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Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United StatesDepartment of Pathology, University of Pittsburgh, Pittsburgh, PA, United StatesMcGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States
Address Correspondence: David A. Vorp, Ph.D, John A. Swanson Professor of Bioengineering, Professor of Cardiothoracic Surgery, Surgery, Chemical and Petroleum Engineering, Mechanical Engineering and Materials Science and the Clinical and Translational Sciences Institute, University of Pittsburgh, 300 Technology Drive, Suite 300, Center for Bioengineering, Pittsburgh, PA 15219 Phone: 412-624-5317 FAX: 412-383-8788 (shared)
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United StatesMcGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United StatesCenter for Vascular Remodeling and Regeneration, University of Pittsburgh, Pittsburgh, PA, United States
To evaluate the mechanical and matrix effects on abdominal aortic aneurysm (AAA) of during the initial aortic dilation and after prolonged exposure to beta-aminopropionitrile (BAPN) in an topical elastase AAA model.
Methods
Abdominal aortae of C57/BL6 mice were exposed to topical elastase with or without BAPN in the drinking water starting 4 days prior to elastase exposure. For the standard AAA model, animals were harvested at 2 weeks after active elastase (STD2) or heat-inactivated elastase (SHAM2). For the enhanced elastase model, BAPN treatment continued for either 4 days (ENH2b) or until harvest (ENH2) at 2 weeks; BAPN was continued until harvest at 8 weeks in one group (ENH8). Each group underwent assessment of aortic diameter, mechanical testing (tangent modulus and ultimate tensile strength) and quantification of insoluble elastin and bulk collagen in both the elastase exposed aorta as well as the descending thoracic aorta.
Results
BAPN treatment did not increase aortic dilation compared to the standard model after 2 weeks (ENH2: 1.65 ± 0.23mm, ENH2b: 1.49 ± 0.39mm, STD2: 1.67 ± 0.29mm, and SHAM2: 0.73 ± 0.10 mm [p<0.001, cf. STD2]) but did result in increased dilation after 8 weeks (4.3 ± 2.0mm, p<0.005). After two weeks, compared to the standard model, continuous therapy with BAPN did not have an effect on UTS (24.84 ± 7.62 N/cm2, 18.05 ± 4.95N/cm2), tangent modulus (32.60 ± 9.83N/cm2 , 26.13 ± 9.10N/cm2), elastin (7.41 ± 2.43%, 7.37 ± 4.00%) or collagen (4.25 ± 0.79%, 5.86 ± 1.19%) content. The brief treatment, EHN2b, resulted in increased aortic collagen content compared to STD2 (7.55 ± 2.48%, p=0.006) and an increase in UTS compared to ENH2 (35.18 ± 18.60N/cm2, p<0.03). The ENH8 group had the lowest tangent modulus (3.71 ± 3.10N/cm2, p<0.005) compared to all aortas harvested at 2 weeks and lower ultimate tensile strength (2.18 ± 2.18N/cm2) compared to both the STD2 (24.84 ± 7.62 N/cm2, p=0.008), and ENH2b (35.18 ± 18.60 N/cm2, p<0.001) groups. No difference in mechanical properties or matrix protein concentrations were seen associated with abdominal elastase exposure or BAPN treatment for the thoracic aorta.The tangent modulus was higher in the STD2 group (32.60 ± 9.83N/cm2, p=0.0456) vs. the SHAM2 group (17.99 ± 5.76 N/cm2) while ultimate tensile strength was lower in the ENH2 group (18.05 ± 4.95N/cm2, p=0.0292) compared to the ENH2b group (35.18 ± 18.60N/cm2). The ENH8 group had the lowest tangent modulus (3.71 ± 3.10N/cm2, p<0.005) compared to all aortas harvested at 2 weeks and lower ultimate tensile strength (2.18 ± 2.18N/cm2) compared to both the STD2 (24.84 ± 7.62 N/cm2, p=0.008), and ENH2b (35.18 ± 18.60 N/cm2, p<0.001) groups. For the abdominal aortic elastin in the STD2 group (7.41 ± 2.43%, p=0.035) was lower compared to the SHAM2 group (15.29 ± 7.66%). Aortic collagen was lower in the STD2 group (4.25 ± 0.79%, p=0.007) compared to the SHAM2 group (12.44 ± 6.02%) and higher for the ENH2b (7.55 ± 2.48%, p=0.006) compared to the STD2 group.
Conclusions
Enhancing elastase AAA model with BAPN does not substantially affect the initial (2 week) dilation phase either mechanically or by altering the matrix content. Late mechanical and matrix effects of prolonged BAPN treatment are limited to the elastase-exposed segment of the aorta.
Introduction
Cardiovascular disease is the leading cause of death in the United States [
], and type I and III fibrillar collagens provide additional mechanical stability, with type I collagen representing two-thirds of the total aortic collagen content [
] and potentially rupture. Vascular smooth muscle cells (VSMCs) specifically play a role in mediating ECM degradation and synthesis, and their associated cell death can lead to further progression of the aneurysm [
], increasing inflammation and degradative activity at the AAA site. The intraluminal thrombus (ILT) can also create a hypoxic environment within the AAA [
] to further elicit an inflammatory response and weaken the aortic wall. To compensate for loss of elastic fibers in AAA, collagen fibers are synthesized, leading to higher concentrations of collagen in AAA tissue compared to non-aneurysmal tissue [
]. While collagen provides some mechanical stability to the vessel wall, it does not provide the elastic resilience needed to prevent mechanical creep (dilation). Previously, other groups [
] have evaluated elastin and collagen levels in AAA mouse models qualitatively through histology. Others have used desmosine, a unique constituent within elastin, as a marker of degradation within human AAA tissue [
] compared to healthy tissue. An optimal AAA model would faithfully recapitulate the disease processes in humans, including the manifestation of changes in mechanical properties and ECM changes similar to that which occurs in human tissue. Topical elastase administration has been utilized to generate an AAA mouse model; however, this model aneurysm typically stabilizes over time while human AAA continues to expand [
], has been used to create a more severe/progressive AAA mouse model. Mice with aortic elastase exposure and systemic BAPN administration typically have larger abdominal aortas and continue to expand past 14 days with typical reported harvest times of 4-5 weeks after elastase exposure [
]. To better understand the potential of this unique model to represent aspects of the human disease process, the focus of this study was to evaluate the effects of BAPN administration within the initial 2 weeks of model development to understand the changes in aortic structure and strength that may precede dilation. We hypothesized that the late progression in the enhanced model was due to failure to establish crosslinked matrix components during the initial 2 weeks of dilation. We also wanted to evaluate the matrix and mechanical effects of temporary administration of BAPN as well as BAPN exposure which was continued until harvest.
To improve the sensitivity of our evaluations compared to prior studies [
], this study employed uniaxial testing and quantitative structural matrix protein analysis. Uniaxial testing provides data on the failure properties of the AAA tissue, while quantitative analysis of structural ECM constituents contained within these AAA mouse tissues provides additional insight to tissue health and composition compared to qualitative or semi-quantitative histology.
From the uniaxial testing we calculated the tangent modulus and UTS. We measured the ECM constituents insoluble elastin and total collagen as a percentage of total aortic protein of abdominal aortic tissue from a standard topical elastase model of AAA harvested as well as models enhanced with BAPN in the drinking water 4 days prior to surgery to varying time points after elastase exposure. Control tissues were taken from the descending thoracic aorta to evaluate the effect of the BAPN treatment on an uninjured segment of the aorta. One hypothesis that we tested was that enhanced models with BAPN would result in changes to the concentration of elastin and collagen in the tissues which would also be reflected in greater changes to aortic mechanical properties than elastase alone. We also hypothesized that short duration treatment with BAPN would reduce collagen and elastin concentration at harvest.
2.0. METHODS
2.1 Experimental animals
All mice for these experiments were C57BL/6 inbred strain, 8 -10 week male mice (Jackson Labs, ME, USA). Animals were housed in a controlled animal facility, and all mouse care and treatment occurred under protocols approved by the Vanderbilt University School of Medicine Animal Studies Committee.
2.2 Generation of AAA mouse models
Mouse abdominal aortas were isolated and exposed according to previous protocols [
] through a midline laparotomy. A volume of 30 μL of type I porcine pancreatic elastase in saline (0.16 U/mL) (Sigma, St. Louis, MO) was applied to the aorta as previously described [
] to initiate the AAA model (STD2). Elastase that had been heat inactivated (100°C for 30 mins) served as a sham control (SHAM2). For BAPN-treated groups, BAPN (0.2% w/v, Sigma-Aldrich) was diluted in drinking water 4 days prior to initial laparotomy until harvest at 2 weeks (ENH2) or 8 weeks (ENH8) for all animals except for one group where BAPN was stopped after day 4 following elastase perfusion (ENH2b) (see schema in Figure 1). Aortas from all groups were harvested at either 2 or 8 weeks following elastase administration. Photographs of the aorta at physiological pressure [
Transforming growth factor β neutralization finely tunes macrophage phenotype in elastase-induced abdominal aortic aneurysm and is associated with an increase of arginase 1 expression in the aorta.
], with an in-plane calibrated marker, were taken at the initial laparotomy prior to elastase exposure, as well as after re-laparotomy and exposure of the aorta with the animal anesthetized and diameters were quantified using ImageJ (NIH, Bethesda, MD). Following the final imaging of the aorta, the animal was sacrificed and the aorta from the proximal descending aorta to the iliac bifurcation was harvested. The lumen was threaded with a suture to aid in preparation for mechanical testing. Any ILT found in any tissue (particularly in ENH8) was left on the aorta and was subsequently mechanically tested but not included for protein quantification. ILT was left adhered to the wall since it could not be removed without mechanically damaging the tissue.
Figure 1Schematic and timeline of experimental events for the five different mouse groups used in this study. Diameter measurements (black circle) were taken at surgery before elastase application and at harvest of the aorta. Surgery and elastase application (heat-inactive = light blue triangle or functional elastase = red triangle) were performed at day 0. All BAPN feedings (light blue rectangle) started 4 days prior to surgery and continued for 4, 14, or 56 days after surgery.
Sections of 2.5 mm axial length were extracted from the thoracic and abdominal aorta as schematized in Figure 2 within 48 hours of sacrifice and was held at 4°C. The thoracic aorta sections served as a way to evaluate a remote effect of the abdominal aortic elastase administration as well as the systemic BAPN. The thoracic segment (Figure 2A) was taken approximately 1 cm superior to the renal arteries while the abdominal segment (Figure 2B) was taken from the most dilated segment between the renal arteries and the iliac bifurcation. The unpressurized outer diameter and width of the samples were photographed using a dissecting microscope with an in-plane ruler to measure pixel size and determine a scaling factor using ImageJ (NIH, Bethesda, MD) [
Mechanical, compositional and morphological characterisation of the human male urethra for the development of a biomimetic tissue engineered urethral scaffold.
]. In short, the samples were then carefully threaded with wire and clamped into an Instron uniaxial extension testing apparatus (Instron 5543A, Norwood, MA) with a 25N load cell. Data was post-processed in Matlab (R2020a MathWorks Inc., Natick, MA) to transform measured force data into stress versus strain curves, stress was calculated as:
where f is measured force, A the total cross-sectional area of the load bearing tissue (width x thickness), and σ the first Piola-Kirchoff stress. Strain calculation was based on deformed length and original length of the specimen according to:
where l is the length, lo the specimen gauge length, and ε the strain.
The tangent modulus and UTS was extracted from the stress-strain data. Specifically, the tangent modulus was measured as the linear slope region of the curve prior to plastic deformation, and UTS was taken as the maximum stress the specimen underwent during mechanical testing.
2.4Ninhydrin (insoluble elastin) and hydroxyproline (collagen) assays:
Immediately following ring testing of mouse aortas, tissues were frozen at -80°C without fixation. According to previous protocols [
Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture.
Initial Fiber Alignment Pattern Alters Extracellular Matrix Synthesis in Fibroblast-Populated Fibrin Gel Cruciforms and Correlates with Predicted Tension.
Annals of Biomedical Engineering.2011; 39: 714-729
], tissue samples were thawed immediately before base hydrolysis (0.1 M NaOH, 1 h, 98°C) followed by subsequent centrifugation (3000 x g) to separate insoluble elastin protein from soluble non-elastin proteins. Acid hydrolysis (6 N HCl, 24 h, 110°C) and assay quantification were then performed on both soluble and insoluble fractions (ninhydrin-based for elastin and hydroxyproline-based for collagen) to quantitatively determine ECM content in the aortic specimens (reported as % elastin or collagen relative to total aortic protein).
2.5 Statistical analysis
Statistical analysis was performed with Prism 9 software (Graphpad, La Jolla, CA). An unpaired t-test (two-tailed) was used to compare the SHAM2 and STD2 mice. A one-way analysis of variance (ANOVA) was used to determine differences between STD2, ENH2, ENH2b, and ENH8 groups in terms of aortic diameter, mechanical properties, and ECM content. Significance between groups was determined as p-values <0.05 with Tukey tests (two-tailed) to determine which individual groups differed. Paired t-tests (two-tailed) were used to determine differences between abdominal and thoracic aortic sections within the SHAM2 group.
3.0. RESULTS
All sample sizes and values (mean ± standard deviation) for tangent modulus, UTS, elastin content, and collagen content for protein and mechanical data can be found for the abdominal sections (Table I) and the thoracic sections (Table II). Representative images of each treatment group of excised aortae are shown in Figure 3 and histology of two ENH8 aortas are shown in Supplemental Data Figure 1. The sample sizes for each group were as follows: SHAM2 = 7, STD2 = 6, ENH2b = 6, SNH2 = 8, and ENH8 = 5. The sample size of ENH8 is slightly smaller due to the excessive dilation of this group and the presence of an intraluminal thrombus, which made it difficult to thread the aorta for the ring testing. While all mouse mortality we experienced may very well have been due to contained rupture of their aneurysm, ENH8 had no uncontained ruptures.
Table 1Overview of abdominal aortic mechanical properties and % ECM content of 5 different treatment groups. All data given in mean ± SD. ENH8 samples were not able to be analyzed for protein content due to presence of ILT which could not be degraded properly during the sodium hydroxide boil.
Figure 3A representative image of each treatment of aorta that was excised with abdominal aorta labelled (scalebar for reference, 1mm): A. SHAM, B. STD2, C. ENH2b, D. ENH2, and E. ENH8.
At the time of initial laparotomy and aortic exposure, there was no statistical difference in aortic diameter among the mice designated for each group [SHAM2 (0.65 ± 0.03mm), STD2 (0.84 ± 0.13mm), ENH2b (0.72 ± 0.16mm), ENH2 (0.78 ± 0.12mm), and ENH8 (0.85 ± 0.11mm)] (Supplemental Data Figure 2). At harvest, there was a significant difference in final aortic diameter at physiological pressure between SHAM2 (0.73 ± 0.10mm) and the STD2 (1.67 ± 0.29mm, p < 0.001) (Figure 4A). There were no differences between active elastase treatment groups harvested at two weeks regardless of BAPN treatment, STD2 (1.67 ± 0.29mm), ENH2b (1.49 ± 0.39mm), and ENH2 (1.65 ± 0.23mm)].
Figure 4A. There is a significant increase in diameter when comparing the in vivo diameters of the SHAM2 and STD2 mice. B. Comparisons of diameter in elastase treatment groups after treatment. There also was a significant increase in diameter between the shorter treatment groups and the ENH8.
Enhanced model aneurysms harvested at 8 weeks were significantly larger than any of the model aneurysm groups harvested at 2 weeks (4.30 ± 2.25mm) (Figure 4B). Intraluminal thrombus was present in the ENH8 group only.
3.2 Aortic Mechanics:
The stiffness of the abdominal aortic segment, as reflected in the tangent modulus, was significantly increased 2 weeks after elastase exposure (STD2 [32.60 ± 9.83 N/cm2, p = 0.007] compared to SHAM2 [17.99 ± 5.76 N/cm2]) (Figure 5A). Elastase exposure did not affect the UTS of the segment after 2 weeks compared to sham exposure (24.84 ± 8.35 N/cm2 vs 24.27 ± 7.00 N/cm2, respectively) (Figure 5B).
Figure 5A. There was a significant difference in the tangent modulus between these the SHAM2 and STD2 groups. B. Comparison of the tangent modulus between the treatment groups where there is a significant change between ENH8 and the shorter treatments. C. Comparison of the tangent modulus between the sham and E treated groups, with a significant change between ENH8 with the STD2, ENH2b, and ENH2 groups. D. Comparison between the ultimate tensile strength of the treatment groups with a significant change between ENH8 with the STD2 and ENH2b groups, additionally there is a difference between ENH2b and the ENH2.
The aortas from the enhanced models harvested at 2 weeks did not show any difference in abdominal aortic stiffness compared to elastase exposure alone regardless of duration of BAPN treatment (tangent modulus: ENH2 [26.13 ± 11.41 N/cm2] and ENH2b [27.12 ± 9.10 N/cm2]) (Figure 5C). The aortas from the ENH2 group (18.05 ± 4.95 N/cm2) had a significantly lower UTS than animals treated with BAPN for only 4 of the 14 days (35.18 ± 18.60 N/cm2, p = 0.029), but did not have significance compared to the STD2 group (24.84 ± 8.35 N/cm2) (Figure 5D).
At 8 weeks, the ENH8 aortas had a very low tangent modulus (3.71 ± 2.77 N/cm2) that was significantly different from all aortas harvested after two weeks. The ENH8 animals also had a very low UTS (2.18 ± 2.18 N/cm2) which was significantly different from the STD and ENH2b animals and trended toward lower UTS compared to ENH2 animals (p = 0.063). (Figure 5C).
The tangent modulus and the ultimate tensile strength of the thoracic aortic segments was not significantly affected by any of the abdominal aortic elastase exposures or systemic treatment with BAPN for any duration. (Supplemental Data Figure 3A and 3B).
3.3 Aortic ECM quantification
At harvest, there was a significant reduction in the concentration of elastin in the abdominal aorta of the STD2 mice (7.41 ± 2.43%, p = 0.035) compared to the SHAM2 mice (15.29 ± 7.66%) (Figure 6A). There were no significant differences in abdominal aortic elastin content among enhanced models or compared to the STD2 mice at harvest (Figure 6B).
Figure 6A. There was a significant decrease in abdominal aortic elastin content with elastase treatment. B. Among all the elastase treated mice, there was no relationship between BAPN treatment and the amount of elastin in the ECM. C. There was a significant decrease in abdominal aortic collagen content with elastase treatment. D. Among the elastase treatment groups, there was a significant elevation of collagen in the ENH2b group vs. STD2 group.
Abdominal aortic collagen concentration was also significantly lower in the STD2 group (4.25 ± 0.79%, p = 0.007) compared to the SHAM2 mice (12.44 ± 6.03%) (Figure 6C). There was higher abdominal collagen in the ENH2b group (7.55 ± 2.48%, p = 0.006) compared to STD2 mice and a trend toward lower collagen in the ENH2 group (5.86 ± 1.19%, p = 0.154) (Figure 6D). Additionally, there was a trend toward higher collagen in the ENH2 group aortas compared to the STD2 group (p = 0.184).
Using the same techniques, we were unable to reproducibly quantify the elastin or collagen in the ENH8 mice, likely related to the presence of substantial adherent luminal thrombus.
Similar to the mechanical data, thoracic aortic elastin and collagen concentration were not affected by the elastase exposure of the abdominal aorta or any of the BAPN treatments at 2 weeks or at 8 weeks. (Supplemental Data Figure 4A and 4B).
3.4 Regional variance of mechanical properties and ECM content
We compared the mechanical properties and matrix of the thoracic and abdominal aorta in the SHAM2 mice. There was a higher tangent modulus in the thoracic vs. abdominal region in the SHAM2 group (34.98 ± 7.81 N/cm2 vs 17.99 ± 5.76 N/cm2, p = 0.0020) (Figure 7A). There was a trend toward higher UTS in the thoracic vs. abdominal region (33.08 ± 16.17 N/cm2 vs 24.27 ± 7.00 N/cm2, p = 0.0720) (Figure 7B). There was no significant difference between elastin concentration in the thoracic and abdominal regions (22.19 ± 7.37% vs 15.29 ± 7.67%, p = 0.1167) (Figure 7C). Collagen concentration was lower in the thoracic region compared to the abdominal region (3.32 ± 1.23% vs. 12.44 ± 6.02%, p = 0.0092) (Figure 7D).
Figure 7Analysis of region-specific differences in mechanical properties and ECM content within SHAM2 control mice. The thoracic aorta demonstrated higher tangent modulus vs. the abdominal aorta (A), with a trend towards higher UTS (B). While the two regions did not differ in terms of elastin content (C), there was significantly more collagen content in the abdominal aorta than in the thoracic aorta (D).
The addition of BAPN has been increasingly used to establish models of AAA that have been proposed to better represent the tissue changes that occur in the human disease process [
]. While these models recapitulate some morphologic features of human AAA, including continued progressive dilation and development of intraluminal thrombus, we proposed to further examine the effects of BAPN on mechanical and matrix properties of the aorta. We focused primarily on the mechanical and matrix changes 2 weeks after topical elastase application, before substantial aortic dilation was seen in prior studies (around 4-5 weeks) [
]. We also examined a remote segment of aorta (thoracic aorta) after both short and long duration exposure of these animals to BAPN.
The standard model of AAA, topical elastase exposure of the mouse aorta initiates a process that results in a greater than 50% decrease in aortic matrix concentration associated with a mean maximum dilation of approximately 25% after 2 weeks (Figure 6); these results were similar to those found by Romary et al. [
]. These changes in diameter (Figure 4) and matrix result in an aorta that is (Figure 5) stiffer (tangent modulus), but not substantially weaker (UTS). These findings are consistent with loss of elasticity due to damage to the elastic fibers [
Overall, and contrary to our initial hypotheses, there was little effect of BAPN treatment on the animals during the initial two weeks after aortic injury. When BAPN was included in the animal’s drinking water until harvest 2 weeks following elastase exposure, there were no statistically significant changes in aortic diameter or concentrations of elastin and collagen compared to the STD2 model (Figure 4, Figure 6). Mechanically, there was a small decrease in the UTS which did not achieve statistical significance compared to the standard model (Figure 5). A very short course of BAPN following elastase perfusion for 4 days also did not affect aortic diameter at 2 weeks, but it did result in an increase in collagen in the aortic tissues as well as a trend toward increased UTS versus the STD2 model (Figure 5, Figure 6). The UTS was significantly reduced by continuing the BAPN for all 14 days compared to only 4 days (Figure 5).
After 8 weeks, the effect of BAPN on aneurysm size and mechanical properties was dramatic. The aneurysms were much larger with intraluminal thrombus (Figure 4) – features previously described [
] that mimic the human disease features in later stages of dilation . The mechanical analysis was remarkable for a very low tangent modulus and with a very low ultimate tensile strength (Figure 5). The very low UTS does indicate an AAA that is prone to rupture, consistent with late human disease. Unfortunately, the aortic alterations at 8 weeks our assays for matrix proteins were technically unreliable likely related to the substantial adherent luminal thrombus.
Prior studies have shown that the standard elastase induced mouse models of AAA rarely dilate substantially after the initial two weeks following elastase induced injury to the aorta [
]. This initial 14-day period has been intensely studied in the STD2 model since it seemed to demonstrate development of histologic findings that reflected findings seen in human AAA such as macrophage infiltration and SMC loss as well as loss of structured elastic fibers. Yet our mechanical analysis demonstrates that the aorta becomes somewhat stiffer but is no more prone to rupture during this period. The lack of any substantial change in the mechanical properties or matrix content during these two weeks with addition of BAPN suggests that there is little matrix repair activity during this period. The paradoxical effect of increased collagen in the tissues at harvest when there is a short exposure of the animal to BAPN is not easily explained by the data acquired during this study and may warrant specific investigation to understand the underlying mechanisms.
The progression of the model AAA in the ENH8 mice suggests that continued dilation of the model AAA depends on interfering with normal matrix production and deposition in the period following the initial 2 weeks. Decreased matrix cross-linking due to reduced lysyl oxidase activity is also a feature seen in human AAA [
], and the continued progression of the initial dilation with BAPN administration more accurately resembles the progression of small AAA toward large AAA that have a propensity for rupture. This may imply that failure to produce sufficient and sufficiently functional matrix may be more relevant to the growth of small AAA than the destructive processes that have been the primary focus of investigation in the animal models over the past 3-4 decades.
There is no significant difference in elastin content between the abdominal and thoracic sections in the sham group (Figure 7), which seems counter-intuitive as blood vessels closer in proximity to the heart typically have higher elastin content to withstand left ventricular ejection forces [
]; however, the sham group had a significantly higher amount of collagen in the abdominal section compared to the thoracic section (Figure 7). This increase in abdominal collagen could potentially be attributed to any fibrosis caused following isolation of the abdominal aorta during surgery, thus agitating the aorta itself.
Because there is such limited turnover of elastin in the post-natal aorta, it is reasonable to expect that BAPN treatment of these animals would have little to no effect on segments of the aorta that were not injured, as we saw in the thoracic aortic analyses. This could reasonably be expected to be true of the human condition where a generalized impairment in matrix synthesis would affect aorta primarily in the infrarenal segment and iliac vessels, possibly due to independent regional injury which fails to stabilize. This feature of inadequate or abnormal systemic matrix repair potential after injury in patients with AAA is reflected in the increased frequency of hernia formation after abdominal procedures [
Our study has several limitations that should be kept in mind. All of our samples exhibited aneurysmal-like dilation. While we did not see any macroscopic evidence of aortic rupture or dissection in any of our samples, a more thorough evaluation with in vivo ultrasound or serial sections of the aorta would more conclusively evaluate this possibility. Variation in blood pressure at harvest may affect aortic diameter measurements but was not assessed, consistent with other publications with this model [
Transforming growth factor β neutralization finely tunes macrophage phenotype in elastase-induced abdominal aortic aneurysm and is associated with an increase of arginase 1 expression in the aorta.
Initial Fiber Alignment Pattern Alters Extracellular Matrix Synthesis in Fibroblast-Populated Fibrin Gel Cruciforms and Correlates with Predicted Tension.
Annals of Biomedical Engineering.2011; 39: 714-729
]. Potential degradation of ECM components could occur, but we attempted to minimize this possibility by keeping the tissue at 4 °C immediately after sacrifice, testing the tissue within 48 hours, and immediately freezing the tissue after testing for subsequent ECM quantification. The hydroxyproline-modified collagen that is detected following aneurysm induction may not be properly bundled and organized for strength, i.e., more scar-like disorganized fibers than cooperative circumferentially aligned fibers [
]. The elastin that is detected is solely mature insoluble elastin, not immature fibrils, and thus not all elastin products are detected. However, mature elastic fibers are the main contributors to elastic properties of aortic tissue and are most relevant to this study on mechanical aortic tissue properties [
]. Histology could also demonstrate inflammation of the tissue, an important part of the pathology of AAA, but only two samples were used for histology because the limited mouse tissue was prioritized for mechanical testing and quantitative ECM analysis. Semi-quantitative histology is limited compared to the fully quantitative analysis performed in our study. Additionally, both the ninhydrin and hydroxyproline assays are reported as a ratio of the elastin or collagen content to the total protein of a tissue. Interpretation of these results in this context must keep in mind that changes to these proportions can be affected by the introduction of other protein sources such as cells (including inflammatory cell infiltration) as the aorta dilates. A potential remedy for this issue in future studies is calculating total cell number through DNA analysis [
] and factoring this number into the ECM content analysis. Additionally, presence of an ILT in the 8-week BAPN group adversely affected the elastin, collagen, and total protein content calculated using this particular assay given the cellularity of this material, making the measurements inconsistent and unreliable for comparison. As the ILT can contribute to the overall cell count of the tissue, this can affect the overall protein concentration depending on the number of cells/cellular protein. The ENH8 had very thick, strongly adherent ILT, which could not be removed without damaging the tissue and, therefore, we did not include ECM content analysis of that group. The inclusion of ILT and its respective thickness in mechanical testing drastically increases the cross-sectional area of the tissue and thus would have artificially lowered the calculated tangent modulus and tensile strength. The removal of ILT from the ring specimen was not performed prior to mechanical testing to minimize potential additional damage to the aneurysm wall because simple means such as irrigation would not be sufficient to separate. Mechanical properties of blood vessels including tangent modulus and UTS are affected by both passive constituents such as elastic and collagen fibers and active components such as VSMCs [
]. In this specific study, only the analysis of passive components (collagen and elastin) was measured via mechanical testing and protein analysis. As none of the solutions used to maintain the tissues for mechanical testing contained calcium, VSMC contraction did not affect mechanical properties [
Inhibition of the mTOR pathway in abdominal aortic aneurysm: implications of smooth muscle cell contractile phenotype, inflammation, and aneurysm expansion.
American Journal of Physiology-Heart and Circulatory Physiology.2017; 312: H1110-H1119
]. Differences in tangent moduli and UTS between models could also be attributed to the contractile state of the VSMCs; Various mechanical models have computationally modeled VSMC-attributed active mechanics in tissues and have investigated the passive mechanics by inhibiting VSMC contraction [
]. Regardless of the number of VSMCs, if they are not contracting, their effect on mechanical properties would be negligible. One limitation of the elastase-only mouse model is that they do not rupture nor form an ILT, two important aspects of human AAA. Thus, since the elastase-only model does not have those significant characteristics of human AAA and our BAPN + elastase models can present these attributes, it is imperative to investigate the enhanced model further. Additionally, our mouse model only uses male mice, which does not address sex as a biological variable. While AAA is historically more prevalent in males than females [
], the differences in the development of induced AAA (with or without BAPN) in mice and clinical AAA between males and females should be the subject of a separate, specific analysis. Thus, our findings may not be generalizable to females.
As there is a significant correlation between elastic fiber density and collagen content and mechanical resilience and strength with human AAA, understanding a potential correlation in vivo can give rise to better AAA models. Trends such as decreased elastin content and increased tangent modulus found in this study of AAA mouse models are similar to that found with human AAA [
]. By capturing certain pathologies of aneurysm disease in mouse models, better treatments of AAA can be developed and tested with higher potential chances of success in the clinic. For example, creating a regenerative treatment that preserves in vivo elastin content and reduces tissue stiffness provides better insight into future treatments of AAA. Previous studies by Anderson et al. utilized an elastase model and a CaCl2 model with an applied pentagalloyl glucose treatment reported no difference in burst pressure and time to failure in either treatment group [
]. Collins et al. characterized the suprarenal and infrarenal abdominal aortas’ elastic properties via biaxial extension testing in healthy mice. They note marked differences in morphological parameters with slight mechanical differences [
]) is important for accurate testing of potential clinical treatments that affect certain aspects of the AAA. Vascular wall mechanical integrity and ECM constituents, both of which were the focus of this study, are largely affected by AAA. The mechanical comparisons made in this paper focus on the uniaxial elastic and failure properties, which provides information on the biomechanics of diseased AAA tissue in a variety of murine models. Understanding the biomechanics in murine AAA models is an important first step to be able to quantify how a potential regenerative approach to repair the wall can restore it to its native state. By comparing ECM content between human and mouse AAA tissue, a better understanding of the severity of the model can occur. Additionally, these results confirm that these treatments did not significantly affect the thoracic aorta, showing an isolation of damage to the abdominal aorta. In the future, the results of these mouse models can be used to induce AAA in mice and test therapeutics for stopping or potentially reversing the mechanical and matrix effects of AAA.
5.0. CONCLUSION
Inhibition of matrix cross-linking with BAPN treatment after aortic elastase injury has little effect on the mechanical properties or matrix content of the aorta within two weeks of the injury. Defective matrix cross-linking does promote late dilation, reduces ultimate tensile strength and other features of the human disease which may be more representative of the process of small aneurysm progressive dilation. Refocusing investigation of the AAA models on enhanced regeneration of matrix in the period following initial matrix injury may result in therapeutic agents more likely to translate to the treatment of patients with small AAA.
Sources of funding
This material is based upon work supported by the National Institutes of Health award no. R21HL129066 (DV). This work was also supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1747452 (PG). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Additionally, this work is also supported by NIH T32 HL076124 (AM) and the Vascular Cures (JC).
Disclosures
Nothing to disclose.
Acknowledgements
We would like to thank Brittany Rodriguez for her contributions to this project in aiding with mechanical testing of tissue specimens. Figure 1 was created with BioRender.com.
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5Department of Surgery, University of Pittsburgh, Pittsburgh, PA, United States
6Department of Chemical and Petroleum Engineering, University of Pittsburgh, PA, United States
7Department of Mechanical Engineering and Materials Science, University of Pittsburgh, PA, United States
8Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA, United States
9Clinical & Translational Sciences Institute, University of Pittsburgh, Pittsburgh, PA, United States
This paper explores the use of short and long-term exposure to beta-aminopropionitrile to create an enhanced topical elastase abdominal aortic aneurysm model in mice. Readouts of aneurysm severity included loss of mechanical stability and vascular extracellular matrix composition reminiscent of what is seen in the course of human disease. Additionally, we show that the thoracic aorta, unlike the findings below the renal arteries, is not damaged in our animal model.
Abdominal aortic aneurysms (AAAs) are manifested with progressive luminal dilatation and a high risk for aortic rupture. In the past 2 decades, mechanisms of this devastating disease have been explored using multiple mouse models. Since early 2000, three AAA mouse models have been commonly used: angiotensin II infusion,1 periaortic application of CaCl2,2 and intraluminal perfusion of elastase.3 In 2012, Bhamidipati et al. modified the intraluminal perfusion of elastase model with an easier surgical procedure involving periadventitial application.
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