In this study, we have shown that loss of one ECM protein produced by vascular SMCs is sufficient to drive upregulation of ACE in the aortic wall of Fbln4SMKO
mice. Because angiotensinogen expression in the liver and ACE expression in the lung were comparable between controls and Fbln4SMKO
mice and AngII concentration in the aorta was increased in Fbln4SMKO
mice, it is likely that local conversion of AngI to AngII contributes to the cellular changes in the mutant aorta. This is consistent with the previous report showing that local ACE serves as a key rate-limiting enzyme for AngII-mediated vascular hypertrophy in the rat carotid artery overexpressing the Ace
). It is noteworthy that Fbln4SMKO
aorta shows marked proliferation and disarray of SMCs. We speculate that the absence of intact elastic laminae and thus loss of physical barriers within the vessel wall further facilitate migration of SMCs across the lamellar unit and expansion of immature SMCs in the Fbln4SMKO
Upregulation of ACE is associated with various pathological conditions, including hypertension, atherosclerosis and diabetic nephropathy (reviewed in (25
)). Although the endothelium is a prominent source of ACE in the vasculature, ACE has also been shown to be upregulated in SMCs of diseased vessels in humans and animal models, such as neointima induced by wire denudation injury, percutaneous transluminal coronary angioplasty (27
), and small pulmonary arteries in hypoxia-induced pulmonary hypertension (29
). Interestingly, SMCs explanted from thoracic aneurysms of patients carrying MYH11
mutations show high levels of ACE
). The exact mechanism of ACE upregulation in SMCs has not been completely understood; however, soluble factors such as bFGF and endothelin were suggested to mediate upregulation of ACE (31
). Mechanical forces such as shear stress and pulsatile pressure, as well as hypoxia, can also increase expression of ACE in SMCs (33
). Our study provides evidence that loss of fibulin-4 in the context of vessel wall is sufficient for upregulation of ACE in SMCs in vivo. The resulting activation of local AngII signaling caused ascending aortic aneurysms in Fbln4SMKO
mice, as evidenced by the complete prevention of aneurysm formation through perturbation of this pathway either at the level of AngII production or receptor activation. The results also suggest that fibulin-4 is required to prevent overactivation of local AngII-induced pathways that result in loss of the SMC phenotype but may be largely overcome by early interruption of AngII signaling.
Our study allowed us to determine the critical therapeutic time window for prevention of aneurysm development in vivo. Inhibition of AngII-mediated pathway by losartan effectively suppressed local proliferative changes in SMCs if treatment was initiated at P7, and these effects persisted even after withdrawal of losartan at P45. In contrast, after P30, mutant SMCs did not respond to losartan and upregulation of p-ERK1/2 continued. Thus, a critical therapeutic time window is between P7 and P30, and proliferation of mutant SMCs is dependent on AngII signaling during this period. The mechanism underlying the irreversible change in SMCs after P30 was not examined in this study; however, it is possible that AngII activates alternative signaling pathways that independently sustain the abnormal SMC phenotype in Fbln4SMKO aorta and/or vessel microenvironment containing high AngII may induce chromatin modification to alter the response to losartan and/or suppress SMC differentiation markers.
Results from genetic studies with angiotensin II receptor-deficient mice demonstrated that inhibition of Agtr1a alone is not sufficient to achieve complete prevention of aneurysms in Fbln4SMKO
mice. The fact that losartan completely prevented aneurysmal phenotypes indicated that Agtr1b also mediates pathological signals in the Fbln4SMKO
aorta. Agtr1a is the dominant receptor in adult tissues in mice, and Agtr1b expression is limited to a few organs, including adrenal glands, testis and brain (21
). Upregulation of Agtr1b, however, was shown to be responsible for augmenting glomerular lesions in the absence of Atgr1a in murine autoimmune nephritis (35
). Therefore, it is possible that AngII-mediated signaling through Agtr1b participates in the development of aneurysms in 1aDKO
aorta and that losartan effectively prevents the aneurysm formation by blocking both Agtr1a and Agtr1b in Fbln4SMKO
mice. Generation of triple knockout mice for Agtr1a
, and Fbln4
in a SMC specific manner will provide information on the role of Agtr1 receptors in the development of aneurysms in SMKO mice. Our results also show that Agtr2 is not required for mediating the protective action of losartan in Fbln4SMKO
mice, unlike its critical role described in Fbn1C1039G
). Prevention of the aneurysm phenotype by captopril also confirms that blocking AngII signals mediated by both Agtr1 and Agtr2 is an effective strategy in prevention of aneurysm formation in Fbln4SMKO
mice and provides evidence against an antagonistic interaction between Atgr1 and Agtr2 in the development of aortic aneurysms in Fbln4SMKO
mice. This finding is of particular importance in designing a strategy to stimulate Agtr2 as a potential treatment for cardiovascular diseases (36
). Recently, direct stimulation of Agtr2 by a non-peptide agonist Compound 21 (C21) was shown to reduce arterial stiffness independent of blood pressure in rats treated with nitric oxide synthase inhibitor (37
). C21-mediated beneficial effects were also reported on scar size and cardiac function in rat myocardial infarction (MI) model (38
), as well as on perivascular fibrotic changes in stroke-prone rats (39
), while another study in a mouse MI model did not support a protective effect of Agtr2 stimulation (40
). Our animal model will be useful to determine the effect of Agtr2 agonists in aneurysm progression in vivo.
A protective association between ACE inhibitors and risk of aneurysm rupture was reported in a large population-based case-control study in abdominal aortic aneurysm patients, in which an ACE inhibitor, but not ARB or other anti-hypertensive reagents, decreased the risk of aneurysm rupture independent of lowering blood pressures (41
). In the study targeted to Marfan patients, an ACE inhibitor reduced arterial stiffness and aortic root diameters compared to placebo-treated group (42
) or ß blocker (43
). The clinical trails with a larger cohort are currently underway to test the effects of ARB on Marfan patients (44
). Our in vivo results provide further support for the idea that inhibition of AngII-Agtr pathway by ACE inhibitors or ARBs are equally effective choices for the treatment of ascending aortic aneurysms during the early postnatal period in human ascending aortic aneurysm patients and warrant further investigation.
By combining hemodynamic and mechanical studies with pharmacological experiments, we were able to assess the relationship between mechanical stress on the vessel wall and aneurysm phenotypes. Although our data indicate that loss of integrity of the vessel wall is coupled with cellular changes in SMCs, the aneurysm phenotype can be prevented without completely reversing increased pulse pressures (for losartan-treated animals) or the altered mechanical properties of the aorta, including decreased compliance and increased stiffness. The absence of pressure changes in Fbln4SMKO mice with losartan treatment implies that signaling through Agtr2 is sufficient to maintain blood pressure in these mice and provides further evidence against the complete dependence on pressure changes for the aneurysm prevention. Our results show that aneurysm in our model is a disease affecting SMC phenotypes and ECM-cell interactions, rather than a condition resulting solely from increased hemodynamic forces and/or mechanical defects of the vessel wall, and that intervention to alter SMC phenotypes by captopril or losartan leads to effective prevention of the aneurysm formation.
Although fibulin-4 is expressed in both ascending and descending aorta, the aneurysms resulting from a lack of this protein are confined to the ascending aorta, suggesting that SMCs in this region may possess unique characteristics that confer susceptibility to external growth signals in addition to the direct influence of outflow pressure. One possibility is that SMCs with a diverse embryonic origin may contribute to the differential response to AngII signals in the vessel wall. SMCs are known to be derived from the neural crest in the ascending aorta (46
) and are distinct from SMCs in the descending/ abdominal aorta derived from the somite (47
). Our data indicating that ACE expression is higher in the ascending aorta than descending aorta support this possibility. Regional differences in ACE activity were also reported in chick embryos, demonstrating significantly increased ACE activity in the ascending aorta compared to the abdominal aorta (48
). Furthermore, endothelial cells incubated with conditioned media harvested from neural crest-derived SMCs exhibited a marked increase in ACE activity compared with those from mesenchyme-derived SMCs. Therefore, basal ACE activity may be regulated in a cell lineage-dependent manner and may contribute to the regional difference in formation of aortic aneurysms.
In summary, our results highlight the AngII pathway as a cause of ascending aortic aneurysms in Fbln4SMKO
mice and provide a basis for the effective prevention of aneurysms with postnatal administration of ACE inhibitor and/or ARB. Our study underscores differences in the pathological basis of aortic aneurysms and suggests that therapeutic regimens need to be tailored according to underlying disease mechanisms in human patients. The limitations of this study include a lack of observations of the aneurysm phenotype over an extended period of time after early losartan treatment, which would have enabled us to evaluate the long-term effects of losartan on aneurysm prevention in Fbln4SMKO
mice. We recognize that our mouse model is a cell-type specific inactivation of Fbln4
, whereas reported human patients have systemic inactivation of FBLN4
) and, as expected, symptoms are more severe than the present mouse model. Thus, additional information regarding tissue/serum levels of fibulin-4 in a large cohort of aortic aneurysms and the correlation with clinical features and severity of the disease will be valuable. In addition, results from this mouse model of aneurysm development indicate that further investigations regarding intracellular signaling pathways downstream of AngII-Agtr that are responsible for induction of proliferative changes in SMCs and identification of specific SMC populations activated by Ang II signals are needed.