Segmental aortic stiffening contributes to experimental abdominal aortic aneurysm development

Abstract

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.

Keywords: aneurysm; aorta; arterial stiffness; stress, mechanical; vascular remodeling.

© 2015 American Heart Association, Inc.

Figures

Figure 1

Concept of Segmental Aortic Stiffness (SAS) generating axial wall stress during systolic aortic expansion. In contrast to a homogenous expandable vessel a segmentally stiff aorta (stiff segment in red) is subjected to axially tethering forces (solid arrows) during the systolic circumferential expansion of the adjacent compliant wall segments.

Figure 2

Analysis of Segmental Aortic Stiffening and aneurysm progression in the PPE model. (A) Temporal development of circumferential cyclic strain of PPE- and saline-treated segments. (B) Diameter development of the PPE- and saline-treated segments (% vs. baseline (d0)). (C) Temporal analysis of Segmental Aortic Stiffness (SAS) of the PPE- or saline-treated segment relative to the adjacent abdominal aorta. (D) Temporal analysis of the circumferential cyclic strain of the adjacent aorta (bold line) in relation to the PPE-treated segment (thin line). (E) Correlation between the Segmental Aortic Stiffness (SAS) at d7 and the consecutive diameter increase of the PPE-treated segment in the following 7 days. (F) Upper panels: Representative immunofluoresence staining for collagen I + III (red) with green autofluorescence of elastin lamellae. Lower panels: Modified Elastin Verhoeff’s Van Gieson (VVG) staining. Data are mean±SEM. n=5–13 for each condition/time point; p values denote differences between PPE and saline groups by permutation F-test (A–C), aortic strain differences in PPE treated animals over time by Friedman’s test (D), or significance level of Spearman correlation (E).

Figure 3

Finite elements model (FEA) based axial stress analysis of segmental aortic stiffening. A simplified model of the murine infrarenal aorta was subjected to various mechanical conditions and resulting axial (longitudinal) stress (N/mm2) was depicted. (A) The stiffness of the stiff aortic segment (SS) was increased (Shear moduli: 500 kPa left vessel, 1100 kPa middle vessel, 1700 kPa right vessel) to demonstrate the impact of segmental stiffness on axial stress generation. (B) The intraluminal pressure was increased (left vessel: 80 mmHg, middle vessel: 130 mmHg, right vessel: 180 mmHg) to visualize the influence of blood pressure on axial stresses in a segmentally stiff aorta. (C) A segmentally stiff aorta (left) is subjected to external stiffening of the adjacent compliant segments (simulating glue treatment; right) to demonstrate axial stress reduction and homogenization induced by the intervention.

Figure 4

Stiffening mechanisms of the AAA-adjacent aorta. (A) Temporal analysis of the Col1a1 and Col3a1 gene expression in the AAA-adjacent aorta compared to the AAA (PPE-treated) segment. (B) Temporal analysis of miR-29b expression in the AAA-adjacent aorta compared to the AAA (PPE-treated) segment. (C)in situ hybridization for miR-29b (purple-blue dye) and red nuclear counterstain in the AAA-adjacent aortic segments (original magnification 400×, scale bar 50 μm) (D) Representative images of the aortic wall taken from AAA-adjacent aortic segments 7 days or 14 days after PPE-treatment stained with Picrosirius Red (upper panels; red: collagen; yellow: muscle) and Elastin VVG staining (lower panels). Original magnification 400×, scale bar 50 μm. * indicates p<0.05 vs. all other time points; # indicates p<0.05 vs. d0 and d28. n=5 for each time point; p values denote differences between expression levels by Kruskal-Wallis test with Dunn’s post test.

Figure 5

Effects of glue-treatment on Segmental Aortic Stiffness and aneurysm progression. (A) Temporal analysis of the circumferential cyclic strain of the glue-treated adjacent aorta (bold line) in relation to the PPE-treated segment (thin line). (B) Temporal analysis of Segmental Aortic Stiffness (SAS) in glue-treated aortas compared to sham-glue-treated conditions. (C) Diameter development of the PPE-treated segment in glue-treated vs. sham-glue-treated conditions. Temporal development of SAS (D) and aortic diameter (E) following delayed glue or sham treatment 7 days after PPE surgery (arrows). Representative Elastin VVG staining (F) or Picrosirius Red staining (G) of the aortic wall taken from native abdominal aortas (control) or PPE-treated segments (d14) after additional treatment of the adjacent aorta with glue or sham-glue (original magnification 400×; scale bars 50 μm). EVG staining was used to depict the integrity of the medial elastin lamellae. Picrosirius Red staining aided the visualization of the aortic wall architecture and collagen remodeling. n=7 for each time point; p values denote differences between aortic segments (A) or treatment groups (B–E) by permutation F-test.

Figure 6

Effects of glue-induced aortic stiffening on ROS generation and parameters of inflammation, apoptosis and ECM remodeling. (A) In situ DHE staining of native abdominal aortas (control) or PPE-treated segments after additional treatment of the adjacent aorta with glue or sham-glue (d7). ROS production was indicated by red fluorescence. Autofluorescence from elastic lamellae (depicted in green in the upper row) was subtracted (bottom row). Original magnification x400, scale bar 50 μm. (B) Average fluorescence was quantified from 3 high power fields of 3 different aortas per group. (C,D,E) Representative co-staining of macrophages (red F4/80 marker) and the green labeled cytokines IL-6 (C), Il-1β (D) and Ccl2 (E) in native abdominal aortas (control) or PPE-infused segments (d7) after additional treatment of the adjacent aorta with glue or sham-glue (original magnification 400×, scale bar 50μm). Colocalization results in orange/yellow color. Nuclei are Hoechst stained (blue). (F) Corresponding immunostaining of activated caspase-3 (red). (G, H) Expression of Il6, Ccl2, and Il1b in the PPE-infused segment (d7) after additional glue or sham-glue treatment of the adjacent aorta, quantified in whole tissue (G) as well as in laser-captured macrophages.(H). Expression analysis of Mmp2 and Mmp9(I) as well as Col1a1 and Col3a1(J) (all vs. native control) in the PPE-infused segment (d7) after additional glue or sham-glue treatment of the adjacent aorta; p values denote differences between treatment groups by Kruskal-Wallis test with Dunn’s post test (B) or Mann-Whitney test (G–J).

Figure 7

Ex vivo aortic mechanical stimulation. (A) Scheme of the experimental setup for differential mechanical stimulation of the cannulated aorta. Cyclic strain is imposed on unrestrained/unstiffened aortas (NoStiff), completely restrained aorta (CompStiff) or segmentally restrained aorta (SegStiff). (B–D) Gene expression results after one hour of mechanical stimulation in the 3 groups. Depicted is the differential expression of inflammation related genes Il6 and Ccl2 (B), matrix metalloproteinases Mmp2 and Mmp9 (C) and collagen genes Col1a1 and Col3a1 (D) (all vs. NoStiff condition). n=5 for each condition; p values denote differences between treatment groups by Kruskal-Wallis test with Dunn’s post test.

Figure 8

Segmental Aortic Stiffening in the aging human abdominal aorta. (A–C) Correlation between age and circumferential cyclic strain in the supra-renal (A), mid-infrarenal (B) and bifurcational segment (C) of the human abdominal aorta. (D) Correlation between age and segmental stiffness (SAS, bifurcational segment vs. mid-infrarenal segment) along the infrarenal abdominal aorta; p denotes significance level of Pearson correlation.