Vascular smooth muscle–specific YAP/TAZ deletion triggers aneurysm development in mouse aorta

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC–specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8–Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

Supplemental Figure 2. Aneurysms in abdominal and thoracic aorta arise without gross pathology in the gastrointestinal tract.Panel A shows aortic whole mounts at two and eight weeks post-tamoxifen.Black and white images below are shown for clarity.Quantification of the aortic diameter between the dashed lines in A is shown in panel B (n≥5).Panel C shows a grossly normal gastrointestinal tract in control (Ctrl) and i8-YT-KO mice at two and eight weeks.We previously reported colonic pseudoobstruction, with enlargement of the caecum and colon following YAP/TAZ knockout using Myh11-CreER T2 .Only one out of 53 i8-YT-KO mice at eight weeks posttamoxifen exhibited colonic pathology consistent with obstruction.Student's t-test was used in panel B ***P<0.001.
Following vacuum centrifugation, samples were resuspended in 2% acetonitrile (Sigma) / 0.1% TFA (Sigma).Samples were analyzed in replicates on a Bruker timsTOF Pro mass spectrometer (Bruker Daltonics) operating in DIA PASEF mode with a 1.1 s cycle time and a TIMS ramp time of 100 ms, and the MS scan range was set to 100-1700 m/z.Peptides were separated using an Aurora column with captive spray insert (C18, 1.6 μM particles, 25 cm, 75 μm inner diameter; IonOptics) under controlled temperature at 60°C.A solvent gradient of buffer A (0.1% formic acid) and buffer B (99.9% acetonitrile/0.1% formic acid) was employed over a 42-minutes time period, at a flow rate of 600 nL/min.Protein identification was done using DIA-NN software (version 1.8.0) and the deep learning-based spectra approach to generate a comprehensive library (58) based on the UniProt FASTA database for mouse (UP000000589_10090, Oct. 2022).Protein intensity values were averaged based on the respective technical replicate.Data was log2 transformed and filtered by three valid values in each group.Two-sided Student's t-test with permutation-based FDR (<0.05) and 250 randomizations was used to identify significantly regulated proteins between groups.
Correlation analysis and volcano plots were generated in GraphPad Prism (v.9.5.1).The GO enrichment analysis was performed with R package clusterProfiler using the enrichGO function.MS raw files have been deposited to the public repository MassIVE with the identifier MSV000091464.

Transcriptomic analysis
After cDNA library construction, sequencing, and filtering, reads were mapped to the Mus musculus reference genome (GRCm38/mm10) using Hisat2.Read numbers mapping to each gene were obtained using FeatureCounts and converted to Fragments Per Kilobase of transcript per Million mapped reads (FPKM).Differential gene expression analysis was done using DESeq2 in R. Enrichment analysis was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) using clusterProfiler.

Flow cytometry
Aortic tissue was placed into tubes containing 1 mL digestion mix containing Collagenase I (450 U/ml), Collagenase XI (125 U/ml), DNAase I (60 U/ml), and Hyaluronidase (60 U/ml in Ca 2+ and Mg 2+ -free PBS).Tissues were finely minced and incubated for 60 minutes at 37°C with shaking (450 RPM).Single-cell suspensions were obtained by passing through 70 µm cell strainers followed by washing.Blood was isolated using cardiac puncture and subjected to red blood cell lysis using Ammonium Chloride lysis buffer for 3 minutes at room temperature.
Spleens were passed through 70 µm cell strainers followed by erythrocyte lysis with Ammonium Chloride lysis buffer for 3 minutes at room temperature.
Single-cell suspensions were stained for viability with LIVE/DEAD™ Fixable Aqua for 10 minutes on ice.Unspecific binding was prevented using CD16/32 for 5 minutes on ice, followed by extracellular antibody staining for 30 minutes on ice.Single-cell suspensions were stained in Mg 2+ and Ca 2+ -free PBS supplemented with 0.5% BSA and 2.5 mM EDTA.The following antibodies were used: Ly-6G (1A8), anti-Fibroblast (mEF-SK4), TCRb (H57-597), Ly-6C (HK1.4),CD8 (53-6.7),CD64 (X54-5/7. -KO female i8-YT-KO male Supplemental Figure 3. Male i8-YT-KO mice display earlier onset of decreased blood pressure than i8-YT-KO female mice.Mean, systolic and diastolic blood pressure (BP) obtained in awake mice by tail-cuff measurement method.Data has been segregated by gender.Student's t-test was used in all graphs; *P<0.05;**P<0.01;***P<0.001.Supplemental Figure 4. Proteoglycan accumulation in the thoracic aorta at two and eight weeks.Aneurysmal lesions were less prevalent in thoracic aorta at two weeks, but medial blue staining (acidic polysaccharides) using Movat pentachrome stain was typically present, and elastic lamellae (black) appeared less wavy (A).Blue staining of the media was further accentuated at eight weeks (B).Ki67 staining, a marker of cell proliferation, was increased in abdominal aorta at two weeks, mainly in the adventitia but occasionally in the media (M) as shown in panel C. DAPI (blue) was used as nuclear stain.L: arterial lumen, and green is autofluorescent elastin.two weeks.Volcano plots for mRNA (n=4) and protein (n=7) in panels A and B, gene ontology enrichment analysis for downregulated transcripts and proteins in C and D, and for upregulated transcripts and proteins in E and F. Panel G shows correlation between proteins and transcripts at two weeks.Student's t-test with permutation-based FDR was used in panel A-B; Pearson R was used in panel G.

Supplemental Figure 8 .
i8-YT-KO mice show arterial remodeling but maintained or increased contractility at two weeks after YAP/TAZ deletion.Caudal artery segments from 2-week control (Ctrl) and i8-YT-KO mice were mounted in myographs and stretched to a basal tension of 5 mN.The arterial circumference at this tension was calculated from outer wire distance and plotted (A, n≥21 mice and 43 arteries).Full circumference tension relationships confirmed outward remodeling (B).Force in response to 60 mM K + (C), cirazoline (D, n≥6 mice and 12 arteries), and vasopressin (E, n≥6 mice and 12 arteries) was unchanged or increased.Panel F shows RT-qPCR for Acta2 (n=6) and Mylk (n=7) in the abdominal aorta at two weeks.Panel G shows immunofluorescence staining for MLCK in abdominal aorta and panel H shows a western blot for MLCK in 2-week thoracic aorta (n=6).In contrast to the clear-cut reduction of MLCK in abdominal aorta at two weeks, no apparent change was seen in caudal artery upon YAP/TAZ deletion (I, bottom).DAPI (blue) was used as nuclear stain.Two-way ANOVA and Bonferroni post-tests were used in panel B, D, E; Student's t-test was used in panel C, F (second graph); *P<0.05;**P<0.01;***P<0.001.Increased neutrophil and inflammatory monocyte percentages in blood and spleen.Panel A shows RT-qPCR for Il6 in thoracic aorta at two weeks (n≥3) and eight weeks (n≥5) after YAP/TAZ deletion.Panels B and C show flow cytometry data for blood and spleen from Ctrl (orange) and i8-YT-KO mice (blue).Panel D outlines the gating strategy in the flow cytometry experiment.Student's t-test was used in panel A (first graph), B, C; Mann-Whitney was used in panel A (second graph); *P<0.05;**P<0.01;***P<0.001.

9 .
Aortae from i8-YT-KO mice display accumulation of macrophages and activation of the STING-pathway.Panel A shows staining for macrophages (CD68, red) in abdominal aorta eight weeks post-tamoxifen.Dashed lines demarcate the media and blue line depicts neointima border.Macrophage staining was seen both in the adventitia and in the neointima.Panel B shows RT-qPCR for Sting1 in thoracic (n≥4) and abdominal (n≥3) aorta.Panel C shows western blots for total (t)-and phospho (p)-TBK1 in thoracic aorta (n=6).The p-TBK1/t-TBK1 ratio was significantly increased in thoracic aorta as shown in the right graph.DAPI (blue) was used as nuclear stain.A: adventitia, NI: neointima, L: lumen.Student t-test was used in panel B, C (second graph); Mann-Whitney was used in panel C (first graph); *P<0.05;** P<0.01; ***P<0.001.

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Figure 10D t-STING