Fig 1: OPN-N had a causal link of pyroptosis and inflammation through NF-KB mediated signaling pathways in VSMCs. (A) qPCR analyses of the mRNA expression level of NLRP3, Caspase-1, IL-1β, and IL-18 at different OPN-N concentrations. (B) Western blotting analyses of the protein levels of NF-κB in VSMCs, and quantification of NF-κB protein expression. (C,D) Western blotting analyses of the protein levels of NLRP3, Caspase-1, IL-1β, and IL-18 in VSMCs (C), and quantification of these pyroptosis-related inflammatory factors protein expression (D). β-actin was used as an internal control. (E) Immunofluorescence staining of NLRP3, Caspase-1, IL-1β, and IL-18 in mice under different treatments (left), quantification of these pyroptosis-related inflammatory factors area (right). Scar bar 50 μm. Data are expressed as means ± SEM; n = 3 per group. ∗P < 0.05 vs the Thrombin + OPN-N + SN50 group; ∗∗P < 0.05 vs the Thrombin and Thrombin + SN50 groups. Abbreviations: IL, interleukin; NF, nuclear factor; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; OPN-N, osteopontin N-terminal fragment.
Fig 2: The extracellular domain of syndecan‐4 is shed and cleavage of osteopontin is increased during advanced stages of extracellular matrix remodeling after pressure overload. A, Left ventricular syndecan‐4 mRNA at different time points after aortic banding (AB). The line indicates sham levels. The 24‐hour, 7‐day, and 21‐day time points were previously published.40 B, Syndecan‐4 mRNA in myocardial biopsies from patients with aortic stenosis. C, Immunoblot (bottom panel) and quantification (top panel) of the 15‐kDa extracellular syndecan‐4 fragment representing the shed ectodomain and full‐length syndecan‐4 (20–25 kDa) in myocardium after 24 hours, 7 days, and 21 days of AB. D and E, Immunoblot and quantification of full‐length osteopontin (FL‐OPN) and thrombin‐cleaved osteopontin (cl‐OPN) after 24 hours (D) and 21 days (E) of AB. Collagen 1a2 (F) and collagen 3a1 (G) mRNA expression in left ventricular tissue lysate from wild‐type (WT) and syndecan‐4 knockout mice subjected to 21 days of AB. mRNA levels were normalized to GAPDH for mouse samples and to Rpl32 for human samples. Student t test was used to determine significant differences in A through E. Two‐way ANOVA with Tukey's multiple comparisons test was used to determine significant differences in F and G. Numbers are indicated in graphs. n.s. indicates not significant. *P<0.05, **P<0.01, ***P<0.005.
Fig 3: M5Ab inhibits MMP2 and MMP9 expression in mice by blocking the expression of OPN-N. (A,B) Immunohistochemistry staining of MMP2 and MMP9 in mice under different treatments (A), quantification of MMP2 and MMP9 area (B). Scar bar 100 μm. (C) Western blotting analyses the protein levels of MMP2 and MMP9 in mice, and quantification of MMP2 and MMP9 protein expression. β-actin was used as an internal control. Data are expressed as means ± SEM; n = 3 per group. ∗P < 0.05 vs the Ang II + M5Ab group; ∗∗P < 0.05 vs the control and control + M5Ab groups. Abbreviations: Ang II, angiotensin II; MMP, matrix metalloproteinase; and L, lumen.
Fig 4: Administration of M5Ab attenuates the expression of OPN-N in ApoE-/- mice with AAA. (A) ELISA analyses of the levels of serum OPN-N in mice under different treatments. (B) Western blotting for OPN-N in mice under different treatments (left), bar graph showing quantification of OPN-N protein expression (right). ß-actin was used as an internal control. (C,D) Representative images of aortic immunohistochemistry staining to detect OPN-N expression in mice (black arrows), quantification of OPN-N area. Scar bar 200 µm, boxed areas; Scar bar 50 µm, main images. (E,F) OPN-N immunofluorescence staining of aortic tissues in mice and quantification of OPN-N intensity (Scar bar 50 µm), boxed areas in F are shown at higher magnification below (Scar bar 20 µm). Data are expressed as means ± SEM; n = 3 per group. *P < 0.05 vs the Ang II + M5Ab group; **P < 0.05 vs the control and control + M5Ab groups. Abbreviations: Ang II, angiotensin II; OPN-N, osteopontin N-terminal fragment. DAPI staining of nuclei: blue; OPN-N staining: red. Ta, indicates tunica adventitia; Int, intima; Med, media; L, lumen; and IT, intraluminal thrombus.
Fig 5: Cardiac expression and cleavage of osteopontin is induced by left ventricular pressure overload. A, Osteopontin mRNA levels in left ventricular tissue after aortic banding (AB) relative to sham-operated animals for each time point. The 24-hour and 7-day time points were previously published.7 B, Osteopontin protein in mouse plasma 3 days after AB. C, Left ventricular weight (LVW) relative to tibia length (TL) in sham and 3-day AB mice. D, Osteopontin mRNA levels after stretching cardiac fibroblasts in vitro. E, Micrographs of full-length osteopontin (FL-OPN) and N-terminal fragment of thrombin-cleaved osteopontin (cl-OPN) detected by immunohistochemistry of left ventricular tissue sections from mice 3 days after AB or sham operation (bar=200 µm). F, Quantification of osteopontin-positive pixels relative to total number of pixels analyzed of micrographs, as represented in E. G, cl-OPN protein levels relative to FL-OPN in plasma from the coronary sinus and radial artery of patients with aortic stenosis (AS) receiving blood cardioplegia or crystalloid cardioplegia. H, Osteopontin mRNA normalized to Rpl32 in myocardial biopsies from patients with AS and controls. Student unpaired t test was used to determine significant changes. Numbers are indicated in graphs. *P<0.05, **P<0.01, ***P<0.005.
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