Targeted inhibition of PTPN22 is a novel approach to alleviate osteogenic responses in aortic valve interstitial cells and aortic valve lesions in mice

Figures

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  Fig. 1. PTPN22 expression is increased in aortic valves from CAVD patients. The aortic valves were collected from CAVD and non-CAVD patients. A-B Volcano plot and transcriptome sequencing analysis of the non-CAVD and CAVD groups indicated that PTPN22 expression is upregulated in tissue from CAVD patients. n = 3. C H&E, Masson’s trichrome, and Von Kossa staining for calcified valve leaflets. Scale bar = 200 μM. D Immunofluorescent staining of PTPN22 (red) in tissue (DAPI: blue, vimentin: green). n = 4. Scale bar = 100 μM. E-F The protein expression of PTPN22 in tissue was detected by immunoblotting. n = 6. G-H Human AVICs were stimulated with OM for different time periods. PTPN22 protein expression in human AVICs was detected by immunoblotting. n = 4. I-J Immunofluorescent staining was used to detect PTPN22 (red) expression in different mouse models of CAVD (DAPI: blue, WGA: green). n = 5. Scale bar = 50 μM. All data are shown as means ± SEM. *p < 0.05; **p < 0.01

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  Fig. 2. PTPN22 mediates OM-induced osteogenic responses in vitro. A-B AVICs were transfected with the PTPN22 expression vector (pCMV3-PTPN22) or empty vector ((pCMV3-control). The immunoblotting results show the expression of ALP and Runx2 in vitro. n = 3. C-D Cells were treated with PTPN22 siRNA following stimulation with OM. Representative western blot and quantitative data on the relative abundance of ALP, Runx2 and BMP2 in the different groups. Cells were transfected with pCMV3-PTPN22. Images of DCFH-DA staining E and TMRM staining (F). n = 4. Scale bar = 25 μm. AVICs were transfected with PTPN22 siRNA. Images of DCFH-DA staining (G) and TMRM staining (H). n = 4. Scale bar = 25 μm. I-J Western blot analysis shows the levels of NF-κB and ERK1/2 phosphorylation after PTPN22 knockdown. n = 4. All data are shown as the means ± SEM. *p < 0.05; **p < 0.01

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  Fig. 3. PTPN22 deletion mitigates aortic valve lesions in vivo. Echocardiographic data in a wire injury-induced CAVD mouse model. A-B Peak transvalvular jet velocity, aortic valve peak pressure, and AVA. C Image of aortic valves in the aortic valve calcification model. H&E staining of aortic valves after wire injury. Scale bar = 200 μm. D Masson’s trichrome staining. Scale bar = 50 μm. E Von Kossa staining. Scale bar = 50 μm. F-G Immunofluorescence images of the aortic valve (DAPI: blue, WGA: green). Scale bar = 50 μm. n = 5. Values are the means ± SEMs. #p < 0.05, ##p < 0.01 vs. sham group; *p < 0.05, **p < 0.01 vs. AVI + PTPN22−/− group

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  Fig. 4. Discovery and characterization of compound S18, a novel inhibitor of PTPN22. A The molecular structure of S18. B The binding affinities of S18/PTPN22 (Kd = 86.8 μM) and S18/GFP control (Kd = no binding) were detected by MST assay. Values are the means ± SEM. n = 3. *p < 0.05. The detailed docking mode of (C) N75/PTPN22 and (D) S18/PTPN22 with docking pock. E Cell viability of human AVICs treated with S18 for 3 days. n = 5. F Cell morphology of human AVICs treated with S18 for 3 days. Scale bar = 25 μm. G-H Alizarin Red S staining of cells cultured under different conditions. n = 4. Values are the means ± SEM. #p < 0.05 vs. the control group, *p < 0.05 vs. the OM group

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  Fig. 5. Piericidin diglycoside S18 exhibited a remarkable anti-osteogenic responses effect. Human AVICs were cultured with OM and treated with or without 0.5 µM S18 for 3 days. A-C Western blotting and quantification analysis showed the expression of ALP, Runx2 and BMP2 in human AVICs. n = 4. D-G Immunofluorescence staining of ALP (green), Runx2 (red) and BMP2 (green) in human AVICs (DAPI: blue). n = 5. Scale bar = 100 μm. H qRT-PCR was employed to measure ALP mRNA and Runx2 mRNA expression. n = 4. Values are the means ± SEM. #p < 0.05 vs. the control group, *p < 0.05 vs. the OM group

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  Fig. 6. Piericidin diglycoside S18 suppresses PTPN22-mediated osteogenic responses in vitro. Human AVICs were pretreated with S18 for 1 h followed by exposure to OM for 3 days. A-B The PTPN22 protein and mRNA levels were decreased in human AVICs treated with S18. n = 5. C Representative micrographs of the immunofluorescence staining of PTPN22 (red) expression in human AVICs treated with S18 (DAPI: blue). n = 5. Scale bar = 100 μm. Values are the means ± SEM. #p < 0.05, ##p < 0.01 vs. the control group, *p < 0.05; **p < 0.01 vs. the OM group. D-E Human AVICs were transfected with pCMV3-PTPN22 or pCMV3-control. Immunoblotting results show ALP, Runx2 and BMP2 expression in human AVICs. n = 3. Values are the means ± SEM. ##p < 0.01 vs. control group, **p < 0.01 vs. pCMV3-PTPN22 group. F-G AVICs were treated with nonspecific control or PTPN22-specific siRNA and treated with OM and S18 for the indicated times. Representative immunoblots and corresponding quantifications show that ALP, Runx2 and BMP2 expression was decreased in PTPN22 knockdown human AVICs. n = 5. Values are the means ± SEM. *p < 0.05, **p < 0.01, n.s. H-I Western blot analysis was used to detect the levels of NF-κB and ERK1/2…

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  Fig. 7. Piericidin diglycoside S18 protects the aortic valve against injury in an established CAVD model. Echocardiographic data in wire injury-induced CAVD moues model. A-B Peak transvalvular jet velocity, aortic valve peak pressure, and AVA. C-D Images of aortic valves in the aortic valve calcification model. H&E staining of aortic valves after wire injury. Scale bar = 200 μm. E Masson’s trichrome staining. Scale bar = 50 μm, F Von Kossa staining. Scale bar = 50 μm. G-H Immunofluorescence images of the aortic valve (DAPI: blue, WGA: green). Scale bar = 50 μm. n = 5. All data are the means ± SEM. #p < 0.05, vs. the sham group, *p < 0.05 vs. the AVI and S18 group