Phospholipid membranes drive abdominal aortic aneurysm development through stimulating coagulation factor activity

Abstract

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE^-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE^-/- deletion, and many were absent in Alox^-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.

Keywords: aneurysm; angiotensin; lipid; lipoxygenase; phospholipid.

Fig. 1.

EoxPL are detected in ApoE^−/− mouse and human AAA tissue. (A) Lipidomics shows eoxPL in murine AAA. Lipids from abdominal aorta harvested from ApoE^−/− mice following Ang II was analyzed using LC/MS/MS, as in SI Appendix, SI Materials and Methods (each sample represents a different aorta). (B) Lipidomics reveals eoxPL in human AAA. Abdominal aorta were harvested from six male patients, and lipids were analyzed using LC/MS/MS. Heatmaps were generated using Pheatmap as described in SI Appendix, SI Materials and Methods (each sample represents a different sample), shown as log10 values for analyte:internal standard normalized to tissue weight (mg). (C) Representative chromatograms show 18:0p/HETE-PE lipids, with a predominance of eoxPL from either Alox15 or Alox12. Middle shows m/z 766.6/319.2, detecting all HETE-PE isomers and revealing two main products. These are confirmed as 15-HETE-PE (13.7 min) or 12-HETE-PE (14.2 min), by detecting internal daughter ions at m/z 219.1 or 179.1, respectively.

Fig. 2.

The 12- and 15-LOX–derived eoxPL are detected in human AAA thrombus, while Alox12 or Alox15 deletion prevents AAA. (A) Representative chromatograms showing a truncated PE and PC both detected in AAA wall. (B) Representative chromatograms of eoxPL generated by leukocyte 5-LOX, in AAA wall. (C and D) Alox15 or Alox12 deletion reduces AAA development in ApoE^−/− mice. Male (C) or female (D) ApoE^−/−, ApoE^−/−/Alox15^−/−, and ApoE^−/−/Alox12^−/− mice aged 19–24 wk were infused with Ang II for 2 wk. Aortae were imaged as described in Materials and Methods. Representative aortae are shown (Left), including a PBS control for each condition. The full datasets are provided in SI Appendix, Figs. S3–S5. Incidence of AAA as percent of group is shown (Right). Data are analyzed using Fisher’s exact test and expressed as percent AAA, ***P < 0.001, **P < 0.01.

Fig. 3.

Alox12 ^−/− or Alox15 ^−/− mice show disruption of the thrombosis/bleeding axis, with increased aPL externalization in circulating blood cells. (A) Alox12 or Alox15 deficiency promotes thrombin generation in vivo in wild-type and ApoE^−/− mice. Plasma from 19-wk-old mice was isolated as in Materials and Methods. Thrombin/anti-thrombin (TAT) complexes were measured using ELISA. (B) Alox12 or Alox15 deficiency reduces clotting in wild-type and ApoE^−/− mice. Mouse plasma obtained as in B was tested for PT as in Materials and Methods. For A and B, data were analyzed using Mann–Whitney nonparametric u test and shown on box plots (median, with whiskers representing interquartile range), ***P < 0.001, **P < 0.01, *P < 0.05. (C) Eosinophils from Alox15^−/− mice externalize more PE than wild type on ADP activation. Eosinophils generated as described in Materials and Methods were activated using 40 μM ADP before PE externalization determined (n = 3, mean ± SEM). (D) Platelets from Alox12^−/− mice externalize more PE than wild type on thrombin activation. Mouse platelets were isolated as described in Materials and Methods, and PE externalization was determined. Data were analyzed using Student’s t test and expressed as mean ± SEM (n = 10). **P < 0.01, *P < 0.05 (n = 10).

Fig. 4.

Ang II infusion induces consumptive coagulopathy in mice, while ApoE and Alox deletion is associated with major changes in eoxPL profile. (A) Ang II increases plasma TATs in mice. ApoE^−/−, ApoE^−/−/Alox12^−/−, and ApoE^−/−/Alox15^−/− 19- to 24-wk-old male mice maintained on a normal chow diet were infused with Ang II for 2 wk. TATs were measured by ELISA. (B) PT of plasma in response to 2-wk infusion of Ang II with/without liposomes. ApoE^−/−, ApoE^−/−/Alox12^−/−, and ApoE^−/−/Alox15^−/− 19- to 24-wk-old male mice maintained on a normal chow diet were administered Ang II for 2 wk. PT was measured as per Materials and Methods. Data were analyzed using Mann–Whitney nonparametric u test and shown on box plots (median, with whiskers representing interquartile range), ***P < 0.001, *P < 0.05. (C) Oxylipidomics of blood clotting from all mouse strains. Blood was harvested and induced to clot as in Materials and Methods. Lipids were extracted from clots and profiled using LC/MS/MS. Heatmaps were generated using Pheatmap (n = 3 per sample) using log10 data values as described in SI Appendix, SI Materials and Methods.

Fig. 5.

Procoagulant liposomes prevent AAA in mice, while modulating Ang II induced coagulation activities, and Alox15 deficiency is associated with decreased Ang II induction of IL6 and Ccl2. (A) eoxPL/aPL liposomes prevent AAA in ApoE^−/− mice. Male ApoE^−/− mice on normal chow diet aged 19–24 wk were administered Ang II as described in Materials and Methods. Liposomes (eoxPL or aPL) were administered every second day (see Materials and Methods for composition and dose). Data are expressed as percentage of mice developing AAA. (B and C) eoxPL/aPL liposomes reduce AAA in ApoE^−/− mice lacking either Alox12 or Alox15. Male mice on normal chow diet aged 19–24 wk were administered Ang II with/without liposomes as in C. Data are expressed as percentage of mice developing AAA. For A–C, the full dataset of aortae are shown in SI Appendix, Figs. S3 and S8–S10). (D) eoxPL liposomes alter coagulation parameters during Ang II infusion into ApoE^−/− mice. ApoE^−/− 19- to 24-wk-old male mice maintained on a normal chow diet were infused with Ang II for 2 wk, and every second day, mice eoxPL or aPL liposomes were administered as described in Materials and Methods. TATs were measured by ELISA. (E) Systemic administration of eoxPL/aPL liposomes promotes bleeding in mice. Male wild-type mice (11 wk old) were anesthetized and i.v. injected with liposomes, and tail bleeding was measured after 1 h, as described in Materials and Methods. Data from A–C were analyzed using Fisher’s exact test, and data from D and E were analyzed using Mann–Whitney nonparametric u test. ***P < 0.001, **P < 0.01, *P < 0.05; NS, no significance. Data are shown on box plots (median, with whiskers representing interquartile range). (F) Alox15 deficiency reduces the Ang II-induced elevation in IL6 and Ccl2 in mouse AAA tissue. RNA was isolated from ApoE^−/− AAA tissues as detailed in Materials and Methods (n = 4 for all groups) and analyzed using the RT² Profiler PCR Array for Mouse Inflammatory Response and Autoimmunity. Data are shown as ΔΔC_T, expressed as fold change between control and Ang II-treated mice, and compared using Student’s t test. ***P < 0.005.