AT2R agonist NP‐6A4 mitigates aortic stiffness and proteolytic activity in mouse model of aneurysm

Figures

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  Figure 1. NP‐6A4 attenuates AngII‐induced aortic stiffness in Apoe−/− mice. A, Graph showing the survival rate of Apoe−/− mice in response to NP‐6A4 and AngII. Five out of 17 mice died of abdominal aortic rupture in NP‐6A4–treated AngII animals, whereas 10 out of 23 mice died in the AngII group. B, Aneurysm severity (type I to type IV) scored using a classification system at day 28. C, Quantification for maximal intraluminal diameter (MILD) as measured by ultrasound at day 28 of AngII infusion. D, Quantification of ex vivo maximal external aortic diameter (MEAD) of suprarenal aorta (mm) by microscopy at day 28. E, Pulse‐wave velocity (PWV) calculated from measurements of abdominal aortic pulse pressure as determined by EKV in response to AngII at day 28. F and G, Distensibility and radial strain at day 28 of AngII and NP‐6A4 treatments as measured by Vevo Vasc analysis. Log‐rank test was used for comparing survival in A. Fisher's exact test was used for analysis in B. Kruskal‐Wallis test was applied for statistics in C and D. Tukey's multiple comparisons test was used for data analysis in E‐G. *P < 0.05; **P < 0.01; ***P < 0.001, ns = non‐significant

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  Figure 2. NP‐6A4 modulates AngII‐induced changes in collagen location in the aortic tissues of Apoe−/− mice. A‐H, Representative images of the transverse sections of aortic sections stained with H&E (haematoxylin and eosin; 4X and 40X), illustrating the extent of aortic thickening at day 28 in indicated experimental groups. I‐L, Representative images of Verhoeff‐van Gieson (VVG) staining demonstrating elastin fragmentation in Apoe−/− mice infused with AngII at day 28. M‐T, Representative collagen staining using picrosirius red in the experimental groups (n = 6 for each group, 5X and 20X). Q, Quantification of medial elastin degradation in experimental groups. Scale bar = 0.5 mm in A‐D, M‐P and 50 µm in E‐L and Q‐T. ***P < 0.001, ns = non‐significant in Tukey's multiple comparisons test

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  Figure 3. NP‐6A4 affects phenotypic changes in aortic tissues. A‐D, Immunohistochemistry (IHC) images showing expression of Mcp1, F4/80, caspase‐3 and osteopontin in the aortic tissue at day 28 of experimental Apoe−/− mice. E‐H, Quantification data of Mcp1, F4/80, caspase‐3 and osteopontin expressions in the aortic tissue of indicated mice. Scale bar = 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001 in Tukey's multiple comparisons test (E‐H)

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  Figure 4. Proteolytic activity is attenuated with NP‐6A4 in AngII‐induced Apoe−/− mice. A and B, mRNA expression of Mmp2 and Mmp9 in abdominal aortae from Apoe−/− mice treated with AngII alone or NP‐6A4 and AngII as measured by qRT‐PCR (n = 4 mice per group). C and D, Detection and quantification of gelatinolytic activity of matrix metalloproteinase (MMP) by in situ zymography (ISZ) (n = 4). Green and blue represent MMP activity and nuclear staining of cells by DAPI, respectively. E and F, Representative gelatine zymography and quantification for pro‐matrix 2 (MMP2), active MMP2 and MMP9 in aortic tissue lysates from Apoe−/− mice with indicated treatment groups (n = 4 mice per group). Pr = protein. Scale bar = 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001 in paired two‐tailed Student's t test

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  Figure 5. NP‐6A4 improves endothelial function and attenuates aortic stiffness. A and B, IHC images showing expression of Cd31 and eNOS in the aortic tissue at day 28 of experimental Apoe−/− mice. C and D, Western blot and its quantification showing expression of phospho‐eNOS, total eNOS and GAPDH in the human umbilical vein endothelial cells (hUVECs) with various treatment. E, Representative flow cytometry histogram measuring nitric oxide (NO) production in the human aortic endothelial cells (hAOECs) pre‐treated with NP‐6A4 using DAF‐FM staining. F and G, Representative ISZ image for measuring NO production using DAF‐FM staining in the aortic tissues from experimental Apoe−/− mice at day 28 of AngII and its quantification. H, Endothelial cell stiffness in the hAOECs with indicated treatment groups measured by atomic force microscopy (AFM). Scale bar = 50 µm. *P < 0.05, ***P < 0.001 in paired two‐tailed Student's t test