Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Western Blot, Infection, BrdU Incorporation Assay

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: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Upregulation of TMEM16A in human PAECs. ( a ) Immunofluorescence staining of TMEM16A in PAECs infected with Ctrl Ad or Ano1 Ad (BP = antibody blocking peptide; scale bar = 20 µm). ( b ) Representative whole-cell I ClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ctrl Ad . ( c ) Representative whole-cell I ClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ano1 Ad and overexpressing TMEM16A. ( d ) Consecutive, calculated Bbr-sensitive current comparing primary PAECs infected with Ctrl Ad or Ano1 Ad . Figures were generated with 8–13 cells from N = 2 healthy donors, data are presented as mean ± s.e.m.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Immunofluorescence, Staining, Infection, Blocking Assay, Transfection, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: TMEM16A-mediated membrane depolarization disrupts Ca 2+ dynamics of human PAECs. ( a ) Fluorometric measurements indicating shift in relative resting membrane potential (E m ) of donor PAECs infected with Ctrl Ad or Ano1 Ad using DiBAC 4 (3) dye. ( b ) Representative traces depict changes in intracellular Ca 2+ detected in PAECs transfected with Ctrl Ad or Ano1 Ad . ( c – e ) The effect of TMEM16A overexpression on cytosolic baseline Ca 2+ concentration ([Ca 2+ ] i ), store depletion and Ca 2+ influx using Fura-2 in donor PAECs infected with Ctrl Ad or Ano1 Ad (BHQ = butylhydroquinone). Figures were generated with 80-116 cells from N = 3 healthy donors. * p < 0.05, *** p < 0.001, paired ( a ) and unpaired t-tests ( c – e ).

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Infection, Transfection, Over Expression, Concentration Assay, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Enhanced TMEM16A mediates metabolic changes of PAECs. ( a – b ) Seahorse mitochondrial stress test profiles of TMEM16A-overexpressing primary PAECs showing the ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) OCR/ECAR. ( c ) Proliferation of human PAECs overexpressing TMEM16A measured with thymidine incorporation ( n = 5). ( d , e ) Cas3/Cas7 apoptosis assay and cell-cycle analysis of human PAECs overexpressing TMEM16A. (STS = staurosporin). ( f ) Western blots of PAECs infected with TMEM16A-overexpressing Ano1 Ad or control Ctrl Ad with quantifications of PCNA, cleaved PARP/PARP and Cyclin D1. Figures were generated with 13 separate sets of experiments. * p < 0.05, ratio-paired ( a ) or paired t-test.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Apoptosis Assay, Cell Cycle Assay, Western Blot, Infection, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Elevated TMEM16A activity disturbs eNOS activation: ( a ) Noninduced nitric oxide levels and ACh-induced nitric oxide production of control (Ctrl Ad ) and TMEM16A-overexpressing (Ano1 Ad ) human PAECs. Figures were generated with 8 sets of experiments with quadruplicate in each group and normalized to protein content. ( b ) Western blots showing ACh-induced changes in eNOS phosphorylation of Ctrl Ad and Ano1 Ad -infected donor PAECs with quantification following the eNOS phosphorylation pattern at activatory Ser1177 and inhibitory Thr495 sites after 5, 15 and 30 min of ACh stimulation. ( c ) Quantification of basal, noninduced level of eNOS phosphorylation at Ser1177 and Thr495 as well as phosphorylation of Ser1177 15 min after ACh stimulation. Figures were generated with 6 samples. * p < 0.05, *** p < 0.001, ratio-paired t-test.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Activity Assay, Activation Assay, Generated, Western Blot, Infection