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PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by <t>ELISA</t> in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test
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PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by <t>ELISA</t> in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test
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PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by <t>ELISA</t> in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test
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PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by <t>ELISA</t> in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test
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PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by <t>ELISA</t> in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test
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PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by ELISA in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test

Journal: Signal Transduction and Targeted Therapy

Article Title: Cyclic guanosine monophosphate-protein kinase G signaling attenuates aortic valve calcification through ULK1-mediated autophagy

doi: 10.1038/s41392-026-02624-5

Figure Lengend Snippet: PKGI haploinsufficiency exacerbates aortic valve calcification both in vivo and in vitro. a – c Echocardiographic assessment of transvalvular peak jet velocity ( a , b ) and LVEF ( c ) in sham-operated mice and mice subjected to AVWI with indicated genotypes (sham, n = 7; AVWI + WT, n = 8; AVWI+ Prkg1 +/− , n = 7). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. d , e Representative Masson’s trichrome staining (d) and quantification of aortic valve leaflet thickness ( e ) from the indicated groups. Scale bar, 500 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. f , g Representative Alizarin Red staining ( g ) and quantification of valve leaflet calcification ( f ) in the indicated groups. Scale bars, 500 μm (5×) and 100 μm (40×). Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. h , i Representative immunofluorescence images ( h ) and quantification ( i ) of RUNX2 (red) in aortic valve leaflets; nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. j Serum cGMP concentrations measured by ELISA in sham, AVWI + WT and AVWI + Prkg1 +/− mice. Data are presented as mean ± SEM, one-way ANOVA. k – m Representative images ( k , l ) and quantification ( m ) of Alizarin Red staining and ALP staining in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM as indicated. Scale bar, 200 μm. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test. n RT-qPCR analysis of RUNX2 and BMP2 mRNA levels in hVICs transfected with control siRNA or siRNA targeting PKGI and cultured in OM. n = 6 independent experiments. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test

Article Snippet: Serum cGMP levels were quantified via a commercial enzyme-linked immunosorbent assay (ELISA) kit (E-EL-0083; Elabscience, Wuhan, China) according to the manufacturer’s instructions.

Techniques: In Vivo, In Vitro, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Transfection, Control, Cell Culture, Quantitative RT-PCR