PTP1B Inhibition Improves Mitochondrial Dynamics to Alleviate Calcific Aortic Valve Disease Via Regulating OPA1 Homeostasis

Figures

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  Figure 1. PTP1B Was Highly Expressed in the Calcified Aortic Valves and the Osteogenic Differentiation of VICs (A) Representative hematoxylin and eosin (H&E) staining, Masson’s trichrome staining (Masson), and Von Kossa staining of the human aortic valve tissues from the normal control (NC) and calcific aortic valve disease (CAVD). Scale bar: 50 μm. (B) Representative PTP1B immunofluorescence staining images of human aortic valves from the NC and CAVD groups. Scale bar: 15 μm. (C) Quantitative analysis of the PTP1B-positive cells showed higher PTP1B expression in CAVD (n = 15) vs NC (n = 21). (D) The protein level of PTP1B in human aortic valve lysates was assessed by Western blot and quantified by normalizing to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level (n = 4). (E) Quantitative analysis of the relative PTP1B protein levels in human valvular interstitial cells (VICs) at passage 3 isolated from normal valves (VICs-NC) and calcified aortic valves (VICs-CAVD; n = 3). (F to H) Relative PTP1B protein levels in VICs after treatment with β-glycerophosphoric acid (β-GA) (F), Na2HPO4(G), and TGFβ (H) (n = 3). (I) Representative immunofluorescence staining images of PTP1B in aortic…

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  Figure 2. Inhibition and Knockdown of PTP1B Prevented β-GA–induced Osteogenic Differentiation of VICs In Vitro (A and B) Calcium nodule formation in human VICs was determined by alizarin red staining (A), and quantitative analysis by 405 nm spectrophotometry (B) showed that the PTP1B-specific inhibitor MSI-1436 (MSI, 20 μmol/L) treatment significantly attenuated β-GA–induced calcium nodule formation in VICs in contrast to dimethyl sulfoxide (DMSO) treatment (n = 4). Scale bar: 50 μm. (C and D) Expression of osteogenic markers, including alkaline phosphatase (ALP) and RUNX2, was assessed by western blot in VICs incubated with β-GA under MSI-1436 or DMSO treatment (n = 6). (E) ALP activity was measured using the ALP activity assay kit and normalized to total protein content (n = 3). (F and G) Alizarin red staining images (F) and quantification results (G) of VICs after β-GA treatment in the presence of PTP1B shRNA (PTP1Bsh) or control vector lentivirus transfection (n = 3). Scale bar: 50 μm. (H and I) Relative ALP and RUNX2 protein levels were decreased in VICs transfected with PTP1B siRNA (PTP1Bsi) in contrast to control vector under β-GA medium (n = 5). Values are mean ± SD. The analysis…

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  Figure 3. MSI-1436 Inhibited TGFβ-Induced Osteogenic and Myofibrogenic Differentiation of VICs In Vitro (A) The protein level of the osteogenic marker RUNX2 was assessed in TGFβ (10 ng/mL)–treated VICs with MSI-1436 (MSI, 20 μmol/L; n = 3) or DMSO. (B and C) Protein levels of the myofibrogenic markers, including fibronectin, collagen1a and αSMA, were assessed in TGFβ-treated VICs with MSI-1436 treatment or DMSO treatment by Western blot (n = 3 or 6). (D and E) Relative protein levels of fibronectin, collagen1a, and αSMA were decreased in VICs transfected with PTP1Bsi or control vector under TGFβ medium (n = 3 or 6). (F) The addition of TGFβ to VICs treated with MSI-1436 increased RUNX2 expression under β-GA medium (n = 3). (G) The relative protein level of p-Smad2 quantified by normalizing to the Smad2 level was decreased in VICs treated with MSI-1436 under β-GA medium (n = 3). Values are mean ± SD. Analysis of variance with Tukey's post hoc test was performed. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Abbreviations as in Figures 1 and 2.

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  Figure 4. MSI-1436 Attenuated Aortic Valve Calcification in LDLR−/− Mice (A) Schematic representation of the animal experiments. LDLR−/− male mice were divided into the following: 1) NC, saline with DMSO; 2) HFD, saline with DMSO; and 3) HFD saline with MSI-1436 (10 mg/kg, intraperitoneally, monthly). (B) Representative echocardiographic images of LDLR−/− mice fed a NC and an HFD with saline containing MSI-1436 or DMSO injection after 24 weeks. (C and D) Continuous-wave Doppler assessment of the aortic valve function indicated that mice in the HFD group (n = 22) showed a significant increase in transvalvular peak jet velocity (C) and mean gradient (D) compared with that of the NC group (n = 13), whereas MSI-1436 treatment (n = 17) reversed these changes. (E to G) Representative H&E staining, Masson’s trichrome staining (Masson), and Von Kossa staining images of aortic valves in LDLR−/− mice from different groups. Scale bar: 150 μm. Quantitative analysis of valve leaflet thickness (E) (n = 11, 10, 11), fibrosis (F) (n = 11, 12, 10), and calcium nodule deposits (G) (n = 10, 13, 12) was assessed by ImageJ. (H to J) Representative immunofluorescence staining images of osteogenic markers,…

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  Figure 5. MSI-1436 Preserved Mitochondrial Homeostasis in the Osteogenic Differentiation of VICs (A) The level of cellular reactive oxygen species (ROS) was detected by flow cytometry with DCFH-DA staining, and quantification of the mean fluorescence intensity of DCFH showed that MSI-1436 treatment decreased ROS generation in VICs cultured in β-GA medium (n = 4). (B) Representative MitoTracker Deep Red (MitoTracker) staining images of VICs treated with β-GA medium in the presence of MSI-1436 were used to observe mitochondrial morphology. Scale bar: 20 μm. (C and D) The level of ROS (C) and representative MitoTracker staining images (D) of VICs under TGFβ medium with or without MSI-1436 treatment. (E) Quantitative analysis of MitoTracker staining by flow cytometry showed that MSI-1436 reversed the mitochondrial mass decrease in VICs caused by β-GA treatment (n = 3). (F) Quantitative real-time polymerase chain reaction was performed to quantify the mitochondrial DNA (mtDNA) mean copy number, which was normalized to nuclear DNA level (n = 3). (G and H) Quantification of the mean fluorescence intensity of tetramethylrhodamine methyl ester (TMRM) staining (G) and the percentage of JC-1…

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  Figure 6. PTP1B Inhibition Preserved Mitochondrial Dynamics In Vivo and Regulated OPA1 Isoform Transition in VIC Osteogenesis (A) Representative immunofluorescence images of mitochondria stained by the mitochondrial marker TOMM20 in aortic valve leaflets of LDLR−/− mice in different groups. Scale bar: 15 μm. (B) Quantification of the percentage of TOMM20-positive cells showed that MSI-1436 treatment (n = 13) reversed the depletion in mitochondrial number in the presence of HFD (n = 15). (C) Representative mitochondrial immunofluorescence staining images of human normal valves and calcified aortic valves. Scale bar: 15 μm. (D) Quantitative analysis showed fewer mitochondria in CAVD vs the normal control subjects (n = 12). (E) Mitochondrial morphology was observed by a transmission electron microscope, and representative images showed significant changes in the mitochondrial number and morphology in VIC osteogenesis after MSI-1436 treatment. Scale bar: 1 μm. (F) Quantification of electronic respiratory chain-related genes by quantitative real-time polymerase chain reaction was normalized to ACTIN level (n = 3). (G and H) Protein levels of mitochondrial dynamics-related proteins, including…

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  Figure 7. PTP1B Inhibition Prevented VIC Osteogenesis by Directly Interacting With the Transition of the OPA1 Isoform (A) OPA1 proteolytic cleavages occurred at S1, S2, and S3 in exons 5, 5b, and 4b. Variant 1 (OPA1v1) contained the S1 site, whereas OPA1 variant 7 (OPA1v7) contained an extra S2 site. Variant 8 (OPA1v8) contained all 3 cleavage sites. (B) Three OPA1 variants were overexpressed in VICs, and Western blot of cell lysates from these cells showed exclusive overexpression of L- and S-OPA1 in VICs expressing OPA1v1 and OPA1v8 in contrast to control vector lentivirus, respectively, and the presence of both L- and S-OPA1 in cells expressing OPA1v7. (C to E) The relative protein level of RUNX2 in VICs transfected with OPA1v1 (C), OPA1v8 (D), and OPA1v7 (E) under β-GA treatment was assessed by Western blot (n = 3). (F) The relative RUNX2 protein level was increased in VICs transfected with YME1L1 siRNA (YME1L1si) or control vector under β-GA medium (n = 3). (G) The protein level of RUNX2 was decreased in VICs overexpressing YME1L1 (YME1L1over) under β-GA medium (n = 3). (H) Immunoprecipitation analysis of VICs with monoclonal anti-PTP1B antibody showed exclusive OPA1 protein…