Age-related differences in elastin content and mechanical properties influence susceptibility to hypoperfusion-induced abdominal aortic aneurysm

Abstract

Abdominal aortic aneurysm (AAA) predominantly affects older individuals, yet the mechanisms by which aging increases susceptibility to aneurysm formation remain incompletely understood. The infrarenal aorta, where most AAAs develop, possesses sparse adventitial vasa vasorum (VV), suggesting limited mural perfusion reserve. We hypothesized that aging reduces the structural and biomechanical resilience of the aortic wall, thereby increasing vulnerability to hypoperfusion-induced degeneration. Using a previously established rat model of AAA induced by hypoperfusion of the adventitial VV, we compared young (12 weeks) and mature adult (24–28 weeks) rats exposed to identical hypoperfusion stress. At baseline, mature adult rats exhibited significantly lower aortic elastin content and reduced tensile strength compared with young rats, suggesting reduced structural reserve. Following hypoperfusion surgery, mature adult rats developed significantly larger aneurysms at postoperative Days 14 and 28 and showed a tendency toward increased rupture-related mortality. Histological analysis demonstrated more severe elastic fiber destruction in mature adult aneurysms. Although baseline VV patency tended to be lower in mature adult rats during aneurysm progression, aneurysm diameter showed an inverse correlation with elastin content. These findings suggest that baseline structural differences in the aortic wall may influence susceptibility to aneurysm formation under conditions of impaired mural perfusion. When considered together with the intrinsically sparse microvascular supply of the infrarenal aorta, these results are consistent with the concept that reduced elastin content and altered wall structure may enhance vulnerability to hypoperfusion-related degeneration. However, the mechanistic relationship between these factors cannot be determined from the present study.

Introduction

Abdominal aortic aneurysm (AAA) is a progressive degenerative disorder characterized by irreversible dilation of the infrarenal aorta and is associated with a substantial risk of rupture and mortality¹. AAA predominantly affects older individuals, and aging is consistently identified as the strongest independent predictor of aneurysm development and expansion^2,3. Despite this well-established epidemiological association, the biological mechanisms by which aging increases susceptibility to AAA remain incompletely understood.

A distinctive feature of AAA is its marked anatomical predilection for the infrarenal abdominal aorta. More than 80% of AAAs arise in this segment^3,4, suggesting the presence of intrinsic regional vulnerability. We previously demonstrated in human autopsy specimens that the density of adventitial vasa vasorum (VV) progressively decreases along the aorta and is lowest in the infrarenal segment⁵. This sparse microvascular network implies limited perfusion reserve in the outer aortic wall. Consistent with this concept, adventitial VV stenosis and mural hypoxia have been observed in human AAA tissues^6,7, implicating impaired mural perfusion as a contributor to aortic wall degeneration.

Experimental models of abdominal aortic aneurysm have traditionally relied on several established approaches, including systemic angiotensin II infusion in hyperlipidemic mice which induces hypertension, inflammation, and medial degeneration leading to aneurysm formation⁸, periaortic calcium chloride application that induces chemical injury to the aortic wall⁹, and intraluminal elastase perfusion that directly degrades medial elastin¹⁰. Although these models have provided important insights into inflammatory and proteolytic mechanisms of aneurysm formation, the severity of the induced injury may obscure baseline structural differences in the aortic wall. Consequently, it remains unclear whether aging itself creates a vulnerable aortic substrate that predisposes the vessel to aneurysm formation under physiologically relevant stress.

Aging profoundly alters the structural and biomechanical properties of the arterial wall through progressive remodeling of the extracellular matrix. Age-associated degradation and fragmentation of elastic fibers, accompanied by compensatory collagen remodeling, lead to substantial changes in arterial wall architecture and mechanics^11–13. These extracellular matrix alterations are particularly pronounced in the abdominal aorta, where elastin content decreases and elastic fibers become increasingly sparse and fragmented with advancing age^14,15. As a consequence, the mechanical resilience of the aortic wall declines, resulting in reduced tensile strength and diminished capacity to withstand hemodynamic stress^16,17. Such age-dependent structural and biomechanical deterioration is thought to reduce the adaptive reserve of the aortic wall. Adaptive reserve refers to the capacity of the aortic wall to maintain structural integrity under hemodynamic stress through its extracellular matrix and cellular responses, therefore increasing its susceptibility to pathological dilation. However, despite these well-recognized age-related alterations, direct experimental evidence linking baseline age-dependent structural vulnerability to aneurysm susceptibility under physiologically relevant stress remains limited.

To investigate the interaction between mature adult stage and impaired mural perfusion without confounding enzymatic or chemical injury, we previously developed a rat model of abdominal aortic aneurysm induced by hypoperfusion of the adventitial vasa vasorum^18,19. This model reproduces key features of degenerative aneurysm remodeling while preserving the native architecture of the aortic wall prior to disease induction²⁰.

Based on these observations, we hypothesized that age-related structural differences in the abdominal aortic wall influence susceptibility to aneurysm formation under identical hypoperfusion stress. Specifically, we hypothesized that mature adult aortas, characterized by lower elastin content and reduced resistance to mechanical deformation, would exhibit greater aneurysmal degeneration than young aortas when exposed to the same experimentally induced mural hypoperfusion. In the present study, we compared young (12 weeks) and mature adult (24–28 weeks) rats exposed to identical hypoperfusion stress. Baseline elastin content and tensile strength were quantified prior to aneurysm induction as a background of both groups, and hypoperfusion stress was added to evaluate the aneurysm growth and structural remodeling for the comparison.

Methods

Ethics statement

All experimental protocols were reviewed and approved by the Animal Care and Use Committee of Hamamatsu University School of Medicine (Approval number: 23-013). All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

All surgical procedures were conducted under general anesthesia induced by intraperitoneal administration of a mixture of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg). All efforts were made to minimize animal suffering.

Animals

Male Sprague-Dawley rats (Japan SLC, Inc., Shizuoka, Japan) were used in this study. Animals were housed under controlled conditions at room temperature (approximately 25 °C) with free access to standard chow and water.

To investigate the effect of susceptibility on hypoperfusion-induced abdominal aortic aneurysm formation, rats were divided into two age groups. Young rats were defined as 12 weeks old, whereas mature adult rats were defined as 24–28 weeks old. All comparative experiments were performed under identical experimental conditions. A total of 88 rats were used in this study, including 43 rats in the young group and 45 rats in the mature adult group.

Measurement of aortic elastin content

Aortic elastin content was quantified using a Fastin Elastin Assay Kit (Biocolor, Carrickfergus, UK) according to the manufacturer’s instructions. Briefly, rat infrarenal aortic samples were homogenized in 0.25 M oxalic acid. After centrifugation, the supernatant was treated with the elastin precipitation reagent and centrifuged again. The resulting elastin pellet was incubated with an elastin-binding dye reagent, and the dye-elastin complex was subsequently dissociated. Absorbance was measured at 513 nm and compared with elastin standards. Elastin content was expressed as micrograms per milligram of dry tissue weight. 12 young rats and 12 mature adult rats were used.

Quantification of medial elastic fiber content

Quantitative analysis of medial elastic fibers was performed using a modified method based on Komutrattananont et al.¹⁵ Abdominal aortic tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 4 μm thickness. Sections were stained with Elastica van Gieson (EVG).

Whole-slide images were obtained using a NanoZoomer digital slide scanner (Hamamatsu Photonics, Japan). Images were analyzed using Fiji software (National Institutes of Health, Bethesda, MD, USA). The entire medial layer was manually delineated as a region of interest (ROI). EVG-positive areas representing elastic fibers were quantified using the Color Threshold function.

Elastic fiber content was expressed as:

To avoid analytical bias, identical threshold parameters were applied to all specimens. Analyses were performed on the entire circumferential cross-section of the aortic media.

Measurement of aortic tensile strength

The abdominal aorta distal to the renal arteries was harvested for tensile testing. Mechanical testing was performed using a custom-made apparatus (Muromachi Kikai Co., Ltd., Tokyo, Japan) previously described by Nishida et al.^21,22.

Aortic segments were cut into ring-shaped sections approximately 0.3 mm in width and immersed in 0.9% saline. Without preconditioning, each ring was mounted on two tungsten rods and stretched uniaxially at a constant rate of 0.1 mm/s. The force applied to the apparatus fulcrum was continuously recorded.

The tensile force measured at + 2 mm elongation was used as an index of aortic wall tensile strength. The + 2 mm elongation was selected because the baseline external diameter of the rat infrarenal abdominal aorta is approximately 2 mm, corresponding to an approximately two-fold increase in diameter.

EVG area quantification and tensile testing were performed in 6 rats per group.

Induction of hypoperfusion of the abdominal aortic wall

Hypoperfusion of the abdominal aortic wall was induced as previously described¹⁸. Following midline laparotomy, the abdominal aorta was carefully dissected from surrounding tissues. Lumbar arteries were ligated using 5 -0 silk sutures to reduce adventitial blood supply.

The aorta was temporarily clamped distal to the renal artery branch using a bulldog vascular clamp. A small transverse arteriotomy was made slightly distal to the clamp, and a pre-cut 8–10 mm 18-gauge ETFE catheter (Terumo, Tokyo, Japan) was inserted into the aortic lumen. The intraluminal catheter was inserted and secured together with the aortic wall to mechanically compress and obstruct the adventitial vasa vasorum, thereby reducing mural blood flow, as previously described by Tanaka et al.¹⁸. This procedure preserves luminal patency and distal perfusion while selectively inducing hypoperfusion of the aortic wall.

The arteriotomy was repaired with 6-0 monofilament sutures (Alfresa Pharma, Osaka, Japan), and the catheter was secured together with the aortic wall using silk sutures. The clamp was then released and distal blood flow was confirmed before closing the abdomen.

Although an identical maneuver was applied to cause mural hypoperfusion, the degree of the hypoperfusion was not directly quantified in each rat because it is technically difficult to assess in living animals.

Ultrasonographic assessment and survival analysis

13 young rats and 15 mature adult rats were used for the assessment. Aneurysm development was monitored using high-resolution ultrasonography (Digital Color Doppler Ultrasound System, E2V, SONOSCAPE, Guangdong, China) at postoperative Days 14 and 28.

All animals were observed daily for survival until Day 28. Rats that died during the observation period underwent immediate necropsy to determine whether death was caused by aneurysm rupture. At Day 28, surviving animals were euthanized by overdose of isoflurane anesthesia. The abdominal aorta was harvested, snap-frozen in liquid nitrogen, and stored at −80 °C.

Histological analyses

Aortic specimens were fixed in 10% formalin, embedded in paraffin, and sectioned using standard procedures. Sections were stained with hematoxylin and eosin (HE) and Elastica van Gieson (EVG).

Assessment of vasa vasorum patency

EVG staining was used to visualize the adventitial vasa vasorum. At identical magnification, the luminal boundary and external elastic lamina (EEL) were traced. The total area enclosed by the EEL was defined as the vessel area⁶.

VV patency was calculated as:

All measurements were performed using Fiji software by investigators blinded to experimental groups.

Separate cohorts were used for baseline structural analyses and for the hypoperfusion-induced aneurysm experiments.

For longitudinal ultrasonographic assessment and survival analysis, a total of 28 rats were initially assigned. Among these, a subset of animals (Young: n = 13; Mature adult: n = 15) was followed throughout the study period and included in the final imaging analysis. For histological analyses, including evaluation of vasa vasorum (VV) patency, separate subsets of animals (n = 6 per group) were followed and euthanized at predefined time points for tissue harvesting. Consequently, sample sizes differ between imaging-based analyses and histological assessments.

Statistical analysis

Data were analyzed using SPSS software (v29; IBM, Armonk, NY, USA). Continuous variables are presented as median (interquartile range). Comparisons between groups were performed using the Mann–Whitney U test. Categorical variables were compared using Fisher’s exact test.

Survival was analyzed using Kaplan-Meier curves and compared using the log-rank test.

Correlations between aneurysm diameter and elastin content or VV patency were evaluated using Spearman’s rank correlation coefficient (ρ).

A p value < 0.05 was considered statistically significant.

Results

Baseline structural vulnerability in mature adult aortas

At baseline (Day 0), mature adult rats exhibited significantly lower elastin content compared with young rats (Young: 37.02 µg/mg [31.65–46.31] vs. Mature adult: 21.18 µg/mg [20.08–25.55], p = 0.004, Fig. 1a).

Fig. 1.

Baseline structural and biomechanical vulnerability of the infrarenal abdominal aorta in young and mature adult rats. (a) Elastin content in the infrarenal abdominal aorta at Day 0. Elastin content per dry tissue weight was significantly lower in mature adult rats than in young rats (n = 12 per group, p = 0.004). (b) Left: Representative Elastica van Gieson (EVG) staining of the abdominal aortic media at Day 0. Although the overall architecture of the medial elastic lamellae appeared preserved in both groups, interlamellar elastic fibers were visibly sparser in mature adult rats. Scale bar, 50 μm. Right: Quantitative analysis of EVG-positive area within the medial layer. The proportion of EVG-positive area (EVG-positive area/medial ROI area × 100) was significantly lower in mature adult rats than in young rats (n = 6 per group, p = 0.026). (c) Tensile strength testing of abdominal aortic rings at Day 0. The force required to elongate the aortic ring by + 2 mm was significantly lower in mature adult rats, indicating reduced biomechanical strength of the aortic wall (n = 6 per group, p = 0.004). Aortic rings were obtained from the infrarenal segment prior to aneurysm induction. From each aortic specimen, two ring segments were harvested from the infrarenal aorta, and tensile testing was performed on both rings. Therefore, although the number of animals was n = 6 per group, a total of 12 measurements were obtained in each group.

Quantitative analysis of EVG-positive area confirmed reduced elastic fiber density in mature adult rats, characterized by sparser interlamellar elastic fibers (Fig. 1b). Biomechanical testing demonstrated that the force required to elongate the aorta by + 2 mm was significantly lower in mature adult rats (Young, 149.8 mN [128.7–159.4] vs. Mature adult, 86.1 [75.0–111.8], p = 0.004, Fig. 1c), indicating reduced tensile strength. Two aortic ring specimens per animal were tested in both groups.

Mature adult stage accelerates hypoperfusion-induced aneurysm formation

The overall experimental design is summarized in Fig. 2. Young (12-week-old) and mature adult (24–28-week-old) rats underwent hypoperfusion surgery at Day 0 and were followed by serial ultrasonographic assessment until Day 28.　Following hypoperfusion surgery　(Fig. 2), ultrasound imaging revealed progressive aneurysm development in both groups (Fig. 3).　Mature adult rats developed significantly larger aneurysms than young rats at Day 14 (Young: 2.80 mm [2.70–3.00] vs. Mature adult: 4.90 mm [4.09–5.51], p < 0.001) and at Day 28 (Young: 3.20 mm [3.00–3.50] vs. Mature adult: 5.40 mm [4.83–7.60], p = 0.004) (Fig. 4a). Three mature adult rats died from aneurysm rupture at postoperative Days 23, 23, and 25, whereas all young rats survived until Day 28 (Fig. 4b). Although the survival difference did not reach statistical significance (p = 0.094), rupture occurred exclusively in the mature adult group.

Fig. 2.

Experimental design of the hypoperfusion-induced abdominal aortic aneurysm model. Schematic illustration of the experimental design. Young (12 weeks) and mature adult (24–28 weeks) rats underwent hypoperfusion surgery at Day 0. Serial ultrasonographic measurements were performed at baseline (Day 0), Day 14, and Day 28. At Day 28, animals were euthanized for tissue harvesting and subsequent histological and biochemical analyses. Outcomes included aneurysm progression, survival, and correlation analyses.

Fig. 3.

Representative ultrasonographic and macroscopic findings during aneurysm progression. (a) Representative ultrasonographic images of the infrarenal abdominal aorta at Days 0, 14, and 28 in young and mature adult rats. (b) Representative macroscopic images of the abdominal aorta at Day 28, demonstrating greater aneurysmal dilation in mature adult rats than in young rats. Scale bar, 5 mm. (c) A schematic image of ultrasonography. The maximal aortic diameter was measured as the maximal short axial diameter perpendicular to the vessel axis, as indicated by the measurement line.

Fig. 4.

Aging accelerates aneurysm enlargement and rupture tendency after hypoperfusion. (a) Time course of maximal infrarenal aortic diameter measured by ultrasonography. Mature adult rats developed significantly larger aneurysms than young rats at both Day 14 and Day 28 (p < 0.01 for both time points). Young: n = 13; Mature adult: n = 15 at Day 0; numbers decreased thereafter because of rupture-related deaths. (b) Kaplan-Meier survival curves through Day 28. Three mature adult rats died from aneurysm rupture, whereas all young rats survived. The difference in survival did not reach statistical significance (log-rank p = 0.094). Sample sizes differ between analyses because separate subsets of animals were used for longitudinal imaging and terminal histological evaluation.

Histopathological changes in mature adult aneurysms

Elastic fiber alterations were qualitatively assessed based on EVG staining, focusing on fragmentation and loss of medial elastic lamellae. Quantitative EVG analysis at baseline (prior to aneurysm induction) is provided in Fig. 1b. However, only histological changes were evaluated at Day 28 (Fig. 5).

Fig. 5.

Histopathological changes in aneurysmal aortic walls at Day 28. Representative hematoxylin and eosin (HE) and Elastica van Gieson (EVG) staining of aneurysmal aortic walls harvested at Day 28 (n = 6 per group). Mature adult rats exhibited more severe elastic fiber disruption and greater degenerative remodeling than young rats. L: Lumen. Scale bar, 250 μm.

Vasa vasorum remodeling during aneurysm progression

At Day 0, VV patency did not differ between the two groups (Young: 65.71% [64.58–67.79] vs. Mature adult: 64.75% [62.32–69.98], p = 0.699, Fig. 6a). By Day 28, mature adult rats showed greater luminal narrowing of adventitial vasa vasorum, reflected by reduced VV patency (luminal area / EEL-enclosed area), although the difference between groups did not reach statistical significance (Young: 28.26% [26.38–32.53] vs. Mature adult: 19.66% [14.41–26.84], p = 0.065) (Fig. 6b). A trend towards inverse correlation was observed between aneurysm diameter and VV patency (Spearman’s ρ = −0.553, n = 12, p = 0.062), indicating that larger aneurysms tended to be associated with reduced VV patency (Fig. 6c). A schematic representation of the delineation used for the measurement of VV patency is shown in Fig. 6d.

Fig. 6.

Adventitial vasa vasorum remodeling during aneurysm progression. (a) Representative EVG-stained sections of adventitial vasa vasorum (VV) at Day 0 and quantitative analysis of VV patency. No significant difference in VV patency was observed between young and mature adult rats at baseline (n = 6 per group). Scale bar, 50 μm. (b) Representative EVG-stained sections of adventitial VV at Day 28 and quantitative analysis of VV patency. Mature adult rats showed greater luminal narrowing of adventitial VV, with a trend toward reduced patency compared with young rats (n = 6 per group, p = 0.065). Scale bar, 50 μm. (c) Correlation between aneurysm diameter and VV patency at Day 28. A weak inverse correlation was observed between aneurysm diameter and VV patency (Spearman’s ρ = −0.553, p = 0.062). (d) A schematic representation of the delineation used for the measurement of VV patency. Sample sizes differ between analyses because separate subsets of animals were used for longitudinal imaging and terminal histological evaluation.

Elastin depletion correlates with aneurysm expansion

At Day 28, elastin content was significantly lower in mature adult rats than in young rats (Young: 10.73 µg/mg [6.03–12.76] vs. Mature adult: 3.85 µg/mg [2.89–4.51], p = 0.026) (Fig. 7a). Pooled analysis revealed a significant inverse correlation between elastin content and aneurysm diameter (Spearman’s ρ = −0.781, p = 0.003, n = 12) (Fig. 7b).

Fig. 7.

Elastin depletion is associated with aneurysm severity at Day 28. (a) Elastin content per dry tissue weight in aneurysmal aortic tissue harvested at Day 28. Elastin content was significantly lower in mature adult rats than in young rats (n = 6 per group, p = 0.026). (b) Correlation between elastin content and aneurysm diameter at Day 28. A significant inverse correlation was observed between elastin content and aneurysm diameter (Spearman’s ρ = −0.781, p = 0.003).

Discussion

The present study suggests that differences in baseline aortic wall structure between young and mature adult rats may influence susceptibility to hypoperfusion-induced aneurysm formation. Under identical hypoperfusion conditions, mature adult rats exhibited greater aneurysm expansion, indicating that pre-existing structural differences may contribute to this response. These findings provide experimental evidence that mature adult stage lowers the biomechanical reserve of the aortic wall and thereby converts mural hypoperfusion from a subclinical stress into a potent trigger for aneurysm formation.

Aging is consistently identified as the strongest independent clinical predictor of AAA development and expansion^3,4,23. Despite this robust epidemiological association, the biological mechanisms linking aging to aneurysm susceptibility remain incompletely understood. Our findings suggest that the mature adult stage reduces the baseline structural reserve of the aortic wall, thereby lowering the threshold at which hypoperfusion stress results in pathological dilation.

A striking feature of AAA is its strong anatomical predilection for the infrarenal abdominal aorta^23,24. In our previous human autopsy study, we demonstrated that the density of adventitial vasa vasorum (VV) progressively decreases from the aortic arch toward distal segments and is lowest in the infrarenal abdominal aorta⁵. This sparse VV distribution implies that the infrarenal aorta possesses intrinsically limited mural perfusion reserve. When perfusion of the outer aortic wall becomes compromised, this segment may therefore be particularly vulnerable to ischemia-related degeneration.

Our findings are also consistent with human segmental composition studies demonstrating that elastin content decreases distally along the aorta. In non-aneurysmal human specimens, elastin area fraction is significantly lower in the abdominal aorta and iliac arteries than in the thoracic and suprarenal segments^25,26. This intrinsic structural heterogeneity suggests that the infrarenal aorta operates with a smaller elastin “buffer” even before disease onset. Consequently, the combination of limited perfusion reserve and reduced matrix resilience may predispose this segment to aneurysm formation. Age-related alterations of the extracellular matrix further amplify this vulnerability. Human studies have demonstrated progressive reductions in elastin content and increasing fragmentation of elastic fibers with aging^14,15. These structural changes are accompanied by deterioration of biomechanical properties of the aortic wall. Mechanical testing studies of human aortas have shown that tensile strength and extensibility decline with age^16,17. Consistent with these findings, the present study demonstrated that mature adult rats exhibited significantly reduced elastin content and tensile strength at baseline, indicating that structural weakening precedes aneurysm formation. Although we used a force–displacement, standardized displacement-based approach to compare relative differences in structural resistance between groups, stress–strain relationships provide a more rigorous characterization of material properties. However, such analyses are technically challenging in small animal models and may introduce additional variability.

Importantly, the present measurements reflect resistance to deformation at a predefined submaximal displacement rather than post-failure mechanical behavior. Lower strain ranges are more physiologically relevant for arterial function; therefore, the results should be interpreted as relative differences in deformation resistance under controlled conditions rather than absolute material properties. Future studies incorporating stress–strain analysis and advanced biomechanical modeling will be required to further characterize the mechanical behavior of the aging aortic wall. The functional implications of altered biomechanical properties were not directly assessed in the present study. Although ultrasonography enabled accurate measurement of aortic diameter, it did not allow evaluation of arterial stiffness or dynamic wall mechanics comparable to clinical assessments. Previous studies using this hypoperfusion-induced AAA model have demonstrated no significant changes in systemic blood pressure, suggesting that aneurysm progression in this model is not driven by hypertension²⁷. Therefore, the observed differences in mechanical response are more likely attributable to intrinsic structural alterations of the aortic wall rather than systemic hemodynamic changes.

The present results also suggest that impairment of the vasa vasorum may contribute to aneurysm progression. In addition to the relationship between elastin depletion and aneurysm size, we observed a trend toward an inverse correlation between aneurysm diameter and VV patency. Although this association did not reach statistical significance, likely due to the limited sample size, animals with larger aneurysms tended to exhibit greater narrowing of the adventitial VV. Histopathological studies of human AAA tissues have similarly demonstrated intimal hyperplasia and luminal narrowing of VV, suggesting that microvascular dysfunction contributes to medial degeneration of the aortic wall⁶. Major clinical risk factors for AAA may further aggravate this process through injury to the vasa vasorum. Smoking and hypertension, two of the strongest risk factors for AAA, have been linked to pathological alterations of the microvasculature supplying the arterial wall^28,29. Experimental studies have shown that nicotine exposure induces abnormal proliferation of smooth muscle cells within the VV, resulting in luminal stenosis and impaired mural perfusion³⁰. Hypertension has also been associated with reduced VV flow and degeneration of the outer aortic wall³¹. These observations suggest that major AAA risk factors may converge on a common pathway of VV injury, mural hypoperfusion, and medial degeneration.

Taken together, the present findings are consistent with a conceptual framework in which AAA susceptibility may be influenced by the convergence of three baseline vulnerabilities: segmental hypovascularity, age-dependent matrix weakening, and vasa vasorum dysfunction. The infrarenal aorta is intrinsically disadvantaged by sparse VV supply, while aging progressively reduces elastin content and biomechanical strength. When mural perfusion becomes impaired, whether through VV stenosis or systemic risk factors, the adaptive capacity of the aortic wall may be exceeded, leading to progressive dilation and aneurysm formation.

Our hypoperfusion model provides a useful experimental framework to examine this interaction between structural vulnerability and impaired mural perfusion. In the elastase-induced AAA model, age has been reported to have no significant effect on aneurysm development, suggesting that the strong enzymatic injury overrides baseline structural differences of the aortic wall³². Similarly, the calcium chloride–induced model relies on perivascular chemical injury that triggers acute inflammation and medial degeneration. Given the intensity of this externally imposed injury, it is plausible that age-dependent differences in baseline wall structure are masked in this model as well. These observations support the concept that traditional AAA models, which depend on acute and substantial wall injury, may obscure intrinsic age-related vulnerability. In contrast, our hypoperfusion model preserves baseline wall architecture and therefore enables age-dependent reductions in elastin content and tensile strength to manifest as differences in aneurysm susceptibility.

Baseline structural vulnerability in this study was assessed by elastin content and tensile strength at Day 0. Under identical hypoperfusion conditions, mature adult rats, which exhibited reduced elastin content and tensile strength, developed larger aneurysms. These findings suggest that baseline structural differences may influence the response of the aortic wall to impaired mural perfusion. Rather than demonstrating a direct interaction, our results indicate an association between reduced structural reserve and increased susceptibility to aneurysm formation, highlighting the potential importance of microvascular perfusion in maintaining aortic wall integrity. However, the mechanistic relationship between these factors cannot be determined from the present study and warrants further investigation.　In the present study, only male rats were used. This decision was based on both clinical and experimental considerations. Clinically, abdominal aortic aneurysm occurs more frequently in males than in females, and male sex is a well-established risk factor for AAA development. Experimentally, previous studies using the hypoperfusion-induced AAA model have demonstrated that sex hormones significantly influence aneurysm formation. For example, ovariectomy increases the incidence and diameter of AAA, likely through degradation of vascular wall components such as elastin and collagen³³. These findings suggest that inclusion of female animals would introduce hormonal effects that could confound the evaluation of age-related structural and biomechanical differences. Therefore, to specifically assess the impact of aging on baseline aortic wall properties and susceptibility to hypoperfusion-induced aneurysm formation, we restricted the study to male rats.

Several limitations should be acknowledged. First, we used mature adult rather than senescent animals, and the effects observed here may be even more pronounced at advanced ages. Second, the sample size limited statistical power for survival and VV analyses. Third, it should be noted that tensile testing in this study was performed under low-load, unpressurized conditions, reflecting total vessel compliance rather than physiological stiffness under pulsatile pressure. In contrast to in vivo conditions, arterial stiffness under physiological pressure is known to increase with age. Therefore, the present findings should be interpreted as reflecting structural integrity under passive mechanical conditions rather than physiological arterial stiffness. Finally, molecular mediators linking hypoperfusion to extracellular matrix degradation, or the mechanisms underlying VV narrowing were not comprehensively evaluated and warrant further investigation.

In conclusion, mature adult rats exhibited reduced elastin content and lower tensile strength under the specific experimental conditions of this study, suggesting decreased structural reserve of the aortic wall. These findings provide mechanistic insight into why AAA predominantly occurs in older individuals and preferentially develops in the infrarenal abdominal aorta. Importantly, these mechanical measurements were obtained under non-physiological conditions and may not reflect in vivo arterial behavior under physiological pressure, where aging is generally associated with increased arterial stiffness. Thus, the present results should be interpreted as reflecting passive structural properties rather than physiological arterial stiffness.

Author contributions

N.U. designed the studies, analyzed the data, wrote and edited the manuscript, and secured the funding. N.I. performed the experiments, analyzed the data and wrote themanuscript. H.Ts., Y.E., Y.M., K.F., M.S., K.K., and M.H. performed the experiments and acquired the data. K.I., N.Z, and K.Y. analyzed the data and edited the manuscript. H.Ta. edited the manuscript.

Funding

This work was supported by Grant-in-Aid for Scientific Research (C) (24K11988) from Japan Society for the Promotion of Science (to N.U.). This study was also funded in part by Sunsho Pharmaceutical Co., Ltd. This does not alter the authors’ adherence to all the journal’s policies on sharing data and materials.

Data availability

All data are available upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.