Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Sep 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Jul 2;35(9):2032–2041. doi: 10.1161/ATVBAHA.115.305537

Platelet Inhibitors Reduce Rupture in a Mouse Model of Established Abdominal Aortic Aneurysm

A Phillip Owens III 1,#, Todd L Edwards 2,3,#, Silvio Antoniak 1, Julia E Geddings 1, Eiman Jahangir 4, Wei-Qi Wei 5, Joshua C Denny 5, Yacine Boulaftali 1, Wolfgang Bergmeier 6, Alan Daugherty 7, Uchechukwu KA Sampson 2,8,9, Nigel Mackman 1
PMCID: PMC4552620  NIHMSID: NIHMS703197  PMID: 26139462

Abstract

Objective

Rupture of abdominal aortic aneurysms (AAAs) causes a high morbidity and mortality in the elderly population. Platelet-rich thrombi form on the surface of aneurysms and may contribute to disease progression. In this study, we used a pharmacologic approach to examine a role of platelets in established aneurysms induced by angiotensin II (AngII) infusion into hypercholesterolemic mice.

Approach and Results

Administration of the platelet inhibitors aspirin (ASA) or clopidogrel bisulfate to established AAAs dramatically reduced rupture. The mechanism of protection appears to be a reduction in abdominal aortic platelet and macrophage recruitment resulting in decreased active matrix metalloproteinases (MMPs) 2 and 9. Platelet inhibitors also resulted in reduced plasma concentrations of platelet factor 4, cytokines, and components of plasminogen activation system in mice. To determine the validity of these findings in human subjects, eligible aneurysm patients were retrospectively analyzed using developed and validated algorithms in the electronic medical record database at Vanderbilt University. Similar to mice, administration of ASA or P2Y12 inhibitors was associated with reduced death amongst AAA patients.

Conclusions

These results suggest that platelets contribute to AAA progression and rupture.

Keywords: Abdominal aortic aneurysm, platelets, angiotensin II, clopidogrel, aspirin, mice

Introduction

Abdominal aortic aneurysms (AAA) affect 5-10% of the male and 1% of the female population over the age of 65 and is the 13th leading cause of death (estimated from 15,000 to 30,000 people, both primary and contributing) in the United States (1, 2). AAA is defined as a permanent localized dilation in the arterial wall with a diameter greater than 50% of normal (3). It is an inflammatory disease that is often associated with formation of an intramural clot. Rupture of AAA frequently causes death (4). In spite of a high incidence and catastrophic consequences, there is limited information regarding the sequence of events that lead to initiation, progression, and the eventual rupture of AAAs. Currently, the only treatment for AAA is surgical intervention after the aneurysm has reached a diameter of >5.5 cm (5, 6). Since AAA is categorized as a peripheral artery disease, it is currently recommended that patients with AAA start a regiment of low-dose aspirin (ASA) therapy (7, 8).

Infusion of AngII into hypercholesterolemic mice induces formation of AAA localized to the suprarenal aorta (9, 10). This model is highly reproducible and has been used to define mechanisms of vascular pathology associated with AAA (11-13). AngII infusion promotes elastin fiber destruction, proteolytic destruction of medial connective tissue, inflammation, atherosclerosis within the aneurysm, and rupture, which are all features that occur in human AAA (4, 14). Other mouse models of chemically-induced AAA have been developed that include exposure of the aorta to elastase (15) or calcium chloride (16). In addition, there is a xenograft rat model of AAA, which results in aortic dilatation and the presence of a mural thrombus in ~20% of rats (17). However, AngII infusion is the only consistent mouse model of aortic dilatation and rupture (3, 4, 18).

Platelets are required for hemostasis but also contribute to thrombosis and inflammation (19). Primary hemostasis results from platelet adherence to selected adhesive glycoproteins in sub-endothelial matrix. Platelet activation, spreading, degranulation, and aggregation leads to formation of a platelet-rich hemostatic plug (reviewed in detail in (20)). Platelet activation occurs through stimulation of a variety of G protein-coupled receptors with soluble agonists, such as thrombin, adenosine diphosphate (ADP), and thromboxane A2 (TXA2) (21). Activation of coagulation and production of thrombin activates platelets by cleavage of protease-activated receptors (Pars). Activated platelets release ADP and TXA2 that are required for sustained platelet activation and accumulation in a thrombus (21). ADP stimulates the ADP receptors P2Y1 and P2Y12 whereas TXA2 activates the thromboxane receptor (TP) (21). The uncontrolled growth of a platelet-rich thrombus can occlude the blood vessel and result in myocardial infarction and stroke. Patients at risk for thrombosis are treated with platelet inhibitors, such as ASA, which blocks TXA generation, and/or clopidogrel bisulfate, which inhibits P2Y12 activation. In addition, the Par1 inhibitor, Vorapaxar, has recently been approved for treatment of patients with cardiovascular disease (22, 23).

Studies in a rat model showed that platelet inhibitors reduced abdominal diameter (dilatation) and incidence of experimental aneurysm, suggesting that platelets may enhance AAA (24, 25). Furthermore, patients with AAAs have an activated coagulation system and levels of thrombin generation correlate with the maximum diameter of the aorta in the patients (26-31). Importantly, platelets and platelet-specific secretions (soluble P-selectin, soluble CD40L, soluble glycoprotein V, and platelet-derived microparticles) are present in plasma of AAA patients and are specifically released from the luminal thrombus of an aneurysm (25). Despite these results, several meta-analyses and retrospective clinical trials reported no significant benefit of platelet inhibitors on aneurysm growth and incidence of rupture (32-34).

In this study, we investigated the effects of pharmacologic inhibition of platelet activation on aneurysms that were established by infusion of AngII. In addition, we evaluated the progression and rupture of AAA in patients with or without ASA or platelet inhibitors.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement

Study approvals

All mouse studies were performed with the approval of the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (IACUC number 13-062.0). All analysis of human data was approved by the Institutional Review Board of Vanderbilt University Medical Center (IRB number 121802). All Clinical data was de-identified and obtained from patients at Vanderbilt University Medical Center Hospital in Nashville, TN.

Results

Effect of platelet inhibition on established AAAs

Most patients are treated with ASA after being diagnosed with an AAA (7, 8). To determine the effect of platelet inhibition on established AAAs, we generated AAAs in mice and then administered ASA or clopidogrel bisulfate. Ldlr−/− mice were fed a HFD for 1 week prior to, and throughout AngII infusion for 28 days. Abdominal aortic diameters were measured by in vivo ultrasound and then mice were implanted with an additional 42 day AngII pump (Figure 1 A and D). Mice were divided into 4 groups: placebo versus ASA, and placebo versus clopidogrel bisulfate. ASA significantly reduced aracadonic acid-mediated integrin activation (data not shown) and completely suppressed plasma TxB2 (Supplemental Figure 1 A). Clopidogrel bisulfate administration resulted in attenuation of ADP-mediated integrin activation (Supplemental Figure 1 B). All mice had similar body weights, cholesterol, lipid fractions, and systolic blood pressures (Supplemental Table 1). We observed a decrease in abdominal aortic diameters with clopidogrel bisulfate or ASA versus placebo controls, but this was not significant (Figure 1 A and B, D and E). Similarly, mice treated with ASA or clopidogrel bisulfate had a non-significant reduction in aortic arch area and diameter of the thoracic aorta (data not shown). Importantly, both platelet inhibitors protected mice with established AAA from rupture-induced death versus placebo controls (Figure 1 C and F; ASA 0% versus placebo 50%; clopidogrel bisulfate 0% versus placebo 47%, P < 0.01). Further, all deaths were due to rupture of the suprarenal abdominal region of the aorta. Interestingly, both platelet inhibitors also reduced the visible thrombi in mice with aortic arch or thoracic aneurysms (data not shown; P < 0.048).

Figure 1. Platelet inhibition was protective against rupture of established AAAs in mice.

Figure 1

Open in a new tab

Ldlr−/− mice were fed a HFD and infused with AngII for 28 days. Mice were then stratified based on in vivo suprarenal abdominal aortic diamters into 4 equal-sized groups and then placed on placebo (n = 14) or ASA (n = 16) and placebo (n = 15) or clopidogrel bisulfate (n = 15) and infused for an additional 42 days. Ultrasonically measured maximal luminal diameters of in vivo suprarenal aortas were measured at days 0, 28, 49, and 70 (A, D luminal diameters over time; B, E luminal diameters at day 70). Survival curves were also determined in these groups between days 28 and 70 (C, F). Circles represent group means ± SEM (A, D). Circles represent individual mice, diamonds represent means ± SEM (B, E). *P < 0.001 treatment groups versus controls. Data was analyzed with a Repeated Measures ANOVA, a Mann-Whitney Rank Sum with Dunn's post hoc, or a Kaplan Meier estimator.

Platelet inhibitors decreased platelet and macrophage accumulation and MMP-2 and 9 activity in abdominal aortas

MMP-2 and 9 have been shown to contribute to the initiation and progression of AAAs (15, 16, 35). Macrophages are a primary source of MMP-9 and platelets contain both MMP-2 and 9 (36-38). Notably, we found that platelet and macrophage accumulation was decreased significantly in mice treated with platelet inhibitors versus placebo controls (Figure 2 A and C). Therefore, we examined whether platelet inhibitors reduced levels of MMP activity in the aorta. Importantly, MMP activity in the aorta was significantly decreased with platelet inhibitors (Figure 2 B and C).

Figure 2. Platelet inhibition decreased platelet and macrophage accumulation and MMP activity in mouse abdominal aortas.

Figure 2

Figure 2

Open in a new tab

Ldlr−/− mice underwent interventional therapy with placebo or ASA and placebo or clopidogrel bisulfate. Platelets (5 days before sacrifice) and macrophages (24 hours before sacrifice) were labelled with anti-GPIX conjugated 700nm fluorophore (red) or MMP 680-sense fluorophore (red) and dextran-coated nanoparticles conjugated to DyLight 800 fluorophore (green), respectively (treatments), or IgG placebo controls (controls). (A) Representative platelet, macrophage, merged, and grayscale images, (B) representative MMP, macrophage, merged, and grayscale images, and (C) subsequent quantification. Histobars represent means ± SEM of 4-8 mice. The abdominal aorta within the dotted yellow lines were analyzed for total fluorescent signal in panel C. *P < 0.001 placebo versus treatment groups. Data were analyzed with a One Way ANOVA on Ranks with Dunn's post hoc.

To further characterize the effect of platelet inhibitors on MMP activity in the aorta, abdominal aortas were removed and levels of pro and active MMP2 and 9 were quantified by ELISA and visualized with gelatin zymography. Platelet inhibitors significantly decreased levels of active MMP-2 and 9 versus placebo controls (Figure 3 A-D). Interestingly, platelet inhibitors also decreased abdominal aortic tissue concentrations of total MMP2, with a non-significant decrease in total MMP9, as measured by ELISA (Figure 3 A-B). Importantly, the decrease in both MMP2 and MMP9 were significantly correlated with decreased macrophage (r2 = 0.835) and platelet (r2 = 0.913) counts in the AAAs of ASA or clopidogrel-treated mice (data not shown; P < 0.001 for all correlations).

Figure 3. Active MMP-2 and MMP-9 was decreased in the abdominal aorta of mice receiving platelet inhibitors.

Figure 3

Open in a new tab

Protein was harvested from pooled aortas obtained from the intervention study (3 aortas pooled for n =1; total different pools analyzed n = 4). Pooled lysates (1 μg total protein/well) were analyzed by MMP-2 (A) and 9 (B) Biotrak Activity Assay ELISAs. Protein (20 μg/lane) from clopidogrel bisulfate (C) and ASA (D) administered mice was also examined using gelatin zymography. Histobars represent means ± SEM of n = 4 pooled aortas. *P < 0.001 clopidogrel bisulfate and ASA total MMP-2, active MMP-2, and active MMP-9 versus placebo controls. Data were analyzed with a One Way ANOVA with a Holm-Sidak Post Hoc.

Platelet inhibition decreased plasminogen activators and plasma cytokines in AngII infused mice

Plasmin generation by the plasminogen activators, uPA and tPA, is associated with increased levels of MMP-2 and 9 (39). We determined that total and active uPA and tPA in both plasma and abdominal aorta were significantly decreased by platelet inhibitors (Figure 4 C-F). Further, the endogenous inhibitor of uPA and tPA, PAI-1, was significantly increased by platelet inhibitors (Figure 4 A and B).

Figure 4. The plasminogen activation system was decreased in plasma and abdominal aorta of mice receiving platelet inhibitors.

Figure 4

Open in a new tab

Protein was harvested from pooled aortas obtained from the intervention study (3 aortas pooled for n =1; total n = 4). Plasma (n = 14-16 each treatment group) was analyzed on total or active (A) PAI-1, (C) tPA, or (E) uPA ELISA plates. Protein (1 μg protein/well) was run on total or active (B) PAI-1, (D) tPA, or (F) uPA ELISA plates. Histobars represent means ± SEM of n = 14-16 plasma samples (A, C, and E) or n = 4 pooled aortas (n = B, D, or F). *P < 0.001 clopidogrel and ASA total and active PAI-1, uPA, and tPA versus placebo controls. Data were analyzed with a Two Way ANOVA with a Holm-Sidak Post Hoc.

Platelets contain and secrete a variety of inflammatory and thrombotic molecules upon activation (40). We ascertained if anti-platelet therapy decreased secretion and circulation of these molecules. Circulating and abdominal aortic PF4 was significantly decreased by anti-platelet therapy (Figure 5 A). In addition, anti-platelet therapy significantly attenuated several platelet-derived cytokines, such as granulocyte colony stimulating factor (G-CSF), interferon gamma (IFN-γ), regulated on activation normal T cell expressed and secreted (RANTES), interleukin-1α and β (IL-1α, β; Figure 5 B-F). Several other plasma inflammatory cytokines were also significantly decreased by anti-platelet therapy, including: IL-4, IL-5, IL-6, IL-7, IL-12(p70), IL-13, IL-17, KC, MCP-1, MIP-1α and β, MIP-2, and TNF-α (data not shown). Importantly, the decreased level of abdominal aortic chemokines, cytokines, and plasminogen activators were all significantly correlated with decreased macrophage and platelet counts in the AAAs of ASA or clopidogrel-treated mice (data not shown; P < 0.05).

Figure 5. Platelet inhibition reduced plasma and tissue cytokines in established AAAs in mice.

Figure 5

Open in a new tab

Protein was harvested from pooled aortas obtained from the intervention study (3 aortas pooled for n =1; total n = 4). Plasma (n = 14-16 each treatment group) or protein (1μg protein/well) was run on a PF4 ELISA (A). Plasma samples (n = 10 each treatment group) were analyzed on a chemokine/cytokine luminex array and (B) G-CSF, (C) IFN-γ, (D) RANTES, (E) IL-1α, or (F) IL-1β were quantitated. Histobars represent means ± SEM of n = 10 plasma samples (A-F) or n = 4 pooled aortas (A). *P < 0.001 clopidogrel and ASA versus placebo controls. Data were analyzed with a One Way ANOVA on Ranks with Dunn's Post Hoc (B-F) or Two Way ANOVA with a Holm-Sidak Post Hoc (A).

Treatment with P2Y12 inhibitors and ASA significantly reduce rupture and dissection in aneurysm patients

A total of 1,578 eligible participants (non-missing data for all covariates) with aortic aneurysms (AAs; defined as either thoracic, abdominal, or thoracoabdominal) were identified totaling 5,592 years of person time with an average follow-up of 2.28 years per individual. In total, 351 AA dissections (227) or ruptures (124) were recorded. Summaries of drug categories, demographic, and vital characteristics are presented in Table 1.

Table 1.

Demographic, vital, and medication characteristics for AA individuals stratified by outcome

Characteristic Events n = 351 Non-Events n = 1524 p-value*
European ancestry (%) 82.1 75.5 0.009
Female (%) 68.7 69.9 0.635
Age (mean [SD]) 66.5(11.1) 67.6(10.9) 0.125
BMI (mean [SD]) 28.0(5.81) 27.9(5.8) 0.667
Type II diabetes (%) 2.85 4.72 0.121
Smoking (%) 89.8 85.5 0.045
Atrial Fibrillation (%) 10.5 15.8 0.012
Heart Failure (%) 12.3 21.0 <0.001
Chronic Kidney Disease 8.83 13.5 0.017
Dialysis 3.42 2.10 0.141
AA Dissection (%) 64.6 NA NA
AA Rupture (%) 35.4 NA NA
Abdominal AA (%) 22.7 NA NA
Thoracic AA (%) 17.7 NA NA
Thoracoabdominal AA (%) 59.5 NA NA
Anti-platelet drugs (%)
    P2Y12 Inhibitors 11.7 25.9 <0.001
    ASA 49.9 74.9 <0.001
Open in a new tab
*

Categorical variables assessed with exact tests, continuous variables with student's t-test

Following a diagnosis of AAs, P2Y12 inhibitors (HR = 0.49, 95% CI: 0.32, 0.74, p-value = 0.001) were significantly associated with decreased dissection or rupture after adjustment for vital (blood pressure, BMI), demographic (age, sex, race), and comorbid factors (diabetes, atrial fibrillation, heart failure, CKD; Table 1). This effect appears to be modified by whether the location of the aneurysm is thoracic versus abdominal, and the effect estimate in thoracic aneurysms is less protective and not statistically significant. ASA also protected against dissection or rupture (HR = 0.50, 95% CI: 0.35, 0.72, p-value = 1×10−4) in adjusted analyses in both the thoracic and abdominal aorta Table 2. Kaplan-Meier plots of survival for each drug exposure are presented in Figure 6 A and B. Participants with at least 30 days of follow-up underwent sensitivity analyses, which did not substantively change event ratios (data not shown).

Table 2.

Cox proportional hazards regression analysis of drugs by class.

Term HR All 95% CI HR Abd. 95% CI HR Thor. 95% CI
P2Y12 Inhibitors (crude) 0.36 (0.24-0.53) 0.21 (0.10-0.42) 0.42 (0.15-1.15)
ASA (crude) 0.35 (0.26-0.45) 0.28 (0.18-0.43) 0.27 (0.17-0.45)
P2Y12 Inhibitors (adjusted) 0.49 (0.32-0.74) 0.24 (0.12-0.51) 0.81 (0.27-2.41)
ASA (adjusted) 0.50 (0.35-0.72) 0.47 (0.22-1.00) 0.30 (0.15-0.60)
Open in a new tab

Adjusted models are fit with terms for age, sex, race, BMI, smoking, diabetes, heart failure, atrial fibrillation, chronic kidney disease, and dialysis.

Abd.— Abdominal; Thor.— Thoracic; HR — Hazard Ratio; CI — Confidence Interval

Figure 6. The effects of ASA and P2Y12 inhibitors on AA rupture in a human cohort.

Figure 6

Open in a new tab

Kaplan-Meier plot of adverse event-free rates stratified by (A) ASA and (B) platelet inhibitors and adjusted for age, sex, race, BMI, smoking, diabetes, CKD, dialysis, heart failure, and atrial fibrillation. *P < 0.001 drug therapy versus control. Data was evaluated with the proportional hazards test (PHtest) within the parameters of a kaplan-meier estimator in STATA and were found to be satisfied. All statistical tests assumed two-tailed distributions.

Discussion

A better understanding of the underlying pathophysiology in aneurysm disease is essential to develop non-surgical therapeutics to reduce the burden of this condition in our aging population. A prominent feature of human AAAs is the accumulation of a laminated mural platelet-rich thrombus that develops along the luminal surface (41, 42). Interestingly, a thrombus is a dynamic biological entity that is balanced between luminal renewal and abluminal fibrinolysis (24, 25, 30, 43). Importantly, clinical studies suggest that thrombus volume or blood displacement caused by presence of mural thrombus may be a predictor of both AAA expansion and rupture (42, 44). Therefore, it is surprising that the role of platelets in the formation of mural thrombi and their potential contribution to both the progression of AAAs has not been investigated systematically in a mouse model. We found that inhibition of platelets reduced rupture of established AAAs. Furthermore, we found ASA or P2Y12 inhibitors administration may protect human AAA patients from rupture. In summary, our results suggest that inhibiting platelet activation slows AAA progression and reduces AAA rupture, which may support the clinical use of ASA and P2Y12 inhibitors in AAA patients.

ASA therapy is recommended for patients with AAA from the time of diagnosis until the perioperative period (7, 8). It is currently hypothesized that the benefit of ASA in reducing cardiovascular morbidity and mortality and potentially AAA progression outweigh the risks of bleeding and AAA rupture (7, 8). However, clinical studies examining the effects of platelet inhibitors (clopidogrel bisulfate and/or ASA) on non-genetically categorized AAA ruptures are extremely limited. Among a large meta-analysis of 6 studies, only one reported data with regard to AAA rupture (33). The UKSAT study reported that ‘anti-platelet’ administration resulted in a rupture rate hazard ratio of 0.83, which was not significant (33, 45). The mechanism by which ASA reduces aneurysm expansion is hypothesized to be via a decrease in thrombus formation, a reduction in aortic wall inflammation, and stabilization of the aortic wall (46). A small retrospective study reported patients with medium-sized AAAs had significantly reduced AAA expansion and time to aneurysm repair on low-dose ASA (46). Other studies have demonstrated a reduction in progression of small AAAs, though a definitive association between platelet inhibitors and aneurysm reduction was not established (34). However, a large meta-analysis demonstrated that ‘anti-platelet’ therapy resulted in a non-significant decrease in AAA growth compared to untreated aneurysm patients (P = 0.241) after adjusting for confounding variables (33). In our study, we observed protective associations between platelet inhibitors and AA rupture or dissection in 351 patients. We also found an inverse relationship between adverse AA events and non-ASA platelet inhibitor use, as well as an independent effect of ASA use. This finding indicates that addition of ASA or P2Y12 inhibitors to standard therapy may be beneficial to AAA patients in addition to effects on other presumed cardiovascular diseases. In support of our findings, a phase 2 clinical trial is examining the efficacy of ticagrelor on patients with small AAAs (Government clinical trial identifier: NCT02070653).

To better mimic this clinical situation, we administered platelet inhibitors to mice with established AAAs. We found that prolonged AngII infusion increased rate of aortic rupture, which was significantly reduced with clopidogrel bisulfate or ASA. These inhibitors also had no effect on abdominal aortic diameter (clopidogrel bisulfate, P = 0.19 and ASA, P = 0.08). Other studies have shown that a GPIIa/IIIb platelet inhibitor (abciximab) and a P2Y12 receptor antagonist, (AZD6140) prevented aneurysm growth in the rat xenograft model of aneurysm (17, 24, 25). While this model does not exhibit rupture, it does exhibit an intraluminal thrombus similar to human aneurysms in a certain percentage of rats (17). We speculate that our results may be different because of the large amount of ruptures in our placebo groups, resulting in a lack of subsequent measurements of aortic diameters. Alternatively, a previous publication demonstrated a distinct difference between the incidence or maximal diameter of AngII-induced AAAs and increased mortality due to rupture (47).

The continued accumulation of platelets and macrophages may result in proteolytic destruction of the aortic architecture via release of MMPs. Platelets (36, 37) and macrophages (38) are a robust source of MMPs. Further, platelet-derived chemokines can regulate the expression of MMPs from VSMCs and macrophages (48, 49). In addition, plasmin production by uPA or tPA is a critical step in fibrinolysis and MMP activation (39). Here, we demonstrate clopidogrel bisulfate and ASA intervention reduce platelet and macrophage infiltration into the vessel wall, circulating platelet-derived cytokines, plasminogen activators, and ultimately the amount of active MMP-2 and MMP-9 in the abdominal aortas. Importantly, MMP-2 and MMP-9 are correlated with increased aneurysmal disease and rupture (16, 50). Further, several of the attenuated cytokines and chemokines play a significant role in AAA progression (51-54). Additionally, there is a role for the uPA, uPAR, tPA, and PAI-1 plasminogen axis in both the progression and rupture of experimental aneurysm (47, 55-57). While it is uncertain whether these MMPs, cytokines, and plasminogen activators/inhibitors are primarily derived from platelets or macrophages, there are significant correlations between platelets or macrophages amongst all of these inflammatory mediators. However, there are several contradictions in the literature regarding the role of Th1/Th2 chemokines and cytokines and MMPs with regard to the outcome of AAA pathogenesis (58, 59). Indeed, the differences in cytokine and MMP profile only reflect a single stage of AAA development/degeneration thus complicating a proper analyses and interpretation of these correlations.

In conclusion, we show that platelet accumulation and activation is detrimental in a mouse model of established AAAs. The pathological role appears to involve macrophage recruitment and the production of MMPs resulting in vessel instability and rupture. We further show a positive association with platelet inhibitors and ASA in the prevention of human AA rupture or dissection. The results indicate that platelet inhibitors are beneficial in pre-existing aneurysms. Future studies will be directed at dissecting the role of platelet signaling in AAAs.

Supplementary Material

ATVB_ATVB-2015-305537-T.xml
Owens AAA Methods and Materials
Supplemental Figure and Table

Significance.

Abdominal aortic aneurysm (AAA) is a progressive expansion of the aorta which may result in catastrophic rupture and death. This cardiovascular disease is estimated to affect almost 10% of people over the age of 50 with an estimated 1 out of every 250 people affected. Despite decades of research, there are no clinically approved drug regimens for this disease with surgical intervention as the only approved therapy. Here, we demonstrate commonly used antiplatelet drugs prevents rupture of advanced AAAs in a mouse model. Antiplatelet therapy dramatically reduces the amount of destructive enzymes and tissue/circulating inflammatory proteins. Lastly, we verify this effect in a retrospective analysis of human aneurysm patients. These results identify platelets as a critical component of aneurysm rupture and suggests utilizing antiplatelet therapy may be beneficial in patients with AAA.

Acknowledgements

We acknowledge helpful suggestions provided by the VTRACC working group that contributed to this work, as well as helpful advice from Dr. Digna Velez Edwards.

Sources of Funding

This work was supported by National Institutes of Health grants PO1-HL006350 (N.M). A. Phillip Owens III was supported by a National Institutes of Health NHLBI K99/R00 award (5K99-HL116786-02). Dr. Antoniak was supported by a Beginning Grant-in Aid from the Mid-Atlantic affiliate of the American Heart Association (14BGIA20380134). Dr. Edwards development resources as well as the Vanderbilt Clinical and Translational Research Scholar Award 5KL2RR024977 from the National Center for Advancing Translational Sciences, the Vanderbilt CTSA award, from the National Center for Advancing Translational Sciences, and the BioVU dataset used for the analyses described was obtained from Vanderbilt University Medical Center's BioVU which is supported by institutional funding and by the Vanderbilt CTSA grant ULTR000445 from NCATS/NIH.

Nonstandard Abbreviations and Acronyms

AA

aortic aneurysm

AAA

abdominal aortic aneurysm

ADP

adenosine diphosphate

AngII

angiotensin II

ASA

acetylsalicylic acid (aspirin)

BMI

body mass index

CKD

chronic kidney disease

G-CSF

granulocyte colony stimulating factor

IFN

interferon

IL

interleukin

Ldlr

low-density lipoprotein receptor

MMP

matrix metalloproteinase

PAI-1

plasminogen activator inhibitor 1

Par

protease-activated receptor

PF4

platelet factor 4

RANTES

regulated on activation, normal T cell expressed and secreted

tPA

tissue plasminogen activator

TP

thromboxane receptor

TxA2

thromboxane A2

uPA

urokinase plasminogen activator

Footnotes

Disclosures

The authors have no actual or potential perceived conflicts of interest to disclose.

References

  • 1.Alcorn HG, Wolfson SK, Jr., Sutton-Tyrrell K, Kuller LH, O'Leary D. Risk factors for abdominal aortic aneurysms in older adults enrolled in The Cardiovascular Health Study. Arterioscler Thromb Vasc Biol. 1996;16:963–970. doi: 10.1161/01.atv.16.8.963. [DOI] [PubMed] [Google Scholar]
  • 2.Bengtsson H, Bergqvist D, Sternby NH. Increasing prevalence of abdominal aortic aneurysms. A necropsy study. Eur J Surg. 1992;158:19–23. [PubMed] [Google Scholar]
  • 3.Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2004;24:429–434. doi: 10.1161/01.ATV.0000118013.72016.ea. [DOI] [PubMed] [Google Scholar]
  • 4.Saraff K, Babamusta F, Cassis LA, Daugherty A. Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23:1621–1626. doi: 10.1161/01.ATV.0000085631.76095.64. [DOI] [PubMed] [Google Scholar]
  • 5.Thompson MM, Bell PR. ABC of arterial and venous disease. Arterial aneurysms. BMJ. 2000;320:1193–1196. doi: 10.1136/bmj.320.7243.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chaikof EL, Brewster DC, Dalman RL, Makaroun MS, Illig KA, Sicard GA, Timaran CH, Upchurch GR, Jr., Veith FJ. The care of patients with an abdominal aortic aneurysm: the Society for Vascular Surgery practice guidelines. J Vasc Surg. 2009;50:S2–S49. doi: 10.1016/j.jvs.2009.07.002. [DOI] [PubMed] [Google Scholar]
  • 7.Moll FL, Powell JT, Fraedrich G, et al. Management of abdominal aortic aneurysms clinical practice guidelines of the European society for vascular surgery. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. 2011;41:S1–S58. doi: 10.1016/j.ejvs.2010.09.011. [DOI] [PubMed] [Google Scholar]
  • 8.Hirsch AT, Haskal ZJ, Hertzer NR, et al. ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease: a collaborative report from the American Association for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines: endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute; Society for Vascular Nursing; TransAtlantic Inter-Society Consensus; and Vascular Disease Foundation. Circulation. 2006;113:e463–e654. doi: 10.1161/CIRCULATIONAHA.106.174526. [DOI] [PubMed] [Google Scholar]
  • 9.Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest. 2000;105:1605–1612. doi: 10.1172/JCI7818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Daugherty A, Cassis L. Chronic angiotensin II infusion promotes atherogenesis in low density lipoprotein receptor −/− mice. Ann N Y Acad Sci. 1999;892:108–118. doi: 10.1111/j.1749-6632.1999.tb07789.x. [DOI] [PubMed] [Google Scholar]
  • 11.Owens AP, 3rd, Rateri DL, Howatt DA, Moore KJ, Tobias PS, Curtiss LK, Lu H, Cassis LA, Daugherty A. MyD88 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation independent of signaling through Toll-like receptors 2 and 4. Arterioscler Thromb Vasc Biol. 2011;31:2813–2819. doi: 10.1161/ATVBAHA.111.238642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Weiss D, Kools JJ, Taylor WR. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in apoE-deficient mice. Circulation. 2001;103:448–454. doi: 10.1161/01.cir.103.3.448. [DOI] [PubMed] [Google Scholar]
  • 13.Martin-McNulty B, Tham DM, da Cunha V, Ho JJ, Wilson DW, Rutledge JC, Deng GG, Vergona R, Sullivan ME, Wang YX. 17 Beta-estradiol attenuates development of angiotensin II-induced aortic abdominal aneurysm in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23:1627–1632. doi: 10.1161/01.ATV.0000085842.20866.6A. [DOI] [PubMed] [Google Scholar]
  • 14.Powell JT, Brady AR. Detection, management, and prospects for the medical treatment of small abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2004;24:241–245. doi: 10.1161/01.ATV.0000106016.13624.4a. [DOI] [PubMed] [Google Scholar]
  • 15.Pyo R, Lee JK, Shipley JM, Curci JA, Mao D, Ziporin SJ, Ennis TL, Shapiro SD, Senior RM, Thompson RW. Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. J Clin Invest. 2000;105:1641–1649. doi: 10.1172/JCI8931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest. 2002;110:625–632. doi: 10.1172/JCI15334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Allaire E, Guettier C, Bruneval P, Plissonnier D, Michel JB. Cell-free arterial grafts: morphologic characteristics of aortic isografts, allografts, and xenografts in rats. J Vasc Surg. 1994;19:446–456. doi: 10.1016/s0741-5214(94)70071-0. [DOI] [PubMed] [Google Scholar]
  • 18.Tsui JC. Experimental models of abdominal aortic aneurysms. The open cardiovascular medicine journal. 2010;4:221–230. doi: 10.2174/1874192401004010221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bhatt DL, Topol EJ. Scientific and therapeutic advances in antiplatelet therapy. Nat Rev Drug Discov. 2003;2:15–28. doi: 10.1038/nrd985. [DOI] [PubMed] [Google Scholar]
  • 20.Jurk K, Kehrel BE. Platelets: physiology and biochemistry. Semin Thromb Hemost. 2005;31:381–392. doi: 10.1055/s-2005-916671. [DOI] [PubMed] [Google Scholar]
  • 21.Woulfe D, Yang J, Brass L. ADP and platelets: the end of the beginning. J Clin Invest. 2001;107:1503–1505. doi: 10.1172/JCI13361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Scirica BM, Bonaca MP, Braunwald E, et al. Vorapaxar for secondary prevention of thrombotic events for patients with previous myocardial infarction: a prespecified subgroup analysis of the TRA 2 degrees P-TIMI 50 trial. Lancet. 2012;380:1317–1324. doi: 10.1016/S0140-6736(12)61269-0. [DOI] [PubMed] [Google Scholar]
  • 23.Morrow DA, Braunwald E, Bonaca MP, et al. Vorapaxar in the secondary prevention of atherothrombotic events. The New England journal of medicine. 2012;366:1404–1413. doi: 10.1056/NEJMoa1200933. [DOI] [PubMed] [Google Scholar]
  • 24.Dai J, Louedec L, Philippe M, Michel JB, Houard X. Effect of blocking platelet activation with AZD6140 on development of abdominal aortic aneurysm in a rat aneurysmal model. J Vasc Surg. 2009;49:719–727. doi: 10.1016/j.jvs.2008.09.057. [DOI] [PubMed] [Google Scholar]
  • 25.Touat Z, Ollivier V, Dai J, et al. Renewal of mural thrombus releases plasma markers and is involved in aortic abdominal aneurysm evolution. Am J Pathol. 2006;168:1022–1030. doi: 10.2353/ajpath.2006.050868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Abdelhamid MF, Davies RS, Adam DJ, Vohra RK, Bradbury AW. Changes in thrombin generation, fibrinolysis, platelet and endothelial cell activity, and inflammation following endovascular abdominal aortic aneurysm repair. J Vasc Surg. 2012;55:41–46. doi: 10.1016/j.jvs.2011.07.094. [DOI] [PubMed] [Google Scholar]
  • 27.Parry DJ, Al-Barjas HS, Chappell L, Rashid T, Ariens RA, Scott DJ. Haemostatic and fibrinolytic factors in men with a small abdominal aortic aneurysm. Br J Surg. 2009;96:870–877. doi: 10.1002/bjs.6632. [DOI] [PubMed] [Google Scholar]
  • 28.Davies RS, Abdelhamid M, Wall ML, Vohra RK, Bradbury AW, Adam DJ. Coagulation, fibrinolysis, and platelet activation in patients undergoing open and endovascular repair of abdominal aortic aneurysm. J Vasc Surg. 2011;54:865–878. doi: 10.1016/j.jvs.2011.04.010. [DOI] [PubMed] [Google Scholar]
  • 29.Wallinder J, Bergqvist D, Henriksson AE. Haemostatic markers in patients with abdominal aortic aneurysm and the impact of aneurysm size. Thromb Res. 2009;124:423–426. doi: 10.1016/j.thromres.2009.01.016. [DOI] [PubMed] [Google Scholar]
  • 30.Touat Z, Lepage L, Ollivier V, Nataf P, Hvass U, Labreuche J, Jandrot-Perrus M, Michel JB, Jondeau G. Dilation-dependent activation of platelets and prothrombin in human thoracic ascending aortic aneurysm. Arterioscler Thromb Vasc Biol. 2008;28:940–946. doi: 10.1161/ATVBAHA.107.158576. [DOI] [PubMed] [Google Scholar]
  • 31.Scott DJ, Prasad P, Philippou H, et al. Clot architecture is altered in abdominal aortic aneurysms and correlates with aneurysm size. Arterioscler Thromb Vasc Biol. 2011;31:3004–3010. doi: 10.1161/ATVBAHA.111.236786. [DOI] [PubMed] [Google Scholar]
  • 32.Sidloff DA, Stather PW, Choke E, Bown MJ, Sayers RD. A systematic review and meta-analysis of the association between markers of hemostasis and abdominal aortic aneurysm presence and size. J Vasc Surg. 2014;59:528–535. doi: 10.1016/j.jvs.2013.10.088. [DOI] [PubMed] [Google Scholar]
  • 33.Sweeting MJ, Thompson SG, Brown LC, Powell JT, collaborators R. Meta-analysis of individual patient data to examine factors affecting growth and rupture of small abdominal aortic aneurysms. Br J Surg. 2012;99:655–665. doi: 10.1002/bjs.8707. [DOI] [PubMed] [Google Scholar]
  • 34.Thompson A, Cooper JA, Fabricius M, Humphries SE, Ashton HA, Hafez H. An analysis of drug modulation of abdominal aortic aneurysm growth through 25 years of surveillance. J Vasc Surg. 2010;52:55–61. doi: 10.1016/j.jvs.2010.02.012. [DOI] [PubMed] [Google Scholar]
  • 35.Basu R, Fan D, Kandalam V, Lee J, Das SK, Wang X, Baldwin TA, Oudit GY, Kassiri Z. Loss of Timp3 gene leads to abdominal aortic aneurysm formation in response to angiotensin II. The Journal of biological chemistry. 2012;287:44083–44096. doi: 10.1074/jbc.M112.425652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Cecchetti L, Tolley ND, Michetti N, Bury L, Weyrich AS, Gresele P. Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events. Blood. 2011;118:1903–1911. doi: 10.1182/blood-2010-12-324517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Mannello F, Medda V. Differential expression of MMP-2 and MMP-9 activity in megakaryocytes and platelets. Blood. 2011;118:6470–6471. doi: 10.1182/blood-2011-07-366195. [DOI] [PubMed] [Google Scholar]
  • 38.Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest. 1995;96:318–326. doi: 10.1172/JCI118037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Deryugina EI, Quigley JP. Cell surface remodeling by plasmin: a new function for an old enzyme. Journal of biomedicine & biotechnology. 2012;2012:564259. doi: 10.1155/2012/564259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Golebiewska EM, Poole AW. Platelet secretion: From haemostasis to wound healing and beyond. Blood reviews. 2015;29:153–162. doi: 10.1016/j.blre.2014.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Harter LP, Gross BH, Callen PW, Barth RA. Ultrasonic evaluation of abdominal aortic thrombus. Journal of ultrasound in medicine : official journal of the American Institute of Ultrasound in Medicine. 1982;1:315–318. doi: 10.7863/jum.1982.1.8.315. [DOI] [PubMed] [Google Scholar]
  • 42.Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: basic mechanisms and clinical implications. Current problems in surgery. 2002;39:110–230. doi: 10.1067/msg.2002.121421. [DOI] [PubMed] [Google Scholar]
  • 43.Houard X, Rouzet F, Touat Z, et al. Topology of the fibrinolytic system within the mural thrombus of human abdominal aortic aneurysms. J Pathol. 2007;212:20–28. doi: 10.1002/path.2148. [DOI] [PubMed] [Google Scholar]
  • 44.Satta J, Laara E, Juvonen T. Intraluminal thrombus predicts rupture of an abdominal aortic aneurysm. J Vasc Surg. 1996;23:737–739. doi: 10.1016/s0741-5214(96)80062-0. [DOI] [PubMed] [Google Scholar]
  • 45.Brady AR, Thompson SG, Fowkes FG, Greenhalgh RM, Powell JT, Participants UKSAT Abdominal aortic aneurysm expansion: risk factors and time intervals for surveillance. Circulation. 2004;110:16–21. doi: 10.1161/01.CIR.0000133279.07468.9F. [DOI] [PubMed] [Google Scholar]
  • 46.Lindholt JS, Sorensen HT, Michel JB, Thomsen HF, Henneberg EW. Low-dose aspirin may prevent growth and later surgical repair of medium-sized abdominal aortic aneurysms. Vascular and endovascular surgery. 2008;42:329–334. doi: 10.1177/1538574408315205. [DOI] [PubMed] [Google Scholar]
  • 47.Uchida HA, Poduri A, Subramanian V, Cassis LA, Daugherty A. Urokinase-type plasminogen activator deficiency in bone marrow-derived cells augments rupture of angiotensin II-induced abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 2011;31:2845–2852. doi: 10.1161/ATVBAHA.111.234997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Risinger GM, Jr., Hunt TS, Updike DL, Bullen EC, Howard EW. Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors. The Journal of biological chemistry. 2006;281:25915–25925. doi: 10.1074/jbc.M513513200. [DOI] [PubMed] [Google Scholar]
  • 49.Stellos K, Seizer P, Bigalke B, Daub K, Geisler T, Gawaz M. Platelet aggregates-induced human CD34+ progenitor cell proliferation and differentiation to macrophages and foam cells is mediated by stromal cell derived factor 1 in vitro. Semin Thromb Hemost. 2010;36:139–145. doi: 10.1055/s-0030-1251497. [DOI] [PubMed] [Google Scholar]
  • 50.Petersen E, Wagberg F, Angquist KA. Proteolysis of the abdominal aortic aneurysm wall and the association with rupture. European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. 2002;23:153–157. doi: 10.1053/ejvs.2001.1572. [DOI] [PubMed] [Google Scholar]
  • 51.Ishibashi M, Egashira K, Zhao Q, et al. Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice. Arterioscler Thromb Vasc Biol. 2004;24:e174–e178. doi: 10.1161/01.ATV.0000143384.69170.2d. [DOI] [PubMed] [Google Scholar]
  • 52.Xiong W, MacTaggart J, Knispel R, Worth J, Persidsky Y, Baxter BT. Blocking TNF-alpha attenuates aneurysm formation in a murine model. Journal of immunology. 2009;183:2741–2746. doi: 10.4049/jimmunol.0803164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Johnston WF, Salmon M, Su G, Lu G, Stone ML, Zhao Y, Owens GK, Upchurch GR, Jr., Ailawadi G. Genetic and pharmacologic disruption of interleukin-1beta signaling inhibits experimental aortic aneurysm formation. Arterioscler Thromb Vasc Biol. 2013;33:294–304. doi: 10.1161/ATVBAHA.112.300432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.King VL, Lin AY, Kristo F, et al. Interferon-gamma and the interferon-inducible chemokine CXCL10 protect against aneurysm formation and rupture. Circulation. 2009;119:426–435. doi: 10.1161/CIRCULATIONAHA.108.785949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Deng GG, Martin-McNulty B, Sukovich DA, Freay A, Halks-Miller M, Thinnes T, Loskutoff DJ, Carmeliet P, Dole WP, Wang YX. Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm. Circ Res. 2003;92:510–517. doi: 10.1161/01.RES.0000061571.49375.E1. [DOI] [PubMed] [Google Scholar]
  • 56.Qian HS, Gu JM, Liu P, Kauser K, Halks-Miller M, Vergona R, Sullivan ME, Dole WP, Deng GG. Overexpression of PAI-1 prevents the development of abdominal aortic aneurysm in mice. Gene Ther. 2008;15:224–232. doi: 10.1038/sj.gt.3303069. [DOI] [PubMed] [Google Scholar]
  • 57.Wang YX, Martin-McNulty B, Freay AD, et al. Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice. Am J Pathol. 2001;159:1455–1464. doi: 10.1016/S0002-9440(10)62532-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Jagadesham VP, Scott DJ, Carding SR. Abdominal aortic aneurysms: an autoimmune disease? Trends in molecular medicine. 2008;14:522–529. doi: 10.1016/j.molmed.2008.09.008. [DOI] [PubMed] [Google Scholar]
  • 59.Meijer CA, Stijnen T, Wasser MN, Hamming JF, van Bockel JH, Lindeman JH, Pharmaceutical Aneurysm Stabilisation Trial Study G Doxycycline for stabilization of abdominal aortic aneurysms: a randomized trial. Annals of internal medicine. 2013;159:815–823. doi: 10.7326/0003-4819-159-12-201312170-00007. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ATVB_ATVB-2015-305537-T.xml
NIHMS703197-supplement-ATVB_ATVB-2015-305537-T_xml.xml (17.6KB, xml)
Owens AAA Methods and Materials
NIHMS703197-supplement-Owens_AAA_Methods_and_Materials.pdf (445KB, pdf)
Supplemental Figure and Table
NIHMS703197-supplement-Supplemental_Figure_and_Table.pdf (330.4KB, pdf)

RESOURCES