Research ArticleVASCULAR BIOLOGY

Abnormal mechanosensing and cofilin activation promote the progression of ascending aortic aneurysms in mice

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Science Signaling  20 Oct 2015:
Vol. 8, Issue 399, pp. ra105
DOI: 10.1126/scisignal.aab3141
  • Fig. 1 Proteomic analysis of ascending aortas during initiation and expansion of aneurysms.

    (A) Representative Western blots of ascending aortas from control (CTRL) and Fbln4SMKO (SMKO) mice. The experiment was performed three times with different pools of animals. The numbers of aortas used per genotype in each time point are as follows: P1, n = 5 to 12; P7, n = 5 to 15; P14, n = 5 to 12; P30, n = 3 to 7; and P90, n = 2 to 6. See fig. S1 for quantification. (B) Quantitative polymerase chain reaction (qPCR) analysis of SMC-specific genes from ascending aortas of CTRL (pooled P1, n = 12; P7, n = 18; P14, n = 9; and P30, n = 12) and Fbln4SMKO (pooled P1, n = 12; P7, n = 18; P14, n = 8; and P30, n = 11) mice performed in technical triplicate. (C) Representative two-dimensional differential gel electrophoresis (2D-DIGE) using entire aortas (for P1, n = 4 per genotype) or ascending aortas (P7, n = 7; P14, n = 5; and P30, n = 3 per genotype). Proteins with increased abundance in the Fbln4SMKO aortas appear in red, proteins with decreased abundance appear in green, and those with similar abundance appear in yellow. Circled spots with numbers indicate more than twofold changes between CTRL and Fbln4SMKO in three independent experiments. (D) Heat map showing identified proteins divided into four clusters according to the expression patterns during postnatal development. Red, increased abundance in Fbln4SMKO aortas; green, decreased abundance in Fbln4SMKO aortas. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p, phosphorylated; t, total.

  • Fig. 2 Activation of cofilin in Fbln4SMKO ascending aorta.

    (A) Representative Western blots of ascending aortas of CTRL and Fbln4SMKO (SMKO). The experiment was performed three times with different pools of animals with similar results. n values as in Fig. 1A. *P < 0.05, **P < 0.01, unpaired t test. SSH, slingshot; CIN, chronophin. (B) Cross sections of the ascending aorta at P30 (n = 5 mice per genotype) immunostained with phosphorylated cofilin (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Elastic laminae are green (autofluorescence). Scale bars, 50 μm. L, lumen. (C) G- and F-actin for ascending (Asc) and descending (Des) aortas at P30. Representative blot (upper) and quantification of G- and F-actin (bottom) are shown (n = 5 aortas per genotype). Means of G-actin (black bar) and F-actin (gray bar) are shown in each bar. **P < 0.01, unpaired t test; NS, not significant. (D) Cross sections of the ascending aorta at P30 from CTRL and Fbln4SMKO (n = 5 mice per genotype), stained with phalloidin (red) and DAPI (blue). Scale bars, 50 μm.

  • Fig. 3 Disruption of elastic lamina–SMC connections and alteration of the mechanical properties of Fbln4SMKO aortas.

    (A) Electron microscopy images from CTRL and Fbln4SMKO (SMKO) ascending aortas at P90 and P7. Elastic lamina (EL)–SMC connections are well formed in CTRL aortas (white arrows), whereas elastic laminae are disrupted and not connected to SMCs in the Fbln4SMKO aorta at P90. Elastic laminae were also abnormal at P7 in Fbln4SMKO vessels, with numerous globules of elastin rather than solid bands of elastin (white arrowhead) and less organized cell-elastin associations (white arrows). Scale bars, 1 μm. Images are representative of at least n = 2 (CTRL) and n = 3 (Fbln4SMKO) mice per age. (B) Upper panel: Aortic pressure–outer diameter curves for P1, P7, P14, and P30 ascending aorta. Fbln4SMKO aortas at P14 have significantly large outer diameter than CTRL. Lower panel: Aortic pressure–compliance curves for CTRL and Fbln4SMKO ascending aortas. Fbln4SMKO aortas show significant differences beginning at P7. n = 5 to 8 mice per group. Bars are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, generalized estimating equation. (C) Western blots showing the abundance of ACE, TSP1 (thrombospondin-1), Egr1 (early growth response 1), and the phosphorylation of ERK are increased by transverse aortic constriction (TAC) in wild-type mice. n = 4 mice for sham and n = 5 mice for TAC. Bars are means ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001. Exact Wilcoxon rank sum test for comparison with TSP1 in sham group. All the other comparisons were done by unpaired t test. (D) Western blots showing Egr1 and TSP1 abundance is increased in postnatal Fbln4SMKO aortas. The experiment was performed three times with different pools of animals with similar results. n values as in Fig. 1A. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test.

  • Fig. 4 Postnatal deletion of Fbln4 in vascular SMCs.

    (A) Gross photos of CTRL and SMA-Cre-ERT2 (iSMKO) aortas at P60. Mice were injected with tamoxifen for five consecutive days beginning at P7. Images are representative of 17 mice per genotype. (B) qPCR analysis on aortas harvested from P60 CTRL and iSMKO mice (n = 6 mice per genotype). ****P < 0.0001, unpaired t test. (C) Representative Western blots of ascending aortas from CTRL and iSMKO mice (n = 6 mice per genotype). Exact Wilcoxon rank sum test for comparison with phosphorylated ERK/total ERK in CTRL group. All the other comparisons were done by unpaired t test. (D) Cross sections of the ascending aorta from CTRL and iSMKO mice at P60 (n = 5 mice per genotype) immunostained with phosphorylated cofilin (red) and DAPI (blue). Elastic laminae are green (autofluorescence). Scale bars, 50 μm.

  • Fig. 5 The effects of losartan on cofilin activity and aneurysm formation.

    (A) Effects of losartan (LSRT) treatment from P7 to P30 on ACE, TSP1, Egr1, phosphorylated ERK, total ERK, phosphorylated cofilin, cofilin, and SSH1 abundance in Fbln4SMKO (SMKO) aortas (pooled three to six aortas per sample; 9 to 17 mice per genotype and treatment). All animals were evaluated at P30. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way analysis of variance (ANOVA). (B and C) Cross sections of the ascending aorta from Fbln4SMKO with losartan treatment from P7 to P30. n = 5 mice per genotype and treatment. Scale bars, 50 μm. (B) Immunostained with phosphorylated cofilin (red) and DAPI (blue). Elastic laminae are green (autofluorescence). (C) Immunostained with phalloidin (red) and DAPI (blue). n = 5 mice per treatment. (D) Effects of postnatal losartan treatment on aneurysm formation. All animals were evaluated at P90. n = 3 mice per genotype and treatment. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA.

  • Fig. 6 The involvement of PI3K in aortic aneurysm formation.

    (A) Gross photos of CTRL and Fbln4SMKO (SMKO) aortas with or without wortmannin treatment. Arrow shows a tortuous descending aorta. Images are representative of 11 to 16 mice per genotype. (B) Histological images of cross sections of the ascending aorta from wortmannin (HWT)-treated CTRL, Fbln4SMKO, and untreated Fbln4SMKO mice stained with hematoxylin and eosin (H&E), Hart’s (elastin), and Masson trichrome (collagen). Scale bars, 500 μm (×5) and 20 μm. Images are representative of four mice per genotype. (C) Western blots showing the effect of wortmannin treatment (pooled three aortas per sample) compared to vehicle-treated Fbln4SMKO (pooled two aortas per sample). Six to nine mice per genotype and treatment. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA.

  • Fig. 7 A model illustrating a potential mechanism of aneurysm formation in Fbln4SMKO aortas.

    Absence of fibulin-4 in SMCs led to loss of elastic lamina–SMC connections and changes in the mechanical properties of the aorta. Abnormal mechanosensing of SMCs is indicated by increased Egr1, TSP1, and ACE abundance. Increased abundance of ACE leads to AngII-mediated signaling and induces downstream events, including (i) increased abundance of Egr1 and establishment of a feed-forward loop of AngII signaling, (ii) increased phosphorylation of ERK and proliferation of SMCs, and (iii) PI3K-dependent activation of cofilin through SSH1, leading to the aneurysm formation.

  • Table 1 Identification of proteins 1 to 35 by Orbitrap Velos or Q Exactive mass spectrometer.

    CPFP (Central Proteomics Facilities Pipeline) version 2.0.3 was used for database searching against the UniProt mouse database. MW, theoretical molecular weight (kD); pI, theoretical isoelectric point.

    SpotProteinAccession
    no.
    SymbolMWpIPeptides*% Coverage
    1Collagen type 1 α2 chainNP_031769Col1a2129.69.375.9
    2Elongation factor 2NP_031933Eef295.36.41414.0
    3MoesinNP_034963Msn66.55.95170.9
    Caldesmon 1NP_663550Cald160.57.04669.2
    4Dihydropyrimidinase-like 3NP_001129558Dpysl361.96.01226.4
    5Serine protease HTRA1NP_062510Htra151.27.82245.0
    6Serpin H1NP_001104514Serpinh146.58.91330.0
    7Serine-threonine kinase receptor–associated proteinNP_035629Strap38.45.0416.9
    8Tropomyosin 3NP_071709Tpm332.84.22660.2
    914-3-3 protein εNP_033562Ywhae29.24.62874.9
    10Protein-lysine 6-oxidaseNP_034858Lox46.78.7615.8
    11Annexin A2NP_031611Anxa238.67.6532.4
    12Rho dissociation inhibitor 2NP_031512Arhgdib22.95.01357.0
    13Carbonic anhydrase 3NP_031632Car329.46.9624.6
    14Peptidyl-prolyl cis-trans isomerase CNP_032934Ppic22.87.0211.8
    15Protein S100-A11NP_058020S100a1111.05.3651.0
    16Phosphodiesterase 4D, cAMP-specific (fragment)NP_035186Pde4d84.54.822.3
    17Protein S100-A13NP_033139S100a1317.76.2543.9
    18Cytochrome c oxidase subunit 6B1NP_079904Cox6b110.19.0211.6
    19Protein S100-A6NP_035443S100a610.15.3216.9
    20Secernin-1NP_081544Scrn146.34.7715.2
    21Connective tissue growth factorNP_034347Ctgf38.67.6312.1
    22Transgelin (SM-22)NP_035656Tagln22.58.91763.2
    23Hemoglobin subunit β1NP_032246Hbb-b115.77.1749.7
    24Myosin light polypeptide kinaseNP_647461Mylk213.65.91415.2
    Protein phosphatase 1 regulatory subunit 12ANP_082168Ppp1r12a111.85.566.3
    25Myh11 proteinNP_038635Myh11227.15.41910.4
    26Leucine zipper transcription factor–like 1NP_201579Lztfl134.75.1621.5
    27Sepiapterin reductaseNP_035597Spr27.95.9637.4
    Heat shock protein β1NP_038588Hspb123.06.1531.4
    28Myosin regulatory light polypeptide 9NP_742116Myl919.94.8633.7
    29Cofilin-2NP_031714Cfl218.77.7951.8
    30Cofilin-1NP_031713Cfl118.68.2851.8
    31Prefoldin subunit 2NP_035200Pfdn216.56.2745.5
    32Fatty acid–binding protein, heartNP_034304Fabp314.86.1854.9
    33DestrinNP_062745Dstn18.58.1849.1
    34Glutathione S-transferase μNP_034488Gstm126.07.71147.2
    35Mimecan (osteoglycin)NP_032786Ogn34.05.5513.8

    *Number of matched peptides.

    †Percentage of sequence coverage.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/8/399/ra105/DC1

      Fig. S1. ACE abundance and ERK phosphorylation in Fbln4SMKO aortas after P7.

      Fig. S2. Ontology analysis and proteins that showed changes in abundance during aneurysm development.

      Fig. S3. qPCR analysis of genes encoding the proteins identified by 2D-DIGE.

      Fig. S4. Phosphorylation of cofilin in Fbln4SMKO descending aortas.

      Fig. S5. Activated RhoA signaling in Fbln4SMKO ascending aortas.

      Fig. S6. Dephosphorylation of cofilin by SSH1 in rat vascular SMCs.

      Fig. S7. Phosphorylation of FAK and ILK in ascending aortas.

      Fig. S8. The effects of PI3K inhibitors on general growth and aneurysm development.

      Fig. S9. The effect of LY294002 on aneurysm development in Fbln4SMKO mice.

      Fig. S10. Evaluation of PKD phosphorylation and calcineurin activity in ascending aortas.

      Table S1. Antibodies used in this study.

      Table S2. qPCR primer sequences.

      Table S3. List of links to raw data for mass spectrometry.

    • Supplementary Materials for:

      Abnormal mechanosensing and cofilin activation promotes the progression of ascending aortic aneurysms in mice

      Yoshito Yamashiro, Christina L. Papke, Jungsil Kim, Lea-Jeanne Ringuette, Qing-Jun Zhang, Zhi-Ping Liu, Hamid Mirzaei, Jessica E. Wagenseil, Elaine C. Davis, Hiromi Yanagisawa*

      *Corresponding author. E-mail: hkyanagisawa{at}tara.tsukuba.ac.jp

      This PDF file includes:

      • Fig. S1. ACE abundance and ERK phosphorylation in Fbln4SMKO aortas after P7.
      • Fig. S2. Ontology analysis and proteins that showed changes in abundance during aneurysm development.
      • Fig. S3. qPCR analysis of genes encoding the proteins identified by 2D-DIGE.
      • Fig. S4. Phosphorylation of cofilin in Fbln4SMKO descending aortas.
      • Fig. S5. Activated RhoA signaling in Fbln4SMKO ascending aortas.
      • Fig. S6. Dephosphorylation of cofilin by SSH1 in rat vascular SMCs.
      • Fig. S7. Phosphorylation of FAK and ILK in ascending aortas.
      • Fig. S8. The effects of PI3K inhibitors on general growth and aneurysm development.
      • Fig. S9. The effect of LY294002 on aneurysm development in Fbln4SMKO mice.
      • Fig. S10. Evaluation of PKD phosphorylation and calcineurin activity in ascending aortas.
      • Table S1. Antibodies used in this study.
      • Table S2. qPCR primer sequences.
      • Table S3. List of links to raw data for mass spectrometry.

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      Citation: Y. Yamashiro, C. L. Papke, J. Kim, L.-J. Ringuette, Q.-J. Zhang, Z.-P. Liu, H. Mirzaei, J. E. Wagenseil, E. C. Davis, H. Yanagisawa, Abnormal mechanosensing and cofilin activation promotes the progression of ascending aortic aneurysms in mice. Sci. Signal. 8, ra105 (2015).

      © 2015 American Association for the Advancement of Science

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