scholarly journals The Effect of Sodium Bicarbonate, a Beneficial Adjuvant Molecule in Cystic Fibrosis, on Bronchial Epithelial Cells Expressing a Wild-Type or Mutant CFTR Channel

2020 ◽  
Vol 21 (11) ◽  
pp. 4024 ◽  
Author(s):  
Ilona Gróf ◽  
Alexandra Bocsik ◽  
András Harazin ◽  
Ana Raquel Santa-Maria ◽  
Gaszton Vizsnyiczai ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Sodium Bicarbonate ◽  
Vascular Endothelial Cells ◽  
Bronchial Epithelium ◽  
Barrier Properties ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Bronchial Epithelial

Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.

scholarly journals Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

2012 ◽  
Vol 303 (2) ◽  
pp. L97-L106 ◽  
Author(s):  
Shilpa Nimishakavi ◽  
Marina Besprozvannaya ◽  
Wilfred W. Raymond ◽  
Charles S. Craik ◽  
Dieter C. Gruenert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Selective Inhibition ◽  
Airway Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na+ channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o−) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o− epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

scholarly journals Augmentation of CFTR maturation by S-nitrosoglutathione reductase

2016 ◽  
Vol 310 (3) ◽  
pp. L263-L270 ◽  
Author(s):  
Khalequz Zaman ◽  
Victoria Sawczak ◽  
Atiya Zaidi ◽  
Maya Butler ◽  
Deric Bennett ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Reductase Activity ◽  
Subcellular Location ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Wild Type ◽  
Bronchial Epithelial ◽  
Human Airway ◽  
Human Airway Epithelial Cells

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

Thrombomodulin-mediated catabolism of protein C by pleural mesothelial and vascular endothelial cells

2007 ◽  
Vol 98 (09) ◽  
pp. 627-634 ◽  
Author(s):  
Alireza Rezaie ◽  
Steven Idell ◽  
Alexei Iakhiaev
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Vascular Endothelial Cells ◽  
Degradation Products ◽  
Cell Types ◽  
Model Systems ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Endothelial Protein C Receptor ◽  
Gla Domain

SummaryPleural mesothelial and vascular endothelial cells express protein C (PC) pathway components including thrombomodulin (TM) and endothelial protein C receptor (EPCR) and activate PC by the thrombin-TM dependent mechanism.We used these cells as model systems to identify molecules involved in endocytosis and degradation of PC. We find that mesothelial and endothelial cells can bind, internalize and degrade PC.Addition of thrombin markedly induced degradation of PC by these cells in a TM-dependent fashion, implicating the involvement of the thrombin-TM complex in internalization and degradation of PC. This observation defines a novel function for the thrombin-TM complex as a degradation receptor for PC and suggests that PC is degraded concurrent with its activation.A PC Gla-domain mutant, which is unable to bind to the EPCR, was degraded by the cells to a lesser extent than wild-type PC, implicating the PC degradation concurrent with its activation. Consistent with the role of thrombin-TM complex as a degradation receptor, the catalytically inactive thrombin-S195A also induced PC degradation though to a lesser extent than wild-type thrombin.This suggests that generation of activated PC (APC) can contribute to accumulation of degradation products, but is not essential for the thrombin-induced degradation of PC. The thrombin-TMmediated degradation of PC by both cell types suggest a previously unrecognized mechanism, which can contribute to PC consumption.This mechanism may be pathophysiologically relevant and can contribute to an acquired PC deficiency in conditions characterized by sustained thrombin generation.

Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells

2000 ◽  
Vol 279 (2) ◽  
pp. L371-L378 ◽  
Author(s):  
Sandrine Boussat ◽  
Saadia Eddahibi ◽  
André Coste ◽  
Virginie Fataccioli ◽  
Mallaury Gouge ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Vascular Endothelial ◽  
Vegf Expression ◽  
Bronchial Epithelial ◽  
Lung Epithelial ◽  
Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor highly expressed in rodent alveolar epithelium after injury and repair. To investigate VEGF synthesis in human lung epithelial cells, we examined VEGF expression by cultured cells under basal conditions and after cytokine treatment or oxidative stress. Basal VEGF expression was detected in transformed human epithelial cell lines (A549 and 1HAEo−) and in primary human bronchial epithelial cells with RT-PCR, Western blot, and immunocytochemistry. Among the cytokines tested, only transforming growth factor-β1 increased the levels of excreted VEGF165 as measured by ELISA. Under hypoxia (0% O2 for 24 h), the VEGF165 level increased fivefold, and this effect was O2 concentration dependent. VEGF concentrations in the medium of all the cell types studied reached values similar to those found in bronchoalveolar lavage fluids from normal patients. Endothelial cells (human umbilical vein endothelial cells) exposed to conditioned medium from primary bronchial epithelial cell cultures showed an increased growth rate, which was inhibited in the presence of a specific neutralizing antibody to VEGF. These results suggest that lung epithelial cells participate in the endothelial repair and angiogenesis that follow lung injury through the synthesis of VEGF.

scholarly journals Extracellular 2′,3′-cAMP and 3′,5′-cAMP stimulate proliferation of preglomerular vascular endothelial cells and renal epithelial cells

2012 ◽  
Vol 303 (7) ◽  
pp. F954-F962 ◽  
Author(s):  
Edwin K. Jackson ◽  
Delbert G. Gillespie
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Cells ◽  
Extracellular Adenosine ◽  
Tubular Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular

Kidneys release into the extracellular compartment 3′,5′-cAMP and its positional isomer 2′,3′-cAMP. The purpose of the present study was to investigate the metabolism of extracellular 2′,3′-cAMP and 3′,5′-cAMP in preglomular vascular endothelial and proximal tubular epithelial cells and to determine whether these cAMPs and their downstream metabolites affect cellular proliferation. In preglomerular vascular endothelial and proximal tubular epithelial cells, 1) extracellular 2′,3′-cAMP increased extracellular levels of 3′-AMP and 2′-AMP, whereas extracellular 3′,5′-cAMP increased extracellular levels of 5′-AMP; 2) extracellular 5′-AMP, 3′-AMP, and 2′-AMP increased extracellular adenosine; 3) α,β-methylene-adenosine-5′-diphosphate (CD73 inhibitor) prevented the 5′-AMP-induced increase in extracellular adenosine in preglomerular vascular endothelial cells, but did not affect the 5′-AMP-induced increase in extracellular adenosine in proximal tubular cells or the 3′-AMP-induced or 2′-AMP-induced increase in extracellular adenosine in either cell type; 4) extracellular 2′,3′-cAMP, 3′-AMP, 2′-AMP, 3′,5′-cAMP, 5′-AMP, and adenosine stimulated proliferation of both preglomerular vascular endothelial and proximal tubular cells; and 5) MRS-1754 (selective A2B receptor antagonist) abolished the progrowth effects of extracellular 2′,3′-cAMP, 3′-AMP, 2′-AMP, 3′,5′-cAMP, 5′-AMP, and adenosine in both cell types. Extracellular 2′,3′-cAMP and 3′,5′-cAMP stimulate proliferation of preglomerular vascular endothelial cells and proximal tubular cells. The mechanism by which the cAMPs increase cell proliferation entails 1) metabolism to their respective AMPs, 2) metabolism of their respective AMPs to adenosine (which for 5′-AMP in preglomerular vascular endothelial cells is mediated by CD73), and 3) activation of A2B receptors. Both extracellular 2′,3′-cAMP and 3′,5′-cAMP may help restore architecture of the preglomerular microcirculation and tubular system following kidney injury.

scholarly journals Exercise Inhibits Doxorubicin-Induced Damage to Cardiac Vessels and Activation of Hippo/YAP-Mediated Apoptosis

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2740
Author(s):  
Rong-Hua Tao ◽  
Masato Kobayashi ◽  
Yuanzheng Yang ◽  
Eugenie S. Kleinerman
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Caspase 3 ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Wild Type ◽  
Major Side Effect ◽  
Cardiac Blood ◽  
Fractional Shortening

Dose-related cardiomyopathy is a major side effect following doxorubicin (Dox). To investigate whether exercise (Ex)-induced vasculogenesis plays a role in reducing Dox-induced cardiotoxicity, GFP+ bone marrow (BM) cells from GFP transgenic mice were transplanted into wild-type mice. Transplanted mice were treated with Dox, Ex, Dox+Ex, or control. We found Dox therapy resulted in decreased systolic and diastolic blood flow, decreased ejection fraction and fractional shortening, and decreased vascular endothelial cells and pericytes. These abnormalities were not seen in Dox+Ex hearts. Heart tissues from control-, Ex-, or Dox-treated mice showed a small number of GFP+ cells. By contrast, the Dox+Ex-treated hearts had a significant increase in GFP+ cells. Further analyses demonstrated these GFP+ BM cells had differentiated into vascular endothelial cells (GFP+CD31+) and pericytes (GFP+NG2+). Decreased cardiomyocytes were also seen in Dox-treated but not Dox+Ex-treated hearts. Ex induced an increase in GFP+c-Kit+ cells. However, these c-Kit+ BM stem cells had not differentiated into cardiomyocytes. Dox therapy induced phosphorylation of MST1/2, LATS1, and YAP; a decrease in total YAP; and cleavage of caspase-3 and PARP in the heart tissues. Dox+Ex prevented these effects. Our data demonstrated Dox-induced cardiotoxicity is mediated by vascular damage resulting in decreased cardiac blood flow and through activation of Hippo-YAP signaling resulting in cardiomyocyte apoptosis. Furthermore, Ex inhibited these effects by promoting migration of BM stem cells into the heart to repair the cardiac vessels damaged by Dox and through inhibiting Dox-induced Hippo-YAP signaling-mediated apoptosis. These data support the concept of using exercise as an intervention to decrease Dox-induced cardiotoxicity.

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