A study of the involvement of endothelins in the effects of hypoxia on the rat lung

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN016249 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Study of the actions of vasoactive substances in the rat isolated perfused lung

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN004152 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Experimentelle Untersuchungen zum pulmonalen Hochdruck : die Bedeutung der Hypoxie-induzierbaren Faktoren für pulmonalvaskuläre Remodellingprozesse in pulmonalarteriellen Fibroblasten

Die Hypoxie induzierte Proliferation der pulmonalarteriellen Fibroblasten in der Adventitia der kleinen intrapulmonalen Arterien ist als frühes Stadium in der Entwicklung der pulmonalen Hypertonie und des Cor pulmonale beschrieben worden. Wir untersuchten die Rolle der Hypoxie-induzierbaren Faktoren (HIF-1alpha und HIF-2alpha) in primären humanen pulmonalarteriellen Fibroblasten (FB-PA) unter hypoxischen Bedingungen. Der Schwerpunkt lag in der Differenzierung zwischen den beiden HIF-Isoformen und die Bedeutung der Inhibition für die pathophysiologische Reaktion der PA-FB unter Hypoxie. Dazu wurde zunächst die RNA interferenz zur Inhibition von HIF an den humanen Adenokarzinomzellen (A549) etabliert. Es zeigte sich eine potente Inhibition von HIF-1alpha, die Auswirkungen auf die Zellzyklusregulation unter Hypoxie hatte. Vor der Anwendung dieser Methode an den primären Zellen wurden zunächst die Reaktion der PA-FB auf Hypoxie und die Expression der Hypoxie-induzierbaren Faktoren untersucht. Unter hypoxischen Bedingungen reagierten die PA-FB mit einer starken proliferativen Antwort. Im Zellzyklus bewirkte die Hypoxie eine Verschiebung der PA-FB in die S-Phase, wobei die Apoptose gleichzeitig supprimiert wurde. Neben der Steigerung der Proliferation induzierte Hypoxie auch die Migration der PA-FB. Die Expression von alpha-Smooth-Muscle-Cell Aktin, als Indikator der Transdifferenzierung zu Myofibroblasten, blieb jedoch unverändert. Die folgende Analyse der Expression konnte HIF-1alpha und HIF-2alpha auf mRNA-Ebene konstitutiv nachweisen. Auf Protein-Ebene zeigten beide HIF-Isoformen eine starke Induktion unter Hypoxie und unter normoxischen konnte nur das HIF-2alpha Protein zytosolisch und perinukleäre nachgewiesen werden. Die Aktivierung der beiden Subtypen und somit die Funktionalität als Transkriptionsfaktor wurde mittel RT-PCR für die Zielgene von HIF-1alpha und HIF-2alpha dargestellt. Durch siRNA für HIF-1alpha und HIF-2 wurde diese Aktivierung sowohl für die Phosphoglycerat Kinase (PGK), als auch für den Vascular endothelial growth factor (VEGF) inhibiert. Weiterhin bedingte die Suppression von HIF-1alpha, wie auch von HIF-2alpha einen Stopp der Hypoxie induzierten Migration, wobei die Proliferation nur durch siRNA gegen HIF-2alpha zu inhibieren war. PA-FB proliferieren und migrieren unter Hypoxie HIF gesteuert, so dass eine Transdifferenzierung in der Media der pulmonalarteriellen Gefäße ermöglicht wird. Die Proliferation ist dabei abhängig von HIF-2alpha während die Migration durch HIF-1alpha und HIF-2alpha gesteuert wird. Die Transdifferenzierung in PA-FB ist weder direkt durch Hypoxie induziert noch HIF abhängig. Diese Ergebnisse demonstrieren die wichtige Bedeutung der Hypoxie induzierten Faktoren in den PA-FB für die Entwicklung der pulmonalen Hypertonie.Proliferation of adventitial fibroblasts of small intrapulmonary arteries (PA-FB) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response to hypoxia. We investigated the role of hypoxia-inducible transcription factors (HIF) in human PA-FB exposed to hypoxia. Particular attention was paid to a putative role of hypoxia-inducible factors, with two major acting subtypes being currently known (HIF-1alpha, HIF-2alpha). Both subtypes regulate their target gene via the hypoxia-responsive elements (HRE). For determination between the different HIF Isoforms RNA-interference for HIF-1alpha and HIF-1alpha was established using the human adenocarcinoma cell line A549. In this system a potent inhibition of HIF-1alpha with consequences in cell cycle regulation was observed. Further investigations with primary cultures of PA-FB displayed a strong mitogenic response to 24 h of hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of PA-FB was strongly increased under hypoxic conditions but not the expression of alpha-smooth muscle cell actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1alpha and HIF- 2alpha, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1alpha or HIF-2alpha was achieved by RNA interference technology, as proven by HIF-1alpha and HIF-2alpha mRNA and protein analysis and expression analysis of HIF downstream target genes. With the use of this approach, the hypoxia-induced proliferative response of the PA-FB was found to be solely HIF-2alpha dependent, whereas the migratory response was significantly reduced by both HIF-1alpha and HIF-2alpha interference. In conclusion, HIF up-regulation is essential for hypoxic cellular responses in human pulmonary artery adventitial fibroblasts such as proliferation and migration, mimicking the pulmonary hypertensive phenotype in vivo. Differential HIF subtype dependency was noted, with HIF-2alpha playing a predominant role, which may offer future intervention strategies

Oxygen sensing, mitochondrial biology and experimental therapeutics for pulmonary hypertension and cancer

The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca2+ and Ca2+-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca2+, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca2+, promoting Warburg metabolism, whilst increasing cytosolic Ca2+, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca2+ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies

Factores de riesgo cardiometabólico y alteraciones cardiopulmonares por exposición a hipoxia intermitente de altura: papel de la AMPK

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Fisiología. Fecha de Lectura: 21-07-2022La Tesis ha sido Financiada por el proyecto “Generación de nuevos productos para potenciar el capital humano y tecnológico en el desarrollo innovativo de la medicina de altura en la región de Tarapacà”, código: BIP30477541-

Examining the functional role of transporters in modulating drug absorption across lung epithelium

P-glycoprotein (P-gp – MDR-1), a 170 kDa glycosylated membrane bound protein, is a member of the ATP-binding cassette transporter family. The potential for P-gp to reduce drug absorption across lung epithelia is of significant interest; this is particularly so, given P-gp’s broad substrate specificity mediating efflux transport of a range of structurally unrelated substrates. Within lung, Pgp expression is evident in bronchial and alveolar epithelia with functional characterisation of P-gp transport capacity within lung epithelial cells currently restricted to respective in-vitro cell culture models. The aims of this project were to establish the relative mRNA expression of several ATP Binding Cassette (ABC), Solute Carrier (SLC) and Solute Carrier Organic Anion (SLCO) drug transporters within rat lung samples through use of RT-PCR; expression suggesting the potential to serve as targets for pulmonary drug delivery. Further, validation of an Isolated Perfused Rat Lung preparation for use in assessment of drug transport across the lung was conducted. In order to assess the functional significance of the ABC drug transporter, P-glycoprotein, on drugs instilled intra-tracheally to the IPRL set-up, use of the P-gp substrates; Rhodamine 123 (Rh123), digoxin, and flunisolide and the P-gp inhibitor, GF120918 was employed. Further, use of kinetic modelling was employed to establish pharmacokinetic parameters involved. Using the IPRL, the P-gp dependent pulmonary absorption of the P-gp substrate, Rh123, was demonstrated. Dose-dependent absorption, consistent with a saturable component in the molecule’s pulmonary absorption, was demonstrated. Further, the absorption of low dose Rh123 was promoted by the presence of the highly selective P-gp inhibitor GF120918, consistent with a functional role of P-gp mediated efflux within an intact lung; an efflux process which may limit the pulmonary absorption of a lung administered molecule. Further studies using this system and extending the range of molecules studied will provide greater understanding of the quantitative significance of P-gp in limiting pulmonary absorption across lung epithelium.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Examining the functional role of transporters in modulating drug absorption across lung epithelium

P-glycoprotein (P-gp – MDR-1), a 170 kDa glycosylated membrane bound protein, is a member of the ATP-binding cassette transporter family. The potential for P-gp to reduce drug absorption across lung epithelia is of significant interest; this is particularly so, given P-gp’s broad substrate specificity mediating efflux transport of a range of structurally unrelated substrates. Within lung, Pgp expression is evident in bronchial and alveolar epithelia with functional characterisation of P-gp transport capacity within lung epithelial cells currently restricted to respective in-vitro cell culture models. The aims of this project were to establish the relative mRNA expression of several ATP Binding Cassette (ABC), Solute Carrier (SLC) and Solute Carrier Organic Anion (SLCO) drug transporters within rat lung samples through use of RT-PCR; expression suggesting the potential to serve as targets for pulmonary drug delivery. Further, validation of an Isolated Perfused Rat Lung preparation for use in assessment of drug transport across the lung was conducted. In order to assess the functional significance of the ABC drug transporter, P-glycoprotein, on drugs instilled intra-tracheally to the IPRL set-up, use of the P-gp substrates; Rhodamine 123 (Rh123), digoxin, and flunisolide and the P-gp inhibitor, GF120918 was employed. Further, use of kinetic modelling was employed to establish pharmacokinetic parameters involved. Using the IPRL, the P-gp dependent pulmonary absorption of the P-gp substrate, Rh123, was demonstrated. Dose-dependent absorption, consistent with a saturable component in the molecule’s pulmonary absorption, was demonstrated. Further, the absorption of low dose Rh123 was promoted by the presence of the highly selective P-gp inhibitor GF120918, consistent with a functional role of P-gp mediated efflux within an intact lung; an efflux process which may limit the pulmonary absorption of a lung administered molecule. Further studies using this system and extending the range of molecules studied will provide greater understanding of the quantitative significance of P-gp in limiting pulmonary absorption across lung epithelium