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

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 induce aortic aneurysm.

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

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 model [19].

#### **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

*Aortic Aneurysm and Aortic Dissection*

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

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,

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

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%

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/

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

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

diameters of abdominal and thoracic aortas relative to the control [41].

indicating that TAA is likely derived from AAA.

DMSO. All groups of mice were treated for 4 weeks.

day Aldo is sufficient to induce AAA in the presence of high salt.

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

**of high salt**

**80**

high doses of reagent.

whether DOCA- or Aldo-salt-induced aortic aneurysms have these pathologic features, paraffin-embedded aortic cross-sections were subjected to Elastic-Van Gieson staining of elastin. Interestingly, elastin degradation was only observed in AAA induced by DOCA- or Aldo-salt [41, 43]. Immunocytochemistry studies revealed that MMP2, MMP9, F4/80 (macrophages), Ly6B2 (neutrophils), caldesmon (smooth muscle cells), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; apoptosis), and dihydroethidium (DHE; oxidative stress) were increased in aortas with AAA compared with that in control aortas [41, 43].

In agreement with these immunocytochemical studies, we determined mRNA expression of several inflammatory genes, including vascular cell adhesion molecule 1 (Vcam-1), chemokine (C-C motif) ligand 2 (Ccl2, also known as MCP-1), tumor necrosis factor (Tnf), and Ncf1 (also known as p47phox) in both abdominal and thoracic aortas from mice-administrated DOCA-salt or control mice. We found that Vcam-1, Ccl2, Ncf1, and Tnf were all markedly upregulated in thoracic aortas from mice-administrated DOCA-salt compared to control mice. Interestingly, Vcam-1, Ccl2, and Ncf1, but not Tnf, were also significantly upregulated by DOCAsalt in abdominal aorta from mice-administrated DOCA-salt compared to control mice [41, 43].

#### **2.8 Unique features of the DOCA- or Aldo-salt mouse model of aortic aneurysm**

The DOCA- or Aldo-salt mouse model exhibited several unique features that may be relevant to the human aortic aneurysm. First, DOCA- or Aldo-salt-induced aortic aneurysm required to use 10-month-old mice [41, 43] rather than 10-weekold mice (mostly used by the Ang II AAA mouse model [13, 19–23] and other chemical-induced mouse modes [17, 18, 24, 25]). Given the fact that human AAA occurs in old peoples [2, 3, 7], the DOCA- or Aldo-salt mouse model of aortic aneurysm may more resemble human AAA than other chemical-induced aortic aneurysms in this regard. Second, DOCA- or Aldo-salt-induced aortic aneurysm used wild-type C57BL/6 mice [41, 43] rather than hyperlipidemia mice (i.e., apolipoprotein E-deficient (ApoE<sup>−</sup>/<sup>−</sup>) used by Ang II infusion mouse models [13, 19–23]), thus avoiding the potential confounding effects of hyperlipidemia on aortic aneurysm. Third, using Aldo, a physiological agonist of MR, rather than chemicals (i.e., calcium chloride or pancreatic elastase) to induce aortic aneurysm, highlights its potential role in the etiology of aortic aneurysm. Moreover, the plasma concentration of Aldo in mice infused with Aldo [41] could be seen in human congestive heart failure and primary aldosteronism [31, 46, 47], suggesting that the Aldo-salt AAA mouse model is a pathological model rather than a pharmacological model that would cause concerns due to the use of high doses of reagent. Finally, high salt intake was required for DOCA to induce aortic aneurysm [41], indicating that high salt intake may be a new risk factor for the development of human AAA.

### **2.9 Significance and potential impact of the DOCA- or Aldo-salt mouse model of aortic aneurysm**

We described a new mouse model of aortic aneurysm induced by administration of MR agonist DOCA or Aldo plus high salt to 10-month-old male mice and provided compelling preclinical evidence that reveals a previously unrecognized, but potentially significant, role of Aldo, MR, and high salt in the pathogenesis of AAA. It is worth pointing out that this new mouse model of aortic aneurysm could be used as a platform to study intervention including medication (i.e., we have tested the effect of ACE inhibitor (enalapril), ARB (losartan), and MR antagonist (eplerenone and spironolactone) [41]). It is also worth pointing out at least three

**83**

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

significance and potential impact of the DOCA- or Aldo-salt mouse model of aortic aneurysm on the current basic research and clinical practice on the etiology, clinic

First, in agreement with the pivotal role of Aldo in cardiovascular diseases (i.e., hypertension and heart failure) [31, 32, 46], our studies highlight a potentially important but previously unrecognized role of Aldo in the etiology of human aortic aneurysm. Our studies suggest that increased plasma concentration of Aldo may be a new risk factor for human aortic aneurysm or may serve as a new plasma bio-

Second, it is well recognized that unfavorably excessive dietary sodium intakes remain prevalent around the world and are associated with an increased risk for cardiovascular diseases including hypertension, stroke, coronary heart disease, heart failure, and renal disease [52–54]. However, it is unknown that excessive dietary sodium intake may also be detrimental to the aorta with respect to aortic aneurysm. Our finding that excessive dietary sodium intake was essential for MR agonist to induce aortic aneurysm in mice suggests that excessive dietary sodium intakes may also be implicated in the etiology of human aortic aneurysm. In agreement with our findings, it was recently reported that high salt intake was associated with an increased prevalence of AAA in older men [55]. Moreover, our findings indicate that lifestyle change such as reduction of dietary sodium intakes may be effective to prevent old people from the development and progression of aortic aneurysm. Third, given the fact that currently there is no approved drug for treatment of AAA, our studies suggest that spironolactone and eplerenone, two clinically approved drugs that have been used for the treatment of human heart failure and essential hypertension [56], may also be effective in the treatment of human aortic aneurysm. Recently, a proof-of-concept randomized controlled clinical trial has been initiated based on our findings and is currently going on in Australia, which aims to test the effect of eplerenone on the progression of AAA (https://clinicaltri-

1.Subcutaneous implantation of MR agonist DOCA pellets to 10-month-old C57BL/6 male mice can potently induce aortic aneurysm formation and rupture in the presence of high salt. Both DOCA and salt, but not DOCA or salt alone, are required to induce aortic aneurysm formation and rupture in mice.

2.Infusion of 10-month-old C57BL/6 male mice by subcutaneous implantation of osmotic pumps to release Aldo to a pathological level can also induce aortic aneurysm formation and rupture, suggesting that increased plasma concentration of Aldo may be implicated in the etiology of human aortic aneurysm.

3.DOCA- or Aldo-salt-induced AAA mimics human AAA with respect to elastin degradation, MMP activation, inflammatory cell infiltration, smooth muscle

4.Treatment of mice with ACE inhibitor enalapril or an ARB losartan has little effect on DOCA-salt-induced aortic aneurysm, suggesting that DOCA-salt-

5.Treatment of mice with MR antagonist spironolactone and eplerenone effectively abolishes or diminishes DOCa- or Aldo-salt-induced aortic aneurysm,

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

diagnosis, evaluation, and treatment of AAA.

als.gov/ct2/show/study/NCT02345590).

cell degeneration, and oxidative stress.

induced aortic aneurysm is independent of Ang II.

**3. Conclusions**

marker for evaluation of aortic aneurysm progression.

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

significance and potential impact of the DOCA- or Aldo-salt mouse model of aortic aneurysm on the current basic research and clinical practice on the etiology, clinic diagnosis, evaluation, and treatment of AAA.

First, in agreement with the pivotal role of Aldo in cardiovascular diseases (i.e., hypertension and heart failure) [31, 32, 46], our studies highlight a potentially important but previously unrecognized role of Aldo in the etiology of human aortic aneurysm. Our studies suggest that increased plasma concentration of Aldo may be a new risk factor for human aortic aneurysm or may serve as a new plasma biomarker for evaluation of aortic aneurysm progression.

Second, it is well recognized that unfavorably excessive dietary sodium intakes remain prevalent around the world and are associated with an increased risk for cardiovascular diseases including hypertension, stroke, coronary heart disease, heart failure, and renal disease [52–54]. However, it is unknown that excessive dietary sodium intake may also be detrimental to the aorta with respect to aortic aneurysm. Our finding that excessive dietary sodium intake was essential for MR agonist to induce aortic aneurysm in mice suggests that excessive dietary sodium intakes may also be implicated in the etiology of human aortic aneurysm. In agreement with our findings, it was recently reported that high salt intake was associated with an increased prevalence of AAA in older men [55]. Moreover, our findings indicate that lifestyle change such as reduction of dietary sodium intakes may be effective to prevent old people from the development and progression of aortic aneurysm.

Third, given the fact that currently there is no approved drug for treatment of AAA, our studies suggest that spironolactone and eplerenone, two clinically approved drugs that have been used for the treatment of human heart failure and essential hypertension [56], may also be effective in the treatment of human aortic aneurysm. Recently, a proof-of-concept randomized controlled clinical trial has been initiated based on our findings and is currently going on in Australia, which aims to test the effect of eplerenone on the progression of AAA (https://clinicaltrials.gov/ct2/show/study/NCT02345590).
