Research ArticleNanomedicine

RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction

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Science Translational Medicine  08 Jun 2016:
Vol. 8, Issue 342, pp. 342ra80
DOI: 10.1126/scitranslmed.aaf1435
  • Fig. 1. Noncirculating factors contribute to expansion of inflammatory plaque leukocytes and acceleration of atherosclerosis.

    (A) Experimental setup. ApoE−/− mice were joined in parabiosis, and one parabiont was infarcted 2 weeks thereafter. Three weeks later, leukocytes were evaluated by flow cytometry. (B and C) Flow cytometric gating and quantification of blood (B) and aortic (C) leukocytes. Data are means ± SEM (n = 5 to 11 per group from two independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed paired Wilcoxon test for comparison between connected parabionts; two-tailed unpaired Mann-Whitney U for comparison to steady-state parabiosis. (D) Masson staining of aortic roots. Data are means ± SEM quantifying total plaque size and necrotic core area per section (n = 5 to 9 per group from two independent experiments). Scale bars, 250 μm (top) and 100 μm (bottom).

  • Fig. 2. Noncirculating signals contribute to expansion of plaque leukocyte recruitment after MI.

    (A) Gating strategy and histograms of leukocyte adhesion molecules on aortic endothelial cells from ApoE−/− mice 3 days after MI or no MI controls. (B) Mean fluorescence intensity (MFI) of adhesion molecules expressed by aortic endothelial cells from animals in (A). Data are means ± SEM (n = 11 to 13 per group from two independent experiments). P values versus no MI are determined by Mann-Whitney U test. (C) Chemokine mRNA in aortic roots from animals in (A). Data are means ± SEM (n = 7 to 8 per group from two independent experiments). P values are determined by Mann-Whitney U test. (D) Experimental setup. ApoE−/− mice were joined in parabiosis, and one parabiont was infarcted 2 weeks thereafter. Three days later, CAM levels on aortic endothelial cells were evaluated by flow cytometry. (E) MFI on aortic endothelial cells from animals in (D) was evaluated by flow cytometry. Data are means normalized to steady-state parabiosis ± SEM (n = 7 per group from two independent experiments). P values compare respective infarcted to noninfarcted parabionts and are determined by two-tailed paired Wilcoxon test.

  • Fig. 3. Aortic sympathetic tone regulates leukocyte adhesion molecule expression after MI.

    These experiments examine the vascular innervation after coronary ligation. (A) Tyrosine hydroxylase (TH) staining of aortic roots in steady-state and infarcted atherosclerotic mice. L, lumen; M, media; A, adventitia; P, atherosclerotic plaque. Scale bar, 70 μm. TH+ area per high-power field was quantified from fluorescent images as means ± SEM (n = 5 ApoE−/− mice per group). P value is determined by Mann-Whitney U test. (B) Th mRNA levels in aortic roots. Data are means ± SEM (n = 7 ApoE−/− mice per group from two independent experiments, normalized to controls without MI). P value is determined by Mann-Whitney U test. (C) Aortic arch noradrenaline content. Data are means ± SEM (n = 4 to 5 ApoE−/− mice per group). P values are determined by one-way analysis of variance (ANOVA). (D) Aortic arch noradrenaline content in permanent ligation versus 45 min of ischemia followed by reperfusion. Data are means ± SEM (n = 8 to 10 wild-type mice per group from two independent experiments). P value is determined by Mann-Whitney U test. (E) CAM expression by aortic endothelial cells in post-MI atherosclerosis (3 days after MI; red) and after MI + 6-OHDA (blue). The bar graph shows MFI on aortic endothelial cells by flow cytometry (n = 7 to 8 ApoE−/− mice per group from two independent experiments), normalized to no MI controls. P values are determined by one-way ANOVA. (F) Adrenoceptor α1–2 and β1–3 mRNA levels (Adra and Adrb) in sorted murine aortic endothelial cells. Data are means ± SEM (n = 8 ApoE−/− mice and 5 B6 mice as steady-state myocardium). P values are determined by Mann-Whitney U test.

  • Fig. 4. siCAM5 results in endothelial CAM knockdown.

    ApoE−/− mice were injected with either siCAM5 or siCtrl for 1 week. (A) Histograms showing target proteins in murine aortic endothelial cells after RNAi. (B) siCAM5 knockdown on protein level measured by flow cytometric MFI in aortic endothelial cells. Data are normalized to siCtrl. (C) mRNA levels after 1 week of treatment with either siCAM5 or siCtrl. RT-qPCR data are normalized to siCtrl. Data in (B) and (C) are means ± SEM (n = 6 to 7 ApoE−/− mice per group). P values are determined by Mann-Whitney U test.

  • Fig. 5. siCAM5 reduces leukocyte recruitment into plaque.

    ApoE−/− mice were injected with either siCAM5 or siCtrl for 2 weeks. (A) Gating and quantification of GFPhigh myeloid cells in plaques and quantification of GFPhigh myeloid cells in blood after adoptive transfer of 2 × 106 GFPhigh Ly6Chigh monocytes and 2 × 106 GFPhigh neutrophils. Data are means ± SEM (n = 6 to 7 ApoE−/− mice per group from two independent experiments. (B and C) Gating and quantification of neutrophils, Ly6Chigh monocytes, and macrophages in atherosclerotic aortae (B) and in the blood (C). Data are means ± SEM (n = 6 to 10 ApoE−/− mice per group from three independent experiments). (D) Immunohistochemical staining of aortic roots for myeloid cells (CD11b) and neutrophils (Ly6G). Bar graphs show percentage of positive area per region of interest (ROI) or number of cells per high-power field (hpf). Scale bar, 250 μm. Data are means ± SEM (n = 5 to 6 ApoE−/− mice per group). P values in (A), (B), and (D) are determined by Mann-Whitney U test. (E) Quantification of neutrophils and Ly6Chigh monocytes in atherosclerotic aortae after treatment with siCtrl, siVcam1, or siCAM5. Data are means ± SEM (n = 7 to 8 ApoE−/− mice per group from two independent experiments). *P < 0.05, ***P < 0.001, ****P < 0.0001, one-way ANOVA.

  • Fig. 6. siCAM5 reduces inflammation and progression of atherosclerotic plaque phenotype.

    ApoE−/− mice were injected with either siCAM5 or siCtrl for 2 weeks. (A) mRNA levels in aortic arches; values normalized to Gapdh with the siCtrl group set to 1. (B) Aortic root protease activity by fluorescence molecular tomography (FMT)/computed tomography (CT). (C) Immunohistochemical evaluation of aortic roots for collagen-1 after 3 weeks of siCtrl (3 mg/kg) or siCAM5 (3 mg/kg). Bar graphs show percentage of positive staining per ROI. Scale bar, 100 μm. (D) Masson staining of aortic roots. Bar graphs show fibrous cap thickness, necrotic core area, and total plaque size per section. Scale bars, 250 μm (low magnification) and 100 μm (high magnification). The arrows point at a fibrous cap. In (A) to (D), data are means ± SEM (n = 5 to 6 ApoE−/− mice per group). P values are determined by Mann-Whitney U test.

  • Fig. 7. siCAM5 reduces inflammation of infarcted myocardium and improves functional recovery after ischemia.

    The baseline cohort of ApoE−/− mice (No MI athero) received no MI and no therapy. Cohorts of ApoE−/− mice after coronary ligation received either siCtrl (MI athero & siCtrl) or siCAM5 (MI athero & siCAM5) for 3 weeks. Treatment started 2 hours after MI. All cohorts are age-matched and received a similar atherogenic diet. (A) Gating and quantification of neutrophils, Ly6Chigh monocytes, and macrophages in atherosclerotic aortae. Data are means ± SEM (n = 8 ApoE−/− mice per group from two independent experiments). P values are determined by one-way ANOVA. (B) Masson staining of aortic roots. Bar graphs show plaque size, fibrous cap thickness, and necrotic core area. Data are means ± SEM (n = 5 ApoE−/− mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA. (C) Wild-type mice received one injection of either siCAM5 or siCtrl 2 hours after coronary ligation. Flow cytometric gating and quantification of inflammatory monocytes in infarct and blood 3 days after MI. Data are means ± SEM (n = 6 mice per group). P values are determined by Mann-Whitney U test. (D) ApoE−/− mice received either siCAM5 or siCtrl for 3 weeks after coronary ligation, starting 2 hours after MI. Left ventricular ejection fraction (LV-EF) was assessed by cardiac MRI 21 days later. Data are means ± SEM (n = 5 to 6 ApoE−/− mice per group). P values are determined by Mann-Whitney U test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/342/342ra80/DC1

    Materials and Methods

    Fig. S1. Identification of aortic endothelial cells by flow cytometry.

    Fig. S2. siRNA candidate screening and relative potency.

    Fig. S3. siRNA delivery.

    Fig. S4. siRNA is taken up by blood neutrophils and monocytes, but not plaque macrophages.

    Fig. S5. The effect of RNAi on blood myeloid cells.

    Fig. S6. Blood cholesterol levels.

    Fig. S7. RNAi targeting either siSele/Selp or siIcam1/2 + Vcam1.

    Fig. S8. Inflammation in atherosclerotic plaques 3 weeks after MI or sham surgery.

    Table S1. siRNA sequences screened for siCAM5.

  • Supplementary Material for:

    RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction

    Hendrik B. Sager,* Partha Dutta, James E. Dahlman, Maarten Hulsmans, Gabriel Courties, Yuan Sun, Timo Heidt, Claudio Vinegoni, Anna Borodovsky, Kevin Fitzgerald, Gregory R. Wojtkiewicz, Yoshiko Iwamoto, Benoit Tricot, Omar F. Khan, Kevin J. Kauffman, Yiping Xing, Taylor E. Shaw, Peter Libby, Robert Langer, Ralph Weissleder, Filip K. Swirski, Daniel G. Anderson, Matthias Nahrendorf*

    *Corresponding author. Email: mnahrendorf{at}mgh.harvard.edu (M.N.); hendrik.sager{at}tum.de (H.B.S.)

    Published 8 June 2016, Sci. Transl. Med. 8, 342ra80 (2016)
    DOI: 10.1126/scitranslmed.aaf1435

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Identification of aortic endothelial cells by flow cytometry.
    • Fig. S2. siRNA candidate screening and relative potency.
    • Fig. S3. siRNA delivery.
    • Fig. S4. siRNA is taken up by blood neutrophils and monocytes, but not plaque macrophages.
    • Fig. S5. The effect of RNAi on blood myeloid cells.
    • Fig. S6. Blood cholesterol levels.
    • Fig. S7. RNAi targeting either siSele/Selp or siIcam1/2 + Vcam1.
    • Fig. S8. Inflammation in atherosclerotic plaques 3 weeks after MI or sham surgery.
    • Table S1. siRNA sequences screened for siCAM5.

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