normal human pulmonary arterial ecs (hpaecs)  (Lonza)

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Single-Cell and Spatial Transcriptomics Identified Fatty Acid–Binding Proteins Controlling Endothelial Glycolytic and Arterial Programming in Pulmonary Hypertension

doi: 10.1161/ATVBAHA.124.321173

Figure Lengend Snippet: Overexpression of FABP (fatty acid–binding protein) 4/5–induced endothelial cell (EC) dysfunction. A , Overexpression of FABP4 and FABP5 fused with Flag tag in human pulmonary arterial ECs (hPAECs) using lentiviruses mediated gene overexpression. B and C , FABP4/5 overexpression promoted ECs glycolysis and proliferation assessed by 5-bromo-20-deoxyuridine (BrdU) incorporation assay. D , Glycolytic inhibitor 2-DG treatment inhibited FABP4/5–induced PAEC proliferation. E , FABP4/5 deletion in vivo reduced lung PAECs proliferation in TKO mice compared with CKO mice. F and G , Overexpression of FABP4/5 promoted fatty acid oxidation (FAO) in hPAECs in the presence of palmitic acid. The double arrows label the OCR generation contributed by FAO. H , Both CPT1α inhibitor etomoxir (ETO, 20 µmol/L) and DNA synthesis inhibitor (5-FU, 20 µmol/L) blocked FABP4/5–induced EC proliferation. I and J , A representative seahorse data showing upregulation of FAO in PAECs isolated from patients with idiopathic PAH (IPAH) compared with control donors. Six lines of control or PAECs of patients with IPAH were tested. C , D , and H , For BrdU assay, each dot represents a biological replicate. The experiments were performed at least 3 times. Student t test ( D ). ANOVA followed by Tukey post hoc analysis was used for statistical analysis ( B , C , E , F , G , H , and J ). Significance levels were denoted as P <0.05. abs indicates absolute; A.U., arbitrary unit; Cap, capillary endothelial cells; DAPI, 4',6-diamidino-2-phenylindole; FD, false discovery; FDR, false discovery rate; Rep, replicate; and WT, wild type.

Article Snippet: The normal human pulmonary arterial ECs (hPAECs) were purchased from Lonza (Alpharetta, GA).

Techniques: Over Expression, Binding Assay, FLAG-tag, BrdU Incorporation Assay, In Vivo, DNA Synthesis, Isolation, Control, BrdU Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Single-Cell and Spatial Transcriptomics Identified Fatty Acid–Binding Proteins Controlling Endothelial Glycolytic and Arterial Programming in Pulmonary Hypertension

doi: 10.1161/ATVBAHA.124.321173

Figure Lengend Snippet: HIF (hypoxia-inducible factor)-2α–mediated glycolysis induced by FABP (fatty acid–binding protein) 4/5 in pulmonary endothelial cells (ECs). A , A diagram showing the predicted transcription factors based on the DEGs and literature. B , Western blotting demonstrated HIF-2α but not p53 or c-Myc was upregulated in CKO lungs and normalized in TKO lungs. The β-actin on c-Myc blot was shared with Figure 1C FABP5. C , A representative heat map showed that glycolytic genes were dependent on HIF-2α using wild-type (WT), CKO mice, and Egln1 Tie2Cre /Hif2a Tie2Cre (EH2) mice. D , HIF-2α knockdown inhibited FABP4/5–induced endothelial proliferation. E , Nitrative stress assessed by protein nitrotyrosine modification was reduced in TKO lungs compared with CKO lungs. F , Overexpression of FABP4 and FABP5 promoted mitochondrial reactive oxygen species (ROS) levels in human pulmonary arterial EC (hPAECs). G , A diagram showing our proposed model. Our study addresses a novel role of lung endothelial FABP4/5 controlling PAEC accumulation through increased glycolysis in the pathogenesis of pulmonary arterial hypertension (PAH). By facilitating fatty acid uptake and translocation into mitochondria, FABP4/5 promote fatty acid oxidation (FAO) and ROS generation, which activates HIF-2α signaling to promote endothelial glycolysis. For 5-bromo-20-deoxyuridine (BrdU) assay in ( D ) and mitochondrial ROS assay in ( F ), each dot represents a biological replicate. The experiments were performed at least 3 times. ANOVA followed by Tukey post hoc analysis was used for statistical analysis ( B , D , E , and F ). Significance levels were denoted as P <0.05. α-NT indicates anti-nitrotyrosine; Adjp, adjusted P value; A.U., arbitrary unit; DAPI, 4',6-diamidino-2-phenylindole; DEG, differentially expressed gene; dNTP, deoxyribonucleotide triphosphate; ETO, etomoxir; 5-FU, 5-fluorouracil; siCTL, small interfering RNA control; siHIF, small interfering RNA targeting hypoxia-inducible factor; TF, transcription factor; and 2-DG, 2-deoxy-D-glucose.

Article Snippet: The normal human pulmonary arterial ECs (hPAECs) were purchased from Lonza (Alpharetta, GA).

Techniques: Binding Assay, Western Blot, Knockdown, Modification, Over Expression, Translocation Assay, BrdU Staining, ROS Assay, Small Interfering RNA, Control

Journal: Circulation

Article Title: Arterial-Lymphatic-Like Endothelial Cells Appear in Hereditary Hemorrhagic Telangiectasia 2 and Contribute to Vascular Leakage and Arteriovenous Malformations

doi: 10.1161/CIRCULATIONAHA.124.070925

Figure Lengend Snippet: In the absence of ALK1, induction of Sox17 causes arterial lineage ECs to acquire lymphatic endothelial characteristics. A , Time-course expression by real-time PCR of Alk1 and Sox17 in HPAECs after transfection of Alk1 siRNA (n=5). B , Immunoblotting of HAECs transfected with Alk1 siRNA in combination with Sox17 siRNA or infection of lentiviral vectors overexpressing Sox17 (n=3). β-Actin was used as loading control. C , Gene expression in Ephrin B2 + cells isolated from VE-cadherin cre/ERT2 Alk1 flox/flox mice and Alk1 flox/flox mice after Sox17 small hairpin RNA administration (n=8). D , Micro-CT imaging with analysis of nidus formation, vessel radius, and total number of branches of the lungs of VE-cadherin cre/ERT2 Alk1 flox/flox mice and Alk1 flox/flox mice after Sox17 small hairpin RNA administration (n=5). Scale bars=1 mm. E , Immunoblotting of HPAECs transfected with combinations of Alk1 siRNA and Flt4 or Flk1 siRNA (n=3). F , Sox17 expression in HPAECs after transfection of Alk1 siRNA treated with or without VEGF-C, VEGF-C (Cys156Ser), or VEGF-165 (n=7). SCR, scrambled siRNA. G , Immunoblotting after coimmunoprecipitation of Ephrin B2 + cell lysates isolated from VE-cadherin cre/ERT2 Alk1 flox/flox mice and Alk1 flox/flox mice after tamoxifen administration using anti-ALK1 (α-ALK1) or anti-FLT4 (α-FLT4, ab15079) (n=3). H , Immunoblotting after coimmunoprecipitation of Ephrin B2 + cell lysates isolated from VE-cadherin cre/ERT2 Alk1 flox/flox mice and Alk1 flox/flox mice after tamoxifen administration using anti-ALK1 (α-ALK1), anti–VEGF-C (α-VEGF-C), anti-FLT4 (α-FLT4, ab15079), or anti-FLT4 (α-FLT4, AFL4) (n=3). A , C , D , and F were analyzed for statistical significance by ANOVA with post hoc Tukey test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is the median. Error bars are maximal and minimal values ( B ). Error bars are mean±SD ( A ). * P <0.01; *** P <0.0001.

Article Snippet: Human pulmonary arterial ECs (HPAECs) (Thermo Fisher Scientific, catalog No. C-008-5C) were cultured as previously described.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Infection, Control, Gene Expression, Isolation, Micro-CT, Imaging