JAGGED1/NOTCH3 activation promotes aortic hypermuscularization and stenosis in elastin deficiency
Jui M. Dave, … , Kathleen A. Martin, Daniel M. Greif
Jui M. Dave, … , Kathleen A. Martin, Daniel M. Greif
Published January 6, 2022
Citation Information: J Clin Invest. 2022;132(5):e142338. https://doi.org/10.1172/JCI142338.
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JAGGED1/NOTCH3 activation promotes aortic hypermuscularization and stenosis in elastin deficiency

Abstract

Obstructive arterial diseases, including supravalvular aortic stenosis (SVAS), atherosclerosis, and restenosis, share 2 important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic, or deletion mutations in the elastin gene (ELN), and SVAS patients and elastin-mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking, as the underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models, and aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the NOTCH pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain, and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln–/– mutants. Eln–/– mice expressed higher levels of NOTCH ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln–/– mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.

Authors

Jui M. Dave, Raja Chakraborty, Aglaia Ntokou, Junichi Saito, Fatima Z. Saddouk, Zhonghui Feng, Ashish Misra, George Tellides, Robert K. Riemer, Zsolt Urban, Caroline Kinnear, James Ellis, Seema Mital, Robert Mecham, Kathleen A. Martin, Daniel M. Greif

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Figure 8

Elastin deficiency in SMCs induces JAG1 upregulation.

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Elastin deficiency in SMCs induces JAG1 upregulation. (A–C) haSMCs were ...
(A–C) haSMCs were treated with Scr or siELN RNA, and then lysates were analyzed. In A, histogram represents ELN and JAG1 transcript levels relative to 18S rRNA as assessed by qRT-PCR and normalized to Scr treatment (n = 3). Western blots for JAG1 and GAPDH are shown in B, with densitometry of protein bands relative to GAPDH and normalized to Scr in C (n = 3). *P < 0.05, ****P < 0.0001 vs. Scr by Student’s t test. (D) Methylated DNA (5mC) ChIP from haSMCs pretreated with Scr or siELN. Histogram represents 5mC levels at promoter regions of JAG1 or THS2B (positive control) by qPCR and normalized to Scr (n = 4). **P < 0.01 vs. Scr by Student’s t test. (E and F) Aortic lysates from WT or Eln–/– mice at P0.5 were analyzed by Western blotting for JAG1 and GAPDH (for each blot, 2 aortas were pooled per genotype), with densitometry of JAG1 protein bands relative to GAPDH and normalized to WT (n = 6 mice). **P < 0.01 vs. WT by Student’s t test. (G) Transverse sections of ascending aorta from WT and Eln–/– mice at P0.5 were stained for JAG1, CD31, SMA, and nuclei (DAPI). n = 3 mice. Lu, lumen. Scale bar: 25 μm. (H and I) Protein levels of JAG1 and GAPDH in iPSC-SMCs derived from control or WBS or SVAS patients as assessed by Western blotting with densitometric analysis of JAG1 normalized to GAPDH (n = 3). ***P < 0.001 by 1-way ANOVA with Tukey’s post hoc test. (J) Aortic sections from a WBS male patient (46 years old) and control male (53 years old) stained for JAG1, SMA, and nuclei (PI). Scale bar: 50 μm. (K) Column scatter plot represents fluorescence intensity of JAG1 and SMA immunostaining in aortic sections of WBS patients (n = 5) normalized to age-matched controls (n = 11). Intensity was quantified on 8 to 10 microscopic fields per patient. **P < 0.01 vs. control by Student’s t test. All data are averages ± SD.