon General’s Report, 2004), a pathological phenotype characterized by the remodeling and expansion of the aorta. Current smokers are up to 9 times more likely to develop AAA than nonsmokers, depending on smoking history (Takagi and Umemoto, 2005).
Cardiovascular remodeling is a complex, active, and dynamic process within the extracellular matrix, where phenotypic changes are determined by interplay between plasmin, matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs)
(Janssens and Lijnen, 2006). Because of the high pressure within the aorta, any rupture can quickly lead to death. The renin-angiotensin system plays a crucial role in cardiovascular remodeling. Angiotensin II (Ang II) not only helps to regulate arterial blood pressure but, as a powerful constrictor of blood vessels, also promotes endothelial dysfunction, inflammation, proatherosclerotic cytokine secretion, and vascular remodeling (Montecucco et al., 2009).
Furthermore, atherogenesis and AAA formation have been described as being initiated and stimulated by Ang II in apolipoprotein E-deficient (ApoE-/-) mice (Weiss et al., 2001;
Daugherty et al., 2000; Zhou et al., 2005; Tham et al., 2002).
Ang II treatment has been shown to increase blood pressure and induce AAA formation in various mouse models (Daugherty and Cassis, 2004a, 2004b; Kon and Kabs, 2004; Brasier et al., 2002; Kim and Iwao, 2000). The ApoE-/- mouse is a well established model of atherosclerosis, which develops fibrous arterial plaques corresponding to human atherosclerotic alterations and distributions (Breslow, 1996). Others have shown that sidestream smoke accelerates atherosclerosis in ApoE-/- mice (Matetzky et al., 2000), and
we have previously demonstrated that mainstream smoke (MS) exposure accelerates atherosclerotic plaque formation in these animals (Schroeter et al., 2008; von Holt et al., 2009).
Our previous results suggested a dose-dependent effect of MS on the overall development of atherosclerosis and showed an altered number of elastin-rich layers in the brachiocephalic artery, indicating changes in plaque morphology at 6 and 9 months
(Schroeter et al., 2008). In the current study, Ang II-treated ApoE-/- mice were exposed for 4 weeks to MS to investigate the impact of MS and Ang II on AAA formation. The underlying mechanisms of AAA occurrence by analysis of several MMPs and TIMPs were investigated.
2. Materials and methods 2.1 Study design
Baseline assessments (Phase 0) of blood pressure measurements were followed by two 4-week experimental periods. Animals were treated with Ang II or NaCl alone for the first 4 weeks (Phase 1) via subcutaneous osmotic mini-pumps. For the next 4 weeks (Phase 2), these animals were treated with either whole-body MS or fresh air exposure (sham) in addition to Ang II or NaCl treatment.
2.2 Mice
Male ApoE-/- (ApoE/Bom, B6.129P2_Apoetm1 Ulnc N11) mice of 7 to 8 weeks of age, and bred under specified pathogen-free conditions, were obtained from Taconics Europe (Denmark) and allocated into four groups. Mice were kept on a 12-hour/12-hour light-dark cycle and allowed food and water ad libitum. Two hours after the last inhalation period, animals were sacrificed. This study was conducted at Philip Morris Research Laboratories bvba, Leuven, Belgium. The study was conducted in an AAALAC-accredited facility (Association for Assessment and Accreditation of Laboratory Animal Care
International), where care and use of the mice were in accordance with the American Association for Laboratory Animal Science Policy (C.o.L.S. Institute of Laboratory Animal Research, 1996). All animal experiments were approved by the Institutional Animal Care and Use Committee.
2.3 Osmotic pumps
For Phases 1 and 2 of the study, mini-pumps (Model 2004; Alzet, Cupertino, CA) containing Ang II (1.44 mg/kg/day [Phase 1] or 0.7 mg/kg/day [Phase 2]) or NaCl (0.91%) were implanted subcutaneously in the neck region of anesthetized mice. Pumps were replaced prior to beginning Phase 2 (exposure) of the study.
2.4 MS generation and exposure
During the 4-week exposure phase of the study (Phase 2), Reference Cigarettes 2R4F (University of Kentucky, 2003) were smoked in basic conformity with International Standardization Organization (ISO) Standard 3308 (2000) with some minor deviations as described previously (Schroeter et al., 2008;von Holt et al., 2009). In short, the MS from all cigarettes was generated on a 30-port rotary smoking machine with active sidestream exhaust (type SM2000) that was equipped with a four-piston pump and diluted with filtered, conditioned air. The generation of MS and the delivery to exposure chambers were
performed in a reproducible manner. Monitoring of the total particulate matter (TPM) concentrations in the test atmosphere during the inhalation period showed that the target concentration of 600 μg/l TPM was achieved. The mean concentration (± standard deviation [SD]) was 591.3 ± 22.6 μg/l TPM. The mean carbon monoxide concentration (±SD) in the test atmospheres was 591.9 ± 16.7 ppm. The butt length, puff count, puff volume, and static burning rate parameters were all as expected for the 2R4F reference cigarette.
Mice were placed in whole-body exposure chambers and acclimated to the smoking regimen during the first week. From the second week on mice were exposed for 4 hours/day and 5 days/week. There was a 30-min fresh air break between the 1st and 2nd hour of MS exposure and the 2nd and 3rd hour of MS exposure. Sham-exposed mice were exposed to filtered, conditioned air, with the exposure conditions being the same as those for the smoke-exposed mice.
2.5 Blood pressure measurements
Blood pressure (BP) measurements were obtained for 6 animals per group in all experimental phases using a tail-cuff blood pressure measurement apparatus (Fysicon, Oss, The Netherlands). In Phase 0, the mice were acclimated to the procedure over two days.
Measurements were taken on two different days in each phase of the study. In Phase 2, the mice used for BP measurements were exposed to MS or sham for at least two hours before measurement. Before and during measurement, the temperature of the BP-apparatus was set to 32°C. Mice were placed into restrainers and the tail cuffs were attached. After 10 minutes, BP measurements were taken at least 8 times.
2.6 Pathology
At the end of Phase 2, animals were anesthetized and sacrificed; subsequently, the aortic arch, the brachiocephalic artery, the right carotid artery, and the abdominal artery were dissected with the aid of a dissection microscope. The abdominal aortas were photographed and minimum and maximum diameters of each vessel were determined by digitalized
measurement (DISKUS 4.6 software, Hilgers, Koenigswinter, Germany). The average of the
maximal dilation of the NaCl / sham-exposed animals was 169 µm ± 9.3 µm (mean ± SD). If dilation of the vessel wall exceeded 150% of this diameter, the vessel was considered to have an AAA (Prisant and Mondy, 2004). Vessels were then dissected into different parts for extraction of RNA and protein, and snap-frozen in liquid nitrogen and stored at -80°C until analysis. Aortic arches were analyzed en face to investigate the number of atherosclerotic lesions in the aortic arch as described previously (von Holt et al., 2009). In short, the intimal area covered by plaques was determined on images of longitudinally sliced aortic arches.
The vessels of the truncus brachiocephalicus and the right carotid artery were fixed in 4%
formalin for 4 h and then embedded in paraffin. Cross sections were prepared (5 µm) and mounted onto glass slides. Serial sections were stained with hematoxylin-eosin. Plaque size, measured as area covered by plaques, was determined by image analysis (DISKUS 4.6 software, Hilgers) in at least 5 hematoxylin-eosin-stained sections 100 µm apart. Results were averaged per mouse.
2.7 Expression and activity of MMPs/TIMPs
RNA from abdominal aorta sections was extracted using RNeasy® Micro Kit (Qiagen, Hilden, Germany), quantified and quality-checked with the Nanodrop-1000
Spectrophotometer (Fisher Scientific GmbH, Schwerte, Germany). RNA with an integrity number (RIN) higher than 7 was reverse-transcribed with the high-capacity cDNA archive kit (Applied Biosystems Inc., Darmstadt, Germany). The quantitative reverse transcriptase- polymerase chain reaction (qRT-PCR) was performed in 10 µl reaction volume with the TaqMan Universal PCR Master Mix (Applied Biosystems) using a 384-well ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). Cycling parameterswere as follows:
initial denaturation at 95°C for 10 min, followedby 40 cycles comprising 15 sec at 95°C and 60 sec at 60°C. Relative mRNA quantificationwas performed using the standard curve method (user bulletin no. 2; AppliedBiosystems, 2001). A dilution row of five serial 1:10 dilutionsof cDNA was used to specify the linear range of each pre-designed TaqMan® Gene Expression Assays from Applied Biosystems. All samples were assayedin triplicate and analyzed using the Sequence Detection SystemSoftware v2.2.2 (Applied Biosystems).
Controlswithout reverse transcriptase were performed using the sameprotocol to ensure absence of contaminating DNA.
Pre-designed TaqMan® Gene Expression Assays were used for MMPs and TIMPs:
Mm00439508_m1 (Mmp2), Mm00440295_m1 (Mmp3), Mm00772335_m1 (Mmp8), Mm00442991_m1 (Mmp9), Mm00500554_m1 (Mmp12), Mm00441818_m1 (Timp1), Mm00441825_m1 (Timp2), Mm00441826_m1 (Timp3), and Mm00446568_m1 (Timp4). As reference gene eukaryotic 18S rRNA (Hs99999901_s1) was used.
Tissues for zymography were homogenized in a mixer mill (Qiagen) for 5 minutes at 20 Hz three times with 50 µL of a non-denaturing, sulfobetaine detergent buffer (10 mM CHAPS [Sigma], 150 mM NaCl, and 20 mM Hepes). After centrifugation, the protein content was determined using the Bradford assay (Bio-Rad GmbH, Munich, Germany). Then, 10 μg of protein was loaded onto a gelatinase zymogram (Bio-Rad) for electrophoresis. Following electrophoresis, the gels were equilibrated, stained and de-stained, and scanned for
semiquantitative analysis using the Typhoon™ 8600 imaging system (Amersham Biosciences, Munich, Germany).
2.8 Statistical analysis
If not indicated otherwise, statistical analysis was performed using a two-way analysis of variance (ANOVA) at a significance level of 0.05. If the interaction was significant, the Bonferroni post-test for pairwise comparison between all groups was
performed. For qRT-PCR analyses, the statistical evaluation was performed on the values of the delta of the cycle threshold (gene of interest minus reference gene). Nonparametric statistics on the rank of the values were performed for analysis of semiquantitative
measurements of zymography and plaque determination. Due to the exploratory nature of this study, no adjustments for multiplicity were made between the different endpoints, as a large number of true effects would be overlooked with these adjustments (Bender and Lange, 1999). Analyses of AAA incidences were performed using the exact Fisher test among Ang II treatment with and without MS only. For blood pressure measurements, a one- way ANOVA was applied for Phase 1 and a two-way ANOVA was applied for Phase 2. If the interaction between the two factors in Phase 2 was statistically significant, the multiple Bonferroni post-test for pairwise comparisons between all groups was performed. For blood pressure measurements, the variances between the animals in Phase 0 were rather high, so a background correction was performed. For each animal, the mean value in Phase 0 was calculated and subtracted from all single values in Phase 1 and Phase 2 and the statistical evaluation was performed on the differences between Phase 0 and the other phases and not on the absolute values. With this calculation method, the variance between single animals was reduced.
As MS is known to influence bodyweight (von Holt et al., 2009), the effect on body weight was measured in Phase 2. A two-way ANOVA was carried out on the differences in weight measured at the beginning and the end of Phase 2 for each animal.
3. Results
3.1 Effects on body weight
The mean body weights of all mice were comparable at allocation and gradually increased over time in all groups. No significant differences in body weight were seen after Ang II treatment (Phase 1). MS-exposure (Phase 2) influenced body weight development slightly (6.6% reduction, P < 0.0001) compared to sham, whereas there is no statistical evidence (P = 0.132) for a difference between NaCl and Ang II (Table 1). This is similar to what has been seen previously (von Holt et al., 2009).
3.2 Effects on blood pressure
The blood pressure of all animals (Fig. 1) was predefined to be similar at baseline (Phase 0) and all values were normalized for group variations. The mean systolic blood pressure (SBP) of all animals in Phase 0 was 147 mmHg. Treatment with Ang II (Phase 1) resulted in a significantly higher mean SBP (152 mmHg) compared to NaCl-treated animals (SBP: 143 mmHg; P = 0.006). In Phase 2, an interaction of NaCl and Ang II was observed (P = 0.005). The effect of MS exposure on SBP (156 mmHg) was statistically significant in NaCl-treated animals compared to sham (SBP: 132 mmHg, P ≤ 0.0001), whereas the effect of MS exposuse (SBP: 165 mmHg) on Ang II-treated animals was not significant compared to sham (SBP: 156 mmHg, P = 0.33).
3.3 Effects on plaque development
Animals treated with Ang II developed plaque areas about six-fold greater in size in the aortic arch than the NaCl-treated groups (P ≤ 0.001), independent of MS exposure (Fig.
2). Ang II-treatment resulted in a median plaque area of 67 mm2*10-5 (N=16)in sham animals and of 115 mm2*10-5 (N=14) in MS-exposed animals. NaCl treatment resulted in a median plaque area of 3 mm2*10-5 (N=19) in sham animals and of 11 mm2*10-5 (N=16) in MS-exposed animals (Fig. 2). A similar effect of Ang II, independent of MS exposure, was found in the truncus brachiocephalicus and the right carotid artery (data not shown). Overall, there was no statistically significant effect of MS exposure on plaque formation in any area of the investigated vasculature. However, the highest plaque load was reached with the
combination of Ang II and MS exposure with 115 mm2*10-5.
3.4 Incidence of abdominal aortic aneurysms
No AAA was observed in any mouse treated with NaCl, independent of MS- exposure. In contrast, Ang II-treated mice showed AAA formation, which was further increased by exposure to MS (Fig. 3A and 3B). AAAs occurred in 4 sham-exposed, Ang II- treated animals and in 9 MS-exposed, Ang II-treated animals. In the Ang II-treated/sham- exposure group, severe AAAs were detected in 3 animals, and a small AAA was detected in 1 animal. Severe AAAs were detected in 9 animals treated with Ang II and exposed to MS.
These results indicate that MS enhances the occurrence of AAA in response to Ang II. The exact Fisher test showed an increase in AAA incidence among MS in Ang II compared to Ang II alone, with borderline statistical significance (P-value of 0.0656).
3.5 Expression of MMPs and TIMPs
Analysis of the abdominal aortas showed statistically significant changes in the expression of MMP-2, -3, -8, -9, -12, and TIMP-1, -2, -3, and -4 after Ang II treatment and/or MS exposure (Table 2 with exact P-values and Fig. 4). MMP-2, -3, -8, -9, -12, and TIMP-1 and -2 were significantly up-regulated and TIMP-4 was significantly down-regulated in sham- exposed animals in response to Ang II treatment. TIMP-3 was significantly down-regulated in MS-exposed animals treated with Ang II compared to NaCl. In addition, MMP-2 and -8 were significantly up-regulated in response to MS exposure.
3.6 Proteolytic activity of MMP-2 and MMP-9
Analyses of proteolytic activity showed a significant interaction for Ang II treatment and MS exposure. For MMP-2 activity (N=13), the interaction of Ang II and MS was statistically significant (P = 0.026), with a borderline effect of Ang II/MS compared to Ang II/sham (P = 0.098) and a high statistically significance in Ang II/MS compared to NaCl/MS (P = 0.0045).
For MMP-9 activity (N=14), the interaction of Ang II and MS was statistically significant (P = 0.003) and both comparisons in the Bonferroni post-test were statistically significant (Ang II/MS compared to Ang II/sham [P =0.018] and Ang II/MS versus NaCl/MS [P = 0.005], Fig.
5). As there was no significant change after single treatment, a synergistic effect of Ang II and MS on the proteolytic activity of MMP-2 and MMP-9 is indicated.
4. Discussion
In the present study, Ang II was found to induce AAA formation in ApoE-/- mice. While MS exposure alone had no effect on AAA formation, it did increase AAA formation in Ang II- treated mice. This observation is in line with epidemiologic evidence showing a strong association of smoking as well as of high SBP with a risk of AAA (Palazzuoli et al., 2008). In particular, the significant synergistic effect of MS and Ang II on MMP-2 and MMP-9
proteolytic activity suggests a crucial role for these MMPs in AAA formation. Both MMP-2 and -9 have been suggested to play pivotal roles in AAA development in humans and in animal models (Xiong et al., 2006). MMP-2 is a gelatinase that has been found to contribute to both the breakdown of the extracellular matrix and to the progression of AAAs in humans (Xiong et al., 2006; Thompson et al., 1995). An increased amount of MMP-2 has been found in smaller aneurysms and a higher expression has been described in atherosclerotic plaques
(Freestone et al., 1995). It is this increased expression of MMP-2 that is thought to be responsible for the early elastolysis and aneurysmal degeneration of the abdominal aorta, whereas MMP-9 is associated with all phases of AAA, from early changes in the aortic wall to the later inflammatory response and progression to larger AAAs (Goodall et al., 2001;
Wilson et al., 2006). In support of this notion, the increased expression and activity of MMPs, specifically MMP-2 and MMP-9, as well as the changes in TIMP expression in this study further support the hypothesis that the combination of Ang II and MS can enhance the risk of aneurysm formation and structural changes in the abdominal aorta.
Previous studies have focused on the gelatinases MMP-2 and -9; however, recent investigations suggest that the neutrophil collagenase MMP-8 may be just as important in AAA pathology (Wilson et al., 2006). In our study, MMP-8 expression was found to be up- regulated in response to both Ang II treatment and MS exposure. The structural collagens of the aortic cell wall are highly resistant to degradation, and MMP-8 may assist in the initial degradation of extracellular matrix components contributing to AAA formation (Abdul- Hussien et al., 2007). The findings of increased MMP-8 expression and activity in both growing and ruptured AAA have supported the importance of this neutrophil collagenase in the degradation of the aortic cell wall (Wilson et al., 2006; Abdul-Hussien et al., 2007). In vitro studies have shown that C-reactive protein up-regulates MMP-2 and -9 (Abe et al., 2006; Doronzo et al., 2005). In a TNF-knockout in vivo study, it was shown that cigarette smoke up-regulates pulmonary vascular matrix metalloproteinases via TNF-signaling (Wright et al., 2007). On the basis of these findings, our results of enhanced MMP expression and activity may indicate a possible underlying inflammatory phenotype in aneurysm formation.
In our study, the SBP measurements observed were slightly higher than those previously reported in the literature (Tham et al., 2002; Sakamoto et al., 2001). However, these measurements appeared to decrease over time, and the higher-than-expected measurements may be attributed to an inadequate adaptation period to the restraining device used for measurements. The significantly higher SBP values for Ang II-treated animals compared to NaCl controls confirm the appropriate delivery of Ang II in this study.
However, the effect of Ang II on AAA formation might also be independent of increasing SBP, as others have reported (Cassis et al., 2009).
Literature data on aneurysm mouse models exposed to cigarette smoke has also shown an effect on AAA, although with apparently different outcomes than reported here. In a study with C57/bl6 mice, Ang II induced AAA with half the effect that was seen in our study and the additional intraperitoneal administration of benzo(a)pyrene, a pro-inflammatory constituent of cigarette smoke, enhanced the AAA formation in 58% of the mice. Enhanced macrophage infiltration by benz(a)pyrene has been proposed as the underlying mechanism (Zhang and Ramos, 2008). In an elastase perfusion model of smoke-exposed 129/SvEv
mice, no differences in the incidence of AAA in response to cigarette smoke exposure were observed. However, the aortic dilatation was reported to be 50% greater in smoke-exposed animals compared to controls (Buckley et al., 2004). One possible explanation for the difference in findings between the two studies is the difference in the smoking protocols.
Others have reported that in an elastase perfusion model of C57bl6 mice, exposure to tobacco smoke after a relatively minor aortic elastase injury produces increases in elastin degradation and aneurysm size without affecting MMP-9 or MMP-12 expression (Bergoeing et al., 2007). In our study, however, we used Ang II to induce aneurysms and saw an effect on both MMP-9 and MMP-12 expression in MS-exposed animals. By this, we have clearly shown that the combined application of Ang II and MS results in a high prevalence of AAA formation with higher expression of several MMPs.
5. Conclusions
In conclusion, the animal model presented here seems to be suitable for future studies investigating the mechanisms of hypertension and MS-induced AAAs. The results suggest that MS potentiates the effect of Ang II on AAA formation in this model.
Furthermore, the expression and activity of MMPs are shown to be involved in the underlying mechanism of MS-induced AAA. Future investigations should clarify the structural
remodeling of aneurysms and the underlying mechanisms in this model in further detail.
Acknowledgements
The authors are grateful to the staff of PHILIP MORRIS Research Laboratories in Leuven, Belgium, and Cologne, Germany, for their skillful technical assistance and support, to Etienne Kaelin for statistical advice, and to Lynda Conroy for editorial assistance. This work was supported in part by Philip Morris USA, Inc. prior to the spin-off of Philip Morris International, Inc. by Altria Group, Inc. on March 28, 2008.
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Fig. 1. Blood pressure measurement of a subset of animals (N=6) with the tail-cuff system.
Values were normalized for individual differences within the group, so no standard deviations are available.
Fig. 2. Plaque area on the luminal surface of the aortic arch. Animals treated with Ang II developed significantly more plaques (***P ≤ 0.001) than controls in MS-exposed mice (115 mm2*10-5 versus 11 mm2*10-5) and in sham-exposed mice (67 mm2*10-5 versus 3 mm2*10-5). The effect of MS in Ang II treated mice was not statistically significant.
Fig. 3. (A) Incidence of aneurysms (representative examples). There were no aneurysms detected in NaCl treated animals, independent of MS-exposure.
(B) Incidence of aneurysms: 4 of 15 animals treated with Ang II alone and 9 of 15 animals treated with the combination of Ang II and MS-exposure developed an aneurysm. The effect of MS on the Ang II-induced aneurysms showed borderline statistical significance (P = 0.0656).
Fig. 4. Relative quantification (ΔΔCt method) of mRNA expression of MMPs and TIMPs. 18S rRNA was used as reference gene. Bars show fold changes of the relative quantification (RQ). Error bars represent the maximum (RQmax) and minimum (RQmin) of the RQ. Exact statistics can be found in Table 2.
Fig. 5. Activity of MMP-2 (A) and MMP-9 (B) by semiquantitative zymographic analysis. Bars represent the median with first and third quartiles; error bars represent the minimum and maximum. Because variances were different, the statistical evaluation was performed by non-parametric two-way ANOVA based on rank. A significant interaction shows that the Ang II treatment is only reactive on one component of the other factors. For both MMP-2 (A) and MMP-9 (B), a significant interaction (P[MMP-2 ]= 0.026; P[MMP-9 ]= 0.003) was observed.
The Ang II-treated, MS-exposed group showed higher activity compared to the Ang II- treated, sham-exposed group as well as compared to NaCl-treated, MS-exposed group.
Table 1. Bodyweight of mice was changed in Phase 2 by MS exposure but not by Ang II treatment
NaCl sham Ang II sham NaCl MS Ang II MS
N 10 8 19 16
Beginning Phase 2 (Day 2)
Mean 33.45 33.18 34.25 33.10
End of Phase 2 (Day 29)
Mean 34.93 34.98 33.24 32.74
Difference Day 29-Day 2
Mean 1.48 1.80 -1.01 -0.36
SD 0.464 1.222 1.106 1.245
Ang II, angiotensin II; MS, mainstream smoke.
Two-way ANOVA shows statistical significance between all groups (P < 0.0001) with no interaction (P = 0.6054); the Bonferroni post-test shows an effect of Ang II between NaCl- and Ang II-treated groups (P = 0.132) and an effect of MS between sham and MS (P < 0.0001).
Table 2. Analyzed genes from the abdominal aorta (qRT-PCR)
Gene NaCl
/Sham
Ang II/Sham
NaCl/
MS
Ang II /MS
ANOVA and interaction Main effect*
Ang II and/or MS
MMP-2 (N) (13) (13) (13) (13)
RQ 1 3.1 2.8 9.5 ANOVA (P < 0.0001) Ang II (P = 0.0001)
RQ Min, Max 0.9; 1.1 2.8; 3.6 2.5; 3.2 8.6; 10.5 INTER (P = 0.906) MS (P < 0.0001)
MMP-3 (N) (12) (13) (11) (13)
RQ 1 2.2 1 2.5 ANOVA (P = 0.0014)
RQ Min, Max 0.9; 1.1 2.0; 2.5 0.9; 1.1 2.2; 2.8 INTER(P = 0.772) Ang II (P = 0.0001)
MMP-8 (N) (9) (12) (11) (13)
RQ 1 26.1 5.3 89.9 ANOVA (P < 0.0001) Ang II (P < 0.0001) RQ Min, Max 0.8; 1.2 20.9; 32.5 4; 6.9 71.4; 113.2 INTER(P = 0.633) MS (P = 0.0021)
MMP-9 (N) (11) (13) (13) (13)
RQ 1 3.8 0.2 8.1 ANOVA (P = 0.0004) Ang II (P = 0.0002)
RQ Min, Max 0.8; 1.3 2.9, 5.2 0.1; 0.3 5.9; 11.3 INTER (P = 0.060)
MMP-12 (N) (10) (13) (13) (13)
RQ 1 11.7 0.2 40.1 ANOVA (P < 0.0001) Ang II (P < 0.0001) RQ Min, Max 0.6;1.7 7.6; 18 0.1; 0.4 25.1; 64.1 INTER (P = 0.090)
TIMP-1 (N) (13) (13) (13) (13)
RQ 1 8.9 2.3 13 ANOVA (P < 0.0001) Ang II (P < 0.0001)
RQ Min, Max 0.9; 1.1 8; 9.8 2.0; 2.6 11.4; 14.8 INTER (P = 0.378) MS (P = 0.0189)
TIMP-2 (N) (13) (13) (13) (13)
RQ 1 2.1 2.0 1.7 ANOVA (P = 0.0311)
RQ Min, Max 0.9; 1.2 1.8; 2.4 1.8; 2.3 1.5; 1.9 INTER (P = 0.0175) NaCl in sham vs.
Ang II in sham (P = 0.0538)
TIMP-3 (N) (13) (13) (13) (13)
RQ 1 1.4 1.7 0.7 ANOVA (P = 0.0117 )
RQ Min, Max 0.9; 1.2 1.2; 1.6 1.5; 1.8 0.6; 0.8 INTER (P = 0.0026) NaCl in MS vs. Ang II in MS (P = 0.0136)
TIMP-4 (N) (13) (13) (13) (13)
RQ 1 0.5 1.9 0.4 ANOVA (P = 0.0006) Ang II (P = 0.0001)
RQ Min, Max 0.9; 1.1 0.4; 0.5 1.7; 2.2 0.4; 0.5 INTER (P = 0.201)
*Other groups were not statistically significantly different at an α-level of 5%.
Ang II, angiotensin II; Max, maximum; Min, minimum; MS, mainstream smoke; RQ, relative quantification.
ANOVA means that the two-way ANOVA is statistically significant.
The interaction INTER is statistically significant when the P-value is smaller than 0.05.
Ang II (P-value) means that a statistically significant difference is observed between NaCl and Ang II.
MS (P-value) means that a statistically significant difference is observed between sham and MS.
