The Senokine Gas6 Promotes Aortic Valve Calcification Through CaMKII–DRP1–Mediated Terminal Scission of Mitochondrial‐Derived Vesicles

Figures

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  FIGURE 1. Mitochondrial‐derived vesicles act as pro‐calcific mediators in valvular calcification. (A) Schematic illustration of the ex vivo hemodynamic simulation system for vesicle collection from human aortic valve leaflets under physiological pulsatile flow conditions. (B) Immunoblot analysis of EV markers (CD9, CD63), mitochondrial outer membrane protein TOM20, and cytoplasmic marker β‐actin (negative control) in EVs isolated from the perfusate of non‐CAVD and CAVD valve leaflets. (C) Nanoparticle tracking analysis (NTA) showing the size distribution of isolated EVs from nonCAVD and CAVD perfusates. (D and E) Representative nano‐flow cytometry plots (D) and quantification (E) of TOM20‐positive mitochondrial‐derived vesicles (MDVs) in EVs isolated from nonCAVD and CAVD valve perfusates. (F) Schematic illustration of the immunoadsorption‐based MDV enrichment strategy using TOM20‐conjugated magnetic beads. (G) Alizarin Red S staining (scale bar, 200 μm) of naïve recipient hVICs following treatment with MDV preparations enriched from nonCAVD (MDVnonCAVD) or CAVD (MDVCAVD) valve tissues. (H) Immunoblot analysis of RUNX2 (runt‐related transcription factor 2), OPN (osteopontin), and BMP2…

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  FIGURE 2. Senescence‐associated features and Gas6 upregulation in CAVD. (A) Representative histological images of nonCAVD and CAVD valve sections stained with von Kossa and Alizarin Red S to detect calcium deposition, and modified Masson's trichrome and Picrosirius Red to assess collagen deposition and fibrotic remodeling. Scale bars, 500 μm. (B, C) Representative immunoblots (B) and densitometric quantification (C) of p16INK4A and p21 protein expression in nonCAVD and CAVD human valve tissues. GAPDH served as the loading control. (D) Representative low‐ and high‐magnification senescence‐associated β‐galactosidase (SA‐β‐gal) staining images of nonCAVD and CAVD human valve tissues. Scale bars, 500 μm (left) and 20 μm (right). (E–G) Representative immunofluorescence co‐staining images of p16INK4A (green) and RUNX2 (magenta) (E), and p21 (green) and RUNX2 (magenta) (F), together with quantification of median nuclear p16INK4A and p21 fluorescence intensity (G), in human non‐CAVD and CAVD valve leaflets. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (H and I) Representative immunoblots (H) and densitometric quantification (I) of p16INK4A and p21 protein expression in hVICs…

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  FIGURE 3. Gas6 promotes hVIC calcification and osteogenic programming. (A–E) Time‐course analysis of Gas6 and osteogenic marker expression during OM treatment. (A) Representative immunoblots of Gas6, RUNX2, BMP2, and OPN in hVICs cultured in control medium (CM) or pro‐osteogenic medium (OM) for 3, 7, and 14 days. GAPDH served as the loading control. (B–E) Densitometric quantification of Gas6 (B), RUNX2 (C), OPN (D), and BMP2 (E). (F–J) Gas6 and osteogenic marker expression in hVICs treated with siCtrl (non‐targeting control siRNA) or siGas6 (Gas6‐targeting siRNA) under CM and OM conditions. (F) Representative immunoblots of Gas6, RUNX2, OPN, and BMP2. GAPDH served as the loading control. (G–J) Densitometric quantification of Gas6 (G), RUNX2 (H), OPN (I), and BMP2 (J). (K–M) Immunofluorescence images and quantification of RUNX2 and BMP2 in siCtrl and siGas6 hVICs under CM and OM conditions. (K) Representative images of RUNX2 (yellow) and BMP2 (magenta) staining in siCtrl and siGas6 hVICs under CM and OM conditions. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (L, M) Quantification of mean fluorescence intensity of RUNX2 (L) and BMP2 (M). (N) Representative Alizarin Red S…

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  FIGURE 4. Gas6 depletion impairs MDV release by inducing a terminal scission defect. (A, B) Representative nano‐flow cytometry plots (A) and quantification (B) of MDVs in conditioned media from hVICs transfected with non‐targeting control siRNA (siCtrl) or Gas6‐targeting siRNA (siGas6) under OM conditions. (C) GSEA of the “Mitochondrial Fission” gene set from MitoCarta3.0 in siGas6 versus siCtrl hVICs under OM conditions. (D–G) Live‐cell imaging, time‐resolved three‐dimensional reconstruction, and quantification of donut‐shaped mitochondria in siCtrl and siGas6 hVICs under OM conditions. (D) Time‐lapse montage showing mitochondrial curling and self‐fusion to form donut‐shaped structures in siGas6 hVICs. Time stamps indicate seconds. Scale bar, 1 μm. See also Video S3. (E) Three selected time points from the time‐resolved three‐dimensional reconstruction of the same mitochondrial‐curling event shown in (D). The continuous reconstruction is provided in Video S4. (F) Representative live‐cell images and (G) quantification of the proportion of donut‐shaped mitochondria in siCtrl and siGas6 hVICs. Scale bars, 30 μm (overview) and 2 μm (inset). (H–L) Time‐lapse imaging, time‐resolved…

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  FIGURE 5. Gas6 promotes MDV scission and calcification through post‐translational activation of the dynamin‐related protein 1 (DRP1) machinery. (A) Schematic illustration of MDV biogenesis from donut‐shaped mitochondria in hVICs. MIRO1/2 facilitate membrane protrusion, MFF recruits DRP1 to the budding neck, and DRP1 executes terminal membrane scission to release the MDV. (B) Immunoblots of p‐DRP1 (Ser616), total DRP1, mitochondrial Rho GTPase 1 (MIRO1), MIRO2, mitochondrial fission factor (MFF), and fission 1 protein (FIS1) in siCtrl and siGas6 hVICs cultured under CM or OM conditions. GAPDH served as the loading control. See Figure S5A–E for densitometric quantification. (C and D) Representative nano‐flow cytometry plots (C) and quantification (D) of TOM20‐positive MDVs in conditioned media from hVICs transduced with control adenovirus (AdCtrl) or Gas6‐overexpressing adenovirus (AdGas6) under the indicated conditions. Mdivi‐1 was used to inhibit DRP1. (E) Schematic illustration of the functional MDV transfer assay. MDVs were collected from conditioned media of donor hVICs under the indicated conditions and applied to naïve recipient hVICs maintained in control medium. (F, G) Alizarin Red…

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  FIGURE 6. A Ca2+/CaMKII signaling axis mediates Gas6‐induced DRP1 activation. (A) GO enrichment analysis dot plot of RNA‐seq data from siGas6 and siCtrl hVICs under OM conditions. (B) Representative live‐cell fluorescence images of intracellular Ca2+ levels detected using Fluo‐8 in siCtrl and siGas6 hVICs under CM and OM conditions. Scale bars, 20 μm. See Figure S6A for quantification. (C) Representative immunoblots of p‐CaMKII and CaMKII in siCtrl and siGas6 hVICs under CM and OM conditions. GAPDH served as the loading control. See Figure S6D for quantification. (D and E) Representative nano‐flow cytometry plots (D) and quantification (E) of MDVs in conditioned media from hVICs under the indicated conditions. KN‐93 was used to selectively inhibit CaMKII. (F) Schematic illustration of the MDV functional transfer assay. (G and H) Alizarin Red S staining (G) and immunoblots of RUNX2, OPN, and BMP2 (H) in naïve recipient hVICs following the MDV transfer assay. GAPDH served as the loading control. See Figure S6G–I for densitometric quantification. Scale bars, 200 μm. (I) Immunoblots of p‐CaMKII, CaMKII, p‐DRP1, DRP1, MIRO1, and MIRO2 in AdGas6‐transduced hVICs treated with or without KN‐93.…

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  FIGURE 7. Gas6 deletion attenuates AWI‐induced aortic valve stenosis and calcification in vivo. (A–D) Echocardiographic assessment of transvalvular haemodynamics in wild‐type (WT) and Gas6−/− mice following sham or aortic wire injury (AWI) surgery. (A) Representative color Doppler echocardiographic images of transvalvular blood flow. (B) Representative pulsed‐wave Doppler tracings of transvalvular flow velocity profiles. (C, D) Quantification of peak transvalvular jet velocity (C) and effective orifice area (D). (E–J) Histological analysis of aortic valve leaflets from WT and Gas6−/− mice following sham or AWI surgery. (E, F) Hematoxylin and eosin (H&E) staining images (E) and quantification of leaflet thickness (F). (G, H) Von Kossa staining images (G) and quantification of calcium deposition area (H). (I, J) Modified Masson's trichrome staining images (I) and quantification of collagen fiber area (J). Scale bars, 100 μm (E, G, I). (K–N) Immunofluorescence analysis of Gas6 and RUNX2 expression in aortic valve leaflets from WT and Gas6−/− mice following sham or AWI surgery. (K and L) Representative immunofluorescence images (K) and quantification of mean fluorescence intensity (L) for Gas6…