Arterial stiffness and cardiac dysfunction in Hutchinson–Gilford Progeria Syndrome corrected by inhibition of lysyl oxidase

Figures

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  Figure 1.. Mechanical properties of isolated carotid arteries display premature circumferential stiffening in Hutchinson–Gilford Progeria Syndrome (HGPS) male and female mice. (B, C, D, E) Carotid arteries from male (panel B, C) and female (panel D, E) 2-mo (n = 7 per sex) and 24-mo (n = 5 per sex) WT and 2-mo Hutchinson–Gilford Progeria Syndrome (n = 6 per sex) mice were analyzed by pressure myography. (A1) Arteries are axially stretched at multiple pressures, and the intersection point of the stretch–force curves defines the “IVS.” The graph displays a representative axial stretch–force curve for a 2-mo WT mouse carotid artery (red arrow shows the IVS). (A2) Arteries are brought to their individual IVS and pressurized. White arrows indicate circumferential deformation with pressure (IR, inner radius; h, wall thickness). (B, D) Circumferential stretch–stress curves for male and female mice, respectively. Each data point corresponds to the circumferential stress and stretch at increments of 10 mm Hg starting from 0 to 140 mm Hg. (C, E) Axial stretch–stress curves, determined at 90 mm Hg, for male and female mice, respectively. Results in panels (B) and (D) show means ± SE and results in…

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  Figure S1.. Biaxial inflation–extension tests and biomechanical properties of WT and Hutchinson–Gilford Progeria Syndrome (HGPS) carotid arteries. (A, B, C, D, E, F) Changes in outer diameter (A, D), inner radius (B, E) and wall thickness (C, F) with pressure for the male (A, B, C) and female (D, E, F) mice in Fig 1. Results show mean ± SE. Statistical significance was determined by two-way ANOVA relative to the 2-mo WT arteries.

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  Figure S2.. Stiffness estimates by tangent modulus. (A, B, C, D) Derived axial (A, C) and circumferential (B, D) tangent modulus versus stretch plots of the carotid arteries from male (A, B) and female (C, D) WT and Hutchinson–Gilford Progeria Syndrome (HGPS) mice shown in Fig 1.

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  Figure 2.. Carotid arteries from 2-mo WT and Hutchinson–Gilford Progeria Syndrome (HGPS) mice display similar histology. (A, B) Carotid artery cross sections from 2-mo WT and Hutchinson–Gilford Progeria Syndrome mice were stained with H&E (scale bar = 50 μm and inset bar = 25 μm), and the number of medial smooth muscle cell nuclei was quantified from H&E images (n = 7–10 carotids per genotype with three sections analyzed per mouse). (C, D) Carotid cross sections were immunostained for the senescence marker, p16INK4A, and the level of medial p16INK4A was normalized to the mean signal intensity of carotid sections from 2-mo WT mice; (n = 6–10 per genotype). Scale bar = 50 μm. Statistical significance in (B) and (D) was determined by Mann–Whitney tests. (E, F) Carotid cross sections were stained with Alizarin Red (n = 5 per genotype) and (F) immunostained for cleaved caspase-3 (n = 5 per genotype). Scale bars in (E) and (F) = 50 μm. The arterial media (M) is outlined with dashed lines.

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  Figure S3.. Positive controls for morphologic analyses and evaluation of elastic laminae of in WT and Hutchinson–Gilford Progeria Syndrome (HPGS) carotid arteries. (A, B) The images presented in Fig 2E and F are reproduced here with corresponding positive controls, bone and tumor tissue, respectively. (C) Representative elastin autofluorescence images of 2-mo WT and HGPS carotid artery cross sections (n = 7–9 per genotype). Scale bar = 50 μm.

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  Figure 3.. Hutchinson–Gilford Progeria Syndrome (HGPS) carotid arteries display alterations in abundance of fibrillar collagens and lysyl oxidase (LOX). (A) Representative images of carotid artery cross sections from 2-mo WT and HGPS mice immunostained for collagen-I (n = 11–12), -III (n = 13–15), and -V (n = 9–10); scale bar = 50 μm. (B) Collagen signal intensities from the immunostained sections were quantified, and results were normalized to the mean signal intensity in the 2-mo WT media. (C) Representative images of LOX immunostaining of carotid artery sections from 1- to 2-mo WT and HGPS mice (n = 7–9 mice per age and genotype). Scale bar = 50 μm. (D) LOX signal intensities from the medial and adventitial layers were quantified, and the results were normalized to the mean signal intensity of the 1-mo WT medial layer. Statistical significance in (B) and (D) was determined by Mann–Whitney tests. The arterial media (M) is outlined with dashed lines.

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  Figure S4.. Immunostaining controls for WT and Hutchinson–Gilford Progeria Syndrome (HGPS) carotid arteries. (A, B) Representative images of background signals for carotid artery cross sections incubated with targeted versus isotype-matched (negative control) primary antibodies from (A) goat (LOX and COL1) or (B) rabbit (COL3 and COL5).

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  Figure S5.. Quantification of collagen abundance in elastin folds. (A) Representative transmission electron microscopy images from a mouse carotid artery cross section processed in ImageJ to quantify the area of collagen within an elastin fold. The boxed region in image A1 is shown in images A2–A6. Scale bar = 2 μm. See Supplemental Data 1 for details. (B) Carotid arteries from 2-mo WT and Hutchinson–Gilford Progeria Syndrome mice were sectioned and imaged by Transmission Electron Microscopy. Representative images are shown at the indicated magnification. Numbers in each image correspond to the boxed ROI in the preceding image. In image 1, the lumen (L), media (M), and adventitia (A) of the artery are noted (scale bar = 6 μm). Image two shows multiple elastin folds, and image three shows a twofold enlargement of an individual fold used to quantify collagen area as described in Supplemental Data 1 (scale bar = 2 μm). In image 4, individual collagen fibrils within the elastin fold are readily distinguishable (scale bar = 200 nm). (C) Collagen area in elastin folds of WT and Hutchinson–Gilford Progeria Syndrome carotid arteries was quantified as described in Supplemental Data 1 (n = 6 mice per…

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  Figure S6.. Mechanical properties of 1-mo WT and Hutchinson–Gilford Progeria Syndrome (HGPS) carotid arteries. Carotid arteries from mixed sex 1-mo WT (n = 5) and HGPS (n = 5) mice were analyzed by pressure myography. (A, B, C) Changes in inner radius, wall thickness, and outer diameter with pressure. Results show mean ± SE. (D) Axial stress–stretch curves performed at 80 mm Hg. Results show mean ± SD. (E) Circumferential stress–stretch curves. Results show mean ± SE. Statistical significance in (A, B, C, D, E) was determined by two-way ANOVA. (F, G) Derived axial and circumferential tangent modulus versus stretch plots. Male 2-mo HGPS tangent curves were reproduced from Fig S2 for reference (dashed red line).

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  Figure S7.. Collagen and lysl oxidase (LOX) abundance in PBS and BAPN-treated mice. (A) Representative Transmission Electron Microscopy images (50,000× magnification) of cross sections from 2-mo Hutchinson–Gilford Progeria Syndrome (HGPS) carotid arteries that had been treated with vehicle (PBS) or BAPN as described in the Materials and Methods section. Scale bar = 200 nm. (B) Quantification of collagen area within the elastin folds as described in Supplemental Data 1 and Fig S5 (n = 3–4 mice per treatment with 10 elastin folds analyzed per mouse). (C, D) Aorta lysates, prepared from 2-mo HGPS mice treated with PBS or BAPN, were analyzed by Western blotting and probed for LOX and FAK (loading control). (C) Representative Western blot; white spaces indicate removal of extraneous data. (D) Quantification of LOX signal intensities, normalized to the loading control, in HGPS mice treated with PBS (n = 4) or BAPN (n = 4).

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  Figure S8.. Effect of BAPN on carotid artery geometry and tangent modulus in mixed sex WT and Hutchinson–Gilford Progeria Syndrome (HGPS) mice. (A, B, C) Changes in outer diameter, inner radius, and wall thickness with pressure for the PBS- and BAPN-treated Hutchinson–Gilford Progeria Syndrome and WT mice shown in Fig 4. Results show mean ± SE. Statistical significance was determined by two-way ANOVA relative to the vehicle treated arteries. (D, E) Derived circumferential and axial tangent modulus versus stretch plots.

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  Figure 4.. Elevated lysyl oxidase expression is linked to premature arterial stiffening and diastolic dysfunction in Hutchinson–Gilford Progeria Syndrome (HGPS) mice. Mixed sex WT and HGPS mice aged to 1-mo were treated with β-aminopropionitrile, BAPN (WT n = 5, HGPS n = 6) or PBS (WT n = 6, HGPS n = 7) for ∼30 d followed by pressure myography of isolated carotid arteries. (A, B) Circumferential and axial stretch–stress curves, respectively, of WT and HGPS mice treated with PBS or BAPN. Results in panels (A) display means ± SE, and results in panel (B) display means ± SD. Statistical significance for the myographic analysis was determined by two-way ANOVA. (C, D) Echocardiography measurements of (C) the E/E′ ratio and (D) the left ventricular mass index (left ventricular mass to body weight ratio) of 64–75 d old HGPS mice (n = 4), 65–75 d old HGPS mice treated with BAPN (n = 4), and 66–75 d WT mice (n = 4). Mouse numbers in (C, D) were accrued from vehicle-injected and uninjected mice, and statistical significance was determined by Mann–Whitney tests.