JAG1-NOTCH4 mechanosensing drives atherosclerosis

Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 (Jag1ECKO) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.


Fig. S3.
Low JAG1 and NOTCH4 expression in unmodified contralateral carotid arteries of mice. Flow-altering, constrictive cuffs were placed on the right carotid arteries of C57BL/6 mice; contralateral left carotid arteries were unmodified. Carotid arteries were harvested after 14 days, and en face staining was performed using anti-JAG1 (A) or anti-NOTCH4 (B) antibodies (red). Endothelium was co-stained (anti-CDH5; EC; green) and nuclei detected using TO-PRO-3 (DNA; blue). Representative images show low expression of JAG1 and NOTCH4 in contralateral carotid arteries.

Validation of HCAEC responses to flow.
HCAEC were seeded on µ-slides and cultured under LOSS or HSS for 72h using the Ibidi system. Expression level of the known shear-sensitive genes KLF4 (A) and MCP-1 (B) was assessed by qRT-PCR. (n=6). Differences were analysed using paired t-tests.

HSS induces NOTCH1 in arterial endothelial cells.
HCAEC were seeded on µ-slides and cultured under LOSS or HSS for 72h using the Ibidi system. Protein levels of NOTCH1 were quantified by immunoblotting. Representative images (A) and mean values normalized to the level of PDHX (n=6) (B) are shown. Differences between means were analysed using a paired t-test. HCAEC express JAG1 and NOTCH4 mRNA levels at low levels in static conditions. HCAEC from individual donors were seeded on µ-slides and cultured under static conditions or under HSS for 72h using the Ibidi system. Levels of NOTCH4 and JAG1 mRNA were quantified by qRT-PCR. (n=3). Data points and mean +/-SEM from static HCAEC are shown, and mean levels under HSS are represented as a broken line.  Validation of Jag1 deletion. Jag1 ECKO and control mice were analysed 2 weeks post-tamoxifen (TAM) injection. The expression of JAG1 protein (red) was visualized in the murine aorta by en face staining. Endothelium was co-stained (anti-CD31; EC; green) and nuclei detected using TO-PRO-3 (DNA; blue).

Fig. S9.
Lipid profiles in Jag1 ECKO mice. Jag1 ECKO mice aged 6 weeks and littermate controls received five intraperitoneal injections of tamoxifen and one injection of PCSK9-AAV virus at specified time points. After 6 weeks fed with high fat diet, total cholesterol, non-HDL cholesterol and triglyceride levels were measured in Jag1 ECKO mice and controls. Differences between means were analyzed using an unpaired t-test.

Expression of aortic endothelial cell cluster markers from previous publications.
Aortas from Jag1 ECKO and control mice were analysed by FACS of CD31 + CD45cells coupled to scRNAseq. Heatmap showing the highest differentially expressed genes in endothelial clusters 0-8 and 10 relative to other EC clusters, in cluster 11 relative to other EC clusters, and in cluster 9 relative to other EC clusters. (A) Expression of 'EC1', 'EC2', and 'EC3' marker genes from Kalluri et al. (27) in clusters 1-11. The arrows indicate enrichment in the specified clusters. On the right, expression of 'EC1', 'EC2', and 'EC3' marker genes are shown on the t-SNE embedding.

tSNE representation of GO terms for EC clusters.
Aortas from Jag1 ECKO and control mice were analysed by FACS of CD31 + CD45cells coupled to scRNAseq. tSNE representation of the scRNA-seq data showing the expression of defined gene sets that determine different GO pathways. Signature score: sum of all features in each GO pathway.

Fig. S13.
Validation of gene silencing. HCAEC were treated with siRNA targeting NOTCH4 (A) or JAG1 (B) or with scrambled (SCR) control while exposed to LOSS for 48h using the Ibidi system. Expression levels of target genes were quantified by qRT-PCR (n=3-4). Differences between means were analysed using a paired ttest.

Notch signalling controls HCAEC proliferation under LOSS conditions.
(A) HCAEC were exposed to LOSS for 72h. To assess the role of JAG1 during endothelium repair, a scratch wound was made in the monolayer and the cells were treated with JAG1 blocking antibodies for 24h. Proliferation rate at the edge of the wound was then tested by using PCNA immunostaining (green). Nuclei were detected using DAPI (DNA; blue). (B, C) HCAEC were treated with either DAPT (γ-secretase inhibitor) or DMSO while exposed to LOSS or HSS for 72h using the Ibidi system. (B) Proliferation was quantified by immunofluorescence staining using antibodies against PCNA (green). Endothelium was co-stained (anti-CDH5; EC; red) and nuclei detected using DAPI (DNA; blue) (n=4). (C) Protein levels of JAG1 and DLL4 were quantified by immunoblotting and normalized to the level of PDHX (n=3). Differences between means were analysed using a paired t-test (A, B) or by ANOVA (C).

JAG1 reduces endothelial migration under LOSS conditions.
HCAEC were treated with siRNA targeting JAG1 or with scrambled (SCR) control while exposed to LOSS or HSS for 48h using the orbital system. A scratch was then made and cell migration was monitored for 24h (n=3). (A) Representative images are shown with the leading edges indicated. (B) Distance migrated was calculated and mean values are plotted over time. Differences between means were analysed by ANOVA.

Jag1 does not regulate endothelial proliferation at a HSS region of the aorta.
Endothelial cell proliferation was quantified at the outer curvature of the aortic arch, a HSS region, in Jag1 ECKO (n=6) mice and control mice (n=5). Mice were analysed 2 weeks post-tamoxifen (TAM) injection by en face immunostaining of the aorta using antibodies against Ki67 (red). Endothelium was co-stained (anti-CD31; EC; red) and nuclei detected using TO-PRO-3 (DNA; blue). Representative images are shown. The proportion of proliferative Ki67-positive cells were calculated. Differences between means were analysed using an unpaired t-test.

JAG1 controls proliferation in neighbouring endothelial cells.
Schematic diagram showing experimental setup (top). HCAECs transfected with JAG1 siRNA or control (SCR) siRNA were mixed with an equal number of SCR siRNA-transfected cells labelled with CellTracker Red. The cells were exposed to LOSS for 72 hours and proliferation of labelled (CellTracker+) and non-labelled (CellTracker-) cells was assessed by PCNA staining (green) (bottom left) and nuclei were co-stained using DAPI (DNA). Quantification of proliferation (n=4) (bottom right). Differences between means were analysed by two-way ANOVA.