Journal: Journal of Cell Science

Article Title: Contribution of protein–protein interactions to the endothelial-barrier-stabilizing function of KRIT1

doi: 10.1242/jcs.258816

Figure Lengend Snippet: Membrane-targeted KRIT1-RE does not rescue endothelial barrier function. (A) Permeability of transduced monolayers to 40 kDa FITC-dextran. Data shown are mean permeability±s.e.m., normalized to scramble shRNA alone (scr), from n =5 independent experiments. (B) TEER of confluent HPAEC monolayers. Resistance reading of an empty FN-coated Transwell was subtracted from resistance values of Transwells containing HPAEC, then multiplied by the growth area of the wells, yielding Ω*cm 2 values. Data shown are mean Ω*cm 2 values±s.e.m., normalized to scramble shRNA alone (negative control). * P <0.05 by Tukey post-hoc testing vs scramble shRNA alone. # P <0.05 by Tukey post-hoc testing vs shKRIT1 alone. P <0.0001 by one-way ANOVA.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC; Cell Applications, Inc., San Diego, CA, Lot #2228) were cultured in Dulbecco's Modified Eagle's Medium DMEM/F-12 (1:1 ratio), supplemented with 5% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (Gibco/Thermo Scientific, Waltham, MA), 1% endothelial cell growth supplement (ECGS; ScienCell, Carlsbad, CA), and 50 μM heparin (Calbiochem, La Jolla, CA).

Techniques: Membrane, Permeability, shRNA, Negative Control

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A, B) Lung sections of mice conditionally lacking Egfr in ECs were stained for CD68 (green), iNOS (white, A) and Arg1 (red, B). (A, B) The numbers of iNOS + (A) and Arg1 + (B) macrophages were quantified by confocal imaging. (C, D, E) Human pulmonary arterial endothelial cells (PAECs) were transfected with either scrambled siRNA (si CTL ) and siRNA against AREG (si AREG ) and placed in a transwell chamber. They were then cultured in hypoxic conditions with or without leukocytes for 24 h. (C) AREG expression was assessed in leukocytes and HPAECs by qPCR. (D) Apoptosis of normoxic and hypoxic HPAECs was quantified by flow cytometry and shown as fold change compared with the level of apoptosis in si CTL PAECs. (E) HPAECs were plated on Matrigel, and tube formation was measured. (F) Human PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. PAECs apoptosis was assessed by measuring caspase 3 + cells and caspase 3 MFI by flow cytometry. Isotype control was used to determine caspase 3 positivity. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Staining, Imaging, Transfection, Cell Culture, Expressing, Flow Cytometry, Recombinant

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: , (A) Schematic representing AREG and its upstream genes including BRCA1 , HLX , NCOA6 , PHB2 , RRP1B , TAF4 , TP63 , and VAV2 was generated using the Ingenuity Pathway Analysis Software. Each arrow represents the activation of AREG by each gene. (B) Schematic depicting HIF-1⍺–binding sites in PHB2 , RRP1B , and NCOA6 gene promoter regions. This schematic was designed using UCSC Genome Browser website ( https://genome.ucsc.edu ) and Snapgene software ( https://www.snapgene.com ). (C, D, E) Pulmonary arterial endothelial cells (PAECs) were transfected with scrambled siRNA (si CTL ) or siRNA against NCOA6 ( siNCOA6 ), PHB2 (si PHB2 ) or RRP1B (si RRP1B ) and placed in normoxia for 24 h. (C) Apoptotic PAECs were quantified by measuring caspase 3 + cells and caspase 3 MFI by flow cytometry. (D) PAECs were plated on Matrigel, and tube formation was assessed. (E) AREG expression was assessed by qPCR. (F) Human PAECs were transfected with either scrambled siRNA (si CTL ), or siRNA against HIF1A and placed in normoxic or hypoxic conditions for 24 h. AREG expression was quantified by qPCR. (G) Human PAECs were transfected with a lentivirus overexpressing HIF1-A for 48 h. The cells were transfected with siRNA against NCOA6 , PHB2 , or RRP1B and cultured in normoxic conditions for 48 h. AREG expression was assessed by qPCR. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Generated, Software, Activation Assay, Binding Assay, Transfection, Flow Cytometry, Expressing, Cell Culture

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A) BAD expression in pulmonary arterial endothelial cells (PAECs) was quantified after AREG silencing (left panel) and recombinant Amphiregulin treatment (right panel). (B, C, F) PAECs were transfected with either scrambled siRNA (si CTL ) or siRNA against BAD (si BAD ) and placed in hypoxia for 24 h. (B, C) PAECs apoptosis was assessed by measuring caspase 3 + and caspase 3 MFI cells by flow cytometry (B), and tube formation ability was determined by a Matrigel assay (C). (D, E) PAECs were transfected with either scrambled siRNA (si CTL ), siRNA against AREG (si AREG ) or siRNA against both AREG and BAD (si BAD/AREG ) and placed in normoxic conditions for 24 h. (D, E) Apoptosis (D) and tube formation (E) were examined. (F) IFNB , IL1B , IL6 , and TNFA expression was assessed by qRT-PCR. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, *** P < 0.005, **** P < 0.001.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Expressing, Recombinant, Transfection, Flow Cytometry, Matrigel Assay, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A) BAD expression and the frequency of BAD + cells were determined by flow cytometry after AREG silencing in normoxic and hypoxic pulmonary arterial endothelial cells (PAECs). (B) PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. BAD expression was measured by RT-qPCR. (C, D) HPAECs were co-cultured in a transwell with leukocytes and then treated with either control or BAD siRNA. (C) Granulocytes, monocytes, and T cells were enumerated by flow cytometry. (D) Cytokine concentrations were assessed by ELISA. (E) Mechanisms of increased PAEC apoptosis and exaggerated inflammation in the absence of AREG and epidermal growth factor receptor (EGFR) in pulmonary hypertension (PH). Our data support a model whereby decreased amphiregulin and EGFR expression in PAECs promote PH. Specifically, in the steady state, amphiregulin binds to the EGFR, which decreases the expression of BCL2-associated agonist of Cell Death (BAD), resulting in PAEC survival and suppressed inflammation. In PH, HIF-1⍺ binds to the promoters of NCOA6 , PHB2 , and RRP1B and increases their expression. These genes down-regulate AREG , resulting in augmented BCL2 expression. This pro-apoptotic gene, in turn, incites apoptosis and chemokine production. Elevated levels of the chemokines recruit inflammatory myeloid cells in lung vasculature. Mechanisms that were not investigated in the present study are labeled with a dotted arrow. The cartoon was designed with the online Biorender software ( https://biorender.com ). n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Expressing, Flow Cytometry, Recombinant, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling, Software

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: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Repurposing rosiglitazone, a PPAR-γ agonist and oral antidiabetic, as an inhaled formulation, for the treatment of PAH

doi: 10.1016/j.jconrel.2018.04.049

Figure Lengend Snippet: The levels of eNOS expression in human PAH PAECs in hypoxic and normoxic condition (A), PPARγ and endothelin-1 in PAH-ECs (B); PPARγ and NOX-4 expression in PAH-SMCs (C). n=3.

Article Snippet: 2.2.10 The effects of rosiglitazone on human PAH-ECs and PAH-SMCs We grew human pulmonary arterials ECs and SMCs (collected from PAH patients), upon receipt from the Pulmonary Hypertension Breakthrough Initiative (PHBI), in gelatin coated dishes using MV2 (PromoCell GmbH, Heidelberg, Germany) and SmGM (Lonza Inc., Williamsport, PA) media, respectively for 5-7 days until their confluency.

Techniques: Expressing