Adipokine Nesfatin‐1 Mediates Endothelial Dysfunction by Suppressing MEF2B / BMPR1A During Pulmonary Vascular Remodeling

Figures

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  Figure 1. Dysfunction of PVAT and key adipokine nesfatin‐1 in PH. A, PVAT detected by immunofluorescent analysis. Tissue sections were immunostained with perilipin 1 and α‐SMA; nuclei were stained with DAPI. B, Expression of UCP‐1, PRDM‐16, CIDEA, and leptin in lung tissues of SuHx–PH mouse model detected by western blot (n=4). C, Bioinformatic analysis of differentially expressed genes in data set GSE113439 and differential analysis of cardiovascular disease–related adipokines. D, Verification of nesfatin‐1 expression levels in lung tissues from control and SuHx mice (n=3). E, Verification of nesfatin‐1 expression levels in differentiated adipocytes (n=3), and levels in human serum samples from healthy controls and patients with PAH. (n=19–25). Student's unpaired t test was used. C4W indicates control at 4 weeks; CIDEA, cell death–inducing DNA fragmentation factor α–like effector A; PAH, pulmonary arterial hypertension; PH, pulmonary hypertension; PRDM‐16, PR domain containing 16; PVAT, perivascular adipose tissue; α‐SMA, α‐smooth muscle actin; SuHx, Sugen 5416 combined with hypoxia; and UCP‐1, uncoupling protein 1.

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  Figure 2. Protective role of browning of white adipose tissue in an SuHx–PH mouse model. A, Representative hematoxylin and eosin staining and CD31/α‐SMA immunofluorescence of pulmonary arterioles from C4W–DMSO (n=6), C4W–β3‐AR (n=7), SuHx‐DMSO (n=5), and SuHx‐β3‐AR (n=7) groups. RVSP was measured in all 4 groups. Pulmonary arteriole medial thickness was quantified and stratified according to external diameter (0–25, 26–50, 51–75, and 76–100 μm). B, Representative WGA staining of the right ventricle derived from the 4 groups, and right ventricular hypertrophy of the above groups indicated by RVHI of mice. C, Echocardiography showing the representative figures and statistical analysis of PV peak velocity, PAT, TAPSE, RVWTs and RVWTd (n=4). One‐way ANOVA was used with a Bonferroni post hoc test. β3‐AR indicates adrenergic receptor‐β3 agonist; C4W, control at 4 weeks; PAT, pulmonary acceleration time; PV, pulmonary valve; RVHI, right ventricular hypertrophy index; RVSP, right ventricular systolic pressure; RVWT, right ventricular free wall thickness; RVWTd, right ventricular free wall thickness during diastole; RVWTs, right ventricular free wall thickness during systole; α‐SMA, α‐smooth muscle…

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  Figure 3. Alleviation effect of nesfatin‐1 silence on SuHx–PH mediated by AAV nesfatin‐1 shRNA. A, Immunofluorescence staining of AAV‐ZsGreen (green) in the adipocytes of lung tissues, showing the successful transfection of AAV vector. B, RVSP of mice from four groups, C4W‐AAV‐NC (n=6), C4W‐AAV‐Nucb2 shRNA (n=8), SuHx‐AAV‐NC (n=8) and SuHx‐AAV‐Nucb2 shRNA (n=7). C, Hematoxylin and eosin staining and CD31/α‐SMA immunofluorescent staining of pulmonary arterioles from the 4 groups and percent medial thickness of pulmonary arterioles of mice grouped by external diameter (0–25, 26–50, 51–75 and 76–100 μm) in different groups. D, Right ventricular hypertrophy of the above groups, including RVHI and RV/body weight ratio. E, Echocardiography showing the representative figures and statistical analysis of PV peak velocity, PAT, TAPSE, RVWTs, and RVWTd (n=4). One‐way ANOVA was used with a Bonferroni post hoc test. AAV indicates adeno‐associated virus; C4W, control at 4 weeks; NC, negative control; PAT, pulmonary acceleration time; PV, pulmonary valve; RV, right ventricular; RVHI, right ventricular hypertrophy index; RVSP, right ventricular systolic pressure; RVWT, right ventricular free wall…

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  Figure 4. 3T3‐L1 adipocyte‐derived nesfatin‐1 secretion promoted by Th17/interleukin‐17/PI3K/AKT/mTOR. A, 3T3‐L1 adipocytes and T lymphocytes coculture model and concentration of nesfatin‐1 detected by ELISA in 3T3‐L1 adipocytes monoculture, 3T3‐L1 adipocytes+T lymphocytes coculture and 3T3‐L1 adipocytes+Th17 cells coculture groups (n=3). B, Western blot analysis (n=4) of PI3K/AKT/mTOR pathway activation in 3T3‐L1 adipocytes treated with or without interleukin‐17 and PI3K/AKT/mTOR inhibitor LY294002 when inducing differentiation. C, Western blot analysis (n=3) of nesfatin‐1 in 3T3‐L1 adipocytes treated with or without interleukin‐17 and PI3K/AKT/mTOR inhibitor LY294002 when inducing differentiation. The experiments were performed at least in 3 independent assays. One‐way ANOVA was used with a Bonferroni post hoc test. IL‐17 indicates interleukin‐17; mTOR, mammalian target of rapamycin; p‐mTOR, phosphorylated mammalian target of rapamycin; and Th17, T helper 17.

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  Figure 5. Endothelial dysfunction induced by nesfatin‐1. A, Spontaneous formation of capillaries by hPAECs treated with or without nesfatin‐1 for 24 hours. The result is expressed as the numbers of tubes and total tube length per field. B, Adhesion of hPAECs to culture plate wells when treated with or without nesfatin‐1 for 24 hours. Adherent cells were counted and expressed as the mean numbers of cells per high‐power field. C, hPAEC apoptosis evaluated by TUNEL analysis. D, hPAECs apoptosis evaluated by western blot analysis of Bcl‐2 and Bax expression (n=3). E, hPAECs apoptosis evaluated by flow cytometry (n=3). The experiments were performed at least in 3 independent assays. Student's unpaired t test (A, B) and 1‐way ANOVA (C–E) were used with a Bonferroni post hoc test. hPAEC indicates human pulmonary artery endothelial cell; NC, negative control; siRNA, small interfering RNA; and TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling

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  Figure 6. Cross‐talk between 3T3‐L1 adipocytes and PAECs mediated by nesfatin‐1. A, 3T3‐L1 adipocyte and PAEC coculture model. B, Concentration of nesfatin‐1 detected by ELISA in PAEC monoculture, 3T3‐L1 adipocyte monoculture and 3T3‐L1 adipocyte+PAEC coculture groups (n=3). C, Spontaneous formation of capillaries by hPAECs cultured with or without 3T3‐L1 adipocytes treated with or without nesfatin‐1 silence for 24 h. The result is expressed as total tube length per field. D, Adhesion of hPAECs to culture plate wells when cultured with or without 3T3‐L1 adipocytes that treated with or without nesfatin‐1 silence for 24 hours. E, Apoptosis of hPAECs detected by PI positive staining when cultured with or without 3T3‐L1 adipocytes that treated with or without nesfatin‐1 silence for 24 hours. The experiments were performed at least in 3 independent assays. One‐way ANOVA was used with a Bonferroni post hoc test. NC indicates negative control; PAEC, pulmonary artery endothelial cell; PI, propidium iodide; and siRNA, small interfering RNA.

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  Figure 7. Interaction between MEF2B and BMPR1A and nesfatin‐1–induced endothelial dysfunction via downregulating MEF2B/BMPR1A. A, Heat map of downregulated genes from RNA‐sequencing analysis of control group and nesfatin‐1 treated group. B, Real‐time polymerase chain reaction and western blot analysis of MEF2B and BMPR1A in control group and nesfatin‐1 treated group (n=3). C, The JASPAR website was used to scan the MEF2B‐bound promoter and predict potential binding sites for MEF2B. D, The band of MEF2B in HEK‐293 T cells is shown in western blot analysis; BMPR1A promoter luciferase activity was detected by dual‐luciferase assays. E, Spontaneous formation of capillaries by hPAECs transfected with MEF2B‐OE constructs vs empty vector controls following nesfatin‐1 treatment or not for 24 hours. The result is expressed as numbers of tubes per field. F, Adhesion of hPAECs to culture plate wells when transfected with MEF2B‐OE constructs vs empty vector controls following nesfatin‐1 treatment or not for 24 hours. The experiments were performed at least in 3 independent assays. Student's unpaired t test (B) and 1‐way ANOVA (D–F) were used with a Bonferroni post hoc test. hPAEC indicates human…

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  Figure 8. MEF2B‐regulated BMPR1A ubiquitination through UBE2C/UBR7. A, Heat map of upregulated genes from RNA‐sequencing analysis of control group and nesfatin‐1 treated group. B, Western blot analysis of UBE2C and UBR7 in the control group and nesfatin‐1 treated group (n=3). C, Half‐life process of BMPR1A in PAEC cells with different treatments. 24 hours after transfection with UBR7 and/or UBE2C or not, PAEC cells were treated with DMSO, MG132, or cycloheximide. Cells were lysed at 0, 8, 12 hours and were evaluated by Western blot for BMPR1A (n=3). The density for each band was measured and normalized so that the number of density at t=0 hour was 1, the ratio of each time point to 0 hour were calculated. D–H, Coimmunoprecipitation analysis of HEK‐293T cells cotransfected with GFP‐tagged MEF2B (GFP‐MEF2B), Myc‐tagged UBE2C (Myc‐UBE2C), V5‐tagged UBR7 (V5‐UBR7), Flag‐tagged BMPR1A (Flag‐BMPR1A) to examine their interactions. D, The protein level of MEF2B, UBE2C, UBR7, and BMPR1A in input was evaluated by western blot. E, Flag antibody was used for immunoprecipitation. F, Myc antibody was used for immunoprecipitation. G, GFP antibody was used for immunoprecipitation. H, V5 antibody was used…