Time-Dependent Pathological Changes in Hypoperfusion-Induced Abdominal Aortic Aneurysm

Simple Summary Abdominal aortic aneurysm (AAA) is a vascular disease that involves gradual dilation of the abdominal aorta and has a high mortality due to rupture. Hypoperfusion due to the obstruction of vasa vasorum, which is a blood supply system in the aortic wall, may be an important factor involved in AAA pathophysiology. A time-dependent analysis is important to understand the pathological cascade following hypoperfusion in the aortic wall. In our study, time-dependent analysis using a hypoperfusion-induced animal model showed that the dynamics of many AAA-related factors might be associated with the increased hypoxia-inducible factor-1α level. Hypoperfusion due to stenosis of the vasa vasorum might be a new drug target for AAA therapeutics. Abstract Hypoperfusion due to vasa vasorum stenosis can cause wall hypoxia and abdominal aortic aneurysm (AAA) development. Even though hypoperfusion is an important contributor toward pathological changes in AAA, the correlation between hypoperfusion and AAA is not fully understood. In this study, a time-dependent semi-quantitative pathological analysis of hypoperfusion-induced aortic wall changes was performed to understand the mechanisms underlying the gradual degradation of the aortic wall leading to AAA formation. AAA-related factors evaluated in this study were grouped according to the timing of dynamic change, and five groups were formed as follows: first group: angiotensin II type 1 receptor, endothelin-1 (ET-1), and malondialdehyde (MDA); second group: matrix metalloproteinase (MMP)-2, -9, -12, M1 macrophages (Mac387+ cells), and monocyte chemotactic protein-1; third group: synthetic smooth muscle cells (SMCs); fourth group: neutrophil elastase, contractile SMCs, and angiotensinogen; and the fifth group: M2 macrophages (CD163+ cells). Hypoxia-inducible factor-1α, ET-1, MDA, and MMP-9 were colocalized with alpha-smooth muscle actin cells in 3 h, suggesting that hypoperfusion-induced hypoxia directly affects the activities of contractile SMCs in the initial stage of AAA. Time-dependent pathological analysis clarified the cascade of AAA-related factors. These findings provide clues for understanding complicated multistage pathologies in AAA.


Introduction
Abdominal aortic aneurysm (AAA) is a vascular disease characterized by progressive dilation of the abdominal aorta. The risk factors for AAA include smoking, hypertension, male sex, and older age [1]. The risk of AAA rupture increases with an increase in AAA diameter [2]. The increased AAA diameter is caused by the continuous breakdown of the Biology 2021, 10, 149 3 of 15 ligated just below the renal artery and just above the bifurcation of the aorta ( Figure S1C). A small incision was created by cutting the aortic wall ( Figure S1D), and a polyurethane catheter (Medikit, Tokyo, Japan) shortened to 9 mm in length, was inserted ( Figure S1E). The incision was repaired with a 6-0 monofilament suture (Alfresa Pharma, Osaka, Japan) ( Figure S1F). The aortic wall was ligated over the inserted catheter using a 5-0 silk suture ( Figure S1G). The 5-0 silk suture to block the blood in the aorta was removed to restore the blood flow ( Figure S1H).

Microscopy
Aortic wall sections were observed under an optical microscope (CX23, Olympus Corporation, Tokyo, Japan) with a ×40 objective, connected to a camera (E-620, Olympus Corporation, Tokyo, Japan) with a 4032 × 3024 pixel resolution. Immunofluorescence staining was evaluated using a fluorescence microscope (ECLIPSE E200, Nikon Corporation, Tokyo, Japan) with a ×10 objective, connected to Basler PowerPack for microscopy with a microscopy pulse (Basler AG, Ahrensburg, Germany). Basler microscopy software (Version 1.1, Basler AG) was used for image acquisition. The microscope was equipped with a fluorescence illumination system (E2-FM, Nikon Corporation, Tokyo, Japan) with a DAPI filter, FITC filter, and Texas red filter.

Statistical Analyses
Values are expressed as mean ± SEM. Statistically significant differences were determined using the steel test and Mann-Whitney U test. Statistical significance was set at p < 0.05. Statistical analyses were performed using StatView software (version 5.0; SAS Institute, Cary, NC, USA) and EZR software [29].

Protein Levels in AAA-Related Factors in the Hypoperfusion-Induced Animal Model
Before the time-dependent pathological analysis of AAA wall formation in the hypoperfusion-induced animal model, immunohistochemical analyses of SMemb, neutrophil elastase, angiotensinogen, AT1 receptor, MDA, and ET-1 were performed to elucidate the precise effect of hypoperfusion on the aortic wall on 28 days after the induction of hypoperfusion ( Figure 1 and Figure S2). Positive areas for SMemb+ cells (synthetic SMCs), neutrophil elastase, angiotensinogen, AT1 receptor, MDA, and ET-1 were significantly increased in the hypoperfusion-induced AAA wall ( Figure 1).

Statistical Analyses
Values are expressed as mean ± SEM. Statistically significant differences were determined using the steel test and Mann-Whitney U test. Statistical significance was set at p < 0.05. Statistical analyses were performed using StatView software (version 5.0; SAS Institute, Cary, NC, USA) and EZR software [29].

Protein Levels in AAA-Related Factors in the Hypoperfusion-Induced Animal Model
Before the time-dependent pathological analysis of AAA wall formation in the hypoperfusion-induced animal model, immunohistochemical analyses of SMemb, neutrophil elastase, angiotensinogen, AT1 receptor, MDA, and ET-1 were performed to elucidate the precise effect of hypoperfusion on the aortic wall on 28 days after the induction of hypoperfusion (Figures 1 and S2). Positive areas for SMemb+ cells (synthetic SMCs), neutrophil elastase, angiotensinogen, AT1 receptor, MDA, and ET-1 were significantly increased in the hypoperfusion-induced AAA wall ( Figure 1). Data are expressed as the mean ± SEM. * P < 0.05 versus control wall. SMemb and neutrophil elastase: control wall (n = 9) and AAA wall (n = 10), AGT and AT1 receptor: control wall (n = 8) and AAA wall (n = 10), MDA: control wall (n = 10) and AAA wall (n = 12), and ET-1: control wall (n = 9) and AAA wall (n = 14).

Time-Dependent Changes in AAA-Related Factors in the Hypoperfusion-Induced Animal Model
Next, we performed a time-dependent pathological analysis of AAA-related factors in the aortic wall in the hypoperfusion-induced animal model. The thickness of the medial wall significantly decreased from the 3rd to the 28th day after aortic wall ligation to induce hypoperfusion (  Data are expressed as the mean ± SEM. * P < 0.05 versus control wall. SMemb and neutrophil elastase: control wall (n = 9) and AAA wall (n = 10), AGT and AT 1 receptor: control wall (n = 8) and AAA wall (n = 10), MDA: control wall (n = 10) and AAA wall (n = 12), and ET-1: control wall (n = 9) and AAA wall (n = 14).

Time-Dependent Changes in AAA-Related Factors in the Hypoperfusion-Induced Animal Model
Next, we performed a time-dependent pathological analysis of AAA-related factors in the aortic wall in the hypoperfusion-induced animal model. The thickness of the medial wall significantly decreased from the 3rd to the 28th day after aortic wall ligation to induce hypoperfusion (Figure 2A-I,S). The collagen-positive area significantly decreased from the 5th to the 28th day after aortic wall ligation ( Figure 2J-S). AAA formation was observed from the 10th to the 28th day after the aortic wall ligation ( Figure 2S and Figure S3).

Colocalization Studies of AAA-Related Factors with α-SMA+ Cells and Mac387+ Cells
To understand the AAA pathology at the initial stage, we investigated the colocalization of AAA-related factors with α-SMA+ cells and Mac387+ cells. Mac387+ cells were significantly detected at 6 h after induction of hypoperfusion and were analyzed in 6 h. HIF-1α was colocalized with α-SMA+ cells in 3 h, but not in Mac387+ cells ( Figure 6). AT1 receptor was colocalized with α-SMA+ cells both at 0 h and 3 h, and with Mac387+ cells in 6 h ( Figure S11). ET-1 and MDA were colocalized with α-SMA+ cells in 3 h, but not in Mac387+ cells (Figures S12 and S13). MMP-2 was colocalized with α-SMA+ cells both at 0 and 3 h, but not with Mac387+ cells ( Figure S14). MMP-9 was colocalized with α-SMA+ cells in both 3 h and Mac387+ cells at 6 h ( Figure S15). MMP-12 did not colocalize with α-SMA+ cells at both 0 h and 3 h, but did with Mac387+ cells in 6 h ( Figure S16). MCP-1 was colocalized with α-SMA+ cells both at 0 and 3 h, but not with Mac387+ cells ( Figure S17). Neutrophil elastase did not colocalize with either α-SMA+ or Mac387+ cells ( Figure S18). Angiotensinogen was colocalized with α-SMA+ cells both at 0 h and 3 h, but not with Mac387+ cells ( Figure S19). The data are summarized in Table 1.

Colocalization Studies of AAA-Related Factors with α-SMA+ Cells and Mac387+ Cells
To understand the AAA pathology at the initial stage, we investigated the colocalization of AAA-related factors with α-SMA+ cells and Mac387+ cells. Mac387+ cells were significantly detected at 6 h after induction of hypoperfusion and were analyzed in 6 h. HIF-1α was colocalized with α-SMA+ cells in 3 h, but not in Mac387+ cells ( Figure 6). AT1 receptor was colocalized with α-SMA+ cells both at 0 h and 3 h, and with Mac387+ cells in 6 h ( Figure S11). ET-1 and MDA were colocalized with α-SMA+ cells in 3 h, but not in Mac387+ cells (Figures S12 and S13). MMP-2 was colocalized with α-SMA+ cells both at 0 and 3 h, but not with Mac387+ cells ( Figure S14). MMP-9 was colocalized with α-SMA+ cells in both 3 h and Mac387+ cells at 6 h ( Figure S15). MMP-12 did not colocalize with α-SMA+ cells at both 0 h and 3 h, but did with Mac387+ cells in 6 h ( Figure S16). MCP-1 was colocalized with α-SMA+ cells both at 0 and 3 h, but not with Mac387+ cells ( Figure S17). Neutrophil elastase did not colocalize with either α-SMA+ or Mac387+ cells ( Figure S18). Angiotensinogen was colocalized with α-SMA+ cells both at 0 h and 3 h, but not with Mac387+ cells ( Figure S19). The data are summarized in Table 1.

Discussion
In this study, we performed time-dependent pathological analyses of the hypoperfusioninduced aortic walls to clarify the molecular mechanisms correlating hypoperfusion and AAA formation. A comparison of the pathological events of a hypoperfusion-induced AAA animal model and human AAA is shown in Table 2. Time-dependent changes in pathologies and proteins, including those in previous studies, were sorted by the dynamically changing time of each factor (Table 3A,B). Five dynamic changes were observed in the evaluated AAA-related factors after the induction of hypoperfusion (Table 3B).  Synthetic SMC ↑ Cardiovasc Pathol [41] ↑ Figure 1C Neutrophil elastase ↑ Cardiovasc Surg. [42] ↑ Figure 1F Angiotensinogen ↑ Atherosclerosis [43] ↑ Figure 1I AT 1 receptor ↑ Atherosclerosis [43] ↑ Figure 1L MDA ↑ Arterioscler Thromb Vasc Biol [44] ↑ Figure 1O ET-1 ? ↑ Figure 1R ' Colocalization studies have suggested the mechanisms underlying the cascades of AAA pathology in the initial stage (0 to 6 h). HIF-1α was colocalized with α-SMA+ cells in 3 h, but not with Mac387+ cells, suggesting that hypoperfusion-induced hypoxia directly affects the activities of contractile SMCs in the initial stage of AAA. The expression of ET-1, MDA, and MMP-9 in contractile SMCs might be associated with hypoperfusioninduced expression of HIF-1α.
In this study, we showed the characteristic dynamics of human AAA-related factors using a hypoperfusion-induced animal model. To our knowledge, this is the first timedependent pathological study of an AAA animal model. Expression of HIF-1α and several AAA-related factors was observed in contractile SMCs in the initial stage after induction of hypoperfusion. In addition, the pathological features of hypoperfusion-induced AAA in this study were consistent with those of human AAA. This study suggested that hypoperfusion could induce AAA-related factors, as reported in human AAA studies. However, the limitations of this study should be noted. Because this study was observational, the interrelation between these factors was not determined. The time lag of the dynamic pattern of HIF-1α-related factors suggests the existence of an unidentified interactional cascade. Further studies are required to elucidate these points.    Levels of the AT1 receptor, ET-1, and MDA initially increased 3 h after the induction of hypoperfusion. The AT1 receptor is required for AngII-induced AAA formation [45]. AT1 receptors, including endothelial cells, SMCs, and macrophages, are detected in several cells of the arterial wall. AngII stimulates MCP-1 secretion [46], ROS production [47], and MMP-2 expression in SMCs [48]. ET-1 is a 21-amino-acid peptide and is one of the most potent endogenous vasoconstrictors. ET-1 overexpression induces aneurysms in apolipoprotein E knockout mice with increased oxidative stress levels and monocyte/macrophage infiltration [49]. Similarly, plasma ET-1 levels [50,51] and aortic tissue AT1 receptor levels [43] are significantly increased in patients with AAA. MDA is an oxidative stress marker in AAA walls. Oxidative stress due to ROS production is involved in vascular injury and AAA development [52]. Oxidative stress is induced by endothelial dysfunction and nicotinamide adenine dinucleotide phosphate oxidase overexpression [53]. In our previous report, HIF-1α levels were also increased 3 h after the induction of hypoperfusion [30]. HIF-1α is reportedly the upstream factor of the molecules involved in AAA development [22], including ET-1 [54]. These data suggest that hypoperfusion in the aortic wall first induces hypoxia and vasoconstriction-related factors (AT1 receptor and ET-1) related to increased oxidative stress and inflammation.
Second, 6 h after the induction of hypoperfusion, the levels of MMP-2, MMP-9, MMP-12, Mac387+ macrophages (M1 macrophages), and MCP-1 significantly increased in the aortic walls. Increased levels of these AAA-related factors are consistent with findings in human AAA [17,23,24,26,37,40]. Oxidative stress due to ROS production in vascular SMCs reportedly induces excess MMP-2 expression [55,56]. In addition, HIF-1α induces MMP expression under hypoxic conditions [57]. As mentioned above, the increased levels in the second group can be associated with increased levels of factors in the first group and HIF-1α.
Third, synthetic SMC, SMemb+ SMC, significantly increased 2 days after the induction of hypoperfusion. SMemb is a non-muscle myosin heavy chain abundantly expressed in SMCs in the immature aorta [58], and an increased level of SMemb+ SMC is consistent with human AAA [41]. The increased level of synthetic SMC may be associated with the decreased level of contractile SMC in the fourth group. A hypoperfusion-induced AAA model may reproduce both the increase in synthetic SMC and the decreased in contractile SMC observed in human AAA. Contractile SMCs regulate the diameter and blood flow of the normal aorta. In response to aortic injury, contractile SMC shifts the aorta from a contractile state to a synthetic state, known as "phenotypic switching" [59]. Synthetic SMCs can secrete various extracellular matrix proteins and MMP molecules. Phenotypic switching is involved in the development of AAA [41,60]. Ailawadi et al. reported that phenotypic switching is an early event in aortic aneurysms formed in an elastase-induced AAA animal model [61]. The same group reported the possibility that Krüppel-like factor 4 (KLF4) regulates SMC phenotype switching [60]. KLF4 is able to induce pluripotent stem cells [62] and HIF-1α reportedly induces its expression in cancer cells [63], suggesting that hypoperfusion might be involved in KLF4 expression in the AAA wall.
Fourth, neutrophil elastase, angiotensinogen, and contractile SMC significantly changed 3 days after the induction of hypoperfusion. Changes in these AAA-related factors are consistent with those observed in human AAA [27,42,43]. Neutrophil elastase, which is released by activated neutrophils, plays an important role in the development of AAA [64,65]. It reportedly regulates the formation of neutrophil extracellular traps (NETs) [66], which play a critical role in the neutrophil-mediated development of AAA [67]. In sepsis-induced thrombus formation, HIF-1α activation is associated with NET formation during thrombosis [68], suggesting that NETs are downstream factors of hypoperfusion. Angiotensinogen is involved in hypertension as a precursor of AngII [69]. The promoter domain of angiotensinogen contains a binding site for HIF-1α, which mediates the transcriptional activation of angiotensinogen [70]. A decreased level of contractile SMC might be associated with the phenotypic switching mentioned above.
Fifth, the number of M2 macrophages (CD163+ cells) significantly increased 21 days after AAA formation. The observation of M2 macrophages is consistent with the findings of a previous human AAA study [25]. M2 macrophages promote anti-inflammatory response [71] and angiogenesis [72]. Adventitial angiogenesis has been observed in human AAA [22,32]. In this experimental model, the VV count in the adventitial wall increased in the AAA wall ( Figure 7 and Table 2A). The increased VV count might indicate a compensatory reaction to attenuate the hypoxic conditions in the AAA wall. HIF-1α stimulates angiogenesis via the NF-E2-related factor 2/heme oxygenase-1 (HO-1) pathway [72]. HO-1 is involved in promoting macrophage differentiation into the M2 phenotype as well as other inducible factors, such as interleukins and micro RNAs [73,74].
Colocalization studies have suggested the mechanisms underlying the cascades of AAA pathology in the initial stage (0 to 6 h). HIF-1α was colocalized with α-SMA+ cells in 3 h, but not with Mac387+ cells, suggesting that hypoperfusion-induced hypoxia directly affects the activities of contractile SMCs in the initial stage of AAA. The expression of ET-1, MDA, and MMP-9 in contractile SMCs might be associated with hypoperfusion-induced expression of HIF-1α.
In this study, we showed the characteristic dynamics of human AAA-related factors using a hypoperfusion-induced animal model. To our knowledge, this is the first timedependent pathological study of an AAA animal model. Expression of HIF-1α and several AAA-related factors was observed in contractile SMCs in the initial stage after induction of hypoperfusion. In addition, the pathological features of hypoperfusion-induced AAA in this study were consistent with those of human AAA. This study suggested that hypoperfusion could induce AAA-related factors, as reported in human AAA studies. However, the limitations of this study should be noted. Because this study was observational, the interrelation between these factors was not determined. The time lag of the dynamic pattern of HIF-1α-related factors suggests the existence of an unidentified interactional cascade. Further studies are required to elucidate these points.

Conclusions
Time-dependent pathological analysis is important to understand the mechanism underlying AAA formation and development. We found that five groups were formed by dynamically changing the time. VV stenosis, as an inducer of increased HIF-1α levels, may be a new drug development target. Our time-dependent pathological analysis clarified the black box of AAA pathology.

Conflicts of Interest:
The authors declare no conflict of interest.