Research ArticlePulmonary Arterial Hypertension

NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension

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Science Translational Medicine  13 Jun 2018:
Vol. 10, Issue 445, eaap7294
DOI: 10.1126/scitranslmed.aap7294
  • Fig. 1 BC analysis identifies NEDD9 as a critical node regulating the transition between adaptive and pathogenic fibrosis.

    (A) Protein-protein interaction network analysis was used to clarify the molecular pathways that regulate two functionally distinct fibrosis subtypes: adaptive and pathogenic. Genes related to dermal and vascular fibrosis were collected from the curated literature. Focusing on PAH, as a human disease correlate to these findings in silico, genes specifically associated with lung fibrosis (n = 362) were excluded from the analysis to limit the probability of analyzing pathways responsible for lung parenchymal fibrosis rather than pulmonary vascular fibrosis. (B) The gene products (proteins) associated with adaptive fibrosis (blue), pathogenic fibrosis (red), or both adaptive and pathogenic fibrosis (blue with red border) were mapped to the CHI (12), resulting in the fibrosome. (C) ALDO-fibrosome protein-protein interactome subnetwork resulting from associations involving fibrosis genes connected to ALDO-regulated genes. Arrow points to the SMAD3 target and Cas protein NEDD9. (D) BC, a measure of importance in information transfer across the network of a node (protein) based on the shortest paths, was used to identify NEDD9 as a critical node in the phenotype transition from adaptive to pathogenic fibrosis. BC of an ALDO-regulated gene (ai) in connecting adaptive and pathogenic fibrosis (F1 and F2, respectively) is the sum of the fraction of all fibrosis gene pairs’ shortest paths in the interactome that pass through this ALDO-regulated gene (t) in the interactome, and σ(s,t|ai) is the number of those shortest paths in the interactome that pass through ALDO-regulated gene ai. The BC score for NEDD9 was ranked 6th of 86 proteins.

  • Fig. 2 Oxidation of NEDD9-Cys18 impairs NEDD9-SMAD3 binding.

    (A) Hill curves and half-maximal effective concentration (EC50) values of purified WT human NEDD9 (0.06 nM to 2 μM) or (B) mutant NEDD9 containing a substitution of cysteine for alanine at position 18 (C18A-NEDD9; 0.03 nM to 1 μM), which is resistant to oxidant stress, incubated with fluorescently labeled SMAD3 (20 nM) in the presence of H2O2 (500 μM; 20 min). Data are expressed as EC50 ± EC50 confidence interval. n = 2. (C) Immunoprecipitation (IP) of COS-7 cells transfected with human DNA coding SMAD3 and WT-NEDD9 or C18A-NEDD9 after treatment with vehicle (V) control, which, in this experiment, was phosphate-buffered saline or H2O2 (500 μM) for 5 min. NEDD9-SMAD3 binding was calculated by comparing the densitometry ratio of SMAD3 to NEDD9 assessed by immunoblot (IB). Data are from representative immunoblots and expressed as the ratio of SMAD3/NEDD9 in densitometry arbitrary units (a.u.). Means ± SE, n = 3 to 4, Student’s unpaired two-tailed t test. (D) In-gel trypsin digestion on NEDD9 immunoprecipitated from HPAECs treated with H2O2 (250 μM) for 20 min. Arrow, doubly charged y8 ion corresponding to NEDD9-Cys18 with the addition of three oxygens (-SO3H) at a retention time of 77.3 min and a mass/charge ratio (m/z) value of 944.425 (n = 3). (E) Immunoprecipitation of NEDD9 from HPAECs treated with vehicle control [dimethyl sulfoxide (DMSO); 10−7 M], H2O2 (250 μM) for 20 min, or ALDO (10−7 M) for 24 hours. Immunoblotting used an antibody against dimedone, detected upon reaction with cysteine sulfenic acid (R-SOH). Data are from representative immunoblots expressed as the ratio of oxidized NEDD9 (NEDD9-SOH)/NEDD9 and the ratio of SMAD3/NEDD9-SOH in arbitrary units. Means ± SE, n = 3, Student’s unpaired two-tailed t test. (C to E) Representative immunoblots and spectra.

  • Fig. 3 NEDD9 modulates pulmonary endothelial cell collagen synthesis and fibrosis.

    (A) Immunoblot and quantitation in densitometry arbitrary units of NEDD9 from HPAECs treated with vehicle (V) control, ALDO (10−7 M) for 24 hours, spironolactone (SP; 10 μM) for 24 hours to inhibit ALDO-induced oxidant stress, or hypoxia (0.2% FiO2) for 24 hours. Means ± SE, n = 3, one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. The orientation of the hypoxia samples was adjusted for consistency with ALDO samples; however, all bands are from the same blot. (B) NEDD9 expression in HPAECs treated with the pan–TGF-β ligand neutralizing antibody TGF-β-RII-FC (2.3 μg/ml) for 24 hours before treatment with ALDO (10−7 M) for 24 hours. Means ± SE, n = 3, Student’s unpaired two-tailed t test. (C) NEDD9 expression in HPAECs pretreated with the proteasome inhibitor MG-132 (50 μM) for 0 to 4 hours before H2O2 (250 μM) for 20 min. Means ± SE, n = 3, Student’s unpaired two-tailed t test. The orientation of the si-Scr and si-NEDD9 samples was adjusted for consistency; however, all bands are from the same blot. (D) Coimmunoprecipitation and quantitation for NEDD9 and the COL3A1-targeting transcription factor NKX2-5 from HPAECs incubated with vehicle or ALDO (107 M) for 24 hours. Means ± SE, n = 3, Student’s unpaired two-tailed t test. IP, immunoprecipitation. (E) HPAECs were transfected with siRNA to SMAD3 (si-SMAD3), and the ratio of NKX2-5/NEDD9 is presented in arbitrary units. Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons test. (F) Chromatin immunoprecipitation of cell lysates using an anti–histone H3 antibody (positive control), immunobeads (negative control), and an anti–NKX2-5 antibody was followed by polymerase chain reaction amplification of the Col3A1 region containing the NKX2-5 binding site. Data were normalized to positive control and expressed as fold change (FC) for NKX2-5 over negative control. Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons test. (G) MMP-2 proteolytic activity by SDS–polyacrylamide gel electrophoresis zymography, collagen III (Col III) expression, and quantitation (N9, NEDD9). (H) Images and quantitation of three-dimensional collagen Matrigel contraction (scale bars, 17.4 mm). (I) Images and quantitation of collagen fiber content area in square micrometers (scale bars, 100 μm). (J) Stiffness assessed by atomic force microscopy from HPAECs transfected with si-NEDD9 or si-scrambled (negative) control (si-Scr) and treated with ALDO (10−7 M) for 24 hours. (G to J) Means ± SE, n = 3 to 6, one-way ANOVA and Tukey’s multiple comparisons test. *P < 0.05 versus vehicle; **P < 0.05 versus ALDO. (A to I) Representative immunoblots, gels, and micrographs. Un, untreated.

  • Fig. 4 Pulmonary endothelial NEDD9–collagen III is increased in PAH in vivo.

    (A) Immunofluorescence for NEDD9 and collagen III in tissue sections from animal models of PAH [IL-6 TG+ (n = 4), Schisto (n = 4), SU-5416/Hypoxia (n = 5), and MCT (n = 5)] and PAH patients (n = 9). Samples from control mice (n = 4) and rats (n = 5) and donor tissue from nondiseased control patients (n = 5) were used for comparison. Insets are merged images for 4′,6-diamidino-2-phenylindole (DAPI; blue) and NEDD9 (green) to show nuclear NEDD9 expression; representative images from samples within each condition are provided. Colocalization of collagen III and NEDD9 is expressed as percent double-positive area/sum total area of stain for each protein in pulmonary arterioles. Means ± SE, Student’s unpaired two-tailed t test. (B) Immunofluorescence for collagen III and NEDD9 in HPAECs isolated from PAH patients (PAH-HPAEC). Number of NEDD9+ cells per high-powered field (h.p.f.; 200×) is quantitated. Coimmunoprecipitation of NEDD9 and NKX2-5 and immunoblot of NEDD9 and SMAD3, with quantitation in densitometry arbitrary units. Means ± SE, n = 3 to 6 per group, Student’s unpaired two-tailed t test. (C) Collagen III–vWF colocalization in remodeled PAH pulmonary arterioles, expressed as percent double-positive area/sum total area of stain for each protein in pulmonary arterioles. Means ± SE, n = 3 to 5 per group, Student’s unpaired two-tailed t test. Scale bars, 50 μm (main images) and 4 μm (insets). Representative micrographs and immunoblots are shown.

  • Fig. 5 Exosomes from ALDO-treated HPAECs increase NEDD9 and fibrillar collagen in HPASMCs.

    (A) Schematic of culture conditions and representative immunoblots of NEDD9 from HPASMCs cocultured with HPAECs and treated with ALDO (10−7 M) for 24 hours. Data are expressed as densitometry arbitrary units. Means ± SE, n = 3, one-way ANOVA and Tukey’s multiple comparisons tests. (B) Quantification of NEDD9 positivity in HPASMCs, NHLFs, and dermal fibroblasts from patients with systemic sclerosis without PAH (SSc-DFBs) that were untreated (Un) or treated with exosomes isolated from vehicle (V) (DMSO; 10−7 M)–treated or ALDO (10−7 M)–treated HPAECs. Data are expressed as percentage of cells that were NEDD9+ per high-powered field (200×) (white arrows). Exo-HPAEC, exosomes isolated from HPAECs. Inset, representative NEDD9+ cell. Means ± SE, n = 3 to 4, one-way ANOVA and Tukey’s multiple comparisons test. Scale bar, 100 μm (representative micrographs) and 10 μm (inset). *P < 0.01 versus NHLF exo-HPAEC ALDO; **P < 0.001 versus HPASMC Un. (C) Colocalization of NEDD9 and collagen III in the perinuclear regions of cells undergoing mitotic division in HPASMCs and NHLFs treated with exosomes from ALDO-treated HPAECs (exo-ALDO-HPAEC). n = 3. Scale bars, 20 μm. (D) Immunoblot of NEDD9 and collagen I in HPASMCs treated with exo-ALDO-HPAEC. Data are expressed in densitometry arbitrary units. Means ± SE, n = 4 for NEDD9; n = 4 for collagen I, Student’s unpaired two-tailed t test. (E) Representative immunoblots of exosomal TGF-β-LAP (TGF-β latent complex) secreted by HPAECs treated with vehicle control and ALDO (10−7 M) for 24 hours. Means ± SE, n = 3, Student’s unpaired two-tailed t test.

  • Fig. 6 Gene ablation or molecular inhibition of NEDD9 prevented vascular fibrosis and PAH in vivo.

    (A) RVSP and RV mass by Fulton index (RV/LV + S) in male and female C57BL (WT), C57BL/NEDD9+/−, and C57BL/NEDD9−/− mice injected with Sugen-5416 (20 mg/kg) every 7 days during a 3-week period of hypoxia treatment (10% FiO2). Means ± SE, n = 6 to 9 mice per condition for RVSP; n = 5 to 10 mice per condition for heart weight, one-way ANOVA and Tukey’s multiple comparisons test. (B) Representative images of Gomori trichrome staining to assess vascular fibrosis burden and vWF–collagen III colocalization, expressed as percent double-positive area/sum total area of stain for each protein, of pulmonary arterioles from WT, NEDD9+/−, and NEDD9−/− mice treated with or without SU-5416/Hypoxia. Means ± SE, n = 6 to 10 mice per condition for trichrome; n = 4 to 7 mice per condition for vWF–collagen III, one-way ANOVA and Tukey’s multiple comparisons test. Scale bar, 50 μm. (C) NEDD9–collagen III and NEDD9-vWF colocalization in lung tissue from male Sprague-Dawley rats administrated MCT (50 mg/kg) and treated with Staramine-mPEG (1.5 mg/kg) formulated with NEDD9 siRNA (si-NEDD9). Means ± SE, n = 4 to 5 rats per condition, one-way ANOVA and Tukey’s multiple comparisons test. Scale bars, 50 μm. (D) The number of hypertrophic vessels/high-powered field (red arrow) and percent vascular fibrillar collagen in paraffin-embedded lung sections was analyzed using anti–α-SMA immunohistochemistry and Picrosirius Red staining, respectively, from vehicle (V)–, MCT-, and MCT + si-NEDD9–treated rats. Means ± SE, n = 4 to 5 rats per condition, one-way ANOVA and Tukey’s multiple comparisons test. Scale bars, 100 μm (main images) and 50 μm (insets, representative hypertrophic vessel magnified). (E) Table of phenotypic changes in vehicle-, MCT-, and MCT + si-NEDD9–treated rats. Means ± SE, n = 5 to 7 rats per condition. P values in column represent one-way ANOVA. *P < 0.05 versus vehicle; **P < 0.001 versus MCT; ***P = 0.02 versus MCT by Tukey’s multiple comparisons test. (F) Representative RV pressure-volume loops are shown to quantify RV-pulmonary artery coupling, assessed by the ratio of end-systolic elastance (Ees)/pulmonary vascular elastance (Ea) in MCT- and MCT + si-NEDD9–treated rats. Means ± SE, n = 3 rats per condition, Student’s unpaired two-tailed t test. HR, heart rate; MAP, mean arterial pressure; RAP, right atrial pressure; CI, cardiac index; CO, cardiac output; PVRi, indexed pulmonary vascular resistance (in Wood units).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/445/eaap7294/DC1

    Materials and Methods

    Fig. S1. Fibrosome and ALDO-fibrosome networks.

    Fig. S2. Computational modeling predicts that NEDD9-Cys18 is highly prone to oxidant stress.

    Fig. S3. Proteasomal inhibition or oxidant stress increases NEDD9 accumulation in HPAECs.

    Fig. S4. ALDO does not affect NEDD9 expression in cell types associated with adaptive fibrosis.

    Fig. S5. NEDD9 is expressed in the nucleus of HPAECs and PAH-HPAECs.

    Fig. S6. NKX2-5 and NEDD9 colocalize to the HPAEC nucleus, and inhibition of NKX2-5 prevents COL3A1 mRNA transcription mediated by ALDO.

    Fig. S7. ALDO increases NEDD9-dependent fibrosis in HPAECs without changes to EndMT marker expression.

    Fig. S8. Nuclear NEDD9 expression in vascular cells from pulmonary arterioles in human PAH.

    Fig. S9. The cysteine sulfonic acid modification at NEDD9-Cys18 is observed in PAH-HPAECs.

    Fig. S10. NEDD9 expression is increased globally in remodeled pulmonary arterioles from patients with PAH.

    Fig. S11. The exosome marker CD9 in cells treated with exosomes from HPAECs.

    Fig. S12. NEDD9 was not detected by LC-MS in the culture medium of ALDO-treated cells.

    Fig. S13. Inhibition of NEDD9 improves vascular fibrosis in PAH in vivo.

    Table S1. The effect of NEDD9 inhibition on differential expression of genes related to fibrosis in HPAECs.

    Table S2. Clinical and demographic information for biological samples used in this study.

    Movie S1. Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in vehicle-treated HPAECs.

    Movie S2. Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in ALDO-treated HPAECs.

    Movie S3. Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in PAH-HPAECs.

    Data file S1. Differentially expressed genes from RNASEQ analysis of HPAECs treated with ALDO (10−7 M) for 24 hours versus ALDO for 24 hours after transfection with si-NEDD9.

    Data file S2. Differentially expressed genes from RNASEQ analysis of HPAECs treated with vehicle control or ALDO (10−7 M) for 24 hours.

    Data file S3. Differentially expressed genes from RNASEQ analysis of HPAECs treated with vehicle control or ALDO (10−7 M) for 24 hours after transfection with si-NEDD9.

    Data file S4. Primary data.

    References (4165)

  • Supplementary Material for:

    NEDD9 targets COL3A1 to promote endothelial fibrosis and pulmonary arterial hypertension

    Andriy O. Samokhin, Thomas Stephens, Bradley M. Wertheim, Rui-Sheng Wang, Sara O. Vargas, Lai-Ming Yung, Minwei Cao, Marcel Brown, Elena Arons, Paul B. Dieffenbach, Jason G. Fewell, Majed Matar, Frederick P. Bowman, Kathleen J. Haley, George A. Alba, Stefano M. Marino, Rahul Kumar, Ivan O. Rosas, Aaron B. Waxman, William M. Oldham, Dinesh Khanna, Brian B. Graham, Sachiko Seo, Vadim N. Gladyshev, Paul B. Yu, Laura E. Fredenburgh, Joseph Loscalzo, Jane A. Leopold, Bradley A. Maron*

    *Corresponding author. Email: bmaron{at}bwh.harvard.edu

    Published 13 June 2018, Sci. Transl. Med. 10, eaap7294 (2018)
    DOI: 10.1126/scitranslmed.aap7294

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Fibrosome and ALDO-fibrosome networks.
    • Fig. S2. Computational modeling predicts that NEDD9-Cys18 is highly prone to oxidant stress.
    • Fig. S3. Proteasomal inhibition or oxidant stress increases NEDD9 accumulation in HPAECs.
    • Fig. S4. ALDO does not affect NEDD9 expression in cell types associated with adaptive fibrosis.
    • Fig. S5. NEDD9 is expressed in the nucleus of HPAECs and PAH-HPAECs.
    • Fig. S6. NKX2-5 and NEDD9 colocalize to the HPAEC nucleus, and inhibition of NKX2-5 prevents COL3A1 mRNA transcription mediated by ALDO.
    • Fig. S7. ALDO increases NEDD9-dependent fibrosis in HPAECs without changes to EndMT marker expression.
    • Fig. S8. Nuclear NEDD9 expression in vascular cells from pulmonary arterioles in human PAH.
    • Fig. S9. The cysteine sulfonic acid modification at NEDD9-Cys18 is observed in PAH-HPAECs.
    • Fig. S10. NEDD9 expression is increased globally in remodeled pulmonary arterioles from patients with PAH.
    • Fig. S11. The exosome marker CD9 in cells treated with exosomes from HPAECs.
    • Fig. S12. NEDD9 was not detected by LC-MS in the culture medium of ALDO-treated cells.
    • Fig. S13. Inhibition of NEDD9 improves vascular fibrosis in PAH in vivo.
    • Table S1. The effect of NEDD9 inhibition on differential expression of genes related to fibrosis in HPAECs.
    • Table S2. Clinical and demographic information for biological samples used in this study.
    • Legends for movies S1 to S3
    • References (4165)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in vehicle-treated HPAECs.
    • Movie S2 (.avi format). Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in ALDO-treated HPAECs.
    • Movie S3 (.avi format). Rotational three-dimensional videos acquired by confocal microscopy demonstrate nuclear NEDD9 expression in PAH-HPAECs.
    • Data file S1 (Microsoft Excel format). Differentially expressed genes from RNASEQ analysis of HPAECs treated with ALDO (10−7 M) for 24 hours versus ALDO for 24 hours after transfection with si-NEDD9.
    • Data file S2 (.csv format). Differentially expressed genes from RNASEQ analysis of HPAECs treated with vehicle control or ALDO (10−7 M) for 24 hours.
    • Data file S3 (.csv format). Differentially expressed genes from RNASEQ analysis of HPAECs treated with vehicle control or ALDO (10−7 M) for 24 hours after transfection with si-NEDD9.
    • Data file S4 (Microsoft Excel format). Primary data.

    [Download Movie S1]

    [Download Movie S2]

    [Download Movie S3]

    [Download Data files S1 to S4]

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