Segmental Aortic Stiffening Contributes to Experimental Abdominal Aortic Aneurysm DevelopmentCLINICAL PERSPECTIVE
Background—Stiffening of the aortic wall is a phenomenon consistently observed in age and in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined.
Methods and Results—Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening precedes aneurysm growth. Finite-element analysis reveals that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) wall stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent aortic segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with the reduced segmental stiffness of the AAA-prone aorta (attributable to equalized stiffness in adjacent segments), reduced axial wall stress, decreased production of reactive oxygen species, attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, and attenuated apoptosis within the aortic wall, as well. Cyclic pressurization of segmentally stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening.
Conclusions—The present study introduces the novel concept of segmental aortic stiffening as an early pathomechanism generating aortic wall stress and triggering aneurysmal growth, thereby delineating potential underlying molecular mechanisms and therapeutic targets. In addition, monitoring segmental aortic stiffening may aid the identification of patients at risk for AAA.
Abdominal aortic aneurysm (AAA) carries a high mortality in the case of rupture.1 Current therapies are limited to open surgical or interventional stent-based exclusion of the aneurysmal sac from the circulation to prevent rupture. However, these treatment options are generally reserved for larger aneurysms (typically AAA diameter >5.5 cm), and there is no effective therapy targeting the evolution of small aneurysms. This lack of treatment options partly derives from an insufficient understanding of early AAA pathogenesis.
Editorial see p 1745
Clinical Perspective on p 1795
Recent evidence suggests that AAA formation is not simply attributable to aortic wall degeneration, resulting in passive lumen dilation, but to active, dynamic remodeling. The latter involves transmural inflammation, extracellular matrix (ECM) alterations including elastin fragmentation and (compensatory) collagen deposition, vascular smooth muscle cell (VSMC) apoptosis, and oxidative stress.1–4
From a pathomechanistic point of view it is essential not only to characterize the particular cellular and molecular alterations involved in AAA formation, but also to identify early triggers of remodeling. In that respect, mechanical wall stress is an intriguing candidate. Biomechanical stress (ie, shear stress, circumferential or axial wall stress) may drive adaptive arterial remodeling in response to altered hemodynamics, but also may induce inflammation and ECM remodeling, and VSMC apoptosis, as well, in vascular disease.4,5
AAA growth is accompanied by increasing wall stress.6,7 Although wall stress attributable to the vessel’s expanding geometry significantly contributes to the eventual rupture of the mature AAA, it might appear that wall stress would be unrelated to the pathophysiology in early, preaneurysmal stages, when aortic size has not yet overtly changed. However, enhanced wall stress may still occur owing to early aortic biomechanical alterations (ie, aortic stiffening).
AAA formation is associated with a substantially increased wall stiffness.8,9 Additionally, pronounced stiffening of the abdominal aorta occurs with aging, a major risk factor for AAA.10 We hypothesize that the existence of a stiff aortic segment adjacent to a more compliant aorta (ie, segmental aortic stiffness [SAS]) generates axial wall stress attributable to nonuniform systolic wall deformations, thereby modulating early aneurysm pathobiology (Figure 1).
Materials and Methods
Details are described in the online-only Data Supplement.
Porcine Pancreatic Elastase Infusion Model
The porcine pancreatic elastase (PPE) infusion model to induce AAA in 10-week-old male C57BL/6J mice was performed as previously described.11 In brief, after placing temporary ligatures around the proximal and distal aorta, an aortotomy was created at the bifurcation and an insertion catheter was used to perfuse the aorta for 5 minutes with saline containing PPE (1.5 U/mL; Sigma Aldrich).
Glue Treatment of the PPE-Adjacent Aortic Segments
To locally enhance aortic mechanical stiffness, a surgical adhesive (BioGlue, CryoLife, Atlanta) was applied to the segments adjacent to the PPE-treated aorta directly after completion of the PPE treatment. Complete polymerization of the 2-component glue (albumin/glutaraldehyde) occurred within seconds. Care was taken to avoid the PPE-treated segment (Figure II in the online-only Data Supplement). For sham-treatment groups, only 1 component of BioGlue was applied.
Mouse Ultrasound Studies
Systolic diameter (Ds) and diastolic diameter (Dd) were quantified in the PPE-treated segment, and in the adjacent untreated segments, as well, by using M-mode ultrasound. Circumferential cyclic strain ε was calculated as ε=(Ds–Dd)/Dd×100%. SAS was defined as a relative index to quantify the stiffness of the PPE-treated segment in relation to the adjacent aorta, calculated as SAS=εadjacent aorta /εPPEsegment. The strain values for adjacent aorta (εadjacent aorta) represent an average strain calculated from the adjacent segments proximal and distal to the PPE-treated segment. For shear-stress calculations, blood flow was assessed as previously described.12
Human Ultrasound Studies
Nineteen male volunteers of different ages (youngest age, 36; oldest age, 71; mean age, 51.9 years) were included in the study. Exclusion criteria were cardiovascular diseases (in particular AAA), diabetes mellitus, and history of smoking. M-mode images tracking the anterior and posterior aortic wall motion were recorded at predefined locations (suprarenal, midinfrarenal, and proximal to the aortic bifurcation).
Systolic diameter and diastolic diameter were quantified in the suprarenal, midinfrarenal, and bifurcational segment of the abdominal aorta to calculate cyclic strain and SAS.
Finite-element analyses (FEA) of the mouse aorta were performed by using the commercial finite-element software package ABAQUS. The artery was modeled as a 2.0-mm-long axisymmetric tube with outer diameter Da=0.9 mm and arterial wall thickness t=0.075 mm. The intima, media, and adventitia were summarized in a single homogeneous layer modeled by using an isotropic Neo-Hookean strain energy function with a shear modulus of 300 kPa. Stiffness of the stiff segment (l=1.0 mm) was modified as indicated.
Total aortic RNA was isolated and processed for real-time quantitative reverse transcription polymerase chain reaction by using standard protocols and methods.
Laser Capture Microdissection
Laser capture microdissection was performed as previously described.13 F4/80-stained macrophages were microdissected from frozen aortic cross-sections (7 μm) by using a PALM MicroBeam System (Zeiss). RNA was subsequently processed for real-time quantitative reverse transcription polymerase chain reaction with the use of the Single Cell-to-CT Kit (Ambion).
Histology, Immunofluorescence, In Situ Dihydroethidium Staining, and In Situ Hybridization
Standardized protocols were used, with details available in the online-only Data Supplement.
Ex Vivo Aortic Mechanical Stimulation
Abdominal aortae were explanted, cannulated, and mounted in the heated vessel chamber of a pressure arteriograph system (model 110P, Danish Myotechnology, Copenhagen, Denmark) and stretched to in vivo length. The aorta was then subjected to an automated pressure protocol, cyclically alternating between 80 mm Hg and 120 mm Hg with a frequency of 4/min for 1 hour. To stiffen/restrain either the complete aorta or just the central segment (to simulate segmental stiffening), a silicone cuff (SILASTIC Laboratory Tubing; inner diameter, 0.51 mm; Dow Corning) was placed around the aorta (Figure I in the online-only Data Supplement). After the conclusion of the experiment, the aorta was removed from the cannula and processed for RNA isolation.
Data are presented as mean±standard error of the mean. For comparison of 2 groups, Mann-Whitney test was performed; multiple groups (≥3 groups) comparison was accomplished by Kruskal-Wallis test with the Dunn post test. Ultrasound data comparing 2 groups/treatments over time were analyzed by permutation F test based on 2-way repeated-measures analysis of variance. For each treatment assignment, we performed a repeated-measures analysis of variance and derived a null distribution of the P value for treatment effect. The P value from the permutation test was then established as the percentage of the null P values less than the P value from the real data. To compare ultrasound parameters within 1 treatment group over time, the Friedman test was used. For correlation analysis of animal ultrasound data, Spearman correlation was used. For correlation analyses of human ultrasound data, Pearson correlation was used after passing D’Agostino-Pearson omnibus normality test. A value of P≤0.05 (2-sided) was considered statistically significant.
All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University (http://labanimals.stanford.edu/) and followed the National Institutes of Health and US Department of Agriculture Guidelines for Care and Use of Animals in Research.
Aortic Stiffening Precedes Aneurysmal Dilation in Experimental AAA
We investigated the temporal relationship between aortic biomechanical alterations and aneurysmal dilation in the PPE-infusion model of murine AAA. Circumferential cyclic aortic strain (as a measure of vascular stiffness) and aortic diameter were monitored over time in the PPE-treated segment and saline-perfused controls via M-mode ultrasound (Figure 2A and 2B).
Although native abdominal aortae exhibited a baseline cyclic strain of ≈12%, PPE infusion rapidly induced a substantial strain reduction of >50% in the treated segment at day 1 (d1) followed by further declines until d14, after which it remained stable until d28. In contrast, saline infusion only resulted in a minor strain reduction in the corresponding segment (Figure 2A).
The aortic diameter, however, displayed insignificant enlargement up to d7 post-PPE and postsaline. The PPE-treated segment then dilated markedly between d7 and d14. Afterward the aortic diameter remained relatively stable up to d28 (Figure 2B).
Investigating possible mechanisms for the rapid stiffening of the PPE-treated segments, we found remarkable elastin fragmentation, whereas profibrotic responses were only moderate (Figure 2F).
SAS Generates Axial Wall Stress in the AAA-Prone Segment
Having identified rapid early mechanical stiffening of the aneurysm-prone segment (ie, reduced cyclic strain), we sought to investigate its role in aneurysm development. We hypothesized that SAS (defined as enhanced stiffness of the aneurysm-prone segment relative to the adjacent aorta) would generate adverse wall stress during cyclic deformation of the aortic wall, eventually resulting in AAA formation. We therefore performed in silico wall stress analysis by using a finite-element model.
With the use of a simplified approach, the infrarenal mouse aorta was modeled as a cylindrical tube. To examine the effects of segmental stiffening we simulated a pressure of 130 mm Hg (approximating systolic blood pressure) and introduced a segment of increasing stiffness adjacent to a nonstiff segment. We found that increasing segmental stiffness progressively induced axial stress in the stiff segment extending from the segmental interface (Figure 3A).
Because hypertension represents a risk factor for AAA, we explored the impact of high blood pressure levels on axial wall stress by pressurizing our FEA model with a fixed stiffness of the stiff segment up to 180 mm Hg. This simulation revealed that high blood pressure augmented segmental stiffness–based wall stresses (Figure 3B).
Taken together, these data suggest that SAS generates substantial axial wall stresses that also are susceptible to a hypertensive environment.
SAS Correlates With Experimental Aneurysm Progression
To further investigate the significance of SAS as an inducer of aneurysm growth, we performed temporal analysis of SAS in vivo and correlated it to aneurysm growth in the PPE model. We found a continuous increase in SAS after aneurysm induction, peaking at d7, which was attributable to increasing stiffness of the PPE-treated segment (5-fold higher than adjacent aorta; Figure 2C and 2D). Of note, the SAS peak coincided with the onset of aneurysm expansion. Moreover, the magnitude of SAS at d7 correlated with subsequent aortic enlargement between d7 and d14 (Figure 2E).
After d7 SAS declined as a result of progressive stiffening of the adjacent aortic segments (Figure 2C and 2D), which was accompanied by decelerating aortic diameter enlargement (Figure 2B). Saline-infused controls did not exhibit significantly enhanced SAS at any point during the entire observation period (Figure 2C).
Profibrotic Mechanisms Accompany Stiffening of AAA-Adjacent Segments, Thereby Reducing SAS
Having detected decreased SAS at d14 owing to stiffening in the AAA-adjacent aorta, we investigated the underlying molecular mechanisms.
Medial collagen deposition, a known determinant of arterial stiffness, was remarkably enhanced in AAA-adjacent segments at d14 after aneurysm induction (in comparison with d7; Figure 4D). Expression of the collagen genes Col1a1 and Col3a1 was increased in the adjacent segments in comparison with the AAA segment itself at d7 (Figure 4A), preceding the histological alterations. In line with this observation, miR-29b, previously shown to be an epigenetic negative regulator of collagen expression in AAA, was similarly downregulated at d7 (Figure 4B). More specifically, in situ hybridization indicated marked miR-29b downregulation within the aortic media (Figure 4C).
In contrast to the marked profibrotic changes, elastin architecture appeared unaffected in the AAA-adjacent aorta (Figure 4D).
Interventional Reduction of Segmental Stiffness Reduces Wall Stress and Aneurysm Progression
To investigate the potential causative role of SAS as a mechanism driving AAA development, we focally stiffened the adjacent aorta next to the PPE-treated segment by periaortic application of BioGlue, a surgical adhesive with a relatively high material stiffness (Figure II in the online-only Data Supplement). Glue application induced rapid and sustained stiffening of the adjacent aortic segments (Figure 5A), resulting in near-equalization of stiffness between the PPE-treated segment and the glue-treated adjacent segments. This was reflected in a significant reduction of SAS in comparison with sham glue–treated controls (Figure 5B).
To exclude the possibility that aortic constriction attributable to segmental glue treatment might lead to alterations of the downstream aortic flow and fluid shear stress, thereby affecting aneurysm formation, we monitored the aortic diameter of the glue-treated segment and the downstream flow profile, as well. We detected neither luminal narrowing (data not shown) nor elevated flow shear stress levels (Figure III in the online-only Data Supplement). Glue treatment of the adjacent aorta did not cause perturbations of its elastin architecture nor an enhanced fibrotic response (Figure IV in the online-only Data Supplement), suggesting that direct mechanical interaction with the aortic wall caused the stiffening effect.
Further, our finite-element model demonstrated that stiffness equalization between all segments (ie, reduction of SAS) resulted in decreased and homogenized axial stress (Figure 3C).
Finally, comparing aortic diameter between glue-treated and sham glue–treated animals, we found that PPE-induced aortic expansion was significantly reduced when adjacent segments were immobilized by glue application. The expected rapid diameter increase between d7 and d14 was suppressed (Figure 5C).
To further test the efficiency of delayed glue treatment on aneurysm progression, we performed additional experiments with glue intervention at d7 post-PPE, when there already is a small dilation combined with a high segmental stiffness (Figure 5D and 5E). As a result, we found that delayed glue stiffening of the AAA-adjacent aorta also significantly reduces SAS and thereby represses the consecutive aneurysmal diameter progression in comparison with sham glue–treated animals (Figure 5D and 5E).
Reduction of Segmental Stiffness Modulates Critical Features of AAA Pathobiology
Because AAA formation is accompanied by extensive ECM remodeling, we performed histological analyses of the aneurysm wall, focusing on elastin and collagen architecture. Extensive destruction of elastin fibers, a hallmark of aneurysm pathology, was present in sham glue–treated mice on d14 after PPE infusion (Figure 5F). Further, Picrosirius Red staining revealed disturbed wall architecture with general wall thickening, loss of layered structure, and diffuse collagen enrichment (Figure 5G). In contrast, elastin structure and wall layering were better preserved in the glue-treated group, whereas collagen accumulation appeared less prominent (Figure 5F and 5G).
AAA pathology includes enhanced reactive oxygen species (ROS) generation, vascular inflammation, VSMC apoptosis, and enhanced matrix metalloproteinase (MMP) activity. To assess the impact of SAS modulations on these end points, we analyzed the PPE-treated aorta at d7, which marks the peak of segmental stiffening but precedes the prominent diameter increase between d7 and d14.
We performed in situ dihydroethidium fluorescence to monitor ROS generation. PPE-treated segments exhibited enhanced nuclear fluorescence in comparison with native controls, whereas glue treatment resulted in a significant decrease in ROS production (Figure 6A and 6B).
Inflammation was quantified by aortic macrophage infiltration and cytokine analysis. Extensive macrophage infiltration of the aortic wall was present 7 days after aneurysm induction as assessed by immunofluorescence (Figure 6C through 6E), accompanied by enhanced aortic gene expression of Il6, Ccl2, and Il1b (Figure 6G). Immunofluorescence additionally revealed macrophage colocalization with each of these cytokines (Figure V in the online-only Data Supplement). Glue treatment reduced macrophage infiltration, and cytokine expression, as well (Figure 6C through 6E).
To further delineate the role of macrophages in vascular cytokine production, we analyzed gene expression profiles of macrophages directly isolated from the PPE-aneurysm sections via laser capture microdissection. To this end, we microdissected macrophages (positive F4/80 staining) from the aortic wall and confirmed macrophage transcript enrichment by enhanced Emr1 expression (encoding for F4/80 protein) in comparison with randomly captured F4/80-negative cells (Figure VI in the online-only Data Supplement). Macrophages isolated from sham glue treatment exhibited significantly higher expression of Il1b, Il6, and Ccl2 than those from glue-stiffened samples (Figure 6H).
Assessing apoptosis, we detected enhanced capase-3 activity in the intimal and medial layer of PPE-treated aortic wall, which was reduced in the glue-treated group (Figure 6F).
MMP2 and MMP9 are essential for matrix macromolecule degradation in AAA. In accordance with the substantial elastin breakdown found in PPE-treated segments, both Mmp2 and Mmp9 were significantly upregulated. Glue stabilization of the adjacent aortic segments, which prevented extensive elastin breakdown and collagen remodeling, minimized Mmp expression (Figure 6I). Additionally, this intervention reduced the enhancement of Col1a1 and Col3a1 expression after aneurysm induction (Figure 6J).
Ex Vivo SAS Induces Upregulation of AAA-Related Genes
We examined the mechanism of SAS as a driver of AAA pathogenesis by validating our in vivo findings ex vivo. We explanted murine abdominal aortic segments and mounted them onto a pressure myograph system. Aortae were then subjected to physiological pressure levels, cyclically alternating between 80 mm Hg and 120 mm Hg. To simulate aortic stiffening, the systolic expansion of either the entire aortic segment (complete stiffening) or just the central aortic segment (segmental stiffening) was restrained by an externally applied silicone cuff (Figure 7A, Figure VI in the online-only Data Supplement). After 1 hour of cyclic pressurization, aortic gene expression was analyzed.
Cuffing the entire aortic segment had minimal-to-no effect on the expression of inflammatory cytokines Il6 and Ccl2. Segmental stiffening, in contrast, induced the upregulation of these genes (Figure 7B). Likewise, the expression of metalloproteinases (Mmp2, Mmp9) and collagen genes (Col1a1, Col3a1), as well, quantified as indicators of active matrix remodeling, was significantly enhanced only in response to segmental stiffening (Figure 7C and 7D).
The Aging Human Abdominal Aorta Exhibits Segmental Stiffening
To test whether SAS occurs naturally in the human aorta, we assessed the aortic stiffness in 3 distinct locations (suprarenal, midinfrarenal, bifurcational) along the abdominal aortas of 19 male patients ranging in age from 36 to 71 years without evident AAA.
A significant negative correlation was observed between age and aortic cyclic strain in the suprarenal and midinfrarenal, and in the aortic bifurcation segments, as well, suggesting generally enhanced stiffness in the aging abdominal aorta (Figure 8A through 8C).
We also detected important differences between the distinct aortic locations. Although both the midinfrarenal aorta and the bifurcation exhibited age-related strain reduction, the slope of strain reduction was significantly steeper in the bifurcation segment, altering the (relative) SAS between 2 regions. In younger patients, the stiffness between both segments was similar (SAS≈1), but doubled (SAS≈2) by 60 years of age (Figure 8D). These results indicate that, in addition to overall stiffening of the abdominal aorta with age, the human abdominal aorta exhibits age-related segmental stiffening.
AAA formation is accompanied by increased stiffness of the aneurysmal vessel segment in comparison with the normal aorta.9,14 Aneurysmal stiffening occurs owing to profound changes in ECM organization, including elastin fragmentation and enhanced adventitial collagen deposition and turnover.14 The current study was designed to investigate aortic stiffening as a potential factor driving early AAA pathogenesis.
To explore the temporal relationship between aortic stiffening and AAA growth, we used the widely used PPE animal model. Because human AAA typically occurs in the aged aorta, which exhibits progressive elastin degeneration and stiffening,10,15 we deliberately chose the PPE model as a nondissection type preclinical model of AAA because it not only phenotypically resembles many aspects of the human disease, but it is also initiated by mild destruction of the elastin architecture (although this is achieved enzymatically by PPE perfusion in contrast to fatigue-related elastin fracture in the human situation). Moreover, our previous studies indicated that this model, in particular, appears sensitive to ECM/stiffness–related interventions.16
Our data confirm that aortic stiffening precedes aneurysmal dilation.17 The rapid stiffening that occurred within 1 day after treatment seems to be attributable to early PPE-induced elastin damage (Figure 2F). However, PPE is biologically active for no more than 24 hours after perfusion.18 Therefore, later structural alterations of the aorta, including the pervasive elastin fragmentation observed after 14 days (Figure 5F), appear to be PPE independent.
Although the observed early and sustained stiffening of the aneurysm-prone aorta may seem counterintuitive, this finding supports aneurysm growth as an active process, as opposed to simple passive dilation. Moreover, segmental stiffening of the abdominal aorta may qualify as a mechanism generating wall stress.
Mechanical stress is a potent inducer of physiological arterial remodeling. High flow–induced shear stress, elevated circumferential stress, and increased axial stress result in increased vessel diameter, wall thickening, and arterial lengthening, respectively, to achieve stress normalization.5 From a pathogenic point of view, mechanical forces induce a multitude of adverse events contributing to vascular disease, including ROS generation, apoptosis, and inflammation.4,19–21
To test the hypothesis that SAS generates wall stress that precedes and triggers early AAA growth, we performed in silico stress analysis using a FEA model. Inclusion of a stiff segment in a more compliant aorta generates axial stress under systolic pressurization. Axial stress increases with enhanced stiffness gradients between stiff and nonstiff segments (Figure 3A). Hypertension, a known AAA-associated risk factor, further increases axial stress in the setting of SAS (Figure 3B). Of note, this simplified model only takes into account static wall stresses, neglecting dynamic effects that may occur owing to cyclic systolic-diastolic wall deformations.
In our animal model, the peak of SAS at d7 coincided with the onset of accelerated aneurysmal enlargement. Delayed AAA formation until 7 days after PPE treatment is consistent with the initial characterization of this model.6 The relationship between increasing SAS and subsequent aneurysmal dilation was further strengthened by a positive correlation between the extent of SAS at d7, and aortic diameter enlargement between d7 and d14.
To clarify the pathophysiologic significance of SAS for AAA growth, we selectively applied rapid-hardening biological glue to the aortic segments adjacent to the PPE-injury site, achieving dramatic stiffening of the adjacent aorta, detectable within 1 day after intervention. Subsequently, the relative segmental stiffness of the PPE-treated aorta in comparison with its adjacent segments (ie, SAS) was instantly and permanently reduced. A major finding of this study is that the (glue-induced) reduction in SAS translated into significantly reduced AAA growth. In a more therapeutic context, we additionally found delayed glue application (day 7 after PPE injury) to reduce subsequent AAA progression.
To elucidate the mechanisms of this process we analyzed factors that contribute to AAA, and that are moreover known to be mechanosensitive: ROS generation, inflammation, ECM remodeling, and apoptosis. ROS levels are locally increased in human AAA in comparison with the adjacent nonaneurysmal aorta.22 ROS may be generated in response to mechanical stress in endothelial cells, and in VSMCs, as well, whereby mechanically activated NADPH oxidases and the mitochondrial electron transport chain seem to be significant sources.23,24 Mechanically generated ROS may subsequently trigger a variety of cellular responses such as VSMC apoptosis25 and vascular inflammation.4 ROS scavengers and NADPH-oxidase inhibition have reduced oxidative stress and aortic macrophage infiltration, and ultimately ameliorated aneurysm growth or decreased aneurysm rupture incidence in various murine AAA models.26–28 We found decreased ROS generation following glue-mediated reduction of SAS and axial stress.
AAA formation is characterized by inflammatory remodeling of the aortic wall, and vascular inflammatory reactions are sensitive to mechanical stress–induced signaling. For example, mechanical stress–induced proinflammatory mechanisms involve enhanced cytokine production via Ras/Rac1-p38-MAPK-NF-κB (leading to enhanced interleukin-6 expression in VSMC),20 and enhanced nuclear factor-κB–dependent expression of vascular chemokines and adhesion molecules that facilitate monocyte adhesion to the vascular wall, as well.21 Interestingly, inflammatory cells such as monocytes/macrophages become mechanosensitive once attached to the vascular ECM.29 We show that interventional stiffening of the adjacent aorta decreases macrophage infiltration in the aneurysm-prone (PPE-treated) segment and reduces the aortic and macrophage-specific expression of various inflammatory cytokines that are known to be critical for AAA pathogenesis, including Il1b, Il6, and Ccl2.30–32
ECM remodeling, with enzymatic breakdown of matrix macromolecules mediated by the metalloproteinases MMP-2 and MMP-9, is another hallmark of AAA. MMP expression is increased in human AAA,33–35 and knockout of MMP-2 and MMP-9 abolishes experimental AAA formation.18,36 MMP-2 and MMP-9 are also responsive to mechanical stress attributable to cyclic stretch and enhanced flow.24,37 More importantly, axial stress induces tissue remodeling and Mmp-2 activation in a model of longitudinal carotid growth.38 As expected, Mmp2 and Mmp9 were significantly upregulated in PPE-treated aortae (Figure 6I). Reducing SAS, and thereby cyclic axial stress, with glue stiffening reduced expression of both MMPs.
VSMC apoptosis is another critical feature of human and experimental AAA,39,40 and is susceptible to enhanced mechanical (axial) stress.38 Signaling mechanisms of mechanical stress-induced VSMC apoptosis include a variety of molecules, such as the endothelin B receptor, integrin-β1-rac-p38-p53 signaling or Bcl-2-associated death factor.19 We identified enhanced medial layer apoptosis in PPE-treated segments, which was decreased by glue-mediated axial stress reduction.
We further investigated the impact of SAS on inflammation and matrix remodeling ex vivo. Segmental stiffening (induced with an external cuff around the cyclically pressurized aorta) resulted in significant upregulation of Mmp2 and Mmp9, Col1a1 and Col3a1, and Il6 and Ccl2, as well. In contrast to the in vivo situation, where enhanced biaxial stiffness results from alterations of the inherent material properties of the vessel wall, our ex vivo model only simulated circumferential stiffening by external cuffing. Owing to technical limitations, our systolic and diastolic pressure levels alternated with a frequency of 3/min (normal C57BL/6 heart rate, ≈450/min41). Nevertheless, the data indicate that cyclic axial mechanical stress may directly control genes governing inflammation and matrix remodeling.
We observed stiffening of the aneurysm-adjacent aorta at d14 after PPE induction, with subsequent reduction of aneurysm growth rate. This might represent an endogenous compensatory mechanism to reduce SAS and contain AAA progression. The stiffening process was paralleled by an enhanced fibrotic response in the AAA-adjacent segments’ media, including upregulated collagen expression. A previous study showed that microRNA (miR)-29b is a repressor of collagen expression in AAA.16 We identified analogous miR-29b downregulation in the (VSMC-dominated) media of the AAA-adjacent aortic segments, consistent with miR-29b–modulated VSMC collagen production and medial fibrosis. We previously demonstrated that forced miR-29b downregulation (via systemic anti-miR administration) is a profibrotic intervention reducing AAA growth.16 This reduction, in light of the present study, may be partially attributable to accelerated miR-29b–dependent stiffening of the AAA-adjacent aorta.
Local aortic PPE infusion is a widely used preclinical AAA model that exhibits many features seen in human AAA, including early disturbance of elastin integrity. However, because of the artificial, invasive nature of the model, including enzymatic injury of the vessel, segmental stiffness might be model specific and not a feature of human AAA. We therefore studied whether the human abdominal aorta exhibits segmental stiffness that, according to our hypothesis, would be a contributing factor for AAA formation. Performing ultrasound-based strain analyses in 3 distinct locations along the abdominal aorta (suprarenal, midinfrarenal, bifurcation), we detected age-dependent reduction of strain (increased stiffness), corresponding to previous observations.42 As a novel finding, we detected relatively more pronounced stiffening of the aortic bifurcation segment with age (Figure 8C), translating into increasing SAS of the aortic bifurcation over time (Figure 8D). This distal part of the aorta has relatively low elastin content in comparison with the more proximal segments,43 a feature that might become functionally relevant with age-dependent loss of elastin.15 These data confirm and refine previous observations of enhanced age-dependent stiffening of the abdominal aorta10 and might explain, in part, the significant influence of age on AAA risk.
Of note, the segmental stiffness we observed in the human abdominal aorta (SAS ≈2) was significantly smaller than the peak segmental stiffness in the PPE-treated aorta (SAS ≈5). The study patients presumably exhibited physiological stiffness segmentation that will most likely not result in AAA formation. However, segmental stiffening may have more dramatic effects in individuals with genetic predilection for aneurysm formation.
In conclusion, the present study introduces the novel concept of SAS as a pathogenetic factor contributing to AAA. We propose that degenerative stiffening of the aneurysm-prone aortic wall leads to axial stress, generated by cyclic tethering of adjacent, more compliant wall segments. Axial stress then induces and augments processes necessary for AAA growth such as inflammation and vascular wall remodeling (Figure VII online-only Data Supplement). Clarification of these biomechanical signaling pathways may lead to additional therapeutic targets.
From a diagnostic point of view, AAA characterization has almost exclusively focused on the dilated segment. In light of the present findings, additional mechanical characterization of the AAA-adjacent aortic segments might provide important insights into the stress status of the aneurysm. This might be of particular relevance in early (even preaneurysmal) stages of disease, when mechanical stress is not yet predominantly driven by large geometric alterations. For instance, ultrasound-derived SAS assessment might help to predict the susceptibility for AAA formation and future AAA growth. Therefore, SAS could practically be useful to individualize risk prediction for patient populations at generally increased risk for AAA (eg, smokers, family history) or to better determine monitoring intervals for patients with small AAA. Having a more sensitive and specific indicator for clinical progression may improve decision making in AAA disease, and help direct resources to those in need in an increasingly resource-constrained environment.
From a therapeutic perspective, this study suggests that mechanically stiffening the AAA-adjacent aorta might provide a stress shield to limit AAA remodeling and expansion. This is supported in principle by recent data suggesting reduced growth rate of suprarenal AAAs in patients having undergone endovascular repair of a concomitant infrarenal AAA (in comparison with control patients without infrarenal repair).44 Of note, protective interventional stiffening of an AAA-adjacent segment may create a distal stiffness gradient along the arterial tree that potentially triggers distal aneurysm formation. However, we did not observe any evidence of this during the 28-day time course of our model. This may indicate that, in addition to stiffness gradients, other predisposing cofactors (eg, a structurally impaired vessel matrix) may be required to trigger AAA formation de novo. Further, we did not detect increased blood pressure levels after interventional stiffening of the abdominal aorta that could potentially point toward negative hemodynamic side effects (Table I online-only Data Supplement). Therefore, interventional stiffening of the aortic segment next to a small aneurysm could be further tested as a novel approach to limit further AAA progression, and forestall surgical repair.
We thank Tiffany K. Koyano, Yanli Wang, Michelle Ramseier, and Brian Deng for expert technical assistance. We further thank Hui Wang, Ying Lu, Balasubramanian Narasimhan, and Bradley Efron for expert statistical consulting.
Sources of Funding
This work was supported by research grants from the NIH (1R01HL105299 to Dr Tsao), the Deutsche Forschungsgemeinschaft (RA 2179/1-1 to Dr Raaz), the Stanford Graduate Fellowship (William R. and Sara Hart Kimball Fellowship to A. M. Zöllner), the University of Erlangen-Nuremberg School of Medicine (to I. N. Schellinger), and the Stanford Cardiovascular Institute (to Dr Spin).
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.114.012377/-/DC1.
- Received April 3, 2014.
- Accepted March 5, 2015.
- © 2015 American Heart Association, Inc.
- Shah PK.
- Hoefer IE,
- den Adel B,
- Daemen MJ.
- Maegdefessel L,
- Azuma J,
- Toh R,
- Merk DR,
- Deng A,
- Chin JT,
- Raaz U,
- Schoelmerich AM,
- Raiesdana A,
- Leeper NJ,
- McConnell MV,
- Dalman RL,
- Spin JM,
- Tsao PS.
- Goergen CJ,
- Azuma J,
- Barr KN,
- Magdefessel L,
- Kallop DY,
- Gogineni A,
- Grewall A,
- Weimer RM,
- Connolly AJ,
- Dalman RL,
- Taylor CA,
- Tsao PS,
- Greve JM.
- Pyo R,
- Lee JK,
- Shipley JM,
- Curci JA,
- Mao D,
- Ziporin SJ,
- Ennis TL,
- Shapiro SD,
- Senior RM,
- Thompson RW.
- Zampetaki A,
- Zhang Z,
- Hu Y,
- Xu Q.
- Riou S,
- Mees B,
- Esposito B,
- Merval R,
- Vilar J,
- Stengel D,
- Ninio E,
- van Haperen R,
- de Crom R,
- Tedgui A,
- Lehoux S.
- Miller FJ.
- Matsushita H,
- Lee KH,
- Tsao PS.
- Grote K,
- Flach I,
- Luchtefeld M,
- Akin E,
- Holland SM,
- Drexler H,
- Schieffer B.
- Gavrila D,
- Li WG,
- McCormick ML,
- Thomas M,
- Daugherty A,
- Cassis LA,
- Miller FJ Jr.,
- Oberley LW,
- Dellsperger KC,
- Weintraub NL.
- Nakahashi TK,
- Hoshina K,
- Tsao PS,
- Sho E,
- Sho M,
- Karwowski JK,
- Yeh C,
- Yang RB,
- Topper JN,
- Dalman RL.
- Johnston WF,
- Salmon M,
- Su G,
- Lu G,
- Stone ML,
- Zhao Y,
- Owens GK,
- Upchurch GR Jr.,
- Ailawadi G.
- Tieu BC,
- Lee C,
- Sun H,
- Lejeune W,
- Recinos A 3rd.,
- Ju X,
- Spratt H,
- Guo DC,
- Milewicz D,
- Tilton RG,
- Brasier AR.
- Moehle CW,
- Bhamidipati CM,
- Alexander MR,
- Mehta GS,
- Irvine JN,
- Salmon M,
- Upchurch GR Jr.,
- Kron IL,
- Owens GK,
- Ailawadi G.
- Freestone T,
- Turner RJ,
- Coady A,
- Higman DJ,
- Greenhalgh RM,
- Powell JT.
- Thompson RW,
- Holmes DR,
- Mertens RA,
- Liao S,
- Botney MD,
- Mecham RP,
- Welgus HG,
- Parks WC.
- Davis V,
- Persidskaia R,
- Baca-Regen L,
- Itoh Y,
- Nagase H,
- Persidsky Y,
- Ghorpade A,
- Baxter BT.
- Castier Y,
- Brandes RP,
- Leseche G,
- Tedgui A,
- Lehoux S.
- Jackson ZS.
- Maegdefessel L,
- Azuma J,
- Toh R,
- Deng A,
- Merk DR,
- Raiesdana A,
- Leeper NJ,
- Raaz U,
- Schoelmerich AM,
- McConnell MV,
- Dalman RL,
- Spin JM,
- Tsao PS.
Abdominal aortic aneurysm (AAA) is a common and potentially lethal disease of the aging aorta. Many AAAs remain asymptomatic until fatal rupture. Current treatment is limited to larger aneurysms (typically AAA diameter >5.5 cm) and consists of open surgery or stent-based intervention. Therefore, there is a need for improved risk stratification and disease monitoring, as well as for early treatment options. The present study mechanistically links segmental aortic stiffening (SAS), a feature of aortic aging, to subsequent AAA development. Moreover, it provides preclinical evidence that interventional stiffening of the AAA-adjacent aorta may provide a mechanical stress shield to limit AAA expansion. Those findings give rise to potential diagnostic and therapeutic benefits. From a diagnostic point of view, ultrasound-based assessment of SAS, for instance, might help to predict the susceptibility for AAA formation and future AAA enlargement. Therefore, the parameter SAS could be of practical use for personalized AAA risk prediction in patient populations at a generally increased risk for the disease (eg, smokers, patients with family history for AAA). Further, SAS could be helpful to better determine monitoring intervals for patients with already established small AAA. From a therapeutic perspective, interventional stiffening of the aortic segment next to a developing aneurysm could be further tested as a novel approach to limit AAA progression and to forestall surgical repair.