Clinical and Experimental Pharmacology and Physiology (2009) 36, 919–924
doi: 10.1111/j.1440-1681.2009.05169.x
PREVENTION OF AORTIC ELASTIC LAMINA DEFECTS BY LOSARTAN IN APOLIPOPROTEIN E-DEFICIENT MOUSE Blackwell Losartan EC Chan prevents et Publishing al. elastic Asia lamina defects
Elsa C Chan,* Gregory T Jones,† Gregory J Dusting,* Srinivasa R Datla*‡ and Fan Jiang* *Bernard O’Brien Institute of Microsurgery, Department of Surgery, University of Melbourne, Melbourne, Victoria, Australia and †Department of Surgery, Otago Medical School, Dunedin, New Zealand
SUMMARY 1. In a previous study, we identified prevalent internal elastic lamina (IEL) defects in the aorta of hyperlipidaemic apolipoprotein E (ApoE)-deficient mice that are thought to provide a structural basis for the development of atherosclerosis and intimal thickening. In the present study, we examined the effects of losartan, an angiotensin AT1 receptor antagonist, on the development of IEL defects. 2. Male 18-week-old ApoE-deficient mice (maintained on a normal diet) were treated with losartan (3 or 30 mg/kg per day) for 10 weeks via the drinking water. The IEL defects were quantified histologically by measuring the continuity of the IEL within the inner curvature of the aortic arch. 3. In untreated animals, there was an age-dependent increase in IEL defects from 7.2 ± 2.1% at 18 weeks to 13.8 ± 4.0% at 28 weeks. Treatment with the high dose of losartan significantly prevented the development of IEL defects (4.7 ± 1.3% at 28 weeks; P < 0.05 vs untreated). This effect was independent of changes in blood pressure or plasma lipid levels. Using quantitative real-time polymerase chain reaction, we found that the effects of losartan were not associated with changes in levels of matrix metalloproteinase (MMP)-2 and MMP-9, tissue inhibitor of matrix metalloproteinase-1 or inflammatory markers in the aorta. 4. The results suggest that the renin–angiotensin system may contribute to the development of aortic IEL defects in a blood pressure-independent manner. Key words: angiotensin II, aorta, apolipoprotein E-deficient mouse, elastic lamina defect, losartan.
INTRODUCTION Structural defects of the arterial internal elastic lamina (IEL) have been implicated in the development of atherosclerosis, intimal thickening and atherosclerotic plaque destabilization.1,2 Using the apolipoprotein E-deficient (ApoE0) mouse, a well-established animal model of hyperlipidaemia and atherosclerosis,3 we demonstrated recently that IEL defects are a prominent early feature of atherosclerotic plaques and Correspondence: Fan Jiang, Bernard O’Brien Institute of Microsurgery, 42 Fitzroy Street, Fitzroy, Victoria 3065, Australia. Email:
[email protected] ‡ Present address: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA. Received 27 November 2008; revision 22 January 2009; accepted 20 February 2009. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd
may represent a nidus upon which intimal lesions develop.4 Limited studies have associated angiotensin (Ang) II with IEL disruption in large arteries,5,6 but the mechanism(s) involved remains unclear. In Brown Norway rats, a strain with genetic susceptibility to spontaneous IEL rupture in the aorta, treatment with losartan, an angiotensin AT1 receptor antagonist, and enalapril, an angiotensin-converting enzyme inhibitor, reduced the number of IEL ruptures in the abdominal aorta, whereas the calcium antagonists mibefradil and amlodipine had little effect.5 These results suggest that the endogenous renin–angiotensin system (RAS) may contribute to the development of arterial IEL defects. A role for AngII in the development of IEL defects was further corroborated by another study, in which chronic infusion of AngII in ApoE0 mice significantly increased breakages of the IEL in the aorta and decreased the elastin content in vessel walls, which were correlated with an increase in arterial stiffening.6 However, with the treatment protocols used in these experiments, neither study could completely exclude an impact of changed blood pressure on development of the IEL defect. In the former study, although the authors used calcium antagonists as a negative control, both losartan and enalapril significantly decreased systolic blood pressure (SBP) by approximately 20% at their highest doses.5 In the latter study, AngII infusion in ApoE0 mice raised blood pressure by 30%.6 Therefore, it remains to be confirmed whether AngII blockade has a direct protective effect on the aortic IEL, and not an effect that is secondary to altered haemodynamic factors. In the present study, we examined the effects of chronic administration of losartan on IEL defects in the aorta of ApoE0 mice. In preliminary experiments, we determined that the selected doses of losartan as used in the present study did not have a significant effect on blood pressure in ApoE0 mice, probably because these mice, when maintained on a normal diet, do not develop obvious hypertension compared with their C57BL/6 wild-type controls. Given that atherosclerosis is an inflammatory disorder and is associated with remodelling of the extracellular matrix,7 we also examined the effects of losartan on the gene expression of elastolytic enzymes and inflammatory markers in the aorta.
METHODS Animal treatments and tissue collection All animal studies were performed in accordance with the guidelines of National Health and Medical Research Council of Australia and were approved by St Vincent’s Hospital (University of Melbourne) Animal Ethics Committee. Male ApoE0 mice (purchased from Animal Resource Centre, Canning Vale, WA, Australia) were maintained on a normal diet throughout the study and were used at 18 weeks of age. Animals were randomly divided into three groups: a control group and two groups of losartan-treated mice (3 and 30 mg/kg per
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Table 1 Effects of losartan on general physiological characteristics of male apolipoprotein E-deficient mice
Bodyweight (g) SBP (mmHg) Heart rate (b.p.m.) Cholesterol (mmol/L) Triglycerides (mmol/L)
Control
3 mg/kg per day Losartan
30 mg/kg per day Losartan
30.3 ± 0.9 105.2 ± 5.8 647.0 ± 22.5 15.5 ± 0.8 1.2 ± 0.2
30.4 ± 0.5 102.9 ± 2.9 566.0 ± 8.1** 14.2 ± 0.7 1.3 ± 0.2
29.8 ± 1.1 104.0 ± 2.3 585.1 ± 15.9* 14.3 ± 1.1 1.3 ± 0.2
Apolipoprotein E-deficient mice were treated with losartan (0, 3 or 30 mg/kg per day) via the drinking water for 10 weeks. Data are mean±SEM (n = 6–10 per group). *P < 0.05, **P < 0.01 compared with control (one-way anova). SBP, systolic blood pressure. day losartan (L3 and L30, respectively) administered in the drinking water for 10 weeks). Age-matched untreated C57BL/6 wild-type (Animal Resource Centre) maintained on a normal diet were used as the wild-type controls. Mice were heparinized and killed by intraperitoneal injection of sodium pentobarbital at 28 weeks of age. Blood samples were taken from the right atria and were snap frozen in liquid nitrogen for lipid analysis. Mice were then briefly perfused with neutral-buffered formalin from the left ventricle at 100 mmHg for morphological analysis. For RNA extraction, fresh aortas were excised and cleaned of fat tissue, preserved in RNAlater solution (Applied Biosystems/ Ambion, Austin, TX, USA) overnight at 4°C and then stored dry at –80°C.
RESULTS Effects of losartan on haemodynamic and plasma lipid parameters
Systolic blood pressure was measured in conscious mice using a non-invasive tail-cuff blood pressure system (IITC, Woodland Hills, CA, USA). Mice were trained to become accustomed to the procedure by several mock sessions 1 week before the study. In each measurement, blood pressure readings were taken at least three times and the averaged values were reported.
Chronic treatment of ApoE0 mice for 10 weeks did not have any effect on SBP (Table 1). In contrast, heart rate was significantly slower in mice treated with both low and high doses of losartan. The bradycardia response induced by losartan is consistent to a previous finding that AngII, via the AT1 receptor in the atrium, may have a positive chronotropic effect in mice.8 We did not observe any effects of losartan on plasma levels of total cholesterol or triglycerides (Table 1). Plasma cholesterol and triglyceride levels of ApoE0 mice were significantly higher than those of wild-type C57BL/6 mice (cholesterol < 2.0 mmol/L; triglyceride 0.6 ± 0.1 mmol/L; n = 6). There was no significant difference in bodyweight between the different groups.
Histology
Effects of losartan on elastic laminar defects in the aorta
The IEL defects were quantified histologically by measuring the continuity of the IEL layer within the inner curvature of the aortic arch, as described previously.4
Consistent with previous observations in ApoE0 mice fed with a fatand cholesterol-enriched diet,19 in the present study, micro defects of the internal and medial elastic laminae were readily observable throughout the aorta of normal diet-fed mice (Fig. 1). Using the inner curvature of the aortic arch as a representative site of IEL defects,4 we found that the severity of the IEL defects was not directly associated with the thickness of overlying atherosclerotic plaques (Fig. 1). Indeed, we observed that severe IEL defects in untreated animals were often associated with thinner lesions, whereas 30 mg losartan tended to increase the overall intimal thickness (see Table 2). We propose that this phenomenon may be related to a plaque-stabilizing effect of losartan (e.g. increased collagen content and smooth muscle cells in the fibrous cap and shoulder areas), as suggested by previous studies.9 In some specimens, migration of medial smooth muscle cells into the intima via the IEL defects could be observed (Fig. 1c). Long-term treatment with the high dose of losartan significantly reduced the prevalence of IEL defects, whereas the effect of the low dose of losartan was not statistically significant (Fig. 2). Losartan treatment did not significantly modify the size of atherosclerotic lesions in the aortic arch, as indicated by the similar values of the total intimal area and the intimal medial ratio in control and treated animals (Table 2). To clarify whether the effect of losartan on IEL defects was due to prevention of lesion progression or reversal of established lesions, we also measured lesion prevalence in ApoE0 mice at 18 weeks of age when losartan treatment started. We found that there was an obvious age-dependent increase in aortic elastic lamina defects in ApoE0 mice (7.2 ± 2.1 vs 13.8 ± 4.0% at 18 and 28 weeks, respectively; n = 8–10), although the
Conscious blood pressure measurement
Quantitative real-time polymerase chain reaction Total RNA was extracted from aorta using the TRI Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The RNA was reversetranscribed into cDNA using the TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time polymerase chain reaction (PCR) was performed out with a 10 ng sample of cDNA in a total volume of 25 L of a reaction mixture containing 12.5 L TaqMan Universal PCR Master Mix (Applied Biosystems), 1.25 L predesigned mouse-specific primer–probe sets (Assays-on-Demand; Applied Biosystems) for matrix metalloproteinase (MMP)-2 (Assay ID Mm00439508_m1), MMP-9 (Mm00600163_m1), tissue inhibitor of matrix metalloproteinase (TIMP; Mm00441818_m1), intercellular adhesion molecule-1 (ICAM-1; Mm00516023_m1), vascular adhesion molecule (VCAM)-1 (Mm00449197_m1) and monocyte chemoattractant protein (MCP)-1 (Mm99999056_m1). The PCR reaction (50 cycles) was performed on an ABI Prism 7300 system (Applied Biosystems) with the following settings: 2 min at 50°C, 10 min at 95°C and 50 cycles of 95°C for 30 s and 60°C for 1 min. Ribosomal RNA (18S) was used as the internal standard for each reaction. Threshold cycle (Ct) values for each test gene were normalized against Ct values for 18S RNA (ΔCt) and then expressed as fold relative to the control sample (2–ΔΔCt).
Data analysis Results are presented as the mean±SEM. Unpaired t-test or one-way analysis of variance (anova) followed by post hoc Newman–Keuls t-test, as indicated, were used for statistical analysis. P < 0.05 was regarded as significant.
© 2009 The Authors Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd
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Losartan prevents elastic lamina defects Table 2 Effects of losartan on morphometric parameters of the aortic arch from apolipoprotein E-deficient mice
Total intimal area (×1000 m2) Total medial area (×1000 m2) Intima : media ratio Mean intimal thickness (m) Mean medial thickness (m)
Control
3 mg/kg per day Losartan
30 mg/kg per day Losartan
71.3 ± 11.4 308.9 ± 18.3 23.3 ± 4.0 31.1 ± 6.2 129.8 ± 5.9
64.6 ± 9.9 297.5 ± 19.2 22.8 ± 4.5 28.0 ± 4.8 126.2 ± 6.6
105.1 ± 29.3 278.6 ± 13.8 39.4 ± 11.2 45.2 ± 12.9 116.3 ± 4.2
Apolipoprotein E-deficient mice were treated with losartan (0, 3 or 30 mg/kg per day) via the drinking water for 10 weeks. Data are the mean±SEM (n = 9–10 per group).
Fig. 2 Effects of losartan at 3 (L3) and 30 mg/kg per day (L30) on internal elastic lamina (IEL) integrity in the aortic arch from apolipoprotein Edeficient (ApoE0) mice. Data are the mean±SEM (n = 8–10 per group). *P < 0.05 compared with control (t-test).
difference did not reach statistical significance (P = 0.145). There were no differences in the prevalence of IEL lesions between young ApoE0 mice and the older animals in the L30 group. These results suggest that the protective effect of losartan is likely to involve prevention of the development of new IEL lesions. We also measured other morphometric parameters of the aorta, including total medial area, mean intimal thickness and mean medial thickness. As summarized in Table 2, none of these parameters differed between control and treated animals.
Effects of losartan on aortic expression of MMPs
Fig. 1 Internal elastic lamina (IEL) defects in the aortic arch from male apolipoprotein E-deficient (ApoE0) mice. (a) Untreated mice, (b) mice treated with 30 mg/kg per day losartan (for quantitative data, see Fig. 2). (c) Higher magnification image of the untreated group showing the migration of medial smooth muscle cells into the intima via the IEL defect (arrow). The integrity of the IEL was analysed in 4 m longitudinal sections with Verhoeff’s van Gieson staining. The IEL defects are indicated by arrows. Note that the severity of the IEL defects was not directly associated with the thickness of the overlying atherosclerotic plaques (a,b). Asterisks indicate foam cells in the atherosclerotic lesions. Bars, 100 m.
To clarify whether the protective effect of losartan on the aortic elastic laminae is related to changes in local proteolytic enzymes, we measured expression levels of MMP-2, MMP-9 and TIMP-1. As shown in Fig. 3, losartan had no effect on the expression of MMP-2, MMP-9 or TIMP-1. We also measured the expression of MMPs and TIMP-1 in the aorta of normal C57BL/6 mice. We found that the expression of MMP-2 was significantly higher in untreated ApoE0 mice compared with that in age-matched wild-type C57BL/6 mice (66.9 ± 4.3% of expression in untreated ApoE0 mice; P < 0.01, t-test; n = 6), whereas levels of MMP-9 or TIMP-1 did not differ between ApoE0 and wild-type mice (126.5 ± 16.2 and 83.1 ± 6.0% of expression in untreated ApoE0 mice for MMP-9 and TIMP-1, respectively; both P > 0.05; n = 5–6).
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Fig. 3 Effects of losartan (30 mg/kg per day; 䊏) on gene expression (mRNA) of (a) matrix metalloproteinase (MMP)-2, (b) MMP-9 and (c) tissue inhibitor of matrix metalloproteinase-1 in aortas from apolipoprotein E-deficient (ApoE0) mice. (䊐), control (untreated) ApoE0 mice. Data are the mean±SEM (n = 4– 6 per group).
Fig. 4 Effects of losartan (30 mg/kg per day; 䊏) on gene expression (mRNA) of inflammatory markers, namely (a) monocyte chemoattractant protein-1, (b) vascular adhesion molecule-1 and (c) intercellular adhesion molecule-1 in aortas from apolipoprotein Edeficient (ApoE0) mice. (䊐), control (untreated) ApoE0 mice. Data are the mean±SEM (n = 4–10 per group).
Effects of losartan on aortic expression of inflammatory molecules We finally examined whether the protective effect of losartan is associated with reduced vascular inflammation. We measured mRNA levels of the chemokine MCP-1 and the adhesion molecules ICAM-1 and VCAM-1. However, we did not find any differences in the expression of these inflammatory markers in the aorta between control and treated groups (Fig. 4). Moreover, we found that the expression of MCP-1, ICAM-1 and VCAM-1 in the aorta did not differ between untreated ApoE0 mice and age-matched wild-type mice (135.3 ± 25.6, 118.5 ± 13.7 and 65.3 ± 12.5% of expression in untreated ApoE0 mice for MCP-1, ICAM-1 and VCAM-1, respectively; all P > 0.05; n = 5–6).
DISCUSSION In the present study, we demonstrated that chronic treatment with losartan reduced spontaneous aortic IEL disruption in hyperlipidaemic ApoE0 mice. Importantly, we showed that this protective effect of losartan was independent of changes in blood pressure, indicating that endogenous AngII, via AT1 receptors, promotes the development of aortic IEL defects in a blood pressure-independent manner. Several lines of evidence suggest that an intact IEL layer, which forms a barrier
between the arterial intima and the media, may have a protective role against the development of arterial lesions such as atherosclerosis and neointima formation.1,10,11 First, IEL may affect the transport of proatherogenic low-density lipoprotein (LDL) particles across the intima– media border and subsequent accumulation of LDL within the arterial wall.10,11 In the human aorta, it was demonstrated that an intact IEL layer provided an almost total barrier to LDL, whereas in aortas with disrupted IEL there was a 25-fold increase in LDL concentration outside the IEL layer.10 In a more recent study using two-photon microscopy, Kwon et al.11 provided evidence that, in freshly isolated arteries, a nearly confluent elastin layer was present throughout except in the atherosclerosis-prone branch areas, where dense collagen–proteoglycan complexes were exposed. In LDL-binding studies, this luminal elastin layer limited LDL penetration, whereas its absence at the branching area resulted in extensive LDL binding to the extracellular matrix.11 Second, IEL may prevent the migration of medial smooth muscle cells into the intima. For example, by comparing the internal thoracic artery with the anterior descending coronary artery, it has been observed that migration of medial smooth muscle cells does not occur via penetration of the cells through normal fenestrations of the IEL, but mainly through major defects in the IEL layer.1 In addition, in a mouse model of vascular neointimal hyperplasia induced by electric injury in femoral arteries, MMP-11 deficiency enhanced neointima formation, which was
© 2009 The Authors Journal compilation © 2009 Blackwell Publishing Asia Pty Ltd
Losartan prevents elastic lamina defects accompanied by an increase in the degradation of IEL, although how MMP-11 could protect elastin from degradation is not known.12 In line with these observations, we have demonstrated that in aortas from ApoE0 mice, IEL defects are present beneath all atherosclerotic plaques, ranging from early fatty streaks to advanced fibrous lesions.4 Moreover, elastic tissue defects were not observed in regions that were atherosclerosis resistant. Taking all these data into consideration, we propose that IEL defects may represent a structural basis for the development of atherosclerosis and neointima formation. However, such a relationship between IEL defects and the development of vascular lesions needs to be validated by more experimental data. Indeed, in the present study we found that reductions in IEL defects produced by losartan were not associated with reduced atherosclerosis. Similar to previous findings,4 in the present study we found an agedependent increase in IEL defects in untreated ApoE0 mice. Together with the finding that the prevalence of IEL defects in younger ApoE0 mice and the losartan-treated older mice was similar, it is suggested that losartan prevents the development of new IEL defects rather than reversing existing lesions. The results indicate that the RAS is involved directly in the development of aortic IEL defects in ApoE0 mice. This point is supported by the finding that the expression of the AT1 receptor in the aorta is elevated in ApoE0 mice compared with wild-type controls.13 Moreover, those authors demonstrated that wild-type mice exhibited less responsiveness to AngII treatment than ApoE0 mice in the development of aneurismal aortic lesions.13 The differential expression of the AT1 receptor in the aorta may provide an explanation for the predisposition of ApoE0 mice to develop IEL defects and for the protective effect of AT1 receptor blockade on IEL lesions. However, the factors by which AngII and AT1 receptors mediate the development of IEL degradation remain unclear. Previously, we found that the development of IEL defects was not necessarily an outcome of the presence of hyperlipidaemia, because IEL defects were also observed in wild-type C57BL/6 mice.4 Moreover, we found that this pathological change may represent a feature of vascular ageing, because the percentage of IEL defects increased progressively with age in both wild-type and ApoE0 mice.4 Nevertheless, in both young and old animals, the presence of hyperlipidaemia markedly increased the prevalence of IEL defects in the aorta,4 suggesting that hyperlipidaemia may have a synergistic action with the underlying pathogenic factor(s) for aortic IEL defects, which is currently unknown. However, our data clearly demonstrate that the protective effect of losartan is not associated with changes in the plasma lipid profile. Conversely, another possible mechanism is that losartan may exert its protective action by modulating vascular inflammation, because hyperlipidaemia may potentiate the development of IEL defects by inducing vascular inflammation.14 Indeed, different lines of evidence suggest that AngII, via AT1 receptors, has significant pro-inflammatory effects in the vasculature.15 To test this possibility, we measured mRNA levels of VCAM-1, ICAM-1 and MCP-1 in the aorta, because these molecules are commonly used as markers of vascular inflammation. We found that only VCAM-1 was increased by approximately 50% in ApoE0 mice compared with age-matched wild-type mice, although this difference did not reach statistical significance. There were no significant changes in ICAM-1 or MCP-1 levels. This low level of vascular inflammation in ApoE0 mice may be because these mice were maintained on a normal (low-fat) diet. Nonetheless, the data demonstrate that losartan treatment has little effect on these inflammatory markers in the aorta, suggesting that modulation of vascular inflammation by losartan is unlikely to be involved in its IEL protective effects.
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We also examined whether the protective effects of losartan were related to altered MMPs. Measurement of MMP-2 and MMP-9 mRNA demonstrated that MMP-2 was upregulated in the aorta of ApoE0 mice compared with wild-type controls, but MMP-9 was not. Morphological studies revealed that both MMP-2 and MMP-9 may be involved in the destabilization of elastic lamina in the arterial wall.16,17 Moreover, the MMP inhibitor doxycycline has been shown to suppress elastolysis in an arterial organ culture study.18 However, in the present study, losartan treatment did not have any significant effect on either MMP2 or MMP-9 expression. In addition, we demonstrated that the level of the endogenous MMP inhibitor, TIMP-1, did not differ between control and losartan-treated groups. These findings seem to be consistent with a previous observation that AngII failed to modify MMP2 or MMP-9 release from mononuclear cells in culture.19 There is evidence that MMPs may be activated by reactive oxygen species (ROS).20 Many experimental studies have shown that AT1 antagonists can reduce ROS production in blood vessels.21–23 In line with this notion, we observed that the NADPH oxidase-dependent superoxide production in the carotid arteries was reduced by approximately 50% in 30 mg losartan-treated animals (data not shown). Although this was not measured directly in the aorta, the data confirm that losartan has a potent vascular anti-oxidant effect. Taken together, the results indicate that modulation of MMP activity by ROS may be the potential mechanism underlying our observation that losartan protected against IEL defects in the absence of MMP expression. Conversely, our data do not exclude the possibility that losartan may modulate the expression of other enzymes with elastolytic activities, such as cathepsins and MMP-3, both of which have been implicated in arterial IEL degradation.24,25 Recently, in an ApoE0 mouse model of renal dysfunction, losartan administration was found to suppress the expression of cathepsin S in the aorta, further suggesting that the RAS may have a role in modulating cathepsin expression.26 A limitation of the present study is that we did not directly measure the local MMP activity in the aorta using gelatin zymography; therefore, it is unclear whether the IEL protective effect of losartan is related to altered MMP activity. As mentioned above, in both our previous studies,4,19 as well as in the present study, we observed that the prevalence of aortic IEL defects increased progressively with age in both ApoE0 and wild-type mice, raising the possibility that IEL disruption may represent a feature of vascular ageing.4 It has been suggested that arterial stiffening is an indicator of vascular ageing and this mechanical change may be due to fatigue and fracture of elastic tissues and the subsequent transfer of stress to the stiffer collagenous components.27 Interestingly, there is evidence that long-term blockade of the RAS may prevent both structural and functional alterations of the arterial wall during ageing.28,29 Based on these observations, we propose that the protective action of losartan on IEL integrity in the present study may be an outcome of a decelerated ageing process of the cardiovascular system following RAS blockade. In conclusion, the results of the present study suggest that the RAS may contribute to the development of aortic IEL defects in hyperlipidaemic mice in a blood pressure-independent manner.
ACKNOWLEDGEMENTS This work was supported by project grants from the National Health and Medical Research Council of Australia (NHMRC) and Grants-inAid from the National Heart Foundation of Australia. GJD receives a Principal Research Fellowship from NHMRC. ECC is supported by
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a Melbourne Research Fellowship from the University of Melbourne. The authors thank Nancy Guo for her excellent assistance in performing the quantitative real-time PCR experiments.
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