Palmdelphin Deficiency Evokes NF-κB Signaling in Valvular Endothelial Cells and Aggravates Aortic Valvular Remodeling

Figures

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  Figure 1. Characteristics of Palmd Knockout Mice (A) Western blot analysis of PALMD expression in lung tissues from Palmd+/+ and Palmd−/− mice (n = 4 mice per group). Two-month-old Palmd+/+ and Palmd−/− mice were used. (B) Representative immunostaining of PALMD proteins in Palmd+/+ and Palmd−/− aortic valves. Two-month-old Palmd+/+ and Palmd−/− mice were used. (C) Body weight of 6- and 11-month-old Palmd+/+ and Palmd−/− mice. (D) Heart weight-to-body weight ratio of 13-month-old Palmd+/+ and Palmd−/− mice. (E and F) Left ventricular ejection fraction (EF) (E) and left ventricular outflow tract velocity (F) assessed by echocardiography in 6- to 13-month-old Palmd+/+ and Palmd−/− mice. (C to F) Data are presented as mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001. P values were calculated using an unpaired Student's t-test.

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  Figure 2. PALMD Deletion Leads to Valve Function Decline in Apoe−/− Mice of Advanced Age (A to C) Plasma triglyceride level (A), total cholesterol level (B), and body weight (C) of Apoe−/− and double knockout (DKO) mice. (D) En face Oil Red O Staining and quantification (E) of aortas from Apoe−/− and DKO mice. (F) Heart weight and the ratio of heart to body weight (G) of Apoe−/− and DKO mice. (H to J) Left ventricular ejection fraction (EF) (H), left ventricular outflow tract velocity (I), and mean gradient (J) were assessed by echocardiography in Apoe−/− and DKO mice. All data are from 14-month-old Apoe−/− and DKO mice and are presented as mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001. P values were calculated using an unpaired Student's t-test.

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  Figure 3. PALMD Deficiency Leads to Valvular Remodeling (A) Representative hematoxylin and eosin staining of Apoe−/− and double knockout (DKO) aortic valves. (B) Leaflet thickness of Apoe−/− and DKO aortic valves. Data are presented as mean ± SD. (C) Representative Movat staining of Apoe−/− and DKO aortic valves. (D) Percentage of glycosaminoglycan (blue)-positive area in Apoe−/− and DKO aortic valves. (E) Representative Movat staining of Palmd+/+ and Palmd−/− aortic valves. (F) Percentage of glycosaminoglycan (blue)-positive area in Palmd+/+ and Palmd−/− aortic valves. (G) Representative Sirius Red staining of Palmd+/+ and Palmd−/− aortic valves. (H) Percentage of collagen (red)-positive areas in Palmd+/+ and Palmd−/− aortic valves. ∗P < 0.05. Apoe−/− and DKO mice were age 14 months, and Palmd+/+ and Palmd−/− mice were at age 13 months. ∗P < 0.05. All P values were calculated using an unpaired Student's t-test.

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  Figure 4. PALMD Affects Endothelial Cell Characteristics and Is Involved in OSS-Induced EndMT (A) Immunofluorescent staining of PALMD (green) and PECAM1 (red) in wild-type mouse aortic valves. Nuclei are stained blue with 4’,6-diamidino-2-phenylindole (DAPI). (B) quantitative real-time polymerase chain reaction analysis of PALMD and PECAM1 mRNA in primary valvular endothelial cells (VECs) and valvular interstitial cells (VICs), all normalized to glyceraldehyde 3-phosphate dehydrogenase. ∗∗∗P < 0.001. P values were calculated by unpaired Student's t-test. (C to E) Violin plot of PALMD (C), PECAM1 (D), and TIE1 (E) expression in VEC-1 and -2 clusters from normal aortic valve leaflets and aortic valve leaflets with calcific aortic valve disease. (F) Quantitative real-time polymerase chain reaction analysis of PALMD, ACTA2, VIM, and PECAM1 mRNA in primary VECs in a static state, all normalized to glyceraldehyde 3-phosphate dehydrogenase. Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01. (G) Quantitative real-time polymerase chain reaction analysis of PALMD, ACTA2, VIM, SNAIL1, and PECAM1 mRNA in primary VECs under OSS flow (4.5 dyn) for 24 hours. Data are presented as mean ± SD. ∗P <…

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  Figure 5. PALMD Deficiency Induces TNF and NF-κB Signaling VECs were transfected with scrambled (siCtrl) or PALMD (siPALMD) siRNA (50 μmol/L) for 48 hours under static conditions. (A) Volcano plots for differentially expressed genes (DEGs) in VECs transfected with siCtrl or siPALMD. (|fold change [FC]|>1.2; P < 0.05). (B) Top 10 up-regulated enriched pathways in VECs transfected with siCtrl or siPALMD. (C) TNF signaling pathway defining genes are shown in heatmap. (D) Quantitative real-time polymerase chain reaction analysis of TNF signaling pathway-related genes in VECs transfected with siCtrl or siPALMD (50 μmol/L) for 48 hours under static conditions. Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01. P was calculated using an unpaired Student's t-test. (E and F) Public sc-sequencing data of human aortic valves were reanalyzed. Gene set enrichment analysis (GSEA) probing for enrichment of the TNF signaling pathway (E) and NF-κB signaling pathway (F) active gene signatures. Abbreviations as in Figure 4.

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  Figure 6. PALMD Interacts With TNIP1 and Regulates NF-κB–Induced EndMT (A) Venn diagram of PALMD binding proteins (top) vs TNF and NF-κB signaling pathway genes from the Pathway Database (bottom). (B) The protein-protein interaction network constructed by the STRING database. (C) Coimmunoprecipitation of PALMD and TNIP1 in VECs. Lysates were immunoprecipitated with an anti-PALMD antibody. The precipitates and lysates were analyzed by immunoblotting with anti-PALMD and anti-TNIP1 antibodies. (D) 293T cells were cotransfected with FLAG-IKBKG, HA-ubiquitin, and HA-TNFAIP3, in addition to either PALMD siRNA-1 or PALMD siRNA-2. Cell extracts were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-HA antibodies. Immunoblotting with anti-FLAG, anti-TNFAIP3, and anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies was performed on the total extracts. (E and F) Western blot analysis (G) and quantification (H) of p-p65 and PALMD expression in total lysates of VECs transfected with si scramble or si PALMD (50 μmol/L) for 48 hours under static conditions. Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01. P was calculated using an unpaired Student's t-test. (G)…

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  Figure 7. PALMD Overexpression Ameliorated Decline of Aortic Valve Function and Valvular Remodeling in HFD Fed Apoe−/− Mice 6-week-old Apoe−/− mice were injected with adeno-associated virus (AAV)-eGFP or -Palmd (5 × 1011 v.g/mouse) and fed a high-fat diet (TD.88137) for 7 months. (A) Immunohistochemical staining of PALMD in aortic valves from Apoe−/− mice injected with AAV-eGFP or AAV-Palmd. (B to D) Left ventricular ejection fraction (EF) (B), left ventricular outflow tract velocity (C), and mean gradient (D) assessed using echocardiography in mice injected with AAV-eGFP and -Palmd. Data are presented as mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001. (E) Representative hematoxylin and eosin staining of aortic valves in mice injected with AAV-eGFP and -Palmd. (F) Leaflet thickness of the aortic valves in mice injected with AAV-eGFP and -Palmd. Data are presented as mean ± SD. ∗P < 0.05. (G) Representative Movat staining of aortic valves in mice injected with AAV-eGFP and -Palmd. (H) Percentage of glycosaminoglycan (blue)-positive area in AAV-eGFP and -Palmd aortic valves. ∗P < 0.05. All P values were calculated using an unpaired Student's t-test.