Research ArticleProgeria

Disrupting the LINC complex in smooth muscle cells reduces aortic disease in a mouse model of Hutchinson-Gilford progeria syndrome

See allHide authors and affiliations

Science Translational Medicine  26 Sep 2018:
Vol. 10, Issue 460, eaat7163
DOI: 10.1126/scitranslmed.aat7163
  • Fig. 1 Mice expressing progerin develop aortic pathology.

    (A) Western blot showing the synthesis of progerin but not prelamin A in aortas from LmnaG609G/+ (G609G/+) and LmnaG609G/G609G (G609G/G609G) mice. Average progerin amounts (relative to tubulin) were 1.15 and 1.43 for heterozygous and homozygous G609G mice, respectively. KO, knockout; Ab, antibody. (B) Representative H&E-stained sections of the ascending aorta from 4-month-old Lmna+/+ and LmnaG609G/G609G mice. Colored arrows identify the aortic media (black) and adventitia (yellow). Scale bars, 50 μm. (C) Bar graph showing SMC nuclei (left) and adventitial area (right) in the ascending aorta (inner curvature) of 4-month-old LmnaG609G/G609G mice (blue), compared with age-matched WT mice (black). Means ± SEM; n = 10 per group. LmnaG609G/G609G versus WT; *P < 0.001 (t test). (D) Thoracic aortas from WT (+/+), LmnaG609G/+ (G609G/+), and LmnaG609G/G609G (G609G/G609G) mice. Numbered white ovals identify locations for sections in (E). (E) H&E-stained sections of four regions of the thoracic aorta from 4-month-old Lmna+/+ and LmnaG609G/G609G mice. Colored arrows identify the adventitia (yellow) and media (black). Scale bars, 40 μm. (F) Enlarged image of the proximal ascending aorta [boxed area in (E)] showing vacuolated SMCs (black arrowheads). Scale bar, 20 μm. (G) Electron micrographs showing SMC nuclei in Lmna+/+, LmnaG609G/+, and LmnaG609G/G609G mice. Yellow arrowheads point to intranuclear vesicles in LmnaG609G/+ aortas. A duplicate image is colorized to show the nucleoplasm (blue) and cytoplasm (yellow). Red arrowhead points to a cytoplasmic vacuole in a LmnaG609G/G609G aortic SMC.

  • Fig. 2 Collagen synthesis is increased in the adventitia of LmnaG609G mice.

    (A) Histochemical studies showing collagen content in the adventitia of aortas from 4-month-old LmnaG609G/G609G and Lmna+/+ mice. H&E: blue, nuclei; pink, cytoplasm. VVG stain: black, elastic fibers and nuclei; red, collagen. MT stain: pink/red, cytoplasm; blue, collagen. Colored arrows identify the media (white) and adventitial (black) layers. Scale bars, 50 μm. (B) Reverse transcription polymerase chain reaction (RT-PCR) studies showing Col1a1 and Col8a1 expression in the adventitia of 12-month-old WT and LmnaG609G/+ mice (means ± SEM; n = 3). LmnaG609G/+ versus WT; *P < 0.05, **P < 0.01, and ***P < 0.001 (t test). (C) Serial frozen sections of the ascending aorta from 12-month-old WT and LmnaG609G/+ mice stained with antibodies against collagen types I, III, IV, V, and VIII (red) and CD31 (cyan), examined by confocal fluorescence microscopy. Scale bars, 50 μm.

  • Fig. 3 Lamin A is expressed at high amounts in the aorta of WT mice.

    (A) Representative Western blot comparing lamin A, lamin C, and lamin B1 in different tissues from WT mice. Tubulin was measured as a loading control. Cortex, cerebral cortex; BAT, brown adipose tissue; WAT, white adipose tissue; Gb, gallbladder; ASC, ascending aorta; DESC, descending aorta; ABD, abdominal aorta. Graphs showing lamin A expression relative to tubulin (B), relative to lamin C (C), and relative to lamin B1 (D). For (B) to (D), tissues are arranged in ascending order of expression from left to right, with the expression in kidney set at a value of 1 (means ± SEM; n = 4 mice). (E) RT-PCR comparing the expression of prelamin A (black) and Lmnb1 (blue) in tissues (means ± SEM; n = 4 mice). (F) RT-PCR comparing the expression of prelamin A (black) and Lmnb1 (blue) in the media and adventitia (Adv) layers of WT mice (means ± SEM; n = 4 mice). Media versus adventitia; **P < 0.001 (t test). (G) Confocal fluorescence microscopy images showing the expression of CD31 (cyan) and lamin B1 (red) in the ascending aorta of a WT mouse. In the merged image, the adventitia is outlined by dashed white lines (see fig. S4G). Scale bar, 50 μm.

  • Fig. 4 Vascular pathology is more severe along the inner curvature of the ascending aorta and branches of the aortic arch.

    (A to C) Composite fluorescence microscopy images of the ascending thoracic aorta stained with antibodies against CD31 (green) and collagen type VIII (red) in 12-month-old Lmna+/+ (A) and LmnaG609G/+ (B) mice and in a 4-month-old LmnaG609G/G609G mouse (C). The white lines identify the location where the measurements were made. BC, brachiocephalic; LCC, left common carotid; LSC, left subclavian. Scale bars, 500 μm. (D) Bar graph showing adventitial area as a percentage of total area in WT (black) and LmnaG609G/+ (white) mice at the outer and inner curvatures of the ascending aorta. (E) Bar graph showing the number of SMC nuclei relative to media area in WT (black) and LmnaG609G/+ (white) mice at the outer and inner curvatures of the ascending aorta. Means ± SEM for WT (n = 12) and LmnaG609G/+ (n = 11) mice; *P < 0.02 and **P < 0.001 (t test). (F) RT-PCR measuring prelamin A, lamin C, and Lmnb1 expression at the inner and outer curvatures in WT mice (means ± SEM; n = 4). Inner versus outer; ns, not significant; P > 0.20 (t test).

  • Fig. 5 Disrupting the LINC complex in SMCs ameliorates phenotypes elicited by progerin.

    (A) Microscopy images of mouse aortic SMCs expressing prelamin A and progerin. Scale bars, 20 μm. Bar graph shows quantification of misshapen nuclei (means ± SEM; n = 3 experiments). *P < 0.02 (t test). (B) Western blot of lamin A (LA), lamin C, progerin (P), phosphorylated p53 (Phos-p53), and H2AXγ expression in cells expressing lamin A or progerin. Cells exposed to ultraviolet (UV) light were included as a control. The bar graph shows the expression of p53 phosphorylation (black) and H2AXγ (red) after 1 or 2 days (means ± SEM; n = 3 experiments). Progerin versus prelamin A; *P < 0.01 and **P < 0.001 (t test). (C) Cell protein expressed relative to static cells (means ± SEM; n = 3 experiments) in prelamin A– and progerin-expressing SMCs exposed to uniaxial strain via stretching (6 mm, 0.5 Hz) or static conditions for 1 day. Stretch versus static; *P < 0.001 (t test). (D) Microscopy images of progerin-expressing cells transduced with enhanced green fluorescent protein (EGFP)–labeled KASH2 or EGFP-labeled ext-KASH2 (both in green). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue) and LAP2β (red; a nuclear membrane marker). Scale bars, 20 μm. Bar graph shows quantification of misshapen nuclei in prelamin A– and progerin-expressing cells (means ± SEM; n = 3 experiments). KASH2 versus ext-KASH2; *P < 0.02 (t test). (E) Western blot showing lamin A, progerin, lamin C, phosphorylated p53, KASH2, and H2AXγ in unstrained, prelamin A (WT)– or progerin (608)–expressing cells. The bar graph shows the average expression of p53 phosphorylation (white) and H2AXγ (black) after 1 day (means ± SEM; n = 3 experiments). KASH2 versus ext-KASH2; *P < 0.05 and **P < 0.01 (t test). (F) Bar graph showing relative cell protein in progerin- and prelamin A–expressing stretched SMCs transfected with KASH2 or ext-KASH2 (means ± SEM; n = 3 experiments). KASH2 versus ext-KASH2; *P < 0.001 (t test).

  • Fig. 6 The expression of the KASH2 domain in SMCs ameliorates aortic disease in LmnaG609G/G609G mice.

    (A to D) Representative photographs of H&E-stained cross sections at the outer (A) and inner (B) curvatures of the ascending aorta and proximal descending aorta (C and D) of WT (Lmna+/+KASH2–EGFP+Sm22-Cre+), MUT (LmnaG609G/G609GKASH2–EGFP+Sm22-Cre), and MUT + KASH2 (LmnaG609G/G609GKASH2–EGFP+Sm22-Cre+) mice. Scale bars, 50 μm (A to C) and 20 μm (D). (E) Adventitial area as a percentage of total area and numbers of SMC nuclei per media area at the outer ascending thoracic aorta (means ± SEM; n = 6 per group). (F) Adventitial area and numbers of SMC nuclei at the inner ascending thoracic aorta (means ± SEM; n = 6 per group). (G) Adventitial area and numbers of SMC nuclei in the proximal descending thoracic aorta (means ± SEM; n = 6 per group). *P < 0.01 and **P < 0.001; ns, not significant defined as P > 0.40 (t test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/460/eaat7163/DC1

    Materials and Methods

    Fig. S1. Pathology in LmnaG609G mice.

    Fig. S2. H&E staining of aortic tissue from young mice and nonaortic tissues from older LmnaG609G/+ mice.

    Fig. S3. Cell and nuclear morphology in LmnaG609G aortic SMCs.

    Fig. S4. Nuclear lamin expression in the aorta of WT and LmnaG609G/+ mice.

    Fig. S5. Analysis of the vascular phenotype in the ascending aorta of LmnaG609G/G609G mice.

    Fig. S6. Control studies for the lamin-inducible SMC system and the Sm22-Cre–dependent activation of KASH2 expression in the aorta.

    Table S1. Antibodies used for Western blotting and immunohistochemistry.

    Table S2. Quantitative RT-PCR primers.

    Table S3. Individual subject-level data.

    Reference (69)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Pathology in LmnaG609G mice.
    • Fig. S2. H&E staining of aortic tissue from young mice and nonaortic tissues from older LmnaG609G/+ mice.
    • Fig. S3. Cell and nuclear morphology in LmnaG609G aortic SMCs.
    • Fig. S4. Nuclear lamin expression in the aorta of WT and LmnaG609G/+ mice.
    • Fig. S5. Analysis of the vascular phenotype in the ascending aorta of LmnaG609G/G609G mice.
    • Fig. S6. Control studies for the lamin-inducible SMC system and the Sm22-Cre–dependent activation of KASH2 expression in the aorta.
    • Table S1. Antibodies used for Western blotting and immunohistochemistry.
    • Table S2. Quantitative RT-PCR primers.
    • Legend for table S3
    • Reference (69)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Individual subject-level data.

Stay Connected to Science Translational Medicine

Navigate This Article