Research ArticleVASCULAR PHYSIOLOGY

Fibulin-4 is essential for maintaining arterial wall integrity in conduit but not muscular arteries

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Science Advances  03 May 2017:
Vol. 3, no. 5, e1602532
DOI: 10.1126/sciadv.1602532
  • Fig. 1 Fbln4E57K/E57K mice develop arterial elongation, vascular tortuosity, ascending aortic aneurysms, elastic fiber fragmentation, and SMC disarray.

    (A) Gross morphology of the thoracic aorta and its branches in adult Fbln4E57K/E57K mice and control (Fbln4+/+ and Fbln4+/E57K) littermates injected with yellow latex to visualize the vasculature. Arrows indicate aortic root dilatation and ascending aortic aneurysm seen in approximately half of Fbln4E57K/E57K mice; arrowheads indicate the different angles at which arterial branches come off the aortic arch. Arterial tortuosity and elongation are noted in all Fbln4E57K/E57K mice. (B to D) VVG (Verhoeff–van Gieson) stain (B) and transmission electron micrographs of the ascending aorta of 3-month-old Fbln4E57K/E57K mice and littermate controls at two different magnifications (C versus D). In addition to severe elastic fiber fragmentation, there is increased medial wall thickness and SMC disarray in the aorta of Fbln4E57K/E57K mice. At higher magnification (D), the moth-eaten or spongy appearance of the fragmented elastic fibers is appreciated in the aorta of Fbln4E57K/E57K mice. In addition, there is loss of smooth muscle contact with elastic fibers in Fbln4E57K/E57K vessels. Scale bars, 8 μm (C) and 4 μm (D). Asterisks indicate vessel lumen.

  • Fig. 2 Elastic fiber fragmentation and SMC disarray in descending aorta of Fbln4E57K/E57K mice.

    Transmission electron micrographs of descending aorta of 3-month-old Fbln4E57K/E57K and littermate mice (Fbln4+/+ and Fbln4+/E57K). (A and B) In addition to severe elastic fiber fragmentation, there is increased medial wall thickness and SMC disarray in the aorta of Fbln4E57K/E57K mice. At higher magnification, the moth-eaten or spongy appearance of the fragmented elastic fibers (B) and variable (arrows = larger) collagen fibril size (C) are appreciated in the aorta of Fbln4E57K/E57K mice. Asterisks indicate vessel lumen.

  • Fig. 3 Fbln4E57K/E57K mice develop large artery stiffness and systolic hypertension.

    Pressure-diameter relationships of ascending aorta (A) and carotid arteries (B) from 3- to 6-month-old Fbln4E57K/E57K male mice and littermates (Fbln4+/+ and Fbln4+/E57K). At each pressure, the vessel outer diameter was compared to that of WT vessels using two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. n = 7 to 10 for ascending aorta except for aneurysmal Fbln4E57K/E57K, where n = 3 (A) and 8 to 9 (B) for carotid arteries. Arterial systolic blood pressure (SBP) (C), diastolic blood pressure (DBP) (D), and pulse pressure (PP) (E) of 3- to 4-month-old Fbln4E57K/E57K male and female mice and littermates. PP was calculated as the difference between measured SBP and DBP. Pressure was compared among all genotypes using one-way ANOVA with Tukey’s multiple comparisons test. n = 10 to 12 per genotype. (F) Heart weight–to–body weight ratios between the three genotypes presented in two distinct age groups. At 6 to 7 months of age, mice with aortic root dilatation developed cardiac hypertrophy. Data are means ± SD. *P < 0.05, ****P < 0.0001.

  • Fig. 4 The E57K mutation in Fbln4 has differential effects on elastic versus resistance artery integrity.

    Alexa Fluor 633 hydrazide staining of carotid (A, a and b) and renal arteries (B, a and b) from 3-month-old Fbln4E57K/E57K (b) and WT littermate mice (a). Red indicates elastic fibers, and blue depicts nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Mutant vessels are indistinguishable from WT vessels using Alexa Fluor 633 hydrazide staining. Transmission electron micrographs of carotid (A, c and d) and renal arteries (B, c and d) from 3-month-old Fbln4E57K/E57K mice are shown. Micrographs in (d) are higher magnification of the inset demarcated in (c). Note the moth-eaten appearance of the inner two elastic fibers compared to the normal appearance of the outer two elastic fibers in the carotid artery (A, d). (C) Transmission electron micrographs of second-order mesenteric arteries from 3-month-old (a and b) and 1-year-old (c and d) Fbln4E57K/E57K and WT littermate mice in addition to 1-year-old renal arteries. Asterisks indicate vessel lumen. (D) Table summarizing the presence or absence of elastic fiber abnormalities in the different Fbln4E57K/E57K arteries examined.

  • Fig. 5 Large artery elastic fiber fragmentation and medial wall thickening occur developmentally in Fbln4E57K/E57K mice.

    Transmission electron micrographs of P1 ascending aorta from Fbln4+/+ (A and B) and Fbln4E57K/E57K (C and D) mice. Asterisks indicate vessel lumen. Scale bars, 10 μm.

  • Fig. 6 E57K mutation in Fbln4 does not alter arterial elastin or collagen content.

    Desmosine (A and C) and hydroxyproline (B and D) content were determined in ascending aortae (A and B) and mesenteric arteries (C and D) of 3- to 4-month-old Fbln4E57K/E57K (closed triangles) male and female mice and littermates (open circles, Fbln4+/+; gray squares, Fbln4+/E57K).

  • Fig. 7 E57K mutation in Fbln4 does not affect arterial elastic fiber gene expression.

    Expression of indicated elastic fiber–associated genes (Fbln2, fibulin-2; Fbln4, fibulin-4; Fbln5, fibulin-5; Eln, elastin; Lox, lysyl oxidase; Fbn1, fibrillin 1) in ascending aortae (A and C) and mesenteric arteries (B and D) of 3- to 4-month-old (A and B) and P7 (C and D) Fbln4E57K/E57K (closed bars) and littermate mice (open bars, Fbln4+/+; gray bars, Fbln4+/E57K). Gene expression was normalized to that of Gapdh. Data are means ± SD and were compared using one-way ANOVA with Tukey’s multiple comparisons test. n = 3 to 8 per genotype.

  • Fig. 8 FBLN4(E57K) protein level is reduced in ascending aortae, but not in mesenteric arteries of adult mice.

    Western blot analysis of urea extracts from ascending aortae (A) and mesenteric arteries (B) of Fbln4E57K/E57K mice and littermate controls using a polyclonal rabbit anti-FBLN4 antibody. FBLN4 level was normalized to that of β-actin. The normalized data are represented in the bar graphs on the right. n = 4 to 7 per genotype. Data are means ± SD and were compared using one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Immunofluorescence of ascending aorta and carotid artery sections of 3-month-old Fbln4E57K/E57K and WT control mice. Green, FBLN4; red, autofluorescence of elastic fibers; blue, DAPI. Asterisks indicate vessel lumen.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602532/DC1

    table S1. Echocardiographic data of Fbln4E57K/E57K and littermate WT mice.

    fig. S1. Fbln4E57K/E57K mice are similar in size to their littermates.

    fig. S2. Fbln4E57K/E57K ascending aortae have increased medial wall thickness.

    fig. S3. Fbln4E57K/E57K ascending aortae do not develop calcification.

    fig. S4. Fbln4E57K/E57K mice have similar heart rates to their heterozygous and WT littermates.

    fig. S5. Internal elastic lamina is unaffected in Fbln4E57K/E57K saphenous arteries.

    fig. S6. Homozygous E57K mutation in Fbln4 does not alter arterial elastin or collagen content.

    fig. S7. Homozygous E57K mutation in Fbln4 does not alter ECM gene expression in lungs.

    fig. S8. FBLN4(E57K) protein level is reduced in P7 ascending aorta, but not in mesenteric arteries.

    fig. S9. FBLN4(E57K) protein secretion is impaired compared to WT FBLN4.

  • Supplementary Materials

    This PDF file includes:

    • table S1. Echocardiographic data of Fbln4E57K/E57K and littermate WT mice.
    • fig. S1. Fbln4E57K/E57K mice are similar in size to their littermates.
    • fig. S2. Fbln4E57K/E57K ascending aortae have increased medial wall thickness.
    • fig. S3. Fbln4E57K/E57K ascending aortae do not develop calcification.
    • fig. S4. Fbln4E57K/E57K mice have similar heart rates to their heterozygous and WT littermates.
    • fig. S5. Internal elastic lamina is unaffected in Fbln4E57K/E57K saphenous arteries.
    • fig. S6. Homozygous E57K mutation in Fbln4 does not alter arterial elastin or collagen content.
    • fig. S7. Homozygous E57K mutation in Fbln4 does not alter ECM gene expression in lungs.
    • fig. S8. FBLN4(E57K) protein level is reduced in P7 ascending aorta, but not in mesenteric arteries.
    • fig. S9. FBLN4(E57K) protein secretion is impaired compared to WT FBLN4.

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