Journal: Journal of Clinical Investigation

Article Title: Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse

doi: 10.1172/jci121297

Figure Lengend Snippet: Figure 1. Characterization of Prog-Tg mice. (A) Immunoblots of indicated lysates using anti– human lamin A/C (detecting transgenes, upper panel), anti–lamin A/C (recognizing human and mouse lamin A/C, lower panel), and anti-tubu- lin and anti–VE-cadherin as loading controls. Numbers show ratio of total lamin A levels in transgenic over Wt animals. Endothelial cells (ECs) from Wt (Wt-EC), Prog-Tg (Prog-Tg-EC) and LA-Tg (LA-Tg-EC) animals; EC-depleted cell mix- ture from Prog-Tg animals (Prog-Tg-non-EC); and HGPS patient fibroblasts (HGPS) were analyzed. (B) Histogram of mean progerin fluorescence intensities in immunofluorescence images of Prog-Tg ECs (see Figure 5C) (n = 5 Prog-Tg mice, 462 cells in total). (C and D) Immunofluorescence images of coronary artery (C) and cardiac tissue (D) from Prog-Tg animals stained with antibodies against progerin, VE-cadherin (note green aut- ofluorescence of elastic lamina), and PECAM1, and Hoechst (representative of n = 3 Prog-Tg animals). Progerin expression is confined to the intimal layer (C, arrowheads) and PECAM1-pos- itive cardiac microvasculature (D, arrowheads). Dashed lines, cardiomyocyte boundaries; arrows, intima (C) and progerin-negative cardiomyocytes (D). Scale bars: 10 μm. (E) Body weight over time for male and female Prog-Tg and LA-Tg versus Wt littermates. Two-way repeated-measures ANOVA revealed a significant impact for the Prog-Tg genotype (females F = 72.6, P < 0.001, males F = 65.3, P < 0.001, n = 6 littermate pairs), but not for the LA-Tg genotype (females F = 0.517, P = 0.493; males F = 0.221, P = 0.651, n = 5 littermate pairs). Comparison of Prog-Tg versus Wt revealed at least P < 0.01 (Holm-Sidak meth- od) for females and males at more than 5 and 8 weeks, respectively. (F) Kaplan-Meier survival plot showing significantly reduced life span of Prog-Tg mice (n = 12) versus Wt littermate con- trols (n = 20) and LA-Tg mice (n = 8). P < 0.0001, log-rank (Mantel-Cox) test; pairwise comparison with Bonferroni’s correction of threshold showed significant difference in survival of Prog-Tg compared with control, Prog-Tg with LA-Tg, but not LA-Tg with control mice. Data presented as mean ± SEM.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody (mAb) against lamin A/C (Santa Cruz Biotechnology, clone E-1, sc-376248), mAb against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam), goat anti-MRTFA (Santa Cruz Biotechnology, sc-21558), mAb against progerin (Alexis Biochemicals, clone 13A4, provided by Egon Ogris, Medical University Vienna, Vienna, Austria), mAb against α-tubulin (MilliporeSigma, clone B-5-1-2, T5168), mAb against emerin (Leica Biosystems, clone 4G5, NCL-emerin), rabbit anti-emerin (for immunoblotting, Atlas Antibodies, HPA000609), goat anti–VE-cadherin antibodies (Santa Cruz Biotechnology, sc-6458), rabbit anti-PECAM1 (LSBio, LS-B4737), mAb against eNOS (BD Biosciences, 610297), rabbit affinity-purified antibodies against actin (MilliporeSigma, A2066), guinea pig anti-SUN1 and -SUN2 antibodies (a gift from Manfred Alsheimer, University of Würzburg, Würzburg, Germany).

Techniques: Western Blot, Transgenic Assay, Fluorescence, Immunofluorescence, Staining, Expressing, Comparison, Control

Journal: Journal of Clinical Investigation

Article Title: Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse

doi: 10.1172/jci121297

Figure Lengend Snippet: Figure 7. Impaired MRTFA signaling in Prog-Tg ECs affects eNOS and profibrotic signaling. (A) Immunoflu- orescence of Wt and Prog-Tg ECs using MRTFA antibody and DAPI (images are representative of n = 3 inde- pendent experiments). Scale bar: 10 μm. (B) Representative confocal average intensity projections of Z stacks from mouse aorta of Wt and Prog-Tg animals stained with DAPI and MRTFA and progerin antibodies. EC nuclei (arrowheads) lie on internal elastic membrane that displays blue autofluorescence. Arrowheads, MRTFA-positive ECs. Note that MRTFA accumulates at the nuclear periphery of progerin-positive (white arrowheads) but not progerin-negative (yellow arrowheads) ECs (n = 3 Wt and Prog-Tg littermate pairs). Scale bar: 10 μm. (C) Nos3 mRNA in Wt and Prog-Tg ECs after 24-hour treatment with 15 μM CCG-203971 MRTFA inhibitor or DMSO vehicle control. Values are normalized to Hprt and Nos3/Hprt values in Prog-Tg cells are shown relative to values in Wt cells after MRTFA-inhibitor and control treatment. Nos3/Hprt levels in Wt cells were arbitrary and set to 1 in both control and CCG-203971 conditions (n = 5 independent experiments). Nos3/Hprt levels in Prog-Tg cells relative to Wt are not significantly different (NS) after drug treatment, in contrast to control conditions (P < 0.01). (D) Chromatin immunoprecipitation using anti-MRTFA or goat IgG control. Precipitated DNA was amplified by qPCR with primers spanning Nos3 promoter or gene body (n = 3 independent experiments). **P < 0.01 by unpaired Stu- dent’s t test (C and D). Data presented as mean ± SEM. (E) Acta2 levels normalized to Hprt in fibroblasts after 3 days of coculture with Wt and Prog-Tg ECs either left untreated (left) or treated with 25 μM MRTFA inhibitor CCG-203971 (right). Values were subtracted from those obtained in untreated or CCG-203971–treated fibroblast single cultures (n = 6 Wt, n = 8 Prog-Tg, and n = 3 inhibitor-treated Prog-Tg samples). *P < 0.05 by Mann-Whitney U test. Data presented as median (middle line) with boxes encompassing 25th to 75th percentile, and whiskers, minimum to maximum values.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody (mAb) against lamin A/C (Santa Cruz Biotechnology, clone E-1, sc-376248), mAb against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam), goat anti-MRTFA (Santa Cruz Biotechnology, sc-21558), mAb against progerin (Alexis Biochemicals, clone 13A4, provided by Egon Ogris, Medical University Vienna, Vienna, Austria), mAb against α-tubulin (MilliporeSigma, clone B-5-1-2, T5168), mAb against emerin (Leica Biosystems, clone 4G5, NCL-emerin), rabbit anti-emerin (for immunoblotting, Atlas Antibodies, HPA000609), goat anti–VE-cadherin antibodies (Santa Cruz Biotechnology, sc-6458), rabbit anti-PECAM1 (LSBio, LS-B4737), mAb against eNOS (BD Biosciences, 610297), rabbit affinity-purified antibodies against actin (MilliporeSigma, A2066), guinea pig anti-SUN1 and -SUN2 antibodies (a gift from Manfred Alsheimer, University of Würzburg, Würzburg, Germany).

Techniques: Staining, Membrane, Control, Chromatin Immunoprecipitation, Amplification, MANN-WHITNEY

Journal: Journal of Clinical Investigation

Article Title: Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse

doi: 10.1172/jci121297

Figure Lengend Snippet: Figure 8. Working model. Progerin accumulation at the nuclear lamina leads to perturbations in F-actin level and actin organization and defects in components involved in nucleocytoskeletal coupling (SUN1/2 and emerin). The defective rigid nucleocytoskeletal links show impaired mechanoresponse and cause MRTFA accumulation at the nuclear periphery. MRTFA or associated complexes exert presumably a positive feedback loop on Actb expression (looped arrow). These changes set off the profibrotic signaling cascade (indicated by pale orange color of the nucleus), such as downregulation of eNOS and reduced secretion of NO. Absence of endothelium-derived atheroprotective NO and presumably other unidentified factors leads to increased collagen production in other cell types as shown for fibroblasts through a switch to myofibroblasts, leading to collagen deposition and cardiac fibrosis.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody (mAb) against lamin A/C (Santa Cruz Biotechnology, clone E-1, sc-376248), mAb against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam), goat anti-MRTFA (Santa Cruz Biotechnology, sc-21558), mAb against progerin (Alexis Biochemicals, clone 13A4, provided by Egon Ogris, Medical University Vienna, Vienna, Austria), mAb against α-tubulin (MilliporeSigma, clone B-5-1-2, T5168), mAb against emerin (Leica Biosystems, clone 4G5, NCL-emerin), rabbit anti-emerin (for immunoblotting, Atlas Antibodies, HPA000609), goat anti–VE-cadherin antibodies (Santa Cruz Biotechnology, sc-6458), rabbit anti-PECAM1 (LSBio, LS-B4737), mAb against eNOS (BD Biosciences, 610297), rabbit affinity-purified antibodies against actin (MilliporeSigma, A2066), guinea pig anti-SUN1 and -SUN2 antibodies (a gift from Manfred Alsheimer, University of Würzburg, Würzburg, Germany).

Techniques: Expressing, Derivative Assay

Journal: Aging Cell

Article Title: Heterochromatin loss as a determinant of progerin‐induced DNA damage in Hutchinson–Gilford Progeria

doi: 10.1111/acel.13108

Figure Lengend Snippet: Progerin‐induced DNA damage is restricted to proliferating cells. (a) Schematic representation of the experimental set up. (b) Western blotting showing doxycycline‐dependent progerin expression in proliferating and confluent NDF. Progerin migrates between lamin A and C as indicated (red arrowhead). Lamin A (LA), lamin C (LC), progerin (PG), lamin B1 (LB1), LAP2α, GAPDH and actin are indicated. (c) Immunofluorescence microscopy showing progerin‐induced 53BP‐1 foci (white arrowheads) in proliferating (left panel) or confluent cells (right panel). V5‐progerin (V5 antibody) and 53BP‐1 foci (53BP‐1 antibody) are indicated. Scale bar: 20 μm. (d) Quantification of DNA damage foci (0, 1, 2, 3 or more 53BP‐1 foci), in proliferating or confluent cells in the absence or presence of progerin (*** p < .001, n = 3, χ 2 test). (e) Immunofluorescence microscopy showing Ki‐67 staining in proliferating (left panels) and confluent cells (right panels). DAPI and Ki‐67 antibody are shown on top and bottom panels, respectively. Scale bar: 50 μm. (f) Quantification of the percentage of Ki‐67‐positive and Ki‐67‐negative cells in proliferating or confluent cultures (grey and black bars, respectively)

Article Snippet: Previous studies showed that progerin‐induced DNA damage can occur during DNA replication, although significant amounts of DNA damage were also apparent in G1 cells (Wheaton et al., ).

Techniques: Western Blot, Expressing, Immunofluorescence, Microscopy, Staining

Journal: Aging Cell

Article Title: Heterochromatin loss as a determinant of progerin‐induced DNA damage in Hutchinson–Gilford Progeria

doi: 10.1111/acel.13108

Figure Lengend Snippet: Preferential accumulation of DNA damage foci in cells with lower levels of H3K9me3 and H3K27me3. (a) Scatter plot analysis of H3K9me3, progerin expression and γH2AX DNA damage foci number per nucleus, in NDF expressing progerin (red) or nonexpressing controls (blue). For each nuclei, H3K9me3 and progerin normalized intensities are plotted on Y and X axis, respectively, while the number of DNA damage foci is represented by colour intensity (light to dark colour: 0, 1, 2, 3 or more DNA damage foci per nucleus). Inset: DNA damage counts of the same data (*** p < .001, Student's t test). A total of ~ 1x10 4 cells from 3 independent experiments were analysed. (b) Box plot of data shown in (a), whiskers represent 10–90 percentile (*** p < .001, one‐way ANOVA with Tukey's post‐test). (c) Scatter plot analysis of H3K27me3, progerin expression and γH2AX DNA damage foci number. Inset: DNA damage counts of the same data (*** p < .001, Student's t test). A total of ~ 4x10 3 cells from 3 independent experiments were analysed. (d) Box plot of the same data, whiskers represent 10–90 percentile (*** p < .001, * p < .05, one‐way ANOVA with Tukey's post‐test). (e,f) 3D‐SIM analysis of DNA damage foci. Illustration of the Eroded Volume Fraction (EVF) index to assess proximity to the nuclear lamina, from closest (0) to furthest (1) (reproduction of Figure b). (f,g) Quantification of the average minimum EVF value for 53BP‐1 (f) and γH2AX (g) DNA damage foci, ± progerin. A total of 225 53BP‐1 and 135 γH2AX DNA damage foci were analysed. (* p < .05, Student's t test)

Article Snippet: Previous studies showed that progerin‐induced DNA damage can occur during DNA replication, although significant amounts of DNA damage were also apparent in G1 cells (Wheaton et al., ).

Techniques: Expressing

Journal: Aging Cell

Article Title: Heterochromatin loss as a determinant of progerin‐induced DNA damage in Hutchinson–Gilford Progeria

doi: 10.1111/acel.13108

Figure Lengend Snippet: Progerin‐induced DNA damage occurs in late S‐phase and is associated with a persistent G2 arrest. (a) Schematic representation of the experimental design. (b) Cell cycle profile of control (black bars) and progerin‐expressing (grey bars) NDF at each time point (in hours) after release from G1‐arrest by FACS (% of cells in G1, S‐phase and G2 are shown in top, middle and lower panels, respectively; * p < .05, ** p < .01, n = 3, two‐way ANOVA with Sidak's post‐test). (c) DNA damage accumulation in control (blue) and progerin‐expressing (red) NDF upon release from G1. The number of 53BP‐1 foci per cell is displayed at each time point. A total of ~1 × 10 5 cells from 3 independent experiments were analysed. (d) Quantification of DNA damage foci in control (blue) and progerin‐expressing (red) TERT‐positive NDF upon G1 release. A total of 7 × 10 4 cells from 2 independent experiments were analysed. (e) Scatter plot analysis of phospho‐H3 (pH3), progerin expression and 53BP‐1 DNA damage foci per nucleus, in control (blue) and progerin‐expressing (red) NDF upon release from G1. Data from all time points post‐G1‐arrest release (0–96 hr) are represented. Phospho‐H3 and progerin normalized intensities are plotted on Y and X axis, respectively, while the number of DNA damage foci is represented by colour intensity. From light to dark colour: 0, 1, 2, 3 or more DNA damage foci. ~9 × 10 4 cells from 2 independent experiments were analysed

Article Snippet: Previous studies showed that progerin‐induced DNA damage can occur during DNA replication, although significant amounts of DNA damage were also apparent in G1 cells (Wheaton et al., ).

Techniques: Control, Expressing