Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling

Figures

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  Fig. 1. Calcification of pAVICs is attenuated with the addition of DETA NONOate (NO donor) or GSNO (S-nitrosylating agent). (A to D) Alizarin red staining and nodule formation of pAVICs cultured for 5 days under control conditions or with the addition of NO donor, 8-Br-PET-cGMP (cGMP analog), or GSNO. (E) Quantification of nodule number. (F to I) RUNX2 (red) expression with nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) is shown in four conditions and quantified in (J). (K to N) Total cellular S-nitrosylated proteins (green) counterstained with DAPI and quantified in (O). (P to S) DAR4M staining (red), which measures NO, is shown with DAPI (blue) counterstaining and is quantified in (T). Scale bars, 50 μm. *P ≤ 0.05 (two-tailed). For all experiments, Mann-Whitney test was performed. F.I., fluorescence intensity.

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  Fig. 2. Gene expression analysis of pAVICs exposed to NO donor or GSNO by scRNA-seq. (A) UMAP plot of scRNA-seq expression data shows two distinct populations of pAVICs cultured in the presence (blue) or absence (red) of NO donor. (B) Violin plots show down-regulation of ACTA2, TAGLN, VIM, and CNN1 with NO donor. Each dot in the violin plots represents gene expression in individual cells. Horizontal lines represent 0.05, 0.5, and 0.95 quantiles of gene expression. (C) Heatmap represents Z-scored expression of top 100 differentially expressed genes in individual cells from the four culture conditions on day 5. # and * indicate ACTA2 and VIM, respectively. (D) Heatmap demonstrating Z-scored, log2-transformed expression of NOTCH1 target genes in pAVICs in the presence and absence of NO donor.

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  Fig. 3. Mass spectrometric analysis of S-nitrosylated proteins in rat AVICs exposed to NO donor demonstrates an enrichment of ubiquitin-specific processing proteases and identifies USP9X. (A) Chord plot illustrates overrepresentation of NO-enriched S-nitrosylated proteins. Top 10 Gene Ontology pathways (GO Slim) and gene sets are shown. (B) Immunoblot shows S-nitrosylation of USP9X in the presence of NO donor, where negative ascorbate serves as a technical negative control. NICD and JAG1 immunoblots with ascorbate serve as biological negative controls. GAPDH, a known S-nitrosylated protein, serves as a biological positive control. Total load represents the initial cell lysates used to purify S-nitrosylated proteins. AU-rich element, adenylate/uridylate-rich elements.

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  Fig. 4. S-nitrosylation activates USP9X to deubiquitinate MIB1. HEK293 cells expressing MIB1-FLAG and HA-Ub were cultured in the presence of DETA NONOate (NO donor), GSNO (S-nitrosylating agent), or WP1130 (USP9X inhibitor). (A) After addition of MG132 and immunoprecipitation (IP) with anti-FLAG antibody, immunoblot with anti-HA antibody shows HA-Ub-MIB1-FLAG of various molecular weights in untreated condition (control) and with WP1130, indicating the presence of poly-Ub-MIB1. This is decreased with NO donor and GSNO and is quantified in (B). (C and D) Quantification of USP9X and MIB1-FLAG expression after normalization against GAPDH. (E) pAVICs were cultured in the presence and absence of NO donor for 5 days, and immunoblot for MIB1 is shown with and without the addition of MG132. (F) Quantification of MIB1 is shown after normalization against GAPDH. *P ≤ 0.05. For Western blots (WB), two-tailed unpaired t test was performed. F.C., fold change.

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  Fig. 5. Inhibition of USP9X causes reduced expression of MIB1 and NICD and increased RUNX2 expression in pAVICs. (A) Immunoblot shows decreased levels of MIB1 and NICD in pAVICs cultured with WP1130 for 5 days as compared to untreated cells (Control), while NO donor results in increased MIB1 and NICD. (B) Quantification of MIB1 and NICD expression is shown. (C) RUNX2 expression (red) is increased with WP1130, while RUNX2 is decreased with NO donor when compared to untreated cells (Control). Costained with nuclear DAPI (blue). (D) Quantification of RUNX2 expression is shown. (E) Addition of USP9X-siRNA to pAVICs reduced protein expression of USP9X and MIB1 as compared to scramble (Scr) control, which is quantified in (F). (G) With USP9x knockdown, nuclear NICD is reduced, and this is quantified in (H) after normalization with LAMIN A/C (nuclear). Cytoplasmic NICD was normalized with GAPDH (G and H). (I) After treatment with USP9X-siRNA, RUNX2 expression (red) is increased when compared to scramble (Scr) control cells, costained with nuclear DAPI (blue). (J) Quantification of RUNX2 expression is shown. Scale bars, 50 μm (C) and 20 μm (I). *P ≤ 0.05 (two-tailed). For immunostaining and WBs,…

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  Fig. 6. Aortic valve disease in mice with Usp9x deletion in endothelial cell lineage. (A) Breeding scheme to generate mice harboring endothelial and endothelial-derived cell deletion of Usp9x (Usp9xfl/fl; Tie2Cre). (B) Experimental scheme for Usp9xfl/Y; Cre+ males and Usp9xfl/Y; Cre− controls. (C) Survival in Usp9xfl/Y; Cre+ male mice at P10. (D) Aortic velocity measured by echocardiogram in Cre+ males at 6, 16, and 24 weeks of age compared to Cre− controls. (E) Doppler image shows aortic valve regurgitation in Cre+ male at 24 weeks of age as compared to Cre− control. Table shows incidence of aortic valve regurgitation in Cre+ and Cre− males. (F) Histological section of aortic valve stained with Russell-Movat pentachrome shows increased proteoglycan deposition in Cre+ males compared to Cre− controls. (G) Graph shows quantification of aortic valve area in Cre+ and Cre− males. (H) Alizarin red staining demonstrates calcification in Cre+ males. (I) Table shows incidence of calcification in Cre+ and Cre− males. Scale bars, 100 μm (pentachrome staining) and 20 μm (Alizarin red staining). *P ≤ 0.05 (two-tailed Mann-Whitney test).

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  Fig. 7. Calcified human aortic valves display reduced S-nitrosylation of USP9X along with decreased expression of MIB1 and NICD. (A) Immunoblot for USP9X shows significantly reduced S-nitrosylation in aortic valves from adults with moderate (AVD-1 and AVD-2) and moderate-severe (AVD-3 and AVD-4) CAVD when compared to uncalcified aortic valve (Control). S-nitrosylation of GAPDH is also shown. Absence of a band in the ascorbate-negative experiment served as the negative control. Total load shows protein expression in the cell lysates before purification of S-nitrosylated proteins where USP9X, MIB1, and NICD expression is reduced in calcified valves while RUNX2 is increased. GAPDH serves as a loading control. (B) Schematic showing the proposed model where S-nitrosylation of USP9X leads to MIB1 stabilization via deubiquitination, which increases ligand-mediated NOTCH1 activation in neighboring cells and the inhibition of calcification in physiological concentrations of NO. This pathway is abrogated under low concentrations of NO.