Endothelial cell-specific progerin expression does not cause cardiovascular alterations and premature death

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by a mutation in the LMNA gene that provokes the synthesis of progerin, a mutant version of the nuclear protein lamin A that accelerates aging and precipitates death. The most clinically relevant feature of HGPS is the development of cardiac anomalies and severe vascular alterations, including massive loss of vascular smooth muscle cells, increased fibrosis, and generalized atherosclerosis. However, it is unclear if progerin expression in endothelial cells (ECs) causes the cardiovascular manifestations of HGPS. To tackle this question, we generated atherosclerosis-free mice (Lmna^LCS/LCSCdh5-CreERT2) and atheroprone mice (Apoe^-/-Lmna^LCS/LCSCdh5-CreERT2) with EC-specific progerin expression. Like progerin-free controls, Lmna^LCS/LCSCdh5-CreERT2 mice did not develop heart fibrosis or cardiac electrical and functional alterations, and had normal vascular structure, body weight, and lifespan. Similarly, atheroprone Apoe^-/-Lmna^LCS/LCSCdh5-CreERT2 mice showed no alteration in body weight or lifespan versus Apoe^-/-Lmna^LCS/LCS controls and did not develop vascular alterations or aggravated atherosclerosis. Our results indicate that progerin expression in ECs is not sufficient to cause the cardiovascular phenotype and premature death associated with progeria.

Keywords: Hutchinson‐Gilford progeria syndrome; atherosclerosis; cardiovascular disease; endothelial cells; progerin.

FIGURE 1

Endothelium‐specific progerin expression in Lmna ^LCS/LCS Cdh5‐CreERT2 mice. (a) En face immunofluorescence staining of thoracic aortas from 22‐month‐old Lmna ^LCS/LCS and Lmna ^LCS/LCS Cdh5‐CreERT2 mice, showing progerin (white) and nuclei (blue). The percentage of progerin‐positive ECs in Lmna ^LCS/LCS Cdh5‐CreERT2 mice is indicated (n = 4). Luminal aortic ECs from Lmna ^LCS/LCS Cdh5‐CreERT2 mice were considered progerin‐positive when nuclear progerin expression was above the mean value in Lmna ^LCS/LCS control mice. Bar, 50 μm. (b, c) Immunofluorescence analysis of cross‐sections from thoracic aorta (b), heart, liver, and kidney (c) of 22‐month‐old mice, showing progerin (white), CD31 (green), αSMA (red), and nuclei (blue) (n = 4). Bars, 50 μm. Yellow arrowheads indicate examples of progerin‐positive ECs. (d) Cell sorting gating strategy for the isolation of viable cardiac ECs (PI⁻CD31⁺CD45⁻) and macrophages (PI⁻CD31⁻CD45⁺CD11b⁺F4/80⁺). PI, propidium iodide. (e) RT‐qPCR analysis of progerin expression in cardiac ECs and macrophages from 22‐month‐old mice (n = 5, two‐way ANOVA plus Tukey's multiple comparisons test). ***p < 0.001.

FIGURE 2

Endothelium‐specific progerin expression in mice does not alter the hematological profile or reduce body weight or lifespan. (a) Hematological profile of 22‐month‐old Lmna ^LCS/LCS and Lmna ^LCS/LCS Cdh5‐CreERT2 mice (n = 12–13, two‐tailed unpaired Student t‐test for all parameters except neutrophils, eosinophils, basophils, and MPV, which were analyzed by two‐tailed Mann–Whitney U‐test). (b) Representative image of 22‐month‐old female mice. Bar, 5 cm. (c) Body weight (n = 8–10, mixed effects model with the Geisser–Greenhouse correction) and Kaplan–Meier survival curves (n = 10–13, Mantel–Cox test). WBC, white blood cells; RBC, red blood cells; PLT, platelets; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscle volume; MPV, mean platelet volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width.

FIGURE 3

Endothelium‐specific progerin expression in mice induces neither heart fibrosis nor alterations in cardiac electrical activity and function. (a) Collagen content in hearts from 22‐month‐old Lmna ^LCS/LCS and Lmna ^LCS/LCS Cdh5‐CreERT2 mice, measured by Sirius red staining (n = 7–8, two‐tailed unpaired Student t‐test). Bar, 50 μm. (b, c) Electrocardiography (b) and echocardiography (c) studies in 18‐month‐old mice [n = 14–17, analyzed by two‐tailed unpaired Student t‐test [QT, QT90, E, A, IVRT, LVID (d), LVID (s), LVPW (d), EF, IVS (d), LV mass, LV vol (d), and LV vol (s)] or two‐tailed Mann–Whitney U‐test (QT90c, T steepness, JT, E/A, and heart rate]. A, mitral valve A velocity; E, mitral valve E velocity; E/A, mitral valve E to A ratio; IVRT, isovolumic relaxation time; LV, left ventricle; IVS (d), inter ventricular septum (diastole); LVID (d), LV internal diameter (diastole); LVID (s), LV internal diameter (systole); LVPW (d), LV posterior wall (diastole); EF, LV ejection fraction; LV vol (d), LV volume (diastole); LV vol (s), LV volume (systole); BPM, beats per minute.

FIGURE 4

Endothelium‐specific progerin expression in mice does not induce medial vascular smooth muscle cell loss and fibrosis or intimal leukocyte accumulation in the aorta. (a, b) Hematoxylin–eosin (a) and Masson's trichrome (b) staining of aortic arch sections from 22‐month‐old mice to quantify the number of nuclei in the media, the adventitia (adv.)/media thickness ratio, and collagen accumulation in the media (n = 7–8, two‐tailed unpaired Student t‐test). Bars, 250 μm. (c) En face immunostaining of the luminal surface of thoracic aortas, showing CD45⁺ leukocytes (red) and nuclei (blue) (n = 4, two‐tailed Mann–Whitney U‐test). Bars, 50 μm. L, lumen.

FIGURE 5

Normal body weight and lifespan in atheroprone mice with endothelium‐specific progerin expression. (a) Progerin expression in Apoe ^−/− Lmna ^LCS/LCS Cdh5‐CreERT2 mice and Apoe ^−/− Lmna ^LCS/LCS controls fed normal chow was analyzed by en face immunofluorescence of thoracic aortas from 4‐month‐old mice (white, progerin; blue, nuclei). The percentage of progerin‐positive ECs in Apoe ^−/− Lmna ^LCS/LCS Cdh5‐CreERT2 mice is indicated (n = 7–8). Luminal aortic ECs in Apoe ^−/− Lmna ^LCS/LCS Cdh5‐CreERT2 mice were considered progerin‐positive when nuclear progerin expression was above the mean value in Apoe ^−/− Lmna ^LCS/LCS control mice. Bar, 25 μm. (b) Immunofluorescence assays in thoracic aorta sections, showing progerin (white), CD31 (green), αSMA (red), and nuclei (blue) (n = 5–6). Bar, 50 μm. Yellow arrowheads indicate examples of progerin‐positive ECs. (c) Representative image of 4‐month‐old male mice. Bar, 2 cm. (d) Body weight (n = 6–9, mixed effects model with the Geisser–Greenhouse correction) and Kaplan–Meier survival curves (n = 6–9, Mantel–Cox test).

FIGURE 6

Endothelium‐specific progerin expression does not induce vascular smooth muscle cell loss, medial fibrosis, or aggravated atherosclerosis in atheroprone mice. Apoe ^−/− Lmna ^LCS/LCS and Apoe ^−/− Lmna ^LCS/LCS Cdh5‐CreERT2 mice were fed a high‐fat diet starting at 2 months of age and sacrificed for analysis at 4 months of age. (a) Hematological profile (n = 6–7, two‐tailed unpaired Student t‐test for RBC and PLT, and two‐tailed Mann–Whitney U‐test for WBC). (b) Serum lipid profile (n = 5, two‐tailed unpaired Student t‐test). (c, d) Hematoxylin–eosin (c) and Masson's trichrome (d) staining of aortic arch sections to quantify the number of nuclei in the media, the adventitia (adv.)/media thickness ratio, and collagen accumulation in the media (n = 5, two‐tailed unpaired Students t‐test). Bars, 250 μm. (e) Representative images of en face oil red O staining of aortas and quantification of atherosclerosis burden in aortic arch and thoracic aorta (n = 5, two‐tailed unpaired Student t‐test). Bar, 5 mm. (f) Representative images of Van Gieson staining of aortic valves and quantification of atherosclerosis burden measured as lesion area (μm²) and aortic perimeter affected by atherosclerosis (%) (n = 5, two‐tailed unpaired Student t‐test). Bar, 200 μm. HDL, high density lipoproteins; LDL, low density lipoproteins; L, lumen; PLT, platelets; RBC, red blood cells; WBC, white blood cells.