Efficient aortic lymphatic drainage is necessary for atherosclerosis regression induced by ezetimibe

Abstract

A functional lymphatic vasculature is essential for tissue fluid homeostasis, immunity, and lipid clearance. Although atherosclerosis has been linked to adventitial lymphangiogenesis, the functionality of aortic lymphatic vessels draining the diseased aorta has never been assessed and the role of lymphatic drainage in atherogenesis is not well understood. We develop a method to measure aortic lymphatic transport of macromolecules and show that it is impaired during atherosclerosis progression, whereas it is ameliorated during lesion regression induced by ezetimibe. Disruption of aortic lymph flow by lymphatic ligation promotes adventitial inflammation and development of atherosclerotic plaque in hypercholesterolemic mice and inhibits ezetimibe-induced atherosclerosis regression. Thus, progression of atherosclerotic plaques may result not only from increased entry of atherogenic factors into the arterial wall but also from reduced lymphatic clearance of these factors as a result of aortic lymph stasis. Our findings suggest that promoting lymphatic drainage might be effective for treating atherosclerosis.

Fig. 1. Remodeling of lymphatic vessels on aortic wall during atherosclerosis.

(A) Lymphatic vessels (LVs) were examined in whole-mount aortic sinuses from 24-week-old WT and Apoe^−/− mice. Scale bar, 200 μm. (B) Lymphatic sprouts (arrowheads) were observed in Apoe^−/− aortic sinus. Scale bar, 20 μm. (C) Adventitial LV number/aortic sinus was quantified across different ages. (D) More LVs (arrowheads) were observed in Apoe^−/− mice. Ad, adventitia. (E) Prox-1 mRNA expression was assessed in WT and Apoe^−/− aortic sinuses. (F) LVs in Apoe^−/− aortic sinus were costained with proliferative marker Ki67. Scale bar, 20 μm. (G) LV number was correlated with % plaque area. (H) LV number/aortic sinus was quantified in WT and Apoe^−/− mice with less or >30% of plaque area. (I) LVs were more dilated in Apoe^−/− than in WT mice. Scale bar, 20 μm. (J) Lymphatic lumen area was quantified in WT and Apoe^−/− aortic sinuses. (K) Whole-mount and (L) cross-sectional abdominal aortic LVs from Apoe^−/− mice were examined. Scale bar, 200 μm. (M) Correlation between LV number and % plaque area was analyzed. (N) LV number/abdominal aorta with less or >30% of plaque area was quantified in Apoe^−/− mice. n = 2 to 8 mice. One-way ANOVA was used in (C) and (H), Mann-Whitney U test in (J), and Student’s t test in (N).

Fig. 2. Amelioration of atherosclerosis-associated lymphatic remodeling after ezetimibe treatment.

(A) Schematic diagram of ezetimibe regime. WT and Apoe^−/− mice were fed an HFD from 6 weeks of age. Ezetimibe was orally administered to 12- and 16-week-old Apoe^−/− mice before and after lymphangiogenesis onset, respectively. Aortic sinuses were analyzed after (B to E) 12 and (F to H) 16 weeks of ezetimibe [treated (T)] or vehicle [nontreated (NT)] treatment. (B) LYVE-1 colorimetric whole-mount staining of aortic sinus was performed in NT and T Apoe^−/− mice. Lymphatic sprouts (arrowheads). Scale bars, 1 mm (top) and 50 μm (bottom). (C) LV was examined in NT and T Apoe^−/− aortic sinus after 12 weeks of treatment. Scale bar, 100 μm. (D) LV numbers and (E) LV lumen area/aortic sinus were quantified in NT WT, NT Apoe^−/−, and T Apoe^−/− mice after 12 weeks of treatment. (F) LVs were examined in NT and T Apoe^−/− aortic sinus after 16 weeks of treatment. Scale bar, 100 μm. (G) LV numbers and (H) LV lumen area/aortic sinus were quantified in NT WT, NT Apoe^−/−, and T Apoe^−/− mice. n = 4 to 7 mice. One-way ANOVA was used for multiple group comparisons.

Fig. 3. Association of aortic lymphangiogenesis with immune cell accumulation and inflammation.

Immunoreactivity for CD3⁺ adventitial T cells was examined in aortic sinus from WT and Apoe^−/− mice treated with ezetimibe [treated (T)] or vehicle [nontreated (NT)] for (A) 12 weeks and (E) 16 weeks. Scale bar, 50 μm. The number of adventitial T cells within or outside a distance of 100 μm from the LVs was quantified in (B) 24-week-old and (F) 32-week-old NT WT, NT Apoe^−/−, and T Apoe^−/− mice. Immunoreactivity for CD11c⁺MHCII⁺ adventitial DCs was examined in aortic sinus from (C) 24-week-old and (G) 32-week-old NT WT, NT Apoe^−/−, and T Apoe^−/− mice. Scale bar, 50 μm. The number of adventitial DCs within or outside a distance of 100 μm from the LVs was quantified in (D) 24-week-old and (H) 32-week-old NT WT, NT Apoe^−/−, and T Apoe^−/− mice. qPCR analysis of IL-1β, IL-6, and TNF-α was determined in aorta from NT WT, NT Apoe^−/−, and T Apoe^−/− mice after (I) 12 weeks and (J) 16 weeks of treatment. n = 4 to 14 mice. One-way ANOVA was used for multiple group comparisons.

Fig. 4. Impaired lymphatic drainage during atherosclerosis.

(A) Immunoreactivity for LYVE-1 and SMA was examined in whole-mount WT abdominal aorta (AA). LVs and iliac lymph nodes (iLNs) were positive for LYVE-1 expression. SMA fluorescence intensity in aorta was lower than in IVC and allowed differentiation between the aorta and the IVC. Scale bar, 500 μm. (B) 3D AA, IVC, and LVs from (A) were reconstructed using Imaris software. Scale bar, 100 μm. (C) Lymphatic transport of FITC-labeled dextran from aortic adventitia to thoracic duct was assessed. (i) FITC-labeled dextran injected from AA adventitia flowed through (ii) afferent lymphatic, (iii) iLNs, (iv) efferent lymphatic, (v) renal LNs (rLNs), and (vi) thoracic duct. Magnification: (i) ×0.7, (ii) ×5, (iii) ×0.7, (iv) ×2, (v) ×1.6, and (vi) ×1.25. (D) Schematic diagram depicts lymphatic transport of macromolecule (red arrow: flow direction). (E) FITC-labeled dextran transported to iLN and rLN in WT mice was quantified at 0, 10, 30, and 60 min after injection. (F) Schematic diagram and (G) images depict LV ligation in WT mice to disrupt LV flow. (H) FITC-labeled dextran transported to iLNs and rLNs in ligated and nonligated WT groups was quantified. (I) Quantification of FITC-labeled dextran transport to iLNs and rLNs was performed in WT, Apoe^−/− mice treated with ezetimibe [treated (T)], or vehicle [nontreated (NT)]. n = 1 to 19 mice. One-way ANOVA was used for multiple group comparisons; otherwise, Mann-Whitney U test was used. Photo credit: Yeo Kim Pin, NUS.

Fig. 5. Consequence of aortic lymphatic ligation on atherosclerosis development.

(A) Total cholesterol levels were measured in plasma and abdominal aortae from nonligated and ligated Apoe^−/− mice. (B) Immunoreactivity for CD68, SMA, and COLI was examined in abdominal aorta cross sections from nonligated and ligated Apoe^−/− mice. DAPI, 4′,6-diamidino-2-phenylindole. (C) Total abdominal aorta atherosclerotic plaque area was quantified. (D) Abdominal aorta cross sections from nonligated and ligated Apoe^−/− mice were costained with CD68 and oil red O. (E) Immunoreactivity for CD3⁺ T cells was examined in aorta cross sections from nonligated and ligated Apoe^−/− mice. (F) The number of adventitial T cells and correlation of plaque area with adventitial and intimal T cell number from nonligated and ligated Apoe^−/− mice were quantified. (G) Immunoreactivity for CD11c⁺MHCII⁺ DCs was examined in aorta cross sections from nonligated and ligated Apoe^−/− mice. (H) The number of adventitial DCs and correlation of plaque area with adventitial and intimal DC numbers were determined from nonligated and ligated Apoe^−/− mice. Scale bar, 200 μm. (I) qPCR analysis of IL-6, IL-1β, and TNF-α was assessed in the aortic adventitia of nonligated and ligated Apoe^−/− mice. n = 4 to 13 mice. Mann-Whitney U test and Student’s t test were both used.

Fig. 6. Requirement of a functional lymphatic drainage for ezetimibe-induced atherosclerosis regression.

(A) Total plasma cholesterol of vehicle-treated [nontreated (NT)] Apoe^−/− and ezetimibe-treated (T) Apoe^−/− mice that had undergone sham-operated (nonligated) or aortic lymphatic ligation (ligated) was measured. (B) Oil red O staining of abdominal aorta demonstrated larger plaque in ligated T Apoe^−/− mice with increased lipid accumulation. Scale bar, 100 μm. (C) Immunoreactivity for CD68 (red), SMA (green), and COLI (white) was examined in abdominal aorta cross sections from nonligated NT Apoe^−/−, nonligated T Apoe^−/−, and ligated T Apoe^−/− mice. Scale bar, 100 μm. (D) The total plaque area of abdominal aorta from nonligated NT Apoe^−/−, nonligated T Apoe^−/−, and ligated T Apoe^−/− was quantified. n = 4 to 6 mice. Each point represents one mouse. Mann-Whitney U test was used.