**2. Development of a new mouse model of aortic aneurysm induced by DOCA- or Aldo-salt**

#### **2.1 Discovery of DOCA-salt mouse model of aortic aneurysm by accidence**

In an independent pilot study using 10- to 12-month-old C57BL/6 male mice to investigate DOCA-salt-induced hypertension, we unexpectedly observed that many mice died from AAA rupture. We were intrigued by this observation since it raised the possibility that activation of the MR by DOCA can cause AAA in the presence of high salt. Given that administration of DOCA and salt to mice or rats have been used extensively as an experimental model of low-renin hypertension [45], it was surprising that DOCA-salt-induced AAA has not been reported in previous studies. While the exact reasons for this discrepancy are unclear, our results suggest that the age of mice (i.e., 10-month old *vs.* 10-week old) may be critical for DOCA and salt to induce AAA (see below).

#### **2.2 Both DOCA and high salt are required to induce aortic aneurysm**

To verify our pilot studies and define whether DOCA, salt, or both is critical for DOCA-salt-induced aortic aneurysm, 10-month-old C57BL/6 male mice received DOCA alone (subcutaneous implantation of DOCA pellets; 50 mg, 21-day release; Innovative Research of America, USA), salt alone (drinking water containing 0.9% NaCl plus 0.2% KCl), DOCA and salt, or no treatment (controls). We used C57BL/6 mice because C57BL/6 mice are more susceptible to chemical (i.e., BAPN/ Ang II)-induced TAD, AAD, and aneurysmal rupture than other strains of mice (i.e., FVB mice) [24, 25]. We used 10-month-old rather than 10-week-old mice because we found that DOCA- or Aldo-salt-induced aortic aneurysm were aging dependent [41, 43]. We used male mice rather than female mice because DOCA- or Aldo-salt-induced aortic aneurysm has sex difference (unpublished data). All mice were euthanized 3 weeks after treatment.

We used three different approaches to quantify DOCA-salt-induced aortic aneurysm. First, the maximal intraluminal diameters of abdominal aortas were

*Aortic Aneurysm and Aortic Dissection*

resembles human aortic aneurysm.

models currently studied in the world.

the failure of these clinical trials.

a high mortality rate (65–85%) if rupture occurs [8]. Unfortunately, repair through open or endovascular surgery is currently the only therapeutic option for aortic aneurysm; no drug has been approved for treatment of this devastating disease [3, 5]. One of the major barriers in the field is a lack of an animal model that fully

Over the last few decades, a number of rodent models of AAA and TAA have been developed and have been increasingly utilized to be used in understanding the etiology of human AAA and TAA [2, 9–11]. Aortic aneurysm animal models can be classified into three groups [2, 9–11]: (1) genetically predisposed animal models (i.e., fibrillin-1 (FBN1) mutation (Marfan syndrome) mouse model [2, 12]), (2) chemical-induced animal models (i.e., Ang II infusion hyperlipidemia mouse model [9, 13]), and (3) physical or surgical animal models (i.e., decellularized aortic xenograft rat model [10, 14]). Among them, calcium chloride adventitial application model [15, 16], porcine pancreatic elastase (PPE) model [17, 18], and Ang II infusion hyperlipidemia mouse model [13, 19–23] are the commonest animal

One of the fundamental pathological characteristics in human TAA and AAA is thoracic aortic dissection (TAD) and abdominal aortic dissection (AAD), both of which can lead to aneurysmal rupture with high mortality [1–7]. Many genetically predisposed animal models have TAD and AAD (i.e., fibrillin-1 mutation mouse model [2, 12]). Some of the chemical-induced animal models also have TAD and AAD (i.e., fibrillin-1 mutation and Ang II infusion hyperlipidemia mouse models [13, 19–23]). Recently, a new chemical-induced mouse model for more potently induction of TAD and AAD was developed by administration of β-aminopropionitrile monofumarate (BAPN) to mice to inhibit lysyl oxidase (LOX) and/or Ang II infusion [24, 25]. Kurihara et al. demonstrated that BAPN/Ang II induced TAD in 100% of FVB mice [24]. Ren et al. confirmed this finding and further demonstrated that BAPN/Ang II induced TAD and AAD in 75% of C57BL/6J

Although no single animal model fully reproduces the histological characteristics and natural history of the human aortic aneurysm, each of these animal models more or less recapitulate human aortic aneurysm and have significantly contributed to the current understanding of clinical management and treatment of patients with AAA and TAA [2, 9–11]. Several clinical trials have begun enrollment to examine whether angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARB) are effective in the treatment of human aortic aneurysm. However, the results from these clinical trials are inconsistent and disappointing: either effective [26], no effect [27], or, even worse [28], indicating that the current understanding about the etiologies of aortic aneurysm is limited and additional unknown signaling and mechanism may underlie aortic aneurysm and account for

In sharp contrast to the well-established role of Ang II in aortic aneurysm [13, 19–23], little is known about the role of aldosterone (Aldo) in aortic aneurysm. Aldo is a steroid hormone primarily synthesized and released by the adrenal glands. Aldo is a downstream effect of Ang II and is well recognized for its critical role in renal sodium reabsorption and water retention and consequently extracellular volume and blood pressure [29, 30]. Accumulated data over the last decade, however, demonstrate that Aldo not only acts on the kidney but also targets many other organelles, including those in the cardiovascular system, where it is critically

Several lines of clinical study implicate Aldo signaling in aortic aneurysm. First, individual case reports demonstrated that primary hyperaldosteronism is associated with aortic dissection [34–36]. Second, a retrospective study demonstrated that

involved in diverse pathophysiological processes [31–33].

mice, whereas BAPN alone induced TAD in 87% of C57BL/6J [25].

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quantified in vivo by a high-resolution ultrasound imaging system (Vevo 2100, Visualsonics, Toronto, Canada). The results showed that both DOCA and salt but not DOCA or salt alone could potently induce abdominal aortic dilation relative to the control [41]. Second, the maximal external diameters of isolated abdominal and thoracic aortas were quantified ex vivo by Nikon SMZ800 Stereo Microscope with a digital camera and NIS-Elements software. Consistently with the ultrasound data, both DOCA and salt but not DOCA or salt alone significantly increased external diameters of abdominal and thoracic aortas relative to the control [41].

Third, we calculated the incidence of DOCA-sat-induced AAA, TAA, and aneurysmal rupture based on the definition that AAA or TAA has at least a 50% increase in diameter compared with the normal diameter of the aorta [1]. Of the 45 mice treated with DOCA-salt, 28 mice developed AAA (62%), 22 mice developed TAA (42%), and 8 mice died of aortic aneurysmal rupture (18%). In contrast, no AAA, TAA, or aortic aneurysmal rupture was observed in control, DOCA, or salt alone. Interestingly, AAA was only found in the suprarenal abdominal aorta, which is similar to that in the Ang II AAA mouse model [13], whereas TAA was mostly associated with AAA and was mostly observed in the descending thoracic aorta, indicating that TAA is likely derived from AAA.

### **2.3 Infusion of mice with Aldo can also induce aortic aneurysm in the presence of high salt**

Since DOCA is a synthetic MR agonist, we wondered whether Aldo, a physiologic ligand of MR in our body, could induce aortic aneurysm in the presence of high salt. To define the concentration of Aldo that is sufficient to induce aortic aneurysm in the presence of high salt, 10-month-old C57BL/6 male mice were infused with three different doses of Aldo (200, 500, and 700 μg/kg/day) for 4 weeks. Aldo was delivered to mice via subcutaneous implantation of osmotic minipump (Alzet model 2004; DURECT, USA) containing Aldo solubilized in 50% DMSO. All groups of mice were treated for 4 weeks.

Infusion of mice with all three doses of Aldo was very similar to implantation of mice with DOCA pellets and markedly increased maximal intraluminal and external diameters of suprarenal abdominal aortas compared to the control mice (without treatment). Similarly, infusion of mice with all three doses of Aldo is also similar to implantation of mice with DOCA pellets and potently induced AAA (over 58%), TAA (over 42%), and aneurysmal rupture (over 25%) compared to the control mice. These data demonstrated that the infusion of mice with 200 μg/kg/ day Aldo is sufficient to induce AAA in the presence of high salt.

We measured the plasma Aldo concentrations by a commercial EIA kit (Enzo Life Science, USA) 4 weeks after Aldo and salt administration. We found that plasma Aldo concentrations were elevated in a dose-dependent manner. Of note, infusions of mice with 200 μg/kg/day Aldo resulted in plasma Aldo concentrations to ~10 nM, which could be seen in some human diseases such as congestive heart failure and primary aldosteronism [31, 46, 47]. These results indicate that the Aldosalt AAA mouse model is a physiopathological model that mimics human diseases rather than a pharmacological model that would cause concerns due to the use of high doses of reagent.

#### **2.4 DOCA-salt-induced aortic aneurysm is independent of Ang II**

Although systematic plasma renin and Ang II concentrations are suppressed in animals administered with DOCA and salt [45], local aortic Ang II concentration can be increased due to activation of vascular RAAS, which was thought to be of

**81**

model [19].

*A New Mouse Model of Aortic Aneurysm Induced by Deoxycorticosterone Acetate or Aldosterone…*

pathophysiological relevance to the development of atherosclerosis [48]. Moreover, there is a synergistic interaction between Ang II and Aldo in VSMCs [49, 50]. Therefore, it is interesting to investigate whether DOCA-salt-induced aortic aneurysm is dependent upon Ang II. To address this important question, 10-month-old C57BL/6 male mice were treated with either an ACE inhibitor (enalapril) or an ARB (losartan) before (1 week) and after (4 weeks) DOCA-salt administration. As expected, enalapril or losartan effectively decreased blood pressure, but enalapril or losartan had little effect on the DOCA-salt-induced aortic dilation, aortic aneurysm formation, and aneurysmal rupture [41]. These results demonstrate that the DOCA-salt-induced aortic aneurysm is independent of Ang II thus provide an alternative mouse model of aortic aneurysm for investigators in the field who need

**2.5 Activation of MR is a prerequisite for DOCA- or Aldo-salt to induce aortic** 

To define the role of MR in DOCA- or Aldo-salt-induced aortic aneurysm, we treated 10-month-old C57BL/6 male mice with an MR antagonist eplerenone 1 week before and 4 weeks after Aldo-salt administration [41]. Eplerenone (Pfizer, USA) was delivered by feeding mice with custom diets (chow supplemented with eplerenone at 2.5 mg/g, Research Diets, Inc., USA). In contrast to the minimal effect of blocking Ang II with enalapril or losartan, treatment of mice with eplerenone completely abolished Aldo-salt-induced aortic dilation, AAA formation, and aortic aneurysmal rupture [41]. A similar but less potent effect on DOCA-salt-induced AAA was also found in mice treated with spironolactone [41]. These results suggest that activation of MR by DOCA or Aldo is a prerequisite for DOCA- or Aldo-salt to

**2.6 DOCA-salt induces aortic aneurysm independent of increased blood** 

**2.7 Vascular pathology of DOCA- or Aldo-salt induced aortic aneurysms**

Human aortic aneurysm is characterized by elastin and collagen degradation, matrix metalloproteinase (MMP), upregulation, inflammatory cell infiltration, vascular smooth muscle cell degeneration, and oxidative stress [51]. To investigate

Administration of DOCA and salt to mice or rats has been used in the field to induce hypertension [45]. Hypertension is recognized as a potential risk factor for aortic aneurysm [3–5]. Thus, it is important to determine whether hypertension contributes to DOCA-salt-induced aortic aneurysm. Blood pressure was measured using a noninvasive tail-cuff system (Coda 6; Kent Scientific Corp., USA). As expected, administration of DOCA or Aldo plus salt to 10-month-old male mice increased both blood pressure and external diameters of the abdominal aorta [41, 43]. However, there was no correlation between blood pressure increase and external diameters of abdominal aorta after DOCA-salt treatment. Similarly, there was also no difference in blood pressure between the mice with aortic aneurysm and the mice without aortic aneurysm. Moreover, treatment of mice with ACE inhibitor enalapril or ARB losartan effectively decreased blood pressure, but both enalapril and losartan had little effect on DOCA-salt-induced aortic aneurysm. Thus, we concluded that DOCA-salt induces aortic aneurysm independent of increased blood pressure. This conclusion is consistent with that in the Ang II infusion AAA mouse

an Ang II-independent mouse model to verify their key findings.

*DOI: http://dx.doi.org/10.5772/intechopen.86477*

**aneurysm**

induce aortic aneurysm.

**pressure**

*A New Mouse Model of Aortic Aneurysm Induced by Deoxycorticosterone Acetate or Aldosterone… DOI: http://dx.doi.org/10.5772/intechopen.86477*

pathophysiological relevance to the development of atherosclerosis [48]. Moreover, there is a synergistic interaction between Ang II and Aldo in VSMCs [49, 50]. Therefore, it is interesting to investigate whether DOCA-salt-induced aortic aneurysm is dependent upon Ang II. To address this important question, 10-month-old C57BL/6 male mice were treated with either an ACE inhibitor (enalapril) or an ARB (losartan) before (1 week) and after (4 weeks) DOCA-salt administration. As expected, enalapril or losartan effectively decreased blood pressure, but enalapril or losartan had little effect on the DOCA-salt-induced aortic dilation, aortic aneurysm formation, and aneurysmal rupture [41]. These results demonstrate that the DOCA-salt-induced aortic aneurysm is independent of Ang II thus provide an alternative mouse model of aortic aneurysm for investigators in the field who need an Ang II-independent mouse model to verify their key findings.
