Fig 2: Elastin deficiency reduces DNA methylation (5mC) epigenetic mark and downregulates DNMT1 levels.(A) Transverse sections of ascending aorta from WT or Eln–/– pups at P0.5 stained for 5-methylcytosine (5mC), SMA, and propidium iodide (PI, nuclei). Lu, lumen. Scale bar: 50 μm. (B) Histogram representing the percentage of 5mC+ SMCs in A (n = 3 mice). **P < 0.01 vs. WT by Student’s t test. (C–E) haSMCs were pretreated with Scr or siELN and then lysates were analyzed. In C, histogram depicts transcript levels of DNMT1, DNMT3A, and DNMT3B from qRT-PCR relative to 18S rRNA and normalized to Scr (n = 6). **P < 0.01 vs. Scr by Student’s t test. (D) Western blots for DNMT1 and GAPDH and densitometry of protein bands in E relative to GAPDH and normalized to Scr (n = 4). ***P < 0.001 vs. Scr by Student’s t test. (F and G) Aortic lysates from WT or Eln–/– mice at P0.5 analyzed by Western blotting for DNMT1 and GAPDH (F) with densitometry of protein bands relative to GAPDH and normalized to WT (G) (n = 3 mice). **P < 0.01 vs. WT by Student’s t test. (H) Genomic DNA was isolated from haSMCs pretreated with Scr or siELN and then subjected to methylated DNA chromatin immunoprecipitation (5mC ChIP). Histogram represents 5mC levels at the promoter regions of PSEN1, PSEN2, or THS2B (constitutive 5mC positive control) as assessed by qPCR and normalized to Scr (n = 4). **P < 0.01, ***P < 0.001 vs. Scr by Student’s t test. All data are averages ± SD.

Fig 3: In elastin-mutant mice, deletion of Jag1 in SMCs but not ECs attenuates muscularization and stenosis.(A and B) Jag1fl/fl pups also carrying no Cre, Cdh5-Cre, or Acta2-CreERT2 and either WT or null for Eln were collected at P0.5. Transverse sections of the ascending aorta from indicated genotypes were stained for SMA and CD31. In B, pregnant dams were injected with tamoxifen at E10.5. Lu, lumen. Scale bars: 100 μm. (C–E) Medial thickness (C), lumen area (D), and medial area (E) of sections of ascending aortas from mice as in B (n = 5 to 6 mice). *P < 0.05; **P < 0.01; ****P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test. Data are averages ± SD. (F) Schematic of study findings is shown. During elastin deficiency, reduction in DNMT1-mediated DNA methylation results in expression of PSEN1, PSEN2, and JAG1 genes. These gene products culminate in increased NICD3 and activation of NOTCH3 pathway members (downstream genes, including JAG1, ITGB3, HES1, and HEY1), leading to SMC hyperproliferation, hypermuscularization, and stenosis. Pharmacological inhibition of γ-secretase complex (via DAPT) or genetic deletion of Notch3 or SMC Jag1 attenuates aortic phenotype of Eln–/– mice. Note that we previously found that pharmacological or genetic inhibition of ITGB3 has similar effects (3).

Fig 4: The effect of D-Pen on APP processing in N2a-sw cells. Representative immunoblots and quantification of the expression of APP-processing proteins in N2a-sw cells treated with medium containing 10 and 25 μM D-Pen. (A–D) APP-processing proteins including fl-APP, sAPPα, and sAPPβ were detected by Western blot analysis. (E–G) sAPPα and sAPPβ protein levels were detected in media by Western blot analysis. (H–M) The enzymes involved in ADAM10, BACE1, PS2, and PS1 expression were determined via immunoblot analysis. β-actin was used as an internal control. Data are presented as the mean ± standard error of the mean (SEM) at least three independent experiments by one-way analysis of variance. Extracellular Aβ1-40 and 1-42 secretions (N,O) were measured via enzymatic cleavage assays. Statistical significance in multiple comparisons was determined by one-way analysis of variance, *p < 0.05, **p < 0.01 compared to the control group.

Fig 5: Enhanced aortic γ-secretase levels with elastin deficiency.(A and C) Lysates were analyzed by Western blotting for γ-secretase subunits NCT, PSEN1, PSEN2, and PEN2 and for GAPDH. In A, haSMCs were pretreated with Scr or siELN RNA, and in C, aortas of 2 WT or Eln–/– mice at P0.5 were pooled. (B and D) Densitometry of protein bands in A and C relative to GAPDH and normalized to Scr in B or WT in D (n = 3). *P < 0.05; **P < 0.01 by Student’s t test vs. Scr (B) or WT (D). Data are averages ± SD. (E and F) Ascending aortic cross sections of WT and Eln–/– mice at P0.5 stained for α-smooth muscle actin (SMA, marker of SMCs), nuclei (DAPI), and either PSEN1 in E or PSEN2 in F. n = 3 mice. Lu, lumen. Scale bars: 25 μm.