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

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Endothelial cells characterization regarding morphological, immunophenotyping, and vessel-like structures assay. ( A ) Phase contrast micrography demonstrating the polygonal morphology of aortic artery endothelial cells (PAEC), coronary artery endothelial cells (CAEC), human umbilical vein endothelial cells (HUVEC), and pulmonary artery endothelial cells (HPAEC) cells (100× magnification). ( B ) Immunophenotyping of ECs by flow cytometry. ( C ) All endothelial cells (PAEC, CAEC, HUVEC, and HPAEC) were able to form vessel-like structures when grown in matrigel, evidencing characteristics typical of CEs (40× and 100× magnification).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Characterization of EndMT induction by TGF-β2 (10 ng/mL) in cell lines ( A ) PAEC, ( B ) CAEC, ( C ) HPAEC, and ( D ) HUVECs (non-treated or treated with TGF-β2). Immunofluorescence microscopy of cell lines induced to EndMT shows a decrease in the fluorescent intensity of CD31 (green) in PAECs, CAECs, and HUVECs cells. The nuclei were stained with DAPI (blue) and F-actin were stained with Phalloidin (red) (scale bar 50 µM; representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: TGF-β2 decrease formation of vessel-like structures in the cell lines (CAEC, PAEC, HPAEC, and HUVEC). The cells were treated with TGF-β2 and evaluated the capacity formation of vessel-like structures. This inhibitory effect was observed mainly in PAECs (representative image of one replicate; n = 3).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Effect of EndMT on the activation of the Erk pathway. The cells (CAEC, PAEC, HUVEC and HPAEC) were cultured for five days in presence TGF-β2 (10 ng/mL). Aliquots were withdrawn after the treatment and evaluated by ( A ) Multiplex technique analysis Array Kit ( n = 3, * p ≤ 0.05) and ( B ) western blotting using phospho-Erk1/2 (Thr202/Tyr204) and ERK1/2. β-actin were used as endogenous controls (representative image of one replicate of each sample). ( C ) Chemical inhibitor against MEK1/2 (U0126; 1 μM) inhibits the increase of ERK1/2 phosphorylation in the PAECs treated with TGF-β2. 1) U0126; 2) U0126-15′ TGF-β2; 3) U0126-30′ TGF-β2; 4) TGF-β2-15′; 5) TGF-β2-30′. GAPDH were used as endogenous controls (representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Activation Assay, Cell Culture, Multiplex Assay, Western Blot, Phospho-proteomics