Role of CARM1 in regulation of alveolar epithelial senescence and emphysema susceptibility

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases world-wide. A major feature of COPD is emphysema- a long-term, progressive condition. The hallmark of emphysema includes the destruction of alveolar structures leading to enlarged air spaces and reduced surface area. Experimental evidence suggests that emphysema development is driven by accelerated senescence of lung cells but the underlying mechanism of senescence is yet to be fully elucidated. Protein arginine methyltransferases (PRMTs) are important for cellular processes, such as the regulation of senescence, cell proliferation, differentiation and apoptosis. The PRMT family includes 11 members classified as type I, II or III enzymes depending on their methylation pattern (asymmetric dimethylation, symmetric dimethylation or monomethylation, respectively). One member of this family is PRMT4, a type I enzyme, which is also called coactivator associated arginine methyltransferase 1 (CARM1). It was originally identified as a coactivator for steroid hormone receptors. CARM1 is known to methylate histone H3 and various non-histone proteins that play essential roles in transcriptional regulation, RNA splicing, and metabolism. Most importantly, complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type-II cells (ATII). Furthermore, CARM1 also plays a role in regulating cellular senescence via CARM1-dependent methylation. Based on these reports, we hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and siRNA-mediated knockdown in ATII-like LA4 cells in vitro. We monitored emphysema progression for 161 days in mice treated with a single oropharyngeal application of elastase. The progression was manifested by the decline in lung function parameters. The mean chord length (Lm) confirmed a time dependent airspace enlargement and was directly correlated with a significant increase in dynamic lung compliance. We also observed that at later time points (day 56 and 161), emphysema progression was inflammation-independent. We demonstrated that emphysema advancement was associated with a time-dependent downregulation of CARM1, specifically in alveolar epithelial cells. Furthermore, the global CARM1 activity was also reduced as reflected by an elevated level of CARM1 phosphorylation in the lung. Most importantly, elastase-treated CARM1 haploinsufficient mice showed significantly increased airspace enlargement (52.5±9.6 µm vs. 38.8±5.5 µm, p<0.01) and lung compliance (2.8±0.32 µl/cmH20 vs. 2.4±0.4 µl/cmH20, p<0.04) compared with wild type controls. Reduced CARM1 contributed to senescence of alveolar epithelial cells evident by the reduction of anti-senescence SIRT1 and the induction of senescence markers p16 and β-galactosidase in alveolar epithelial cells. We further demonstrated that CARM1 haploinsufficiency impaired trans-differentiation of ATII into ATI cells. Elastase treatment in CARM1 deficient mouse lungs led to the accumulation of SP-C positive ATII cell. In our in vitro studies, we detected that the knockdown of CARM1 in the ATII-like cell line LA-4 led to decreased SIRT1 expression (0.034±0.003 vs. 0.022±0.001, p<0.05), but increased expression of p16 (0.27±0.013 vs. 0.31±0.010, p<0.5), p21 (0.81±0.088 vs. 1.28± 0.063, p<0.01) and induced a higher number of β-galactosidase-positive senescent cells (50.57%±7.36 vs. 2.21%±0.34, p<0.001), compared with scrambled siRNA. The senescence in CARM1 deficient cells was further augmented after CSE stimulation. Furthermore, we demonstrated that CARM1 deficiency impaired wound healing (32.18%±0.9512 vs. 8.769%±1.967 wound gap closure, p<0.001) of alveolar epithelial cells. The data confirmed that CARM1 reduction induced an accelerated senescence in LA-4 cells by attenuating the effect of SIRT1. Overall, both our in vivo and in vitro results revealed a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence, as well as impaired regeneration, repair and differentiation of ATII cells.Die chronisch obstruktive Lungenerkrankung (COPD) zählt zu den häufigsten schweren Krankheiten weltweit und ist gekennzeichnet durch eine irreversible Abnahme der Lungenfunktion. Ein Hauptcharakteristikum von COPD ist das Emphysem, das sich stetig über einen langen Zeitraum fortschreitend entwickelt, und durch die Zerstörung alveolärer Strukturen, einhergehend mit vergrößerten Lufträumen und verringerter Alveolaroberfläche, gekennzeichnet ist. Ergebnisse aus Studien legen nahe, dass die Entwicklung des Emphysems durch die erhöhte Seneszenz von Zellen der Lunge beschleunigt wird, doch die genauen zugrundeliegenden Mechanismen sind noch nicht aufgeklärt. Protein-Arginin-Methyltransferasen (PRMTs) spielen eine bedeutende Rolle bei zellulären Prozessen wie Regulation von Seneszenz, Zellproliferation, Differenzierung und Apoptose. Die Familie der PRMTs umfasst 11 Enzyme, die als Typ I, II, oder III klassifiziert werden, abhängig von ihrem Methylierungsmuster (asymmetrische Dimethylierung, symmetrische Dimethylierung oder Monomethylierung). Ein Mitglied der Familie ist PRMT4, ein Typ I Enzym, das auch unter dem Namen Coactivator-assoziierte Arginin-Methyltransferase 1 (CARM1) bekannt ist. Ursprünglich wurde CARM1 als Coactivator für Steroid-Hormon-Rezeptoren beschrieben. CARM1 methyliert Histon 3 und verschiedene weitere Nicht-Histon Proteine, die eine wichtige Rolle für die Transkriptionsregulation, das RNA-Splicing und den Stoffwechsel spielen. Darüberhinaus führt der komplette Verlust von CARM1 zu gestörter Differenzierung und Maturation von Typ II Alveolarepithelzellen (ATII). Weiterhin ist CARM1 von Bedeutung für die Regulation von zellulärer Seneszenz mittels CARM1-abhängiger Methylierung. Auf Grundlage dieser Arbeiten wurde die Hypothese aufgestellt, dass CARM1 die Entwicklung und das Voranschreiten des Emphysems reguliert. Um dies aufzuklären, wurde die Rolle von CARM1 am Verlust der Alveolen in einem Elastase-induzierten Emphysem-Mausmodell in vivo sowie in vitro mittels siRNA knockdown in ATII-ähnlichen LA4-Zellen untersucht. Die Entwicklung und das Voranschreiten des Emphysems wurden über einen Zeitraum von 161 Tagen beobachtet. Dabei zeigte sich eine progressive Abnahme verschiedener Lungenfunktionsparameter. Die mittlere Alveolarweite bestätigte die zeitabhängige Vergrößerung des Luftraumes und war direkt mit einer signifikanten Zunahme der dynamischen Lungencompliance korreliert. Außerdem wurde an späteren Zeitpunkten (Tag 56 und 161) eine entzündungsabhängige Progression des Emphysems beobachtet. Das Voranschreiten des Emphysems war mit einer zeitabhängigen Herunterregulierung von CARM1 insbesondere in Alveolarepithelzellen assoziiert. Die Gesamtaktivität von CARM1 war ebenso reduziert, erkennbar anhand von erhöhter CARM1-Phosphorylierung in der Lunge. Weiterhin zeigten mit Elastase behandelte CARM1 haploinsuffiziente Mäuse eine signifikant erhöhte Vergrößerung des Luftraumes (52.5±9.6 µm vs. 38.8±5.5 µm, p<0.01) sowie der Lungencompliance (2.8±0.32 µl/cmH20 vs. 2.4±0.4 µl/cmH20, p<0.04) verglichen mit Wildtyp-Kontrollmäusen. Das verringerte Level an CARM1 trug zur Seneszenz von Alveolarepithelzellen bei, bestätigt durch eine Reduktion des Anti-Seneszenz-Markers SIRT1 und durch die Induktion der Seneszenz-Marker p16 und β-Galactosidase in Alveolarepithelzellen. Des Weiteren wurde gezeigt, dass CARM1 Haploinsuffizienz die Transdifferenzierung von ATII zu ATI Zellen verhindert. Dabei führte die Elastase-Behandlung von CARM1-defizienten Mäusen zu einer Akkumulation von SP-C-positiven ATII-Zellen in der Lunge. Die in vitro Experimente haben gezeigt, dass der knockdown von CARM1 in ATII-ähnlichen LA4-Zellen zu einer verringerten SIRT1-Expression (0.034±0.003 vs. 0.022±0.001, p<0.05), und gleichzeitig zu einer erhöhten Expression von p16 (0.27±0.013 vs. 0.31±0.010, p<0.5), p21 (0.81±0.088 vs. 1.28± 0.063, p<0.01) führte. Weiterhin verursachte der CARM1 knockdown eine erhöhte Anzahl an β-Galactosidase-positiven seneszenten Zellen verglichen mit Kontroll-siRNA-behandelten Zellen (50.57%±7.36 vs. 2.21%±0.34, p<0.001). Die erhöhte Seneszenz in CARM1-defizienten Zellen wurde durch Stimulation mit Zigarettenrauchextrakt noch verstärkt. Weiterhin zeigte sich in den Alveolarepithelzellen eine verhinderte Wundheilung aufgrund CARM1-Defizienz (32.18%±0.9512 vs. 8.769%±1.967 Wundschließung, p<0.001). Diese Daten bestätigten, dass die Reduzierung von CARM1 eine beschleunigte Seneszenz in LA4-Zellen mittels Abschwächung von SIRT1-Effekten verursachte. Zusammenfassend zeigen die in vivo und vitro Ergebnisse eine neue Rolle von CARM1 bei der Regulierung der Emphysem-Entstehung und der vorzeitigen Lungenalterung, in erster Linie mittels Seneszenz von Alveolarepithelzellen, sowie beeinträchtigter Regenerierung, Reparatur und Differenzierung von ATII-Zellen

Biomechanical determinants of emphysema progression in chronic obstructive pulmonary disease

Emphysema is a disease of the lung parenchyma associated with chronic obstructive pulmonary disease (COPD) and characterized by progressive, irreversible tissue destruction. While chronic inflammation due to repeated noxious particle exposure is the most common environmental risk factor, biomechanical stresses are also known to contribute. It is thought that inflammation-related enzymatic weakening predisposes tissue to mechanical failure, leading to self-propagating parenchymal destruction. However, essential questions regarding the underlying disease mechanisms and their link to overall lung decline remain unanswered. The overarching goals of this dissertation were to relate changes at the cell and tissue level to lung structure and function, and to determine how clinical interventions impact the mechanical balance of parenchymal tissue stresses. First, we use a computational network model of lung volume reduction, a palliative treatment for end-stage emphysema, to demonstrate how recent bronchoscopic, biomaterial-based treatments can achieve similar outcomes as traditional surgical procedures. Next, in a cohort of COPD patients with follow-up computed tomography (CT) imaging, we identify a previously unrecognized structural feature of emphysema that suggests a fundamentally new mechanism of disease progression and potential target for tissue engineering solutions. Finally, we describe the design and implementation of an ex vivo platform for cyclic stretching of precision-cut lung slices, demonstrating a stretch-dependent inflammatory response to acute cigarette smoke extract exposure. In summary, this work combines computational modeling, clinical imaging, and ex vivo measurements to characterize the biomechanical stresses driving emphysema progression and provide new insight that may inform more rational, patient-specific treatment strategies.2020-07-02T00:00:00

Novel molecular pathologies in asthma and COPD

Both asthma and COPD are respiratory diseases and a major global health problem with increasing prevalence. Airway inflammation is a characteristic and important hallmark in both diseases and therefore, in the past, investigations focused strongly on the immunological aspect of these disorders. In recent years, it has been shown that resident cells of the airways, in particular airway smooth muscle (ASM) cells, would be pivotal in understanding the mechanisms underlying asthma, since they are able to secrete pro-inflammatory cytokines and exert a major effector function in airway constriction. Especially the abnormal expression in ASM cells in asthmatic patients of the cell cycle regulator and pro-inflammatory gene transcription factor C/EBPα may account for many asthma-specific phenotypes (increased proliferation and increased bulk of ASM cells, increased release of inflammatory mediators). In a first phase, we analyzed the translation of the CEBPA mRNA with a translation control reporter system (TCRS), which is able to monitor translation regulation of the C/EBPα. We found an impaired translation re-initiaion in ASM cells of asthmatic patients, which coincided with decreased levels of eIF4E, an important protein for translation initiation. In a second part of this thesis, we investigated the interaction of ASM cells with house dust mite extract, a potent airborne allergen. We found that HDM extract (i) reduces C/EBPα expression in ASM cells of asthma patients, (ii) enhances the release of IL-6 and (iii) induces cell proliferation. The reduction of the C/EBPα protein is achieved trough up-regulation of calreticulin, a repressor of CEBPA mRNA translation. Therefore, the direct, not immune-mediated interaction of HDM extract with the ASM cells is able to trigger an inflammatory response in these cells and to induce an enhanced proliferation, which may finally lead to the characteristic increased muscle mass observed in the airway of asthmatic patients. These findings may be of particular importance to explain non-atopic, intrinsic asthma, which affects 30% - 50% of asthmatic subjects. In the light of these findings, new therapeutic strategies targeting regulatory mechanisms of CEBPA mRNA translation should be considered in order to restore a balanced expression of the C/EBPα protein. In a third part of this thesis, we investigated the effect of cigarette smoke on the expression levels of C/EBPα and C/EBPβ in primary lung fibroblasts. Cigarette smoke affects both C/EBPα and C/EBPβ expression via translational control mechanisms in primary lung fibroblasts. In serumfree environment, cigarette smoke increased both C/EBPα and -β expression at the translational level via the uORF mechanism. In the presence of FCS, cigarette smoke increased the levels of hnRNP E2, an inhibitor of C/EBPα translation. As a consequence, both C/EBPα and -β expression decreased with increasing concentration of cigarette smoke. In both conditions, cigarette smoke had a potent antiproliferative effect on fibroblasts. Furthermore, cigarette smoke increased the release of IL-8. We postulate that the cigarette smoke-induced imbalance of proand anti-proliferative signals provides a novel mechanism to explain many pathologies of COPD and emphysema, especially the tissue destruction defined as an imbalance between tissue injury and tissue repair. Furthermore, we showed that that the direct interaction of lung fibroblast with cigarette smoke triggers the release of pro-inflammatory mediators, contributing to the inflammatory environment that characterizes COPD

Role of CARM1 in regulation of alveolar epithelial senescence and emphysema susceptibility

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases world-wide. A major feature of COPD is emphysema- a long-term, progressive condition. The hallmark of emphysema includes the destruction of alveolar structures leading to enlarged air spaces and reduced surface area. Experimental evidence suggests that emphysema development is driven by accelerated senescence of lung cells but the underlying mechanism of senescence is yet to be fully elucidated. Protein arginine methyltransferases (PRMTs) are important for cellular processes, such as the regulation of senescence, cell proliferation, differentiation and apoptosis. The PRMT family includes 11 members classified as type I, II or III enzymes depending on their methylation pattern (asymmetric dimethylation, symmetric dimethylation or monomethylation, respectively). One member of this family is PRMT4, a type I enzyme, which is also called coactivator associated arginine methyltransferase 1 (CARM1). It was originally identified as a coactivator for steroid hormone receptors. CARM1 is known to methylate histone H3 and various non-histone proteins that play essential roles in transcriptional regulation, RNA splicing, and metabolism. Most importantly, complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type-II cells (ATII). Furthermore, CARM1 also plays a role in regulating cellular senescence via CARM1-dependent methylation. Based on these reports, we hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and siRNA-mediated knockdown in ATII-like LA4 cells in vitro. We monitored emphysema progression for 161 days in mice treated with a single oropharyngeal application of elastase. The progression was manifested by the decline in lung function parameters. The mean chord length (Lm) confirmed a time dependent airspace enlargement and was directly correlated with a significant increase in dynamic lung compliance. We also observed that at later time points (day 56 and 161), emphysema progression was inflammation-independent. We demonstrated that emphysema advancement was associated with a time-dependent downregulation of CARM1, specifically in alveolar epithelial cells. Furthermore, the global CARM1 activity was also reduced as reflected by an elevated level of CARM1 phosphorylation in the lung. Most importantly, elastase-treated CARM1 haploinsufficient mice showed significantly increased airspace enlargement (52.5±9.6 µm vs. 38.8±5.5 µm, p<0.01) and lung compliance (2.8±0.32 µl/cmH20 vs. 2.4±0.4 µl/cmH20, p<0.04) compared with wild type controls. Reduced CARM1 contributed to senescence of alveolar epithelial cells evident by the reduction of anti-senescence SIRT1 and the induction of senescence markers p16 and β-galactosidase in alveolar epithelial cells. We further demonstrated that CARM1 haploinsufficiency impaired trans-differentiation of ATII into ATI cells. Elastase treatment in CARM1 deficient mouse lungs led to the accumulation of SP-C positive ATII cell. In our in vitro studies, we detected that the knockdown of CARM1 in the ATII-like cell line LA-4 led to decreased SIRT1 expression (0.034±0.003 vs. 0.022±0.001, p<0.05), but increased expression of p16 (0.27±0.013 vs. 0.31±0.010, p<0.5), p21 (0.81±0.088 vs. 1.28± 0.063, p<0.01) and induced a higher number of β-galactosidase-positive senescent cells (50.57%±7.36 vs. 2.21%±0.34, p<0.001), compared with scrambled siRNA. The senescence in CARM1 deficient cells was further augmented after CSE stimulation. Furthermore, we demonstrated that CARM1 deficiency impaired wound healing (32.18%±0.9512 vs. 8.769%±1.967 wound gap closure, p<0.001) of alveolar epithelial cells. The data confirmed that CARM1 reduction induced an accelerated senescence in LA-4 cells by attenuating the effect of SIRT1. Overall, both our in vivo and in vitro results revealed a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence, as well as impaired regeneration, repair and differentiation of ATII cells.Die chronisch obstruktive Lungenerkrankung (COPD) zählt zu den häufigsten schweren Krankheiten weltweit und ist gekennzeichnet durch eine irreversible Abnahme der Lungenfunktion. Ein Hauptcharakteristikum von COPD ist das Emphysem, das sich stetig über einen langen Zeitraum fortschreitend entwickelt, und durch die Zerstörung alveolärer Strukturen, einhergehend mit vergrößerten Lufträumen und verringerter Alveolaroberfläche, gekennzeichnet ist. Ergebnisse aus Studien legen nahe, dass die Entwicklung des Emphysems durch die erhöhte Seneszenz von Zellen der Lunge beschleunigt wird, doch die genauen zugrundeliegenden Mechanismen sind noch nicht aufgeklärt. Protein-Arginin-Methyltransferasen (PRMTs) spielen eine bedeutende Rolle bei zellulären Prozessen wie Regulation von Seneszenz, Zellproliferation, Differenzierung und Apoptose. Die Familie der PRMTs umfasst 11 Enzyme, die als Typ I, II, oder III klassifiziert werden, abhängig von ihrem Methylierungsmuster (asymmetrische Dimethylierung, symmetrische Dimethylierung oder Monomethylierung). Ein Mitglied der Familie ist PRMT4, ein Typ I Enzym, das auch unter dem Namen Coactivator-assoziierte Arginin-Methyltransferase 1 (CARM1) bekannt ist. Ursprünglich wurde CARM1 als Coactivator für Steroid-Hormon-Rezeptoren beschrieben. CARM1 methyliert Histon 3 und verschiedene weitere Nicht-Histon Proteine, die eine wichtige Rolle für die Transkriptionsregulation, das RNA-Splicing und den Stoffwechsel spielen. Darüberhinaus führt der komplette Verlust von CARM1 zu gestörter Differenzierung und Maturation von Typ II Alveolarepithelzellen (ATII). Weiterhin ist CARM1 von Bedeutung für die Regulation von zellulärer Seneszenz mittels CARM1-abhängiger Methylierung. Auf Grundlage dieser Arbeiten wurde die Hypothese aufgestellt, dass CARM1 die Entwicklung und das Voranschreiten des Emphysems reguliert. Um dies aufzuklären, wurde die Rolle von CARM1 am Verlust der Alveolen in einem Elastase-induzierten Emphysem-Mausmodell in vivo sowie in vitro mittels siRNA knockdown in ATII-ähnlichen LA4-Zellen untersucht. Die Entwicklung und das Voranschreiten des Emphysems wurden über einen Zeitraum von 161 Tagen beobachtet. Dabei zeigte sich eine progressive Abnahme verschiedener Lungenfunktionsparameter. Die mittlere Alveolarweite bestätigte die zeitabhängige Vergrößerung des Luftraumes und war direkt mit einer signifikanten Zunahme der dynamischen Lungencompliance korreliert. Außerdem wurde an späteren Zeitpunkten (Tag 56 und 161) eine entzündungsabhängige Progression des Emphysems beobachtet. Das Voranschreiten des Emphysems war mit einer zeitabhängigen Herunterregulierung von CARM1 insbesondere in Alveolarepithelzellen assoziiert. Die Gesamtaktivität von CARM1 war ebenso reduziert, erkennbar anhand von erhöhter CARM1-Phosphorylierung in der Lunge. Weiterhin zeigten mit Elastase behandelte CARM1 haploinsuffiziente Mäuse eine signifikant erhöhte Vergrößerung des Luftraumes (52.5±9.6 µm vs. 38.8±5.5 µm, p<0.01) sowie der Lungencompliance (2.8±0.32 µl/cmH20 vs. 2.4±0.4 µl/cmH20, p<0.04) verglichen mit Wildtyp-Kontrollmäusen. Das verringerte Level an CARM1 trug zur Seneszenz von Alveolarepithelzellen bei, bestätigt durch eine Reduktion des Anti-Seneszenz-Markers SIRT1 und durch die Induktion der Seneszenz-Marker p16 und β-Galactosidase in Alveolarepithelzellen. Des Weiteren wurde gezeigt, dass CARM1 Haploinsuffizienz die Transdifferenzierung von ATII zu ATI Zellen verhindert. Dabei führte die Elastase-Behandlung von CARM1-defizienten Mäusen zu einer Akkumulation von SP-C-positiven ATII-Zellen in der Lunge. Die in vitro Experimente haben gezeigt, dass der knockdown von CARM1 in ATII-ähnlichen LA4-Zellen zu einer verringerten SIRT1-Expression (0.034±0.003 vs. 0.022±0.001, p<0.05), und gleichzeitig zu einer erhöhten Expression von p16 (0.27±0.013 vs. 0.31±0.010, p<0.5), p21 (0.81±0.088 vs. 1.28± 0.063, p<0.01) führte. Weiterhin verursachte der CARM1 knockdown eine erhöhte Anzahl an β-Galactosidase-positiven seneszenten Zellen verglichen mit Kontroll-siRNA-behandelten Zellen (50.57%±7.36 vs. 2.21%±0.34, p<0.001). Die erhöhte Seneszenz in CARM1-defizienten Zellen wurde durch Stimulation mit Zigarettenrauchextrakt noch verstärkt. Weiterhin zeigte sich in den Alveolarepithelzellen eine verhinderte Wundheilung aufgrund CARM1-Defizienz (32.18%±0.9512 vs. 8.769%±1.967 Wundschließung, p<0.001). Diese Daten bestätigten, dass die Reduzierung von CARM1 eine beschleunigte Seneszenz in LA4-Zellen mittels Abschwächung von SIRT1-Effekten verursachte. Zusammenfassend zeigen die in vivo und vitro Ergebnisse eine neue Rolle von CARM1 bei der Regulierung der Emphysem-Entstehung und der vorzeitigen Lungenalterung, in erster Linie mittels Seneszenz von Alveolarepithelzellen, sowie beeinträchtigter Regenerierung, Reparatur und Differenzierung von ATII-Zellen

Injury to the Developing Lung: experimental and clinic al aspects

Injury to the developing lung or disturbance of normal lung development may lead to a chronic lung disease, bronchopulmonary dysplasia (BPD), which may have long-term effects. BPD is characterized by an arrest of development of the lung and the pulmonary vascular system and occurs in around 20% of ventilated newborns. In the first part of this thesis, different factors that influence the development of BPD are studied, both in an experimental and a clinical setting. We found that components of the TGF-b/BMP signalling pathway play a central role in normal and abnormal late lung development. In addition, angiogenic factors, inflammatory cytokines and the nitric oxide system are shown to be of influence. Another factor in the aetiology of BPD is intra-uterine growth retardation. The second part of this thesis focuses on surfactant treatment in acute neonatal lung disorders. Surfactant therapy has become the standard therapy for respiratory distress syndrome in premature infants. There is a significant amount of evidence that near-term or term infants, as well as children, with acute respiratory failure have a profound functional surfactant deficiency. In an experimental setting, we compared synthetic versus natural surfactant and we demonstrated for the first time that both types of surfactant have a positive influence on lung function. However, synthetic surfactant had a significantly stronger effect on inflammatory cytokines. Thus, synthetic surfactant may cause less chronic lung disease and it may be an important part of treatment of these infants in the future

Individual and population based VEGF-endothelial cell processing is modulated by extracellular matrix stiffness

Vascular endothelial growth factor (VEGF) is required for the development, growth and survival of blood vessels. Endothelial cell behavior is altered by cell substrate stiffness, suggesting that VEGF activity might also be influenced by cell-substrate mechanics. We studied VEGF binding, internalization, and signaling as a function of substrate stiffness using endothelial cells cultured on fibronectin (fn) linked polyacrylamide gels. Individual cell analysis of VEGF-induced calcium fluxes in endothelial cells on various stiffness extracellular matrices (ECM) revealed heterogeneity in our cell population that would have been lost using population based averaging. Cluster analysis of individual cells identified two key groups of reacting cells- a minor fraction of highly reactive cells and the bulk of the cells with minimal activation. At subsaturating VEGF doses, highly active cells were phenotypically smaller and thinner than the bulk population. Overall, cells on our softest substrates (4 kPa) were most sensitive to VEGF. To better understand the mechanisms underlying the changes in VEGF signaling due to stiffness, we explored how matrix binding of VEGF and tethering of cells to the matrix modulates VEGF processing. VEGF-ECM binding was enhanced with heparin pre-treatment, which exposed a cryptic VEGF binding site in the fn ECM. Cell produced ECM on the softest substrates were least responsive to heparin, but the cells internalized more VEGF and showed enhanced VEGF signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF in all conditions. β1 integrin, which connects cells to fn, modulated VEGF uptake in a stiffness dependent fashion. β1 protein levels were consistent with stiffness, yet cells on hard surfaces showed greater decreases in VEGF internalization than cells on softer matrices after β1 inhibition. Stiff matrices facilitate the unfolding of fn, which may reduce the binding capacity of β1 integrin. Thus a greater proportion of activated β1 integrin may be sensitive to inhibition in the stiff condition as compared to the soft. Ultimately, through analysis of individual and population-based VEGF-cell responses to stiffness, this study provides insight into how signaling dynamics, cell heterogeneity, and microenvironment influence tissue regeneration and response to injury and disease

Retinoic acid signalling in human lung disease and repair

The normal adult mammalian lung has a robust capacity to regenerate following injury, and evidence for alveolar regeneration was recently demonstrated in adult man. In contrast, mounting evidence suggests COPD/emphysema represents a failure of regeneration. COPD represents an enormous worldwide clinical and social burden, with currently no cure besides lung transplantation. An appealing therapeutic option is induction of endogenous lung regeneration using retinoic acid (RA), demonstrated to stimulate alveolar regeneration in animal models of emphysema. However, clinical trials investigating retinoids for chronic lung diseases have been disappointing. Thus, there is a profound stimulus to understand how the regeneration-inducing effects of RA in animal models translate to man. The molecular regulation of RA signalling in emphysema has not been investigated hitherto, and the role of RA in repair of specific human alveolar cell types, alveolar type 2 cells and lung microvascular endothelial cells, is unknown. Work in this thesis was conducted to address these questions. We demonstrated that CYP26A1, which breaks down RA, is enriched on an mRNA and protein level in emphysematous lung tissue. We also demonstrated using in vitro cell culture assays that RA is unlikely to directly regulate alveolar epithelial wound healing. In contrast, RA stimulated lung microvascular endothelial angiogenesis, likely via retinoic acid receptor alpha, and was associated with induction of angiogenic genes. Further work presented herein involved development of an ex vivo model of lung regeneration using precision cut lung slices (PCLS) derived from adult human distal lung tissue. We demonstrated that human PCLS retain architecture and viability through slicing, that 10% serum supplementation is inappropriate for long-term PCLS culture, and that human PCLS remain viable for at least 4 days in culture, suggesting they are amenable to development of an injury/repair model within this time frame.Open Acces