human pulmonary artery smooth muscle cells pasmcs  (ATCC)

Journal: bioRxiv

Article Title: Impaired Lung BCAA Metabolism Promotes Ferroptosis and Resultant Pulmonary Arterial Hypertension-Associated Hepatopathy

doi: 10.1101/2025.09.03.672819

Figure Lengend Snippet: Excess BCAAs promoted a pro-ferroptotic phenotype in human PASMC . (A) Representative confocal micrographs of PASMC stained with MitoTracker Orange and quantification of mitochondrial organization in control and BCAA-treated PASMC (Control PASMC [Con]: 1.1±0.6, BCAA-Treated PASMC [BCAA]: 0.7±0.3); p -values determined by Mann-Whitney U-test. (B) Confocal micrographs of TRME-stained PASMC in control and BCAA-treated media, with corresponding quantification of mitochondrial membrane hyperpolarization, indicated by fluorescence intensity (Con: 8.3±17.2, BCAA: 42.7±9.7). p -values determined by Mann-Whitney U-test. (C) Excess BCAAs increase mitochondrial ROS, demonstrated by confocal micrographs of control and BCAA-treated PASMCs stained with MitoSox Red (Con: 6.4±2.1, BCAA: 14.5±6.9 MFI). p -values determined by Mann-Whitney U-test. (D) Representative confocal micrographs of control, BCAA-treated, and BCAA and ferrostatin-1-treated PASMCs incubated with 50 μM oleate and 50 μM palmitate and stained for lipid peroxidation using BODIPY (Con: 1.8±0.3, BCAA: 1.4±0.1, BCAA-treated with 5 μM ferrostatin-1 [BCAA-FER1]: 1.7±0.3). p -values determined by Kruskal-Wallis test and Dunn’s multiple comparisons test. (E) Incubation with BCAAs induces ferroptotic cell death, demonstrated by viability staining of control, BCAA-treated, and BCAA-treated with ferrostatin-1 PASMCs via Trypan Blue (Con: 9.4±7.5, BCAA: 31.7±11.7, BCAA-FER1: 13.0±11.1). White arrows indicate trypan blue-positive cells. p -values determined by ordinary one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Human PASMC (ATCC PCS-100-023) were grown with Sigma Basic Eagle Medium (Sigma, B1522-500) with supplements (Lonza, CC-3182) and passaged with subculture reagents (Lonza CC-5034).

Techniques: Staining, Control, MANN-WHITNEY, Membrane, Fluorescence, Incubation, Comparison

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. Dose- and time-course effect of Fragment 1 on NEDD9 expression in cultured human pulmonary artery endothelial cells (HPAECs) ( n =3). b. Anti-NEDD9 immunoprecipitation (IP), anti-SMAD3 immunoblot (IB) was performed on HPAECs treated with H 2 O 2 (500 μM) for 20 min in the presence or absence of 1 ( n =3). c. The effect of 1 on the NEDD9-SMAD3 protein-protein interaction in H 2 O 2 -treated HPAECs measured by proximity ligase assay and quantitated relative to overall NEDD9 expression ( n =3). Scale bar, 40 μm; N9, NEDD9. d. The effect of 1 for 24 hr on cellular NEDD9-positivity (N9+) and collagen III (Col III) deposition in HPAECs measured by immunofluorescence ( n =3). Scale bar, 50 μm. e. Intact mass spectra demonstrating a modification of purified NEDD9-SH3 by 1 at Cys18. f. Dose-response and time-course effect of treatment with 2 on NEDD9-SH3 shows occupancy at Cys18 measured by MS2. g. [left] Electrostatic surface representation of the 2 – NEDD9-SH3 complex. The associated electron density is displayed at the 1α level. The compound binding site is labeled. [right] Stereoscopic ribbon diagram of 2 in complex with NEDD9-SH3, highlighting the covalent bond between the acyl amide of 2 and Cys18. Hydrogen bonds between 2 and residues Trp43 and Glu20 are also shown. h. 3-D alignment of all 7 protein molecules in the asymmetric unit demonstrates three poses of 2 shown in stick format. i. Sequence alignment includes Site-1 and Site-2 of human FAK1 centered on Pro715 and Pro876, respectively, highlighting the consensus motif (PxKPxR). All synthetic peptides are N- and C-terminally capped. j. Fluorescence polarization measurements showing binding of FITC-labeled NEDDtide probe (FITCylated pep01) to GST-fused construct of the NEDD9-SH3 domain. The sigmoidal curve reflects the fitted binding model, indicating a dissociation constant (K D ) of 4 µM. k. Isothermal titration calorimetry (ITC) experiments were conducted to determine the binding affinity of two peptides (pep01 and pep02) to NEDD9. (Top) Raw ITC thermograms display heat release over time upon titration of each peptide into the NEDD9 solution. (Bottom) Integrated binding isotherms with the fitted binding model (red curve) used to determine the dissociation constant (K D ). The binding affinity for pep01 was determined to be approximately 4 µM, whereas pep02 exhibited a weaker interaction with a K D of approximately 14 µM. These results suggest that pep01 has a higher binding affinity to NEDD9 compared to pep02, supporting its potential as a more effective inhibitor. l. Determination of the IC50 of the NEDDtides pep01 and pep02 for disrupting the interaction between FITC-labeled pep01 and GST-NEDD9. m. The effect of HPAEC transfection for 10 min with NEDDtide pep05 on NEDD9 and GBR2, which is an SH3 protein that lacking the PxKPxR consensus motif and therefore served as a negative control ( n =3-4/condition). n. Fluorescence polarization profiles the competitive displacement of FITC-labeled pep01 (50 nM) from NEDDtides pep01, pep10, and pep11 (sequences detailed in panel i ).

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Expressing, Cell Culture, Immunoprecipitation, Western Blot, Immunofluorescence, Modification, Purification, Binding Assay, Labeling, Sequencing, Fluorescence, Construct, Isothermal Titration Calorimetry, Titration, Transfection, Negative Control

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. Results of a bioinformatics analysis profiling proteins in the proteome that express the PxKPxP consensus motif. Proteome sequence analysis using Motif2 revealed human genes with three, two and one occurrences of the motif, including FAK1 and FAK2. b. Human pulmonary artery endothelial cells were incubated with NEDDtide pep01 modified with a biotin tag (which is pep03) or a scrambled control (Scr) peptide for 2 hr. Anti-biotin (red) and anti-NEDD9 (green) immunofluorescence was performed. Blue, Dapi. The results of this analysis demonstrated biotin-NEDD9 colocalization (yellow) at subcellular structures resembling focal adhesions in pep03-transfected but not Scr-transfected cells ( n =3). Scale bar, 20µm. c. Structure of NEDDtide pep05 including thalidomide moiety.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Sequencing, Incubation, Modification, Control, Immunofluorescence, Transfection

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. The pep10 was docked in silico to produce multiple poses, the highest ranked of which is shown with a surface representation of the NEDD9-SH3 domain in light-blue and Cys18 in yellow. b. The structure of the SH3 domain of human BCAR1 (92% homology with NEDD9-SH3) was determined in complex with pep10, which contains the first PxKPxR motif of human FAK1. This electrostatic surface representation shows the complementarity of the charges between the positively charged basic peptide ligand and the negatively charged acidic BCAR1 binding pocket. c. Atomic closeup of the interaction between charged side chains (yellow, carbon atoms) and the SH3 domain showing ionic bonds and revealing the proximity of Arg to Ser64. Positively charged regions of BCAR1 are colored in blue, negatively charged regions in red, and neutral regions in white (−70 to +70 kT/e gradient). d. Sequence alignment of the covalent peptides pep31, pep32, and pep33 where X represents a bromoacetamide warhead. The chemical structure of pep33 is shown with positional labels below. e. Stereoscopic representation of the complex structure of NEDD9 with covalent pep33 in a cartoon format. Peptide side chains are shown as sticks, and nearby residues in NEDD9 are represented as lines. f. Surface representation of pep33 in complex with the SH3 domain of NEDD9. g. Ribbon overlay of the NEDD9-SH3 RT loop in the Apo WT structure (yellow), in the Apo D12A mutant that mimics oxidative modification at position 18 C18D (cyan), and in the pep33 NEDDtide complex structure (green). Pep33 is shown in ribbon format and Cys18 and Asp18 are shown in stick format. h. Deconvoluted intact mass spectra of the protein are shown after treatment with DMSO (vehicle control), pep31, pep32, and pep33. The DMSO-treated sample shows a single peak corresponding to the unmodified (baseline) protein mass. In contrast, samples treated with pep31, pep32, and pep33 exhibit additional peaks corresponding to mass shifts of +708.3 Da, +805.4 Da, and +1117.6 Da, respectively, indicating covalent adduct formation between the protein and each peptide. These mass increases are consistent with the expected mass additions for each peptide, suggesting specific and efficient covalent modification. Relative intensity is normalized across all conditions. The mass addition induced by NEDDtide modification of SH3 is provided adjacent to the spectra in blue. i. NEDDtide pep31 was analyzed using LC-MS against control proteins showing exceptional specificity for NEDD9. j. Determination of the IC50 of the NEDDtides pep31, pep32, and pep33 for disrupting the interaction between FITC-labeled probe and GST-NEDD9. k. Compared to vehicle (V) control, transfecting human pulmonary artery endothelial cells with NEDDtides pep31, pep32, and pep33 decreased cell migration proportionally to peptide length, assessed by wound healing assay ( n =3). Blue, dapi; Scale bar, 400 μm. l . The effect of NEDDtides pep31, pep32, and pep33 on multi-cell cluster formation (arrows) ( n =6-8). Box, representative cluster at high magnification. Scale bar, 600 μm.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: In Silico, Binding Assay, Sequencing, Mutagenesis, Modification, Control, Liquid Chromatography with Mass Spectroscopy, Labeling, Migration, Wound Healing Assay

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. The chemical formula for the pep31, pep32, and pep32 series NEDDtides modified with a bromoacetamide residue. b. Structure of NEDDtide pep41 including thalidomide moiety. c. Cultured human pulmonary artery endothelial cells (HPAECs) were transfected with increasing doses of thalidomide-modified pep41 for 2 hr and NEDD9 expression was measured by immunoblot. The graph shows NEDD9/β-actin ratio for different concentrations of the peptide ( n =3-5/condition).

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Modification, Residue, Cell Culture, Transfection, Expressing, Western Blot

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: Transcriptional regulation of Sulf1 by NEDD9 is a reversible cause of PAH in vivo . a. Disease reversal protocol using the rat SU-5416-hypoxia-normoxia experimental model of PAH and timepoints for intratracheal administration of si-Scrambled (negative) control (Scr) or siRNA against NEDD9. b. Representative hemodynamic tracings for right ventricular pressure (RVSP) measured by invasive cardiac catheterization for each treatment condition. Un, untreated. c. The effect of si-NEDD9 on RVSP ( n =3-5 rats/condition) and d. RV mass measured by Fulton index ( n =3-4 rats/condition). Triangle, si-NEDD9 initiated at day 14; square, si-Scr initiated at day 15; circle, untreated. IVS, interventricular septum; LV, left ventricle. e. Paraffin-embedded lung sections isolated at key timepoints over the course of the PAH reversal protocol in each treatment condition were stained using anti-NEDD9, von Willebrand Factor (vWF), and α -smooth muscle actin (SMA) antibodies, and the immunofluorescence expression profile was analyzed for distal pulmonary arterioles ( n =3-5 rats/condition). These data were used to quantify vascular NEDD9 expression and the lumen/vessel wall ratio ( n =3-5 rats/condition). arb, arbitrary. Scale bar, 20 μm. f. Cultured human pulmonary artery endothelial cells were transfected with vehicle (V) control, si-scrambled (negative) control (Scr), or si-NEDD9 for 24 hr and Sulf1 expression was analyzed by immunoblot, standardized to total protein level ( n =3). g. Paraffin-embedded lung sections from control, rat SU-5416-Hypoxia-Normoxia-PAH, and rat monocrotaline (MCT)-PAH ( n =3-5 rats/condition) or isolated from idiopathic PAH (iPAH) patients ( n =5/condition) at the time of lung explant were stained using anti-PECAM1 and anti-Sulf1 antibodies and the immunohistochemical profile in distal pulmonary arterioles was analyzed. Scale bar, 20 μm. h. Paraffin-embedded lung sections from control, rat SU-5416-Hypoxia-Normoxia-PAH, and rat monocrotaline (MCT)-PAH ( n =3-5 rats/condition) were stained using anti-Sulf1 and anti-HS-6-OS antibodies and the immunofluorescence ratio in distal pulmonary arterioles was analyzed. Scale bar, 20 μm. HS, heparan sulfate.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: In Vivo, Negative Control, Isolation, Staining, Immunofluorescence, Expressing, Cell Culture, Transfection, Control, Western Blot, Immunohistochemical staining

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. Sprague Dawley rats were untreated (Un) or administered SU-5416-Hypoxia-Normoxia to induce PAH and treated with si-NEDD9 or si-scrambled (negative) control (Scr) per the disease reversal protocol illustrated in . Paraffin-embedded lung sections were harvested and analyzed using anti-Sulf1 and anti-collagen III immunofluorescence ( n =5-6 rats/condition). Blue, Dapi. Scale bar, 10 μm. b. Human pulmonary artery endothelial cells were treated with vehicle (V) control or transfected with siRNA against NEDD9 (si-NEDD9) for 24 hr and Sulf1 mRNA transcript quantity was analyzed by RT-qPCR. These data are extracted from findings reported originally in reference 8.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Negative Control, Immunofluorescence, Control, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet:

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Transfection, Derivative Assay

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: Human pulmonary artery endothelial cells were analyzed using double immunofluorescence after probing with a anti-NEDD9 and anti-FAK, b anti-NEDD9 and anti-Sulf1, c anti-Sulf1 and anti-FAK, and d anti-Paxillin and anti Sulf1 antibodies ( n =3-4/condition for all analysis). Blue, Dapi. FAK, focal adhesion kinase; Sulf1, sulfatase-1. Scale bar, 40 µm.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. Paraffin-embedded lung sections from control, rat SU-5416-Hypoxia-Normoxia-PAH, and rat monocrotaline (MCT)-PAH ( n =4-5 rats/condition) or isolated from idiopathic PAH patients ( n =5-8/condition) at the time of lung explant were stained using anti-PECAM1 and anti-NRP-1 antibodies and the immunohistochemical profile in distal pulmonary arterioles was analyzed. Scale bar, 20 μm. b. Human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells were co-cultured in a barrier-free system to recapitulate the direct cell-cell contact conditions in PAH lesions. Double immunogold-labeled staining was performed using anti-NRP-1 and anti-Heparan sulfate antibodies, and imaged using transmission electron microscopy. Scale bar, 500 nm. Inset scale bar, 100 nm. c. Disease prevention protocol using the rat SU-5416-hypoxia-normoxia and monocrotaline (MCT) experimental models of PAH and timepoints for intratracheal administration of si-Scrambled (negative) control (Scr) or siRNA against NEDD9. d. The right ventricular systolic pressure (RVSP) measured by cardiac catheterization ( n =3-7 rats/condition) and e. RV mass measured by Fulton Index. IVs, interventricular septum; LV, left ventricle. f. Human pulmonary artery endothelial cells were untreated or treated with VEGF-C (100 nM) for 6 hr and transfected with vehicle (V) control or adenovirus (Ad) containing green fluorescent protein (GFP), Sulf1 cDNA, or siRNA-Sulf1, and cell migration was quantitated using the wound healing assay. Black line, separate experiment iteration without VEGF-C or other treatment ( n =4-7). Blue, DAPI. Scale bar, 400 μm. g. HPAECs were untransfected or transfected with an Ad carrying vehicle (V) control, Scr, Sulf1, or si-Sulf1 and stained with an anti-FAK-Y861 antibody. The number of podosome rosette-positive cells (podosome+) per low power field (40x) and the number of cells with >2 podosomes was quantitated ( n =3). Arrows indicate podosome structures. Scale bar, 50 μm. h. The number of tubes formed per 10x field was counted for untreated HPAECs and cells transfected with Ad carrying V, Sulf1, and si-Sulf1 ( n =3). Scale bar, 300 μm. i . The effect of Ad-Sulf1 on collagen III deposition in HPAECs ( n =5-10). PFE, plaque forming unit (N=1-3 PFEs). Scale bar, 50 μm.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Control, Isolation, Staining, Immunohistochemical staining, Cell Culture, Labeling, Transmission Assay, Electron Microscopy, Negative Control, Transfection, Migration, Wound Healing Assay

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. Compared to human pulmonary artery endothelial cells (HPAECs) from normal control donors, cell migration is decreased in PAH-HPAECs assessed by wound healing assay ( n =3-4/condition). Scale bar, 400 μm. b. Cultured HPAECs were untreated or transfected with adenovirus (Ad) carrying vehicle (V) control or siRNA-against Sulf1 (si-Sulf1) and probed with antibodies against vinculin, F-actin, Sulf1, contracting, FAK-Y576, and NRP1. Scale bar, 20 μm. FAK, focal adhesion kinase; NRP1, neuropilin-1. ( n =3-4/condition) c. Compared to HPAECs from normal control donors, endothelial tube formation is decreased in PAH ( n =3-4/condition). Scale bar, 300 μm.

Article Snippet: Human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells (all from Lonza) were grown to confluence using EBM-2 and SmGM-2 medium, respectively.

Techniques: Control, Migration, Wound Healing Assay, Cell Culture, Transfection