Angiotensin II Infusion Leads to Aortic Dissection in LRP8 Deficient Mice

Abstract

Myeloid cells are crucial for the development of vascular inflammation. Low-density lipoprotein receptor-related protein 8 (LRP8) or Apolipoprotein E receptor 2 (ApoER2), is expressed by macrophages, endothelial cells and platelets and has been implicated in the development of cardiovascular diseases. Our aim was to evaluate the role of LRP8, in particular from immune cells, in the development of vascular inflammation.

Methods: LRP8^+/+ and LRP8^-/- mice (on B6;129S background) were infused with angiotensin II (AngII, 1 mg/kg/day for 7 to 28 day) using osmotic minipumps. Blood pressure was recorded using tail cuff measurements. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging. Histological analysis of aortic sections was conducted using sirius red staining.

Results: AngII infusion worsened endothelial-dependent vascular relaxation and immune cells rolling and adherence to the carotid artery in both LRP8^+/+ as well as LRP8^-/- mice. However, only LRP8^-/- mice demonstrated a drastically increased mortality rate in response to AngII due to aortic dissection. Bone marrow transplantation revealed that chimeras with LRP8 deficient myeloid cells phenocopied LRP8^-/- mice.

Conclusion: AngII-infused LRP8 deficient mice could be a useful animal model to study aortic dissection reflecting the lethality of this disease in humans.

Keywords: angiotensin II; aortic dissection; low-density lipoprotein receptor-related protein 8.

Figure 1

Vascular function and immune cell infiltration in LRP8^+/+ and LRP8^−/− mice in response to AngII. LRP8^+/+ and LRP8^−/− mice were infused with AngII (1 mg/kg/d for 7 d) vs. sham treatment. (A) Epifluorescence intravital epifluorescence video microscopy (IVM) of endothelial adherent and rolling leukocytes in the common carotid artery. Nucleated cells were visualized with acridine orange (green fluorescence) (scale bar 200 µm). (B) Quantification of adherent and rolling leukocytes. Cell recruitment was quantified in four fields of view (100 × 150 μm) per carotid artery (8 measurements per mouse). Adherent cells were defined in each vessel segment as cells that did not move or detach from the endothelial lining within an observation period of 10 s and presented per mm². One dot corresponds to the mean of 8 measurements in one animal. n = 4–5 animals/group. Data are presented as mean ± SEM; * p < 0.05; vs. sham treatment of the same strain. One-way ANOVA and Bonferroni’s multiple comparison test. (C,D) Flow cytometry of aortic lysates. (C) Representative original plots. (D) absolute numbers of viable CD45⁺ , CD45⁺ CD11b⁺ Ly6G⁺ Ly6C⁻NK1.1⁻, CD45⁺ CD11b⁺ Ly6G⁻Ly6C^lowNK1.1⁻ and CD45⁺ CD11b⁺ Ly6G⁻Ly6C^hiNK1.1⁻ cells. Results are expressed as the percentage of positive cells per total living cells. One dot corresponds to one aorta of one animal. n = 6–8 animals/group. Data are presented as mean ± SEM; * p < 0.05; vs. sham treatment of the same strain. One-way ANOVA and Bonferroni’s multiple comparison test. (E) Concentration—relaxation curves in response to Acetylcholine (ACh) (endothelium dependent) of isolated aortic segments. One dot corresponds to one aortic ring of one animal. n = 5 animals/group. Data are presented as mean ± SEM; * p < 0.05; vs. sham treatment of the same strain. Two-way ANOVA and Dunn’s multiple comparison test. (F) Systolic blood pressure after one week of AngII-infusion or sham treatment. n = 8–14 animals/group. Data are presented as mean ± SEM; * p < 0.05; vs. sham treatment of the same strain; one-way ANOVA and Bonferroni’s multiple comparison test.

Figure 2

Formation of aortic dissections in LRP8^−/− mice in response to AngII. LRP8^+/+ and LRP8^−/− mice were infused with AngII (1 mg/kg/d for 7day) vs. sham treatment. (A) Survival curves during 28 days of AngII infusion. n = 3–5 animals/group (n = 3 in control groups and n = 5 in AngII infused groups). ** p < 0.01; LRP8^+/+ + AngII vs. LRP8^−/− +AngII. Kaplan—Meier curves were compared using a log-rank test. (B) Number of aortic dissection and aneurysm formations in LRP8 deficient and control mice infused with AngII. (C) Representative images of isolated aortas in control mice and after AngII infusion. (D) Representative images of sirius red staining of aortic sections (scale bar 200 µm).

Figure 3

Critical role of myeloid cells to drive aortic dissection in AngII infused LRP8^−/− mice. (A) Aortic mRNA expression of Ccl2, Ccr2, Col1a1, Col1a2 and Eln. One dot corresponds to one aorta of one animal. n = 6–10 animals/group. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01; vs. sham treatment of the same strain. One-way ANOVA and Bonferroni’s multiple comparison test. (B) Aortic dissection development following bone marrow transfer and AngII infusion (Bone marrow from LRP8^+/+ to LRP8^+/+, from LRP8^+/+ to LRP8^−/− and from LRP8^−/− to LRP8^+/+). Six LRP8^+/+ received LRP8^+/+ BM, 8 LRP8^−/− received LRP8^+/+ BM and 10 LRP8^+/+ received LRP8^−/− BM. (C) Representative images of macroscopic inspection of the aorta as well as sirius red staining of aortic section of LRP8^−/− → LRP8^+/+ bone marrow transfer mice, infused with AngII (scale bar 200 µm).