Journal: Scientific Reports

Article Title: Endothelial Twist1-PDGFB signaling mediates hypoxia-induced proliferation and migration of αSMA-positive cells

doi: 10.1038/s41598-020-64298-5

Figure Lengend Snippet: Endothelial Twist1 stimulates SMC DNA synthesis and migration through PDGFB in vitro . ( a ) Graph showing BrdU-positive HPASMCs treated with CM collected from HPAE cells treated with Twist1 siRNA or control siRNA with irrelevant sequences or in combination with PDGFB (n = 4, *p < 0.05). Error bars represent s.e.m. ( b ) Graph showing HPASMCs migrating towards CM collected from HPAE cells treated with Twist1 siRNA or control siRNA with irrelevant sequences or in combination with PDGFB (n = 5, *p < 0.05). Error bars represent s.e.m. ( c) Graph showing BrdU-positive HPASMCs treated with CM collected from HPAE cells treated with lentivirus expressing Twist1 or in combination with PDGF inhibitory antibody or PDGFB (n = 4, *p < 0.05). As a control, CM was collected from HPAE cells treated with control virus (vector alone). Error bars represent s.e.m. ( d ) Graph showing HPASMCs migrating towards CM collected from HPAE cells treated with lentivirus expressing Twist1 or in combination with PDGF inhibitory antibody or PDGFB (n = 5, *p < 0.05). As a control, CM was collected from HPAE cells treated with control virus (vector alone). Error bars represent s.e.m.

Article Snippet: Human pulmonary arterial endothelial (HPAE) cells (Lonza) were cultured in EBM2 medium containing 5% FBS and growth factors (VEGF, bFGF and PDGF).

Techniques: DNA Synthesis, Migration, In Vitro, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: Endothelial Twist1-PDGFB signaling mediates hypoxia-induced proliferation and migration of αSMA-positive cells

doi: 10.1038/s41598-020-64298-5

Figure Lengend Snippet: Endothelial Twist1 mediates hypoxia-induced DNA synthesis and accumulation of αSMA-positive cells in the gel implanted on the mouse lung through PDGFB. ( a ) Graph showing the mRNA levels of Twist1 in HPAE cells treated with hypoxia or in combination with control siRNA or Twist1 siRNA. As a control HPAE cells were treated with control siRNA with irrelevant sequences under normoxia (n = 4, *p < 0.05). Error bars represent s.e.m. ( b ) Graph showing the protein levels of PDGFB in CM collected from HPAE cells treated with hypoxia or in combination with control siRNA or Twist1 siRNA. As a control HPAE cells were treated with control siRNA with irrelevant sequences under normoxia (n = 3, *p < 0.05). ( c ) Graph showing BrdU-positive HPASMCs treated with CM collected from HPAE cells treated with hypoxia or in combination with control siRNA or Twist1 siRNA. As a control HPASMCs were treated with CM collected from HPAE cells treated with control siRNA with irrelevant sequences under normoxia (n = 4, *p < 0.05). Error bars represent s.e.m. ( d ) IF micrographs of fibrin gel supplemented with GFP-labeled B6-GFP mouse lung ECs implanted on C57BL6 mouse lung for 7 days and treated with hypoxia for the last 3 days; GFP-labeled blood vessel lumen structure, HIF1α expression and DAPI ( top ), GFP-labeled blood vessel lumen structure, αSMA expression and distribution and DAPI ( middle ), and GFP-labeled blood vessel lumen structure, PDGFB expression and distribution and DAPI ( bottom ) in the fibrin gel. Scale bar, 10 μm. ( e ) Graphs showing integrated fluorescent density of HIF1α (n = 7, *p < 0.05), αSMA(n = 6, *p < 0.05), and PDGFB (n = 6, *p < 0.05). Error bars represent s.e.m. ( f ) IF micrographs of fibrin gel supplemented with GFP-labeled HPAE cells treated with control shRNA or Twist1 shRNA, implanted on mouse lung for 7 days or in combination with exposure to hypoxia for the last 3 days and/or treatment with PDGF neutralizing antibody showing GFP-labeled blood vessel lumen structure, αSMA expression and distribution and DAPI in the fibrin gel. Scale bar, 10 μm. Graph showing the integrated fluorescent density of αSMA (n = 6, *p < 0.05). Error bars represent s.e.m. ( g ) Graph showing the mRNA levels of Twist1 in HPAE cells treated with lentivirus targeting Twist1 (Twist1 shRNA) or control virus (n = 3, *p < 0.05).

Article Snippet: Human pulmonary arterial endothelial (HPAE) cells (Lonza) were cultured in EBM2 medium containing 5% FBS and growth factors (VEGF, bFGF and PDGF).

Techniques: DNA Synthesis, Labeling, Expressing, shRNA

Journal: bioRxiv

Article Title: Aging-regulated TUG1 is dispensable for endothelial cell function

doi: 10.1101/2022.02.27.482212

Figure Lengend Snippet: (A) Top 10 expressed lncRNAs based on transcript counts from HUVEC bulk RNA sequencing data (n = 4). TUG1 is highlighted in green. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) and Kinase Insert Domain Receptor ( KDR ) were used as controls. (B) RNA expression levels of TUG1 in different human cell types of the cardiovascular system (n=3). Vascular ECs are highlighted by grey bars. AoEC: Aortic ECs, PAEC: Pulmonary Artery ECs, CAEC: Coronary Artery ECs, CMEC: Cardiac Microvascular ECs, DMEC: Dermal Microvascular ECs, PMVEC: Pulmonary Microvascular ECs, SaVEC: Saphenous Vein ECs, HUVEC: Human Umbilical Vein ECs, DLEC: Dermal Lymphatic ECs, MSC: Mesenchymal Stem Cells, AoAF: Aortic Arterial Fibroblasts, AoSMC: Aortic Smooth Muscle Cells, CM: Cardiomyocytes (C) TUG1 expression levels in low (P3) vs. high (P16) passage HUVECs as determined by RT-qPCR. Expression is relative to GAPDH (n = 5-6; SEM; Mann-Whitney-test). (D) Tug1 expression from bulk RNA-sequencing data of the intima of the carotid arteries of young (10 weeks) vs. aged mice (18 months) (n = 3; SEM; Mann-Whitney-test).. (E) Quantification of the expression levels of the lncRNAs Differentiation Antagonizing Non-Protein Coding RNA ( DANCR ), TUG1 and Metastasis Associated Lung Adenocarcinoma Transcript 1 ( MALAT1 ) in subcellular fractions of wild type HUVECs using RT-qPCR (n=3). Results are expressed as percentages of the subcellular fractions associated to cytoplasm, nucleoplasm and chromatin. Expression is normalized to GAPDH as determined by RT-qPCR.

Article Snippet: Alternatively, total RNA was isolated from cell pellets from cardiomyocytes, aortic fibroblasts, pericytes, aortic smooth muscle cells, mesenchymal stem cells, dermal lymphatic endothelial cells, umbilical vein endothelial cells, saphenous vein endothelial cells, pulmonary microvascular endothelial cells, dermal microvascular endothelial cells, cardiac microvascular endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells and aortic endothelial cells (all human; Promocell) with the miRNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions including DNase digest.

Techniques: RNA Sequencing Assay, RNA Expression, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: TSP1–CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

doi: 10.1093/cvr/cvw218

Figure Lengend Snippet: TSP1, via CD47, suppresses cMyc to increase ET-1/ETA signaling in pulmonary vascular cells. (A) Representative immunofluorescence image of cultured rat-1a Myc/ER fibroblast cells expressing the c-Myc/ER fusion protein display nuclear c-Myc (green), DAPI (blue), phalloidin (red); scale bar 50 μm, original magnification ×63. Rat-1a fibroblasts were treated with 4-hydroxytamoxifen at the designated concentrations for 12 h. mRNA expression of CD47, ET-1, and ETA was determined. Data from n = 4 experiments with each run in triplicate are presented as mean ± SD, ****P < 0.0001. (B) Murine pulmonary endothelial cells were isolated from WT and CD47−/− mice. mRNA expression for CD47, cMyc, and ET-1 was determined. Data from n = 6 independent experiments are presented as mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Human pulmonary arterial endothelial cells (hPAEC) (passage 3–6) were cultured to 80% confluence then treated with normoxia (Nx), hypoxia (Hx, FiO2 1%), or hypoxia + CD47 blocking antibody (1 μg/ml) for 24 h. mRNA expression of CD47, ET-1, and ETA was determined. ET-1 level in cell culture supernatants was measured by ELISA. Representative data from four experiments are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) hPAEC were transfected with control (CTRL) or cMyc siRNA for 72 h and transcript levels of CD47, cMyc, ET-1, and ETA measured. ET-1 level in cell culture supernatants was measured by ELISA. Data are represented as mean ± SD from n = 5 experiments; **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Human pulmonary vascular smooth muscle cells (hPVSMC) were subjected to Nx or Hx (FiO2 1%, 24 h) and protein expression for TSP1 and cMyc determined. Representative blots are shown and data from n = 4 independent experiments are presented as mean ± SD, *P < 0.05. (F) hPVSMC were subjected to Nx or Hx (FiO2 1%, 24, 48, and 72 h) and mRNA transcript levels determined for TSP1, CD47, and ETA. Data from n = 4–5 experiments are presented as mean ± SD, *P < 0.05 Nx versus Hx at indicated time point. (G) Pulmonary VSMC from WT, TSP1−/−, and CD47−/− mice were isolated and subjected to Nx or Hx (FiO2 1%, 24 h). Cell lysate was prepared and protein and mRNA analysis performed. Representative western immunoblots for the indicated proteins are shown. Densitometry from n = 4 independent experiments for cMyc and n = 4–5 independent experiments for TSP1 is presented as the mean ratio of target protein to tubulin ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001. mRNA data are presented as the mean ratio of target protein to the housekeeping gene (± SD), *P < 0.05 Nx versus Hx. (H) hPVMSC were incubated with ET-1 (1 μM) with or without a CD47 blocking antibody (clone B6H12, 1 μg/ml) for 24 h. Cell size was analyzed by FACS. Data from n = 3–5 independent experiments are presented as mean ± SD, *P < 0.05, **P < 0.01.

Article Snippet: Human pulmonary arterial smooth muscle cells (PA VSMC) and endothelial cells (hPAEC) were purchased from Lonza (Basel, Switzerland) and grown in proprietary medium.

Techniques: Immunofluorescence, Cell Culture, Expressing, Isolation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Incubation

Journal: The FASEB Journal

Article Title: Primary endothelial cell–specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia

doi: 10.1096/fj.201802650RR

Figure Lengend Snippet: Hypoxia results in accumulation of HIF-1α and HIF-2α in human ECs. Cells were exposed to hypoxia for the time periods specified, and total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by Western blot normalized to β-actin and total protein levels and related to the normoxic control. The densitometry analyses are provided in Supplemental Fig. S1. HIAEC, human iliac EC; HPAEC, human pulmonary artery EC.

Article Snippet: Primary human aortic ECs (HAECs), primary human cardiac artery ECs (HCAECs), primary human iliac ECs, and primary human pulmonary artery ECs were purchased from Lonza and cultured in EGM-2 medium.

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Primary endothelial cell–specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia

doi: 10.1096/fj.201802650RR

Figure Lengend Snippet: Hypoxia reduces HIF1A and EPAS1 mRNA levels in human ECs. Cells were exposed to hypoxia for the time periods specified, and total RNA lysates were collected. HIF1A and EPAS1 mRNA levels were quantified by quantitative real-time PCR and normalized to TBP and 18S rRNA levels and expressed as a fold change over normoxic samples. Data represent the mean ± sd of 2 independent experiments (3 replicates each). *P < 0.05 was considered significant. HIAEC, human iliac EC; HPAEC, human pulmonary artery EC.

Article Snippet: Primary human aortic ECs (HAECs), primary human cardiac artery ECs (HCAECs), primary human iliac ECs, and primary human pulmonary artery ECs were purchased from Lonza and cultured in EGM-2 medium.

Techniques: Real-time Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Primary endothelial cell–specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia

doi: 10.1096/fj.201802650RR

Figure Lengend Snippet: Hypoxia reduces mRNA levels and half-lives of HIF1A and EPAS1. A) The mathematic representation of HIF1A and EPAS1 mRNA levels during hypoxia in human ECs. The changes in mRNA levels obtained from all ECs tested during hypoxia time course were analyzed using the natural exponential function. The dashed lines represent mathematically predicted reduction curves, whereas solid lines represent experimental data. B) HIF1A and EPAS1 mRNA half-life measurements were taken in HUVECs exposed to hypoxia and cultured in normoxia. Actinomycin D was added to stop transcription, after which the RNA was isolated, and total HIF1A and EPAS1 mRNA levels at each time point were measured by real-time PCR and normalized to endogenous 18S rRNA levels. mRNA values for each time point were calculated from 2 individual samples generated in at least 2 independent experiments. Relative HIF1A and EPAS1 mRNA levels at the time points indicated were plotted as percent differences from HIF1A and EPAS1 mRNA levels at the initial time point (t = 0). The mRNA half-lives were calculated from the exponential decay using the trend line equation C/C0 = e–kdt (where C and C0 are mRNA amounts at time t and at the t0, respectively, and kd is the mRNA decay constant). The error bars represent sd. P < 0.05 was considered significant.

Article Snippet: Primary human aortic ECs (HAECs), primary human cardiac artery ECs (HCAECs), primary human iliac ECs, and primary human pulmonary artery ECs were purchased from Lonza and cultured in EGM-2 medium.

Techniques: Cell Culture, Isolation, Real-time Polymerase Chain Reaction, Generated