Inhibition of smooth muscle cell death by Angiotensin 1-7 protects against abdominal aortic aneurysm

Figures

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  Figure 1. Ang 1-7 abrogates the development of Ang II-induced abdominal aortic aneurysm and associated mortality in ApoE knockout mice (A) Representative photographs of the aorta showing macroscopic features of aortic dilatation and (B) quantification of abdominal aortic diameter in the ApoEKO mice in response to saline (n=25), Ang II (n=7) and Ang 1-7 (n=8) for 4 weeks. (C) The administration of Ang II leads to a higher incidence of abdominal aortic rupture (4/12 = 33.33%; 1: unknown cause; D), resulting in significantly increased mortality (n=5) in ApoEKO mice compared to the saline group (0/25 = 0.00%). (C,D) Ang 1-7 prevents aortic dilatation and reduces the incidence of aortic rupture (1/9 = 11.11%), resulting in improved survival. The arrow points to the aneurysmal region. Each data point on graph represents biological replicate. *P<0.05 compared with the saline group; #P<0.05 compared with the Ang II group using one-way ANOVA.

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  Figure 2. Ang 1-7 attenuates Ang II-induced structural and functional alterations in abdominal aorta in ApoE knockout mice Representative (A) B-mode and (B) M-mode echocardiography images of the abdominal aorta and quantification of abdominal aortic (C) systolic and (D) diastolic diameters showing increased aortic dilatation in Ang II-infused ApoEKO mice; Chronic Ang 1-7 administration markedly reduced (A–D) the aortic dilatation. Quantification of (E) aortic expansion index and (F) aortic distensibility exhibits markedly reduced aortic wall elasticity and distensibility in response to Ang II-infusion in ApoEKO mice; chronic Ang 1-7 administration preserved the aortic (E) expansion index and (F) distensibility. Each data point on graph represents biological replicate. *P<0.05 compared with the saline group; #P<0.05 compared with the Ang II group using one-way ANOVA. DD: diastolic diameter, SD: systolic diameter.

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  Figure 3. Ang 1-7 suppresses Ang II-induced adverse aortic remodeling in the abdominal aorta Representative images of (A) Verhoeff-Van Gieson (elastin) staining and brightfield microscopy and (B) quantification of aortic media thickness show significantly decreased aortic medial thickness and increased medial degeneration with elastin breaks in the abdominal aorta of ApoEKO mice in response to Ang II-infusion; chronic Ang 1-7 administration reduced medial degeneration and preserved abdominal aortic medial thickness. Representative images of (C) Gomori trichrome staining and brightfield microscopy and (D) picrosirius red (collagen) staining and confocal microscopy along with (E) quantification of collagen content indicate increased ECM remodeling in response to Ang II administration and (C–E) abridged adventitial fibrosis resulting from chronic Ang 1-7 administration. Each data point on graph represents biological replicate. *P<0.05 compared with saline group; #P<0.05 compared with Ang II group using one-way ANOVA.

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  Figure 4. Ang 1-7 attenuates Ang II-induced apoptosis and phenotypic switching in abdominal aortic SMCs Representative (A) immunofluorescence and confocal microscopy images of α-SMA (green) and wheat-germ agglutinin (red) staining of the abdominal aorta and (B) quantification of α-SMA-immunoreactivity exhibit reduced α-SMA fluorescence suggesting loss of VSMCs density in aortic media by Ang II administration and (A,B) preservation of α-SMA levels by Ang 1-7 administration in ApoEKO mice. (C) Flow cytometric analysis of annexin V/propidium iodide–stained vascular smooth muscle cells (VSMCs) and (D) quantification of % apoptotic cells showing increased apoptosis in the aortic VSMCs in response to Ang II and suppressed by Ang 1-7 stimulation. TaqMan-qPCR based mRNA expression analyses of α-smooth muscle actin (Acta2; E), myosin heavy chain 11 (Myh11; F), and interleukin-6 (Il-6; G) shows decrease expression of contractile genes and increased expression of the inflammatory gene in Ang II-treated abdominal SMCs. mRNA expression analyses of collagen I (Col1a1; H), collagen III (Col3a1; I), matrix metalloproteinase 2 (Mmp2; J), and matrix metalloproteinase 9 (Mmp9; K), indicate pathological…

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  Figure 5. Ang II potentiates mitochondrial fission and oxidative stress in abdominal aortic VSMCs and the critical role of Ang 1-7 Representative (A) confocal images of Mitotracker red staining and (B) their analysis using the Mitochondrial Network Analysis program (MiNA plugin; ImageJ) show smaller and more fragmented mitochondria in response to Ang II-treated abdominal aortic SMCs compared with the control group. Quantification of Mitotracker red-staining images using MiNA showing decreased (C) mean branch length, (D) mean network branches, and (E) decreased mitochondrial footprint in Ang II-treated VSMCs compared with the control group. (A–E) Ang 1-7, treatment attenuates Ang II-induced mitochondrial fission. Representative (F) brightfield microscopy and confocal microscopy images of DHE-stained abdominal SMCs, along with (G) quantification of DHE fluorescence intensity, show increased cellular ROS levels in Ang II-treated VSMCs compared with control group. (H) Flow cytometric analysis of MitoSOX-stained abdominal aortic SMCs and (I) quantification of MitoSOX fluorescence indicate increased mitochondrial ROS levels in Ang II-treated VSMCs. (F–I) Ang 1-7, treatment mitigates Ang…

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  Figure 6. Effect of Ang II (± Ang 1-7) treatment on apoptosis-associated proteins in aortic VSMCs and functional protein–protein network analysis of differentially expressed proteins using STRING database (A) Protein array for assessment of apoptosis-associated protein expression in VSMCs and quantification of fold change showed (B) elevated and (C) reduced apoptosis-associated proteins in response to Ang II. Ang 1-7 attenuates VSMCs apoptosis via suppression of these regulatory proteins. Mouse apoptosis array coordinates: A1,2: Reference spots; A3,4: Bad; A5,6: Bcl-2; A7,8: Reference spots; B3,4:Bcl-x; B5,6: Caspase-3, cleaved; C1,2: Catalase; C3,4: Claspin; C5,6: Cytochrome c; C7,8: Fas/TNFRSF6/CD95; D1,2: HIF-1α; D3,4: HO-1/HMOX1/HSP32; D5,6: HO-2/HMOX2; D7,8: HSP27; E1,2: HSP60; E3,4: HSP70/HSPA1A; E5,6: MCL-1; E7,8: p27/Kip1; F1,2: p53; F3,4: SMAC/Diablo; F5,6: TNF RI/TNFRSF1A; F7,8: TRAIL R2/ TNFRSF10B; G1,2: Reference spots; G5,6: XIAP; G7,8: Negative control. (D) Representative images of the whole interaction network of the proteins significantly altered in Ang II(±Ang1-7) groups in the apoptosis array. Proteins identified by the apoptosis array are highlighted in blue in the…

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  Figure 7. Antagonizing effects of Ang 1-7 against Ang II protects pathological alterations associated with the onset and progression of AAA Chronic infusion of Ang II results in the onset and progression of abdominal aortic aneurysm (AAA) in ApoEKO mice. Ang II triggers a pathological cascade comprising excessive mitochondrial fission, oxidative stress, vascular remodeling, and VSMCs apoptosis. The Ang II-mediated pathological mechanisms lead to the loss of structural integrity and, thus, the development of AAA. Ang 1-7 attenuates Ang II-mediated effector pathological pathways and prevents the progression of AAA. Figure created using Biorender.com.