Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4-null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.Peer reviewe

Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4-null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.Peer reviewe

Reliability of Therapist Effects in Practice-Based Psychotherapy Research : A Guide for the Planning of Future Studies

This paper aims to provide researchers with practical information on sample sizes for accurate estimations of therapist effects (TEs). The investigations are based on an integrated sample of 48,648 patients treated by 1800 therapists. Multilevel modeling and resampling were used to realize varying sample size conditions to generate empirical estimates of TEs. Sample size tables, including varying sample size conditions, were constructed and study examples given. This study gives an insight into the potential size of the TE and provides researchers with a practical guide to aid the planning of future studies in this field

Strukturelle und Funktionelle Analyse von LTBP 4 als Faktor der Pathogenese des Lungenemphsems

Table of contents 4 List of tables and figures 9 Abbrevations 12 1\. Introduction 17 1.1. Lung organogenesis 17 1.1.1. Early lung development 19 1.1.2. Late lung development 20 1.2. Key factors of lung development 21 1.2.1. Cell differentation and ECM deposition during lung development 22 1.2.1.1. Myofibroblasts 23 1.2.1.2. Myofibroblast transdifferentation 25 1.2.1.3. Myofibroblasts in alveolarisaton 26 1.3. Biochemical regulation of lung development 29 1.3.1. TGFβ in mammalian lung tissue 30 1.3.2. TGFβ signalling 32 1.2.2. TGFβ signalling in lung development 33 1.4. Latent TGFβ Binding Protein (LTBP) 35 1.4.1. Regulation oft he bioavailability of TGFβ Through LTBPs 35 1.4.2. Structure of LTBPs 37 1.5. Diseases associated with LTBP4 41 Ltbp4s knockout mice (3C7) 41 Ltbp4 -/- mice (E301B04) 42 Urban Rifkin Davis syndrome (URDS) 43 1.6. Outline of the thesis 47 2\. Material and Methods 49 2.1. Materials 49 2.1.1. Standard solutions 49 2.1.3. Antibodies 53 2.1.4. Primers for qRT-PCR 54 2.1.5. siRNA 56 2.2. Methods 57 2.2.1.. Animal work 57 2.2.1.1. Trunc blood analyses 57 2.2.1.2. Tissue processing 58 2.2.1.3. Histology 59 H&E; staining of lung sections 59 Histomorphometric analysis of lung sect. 60 Ltbp4 staining of lung sections 61 Hart staining of lung sections 62 Anti sma staining of lung sections 62 2.2.2. Cell culture 63 2.2.2.1. Human embryonic lung fibroblasts (HEL) 63 Transfection of HEL 64 2.2.2.2. Isolation of primary murine lung fib. 65 Immunocytochemistry 66 Stimulation of primary murine lung fibroblasts with TGFβ 67 2.2.2.3. Relaxed collagen lattices 67 PCNA staining of relaxed collagen lattices 68 2.2.2.4. Stressed collagen lattices 69 SMA staining of stressed collagen lattices 70 2.2.3. Molecular biological methods 71 2.2.3.1. RNA extraction and qRT-PCR 71 2.2.4. Protein biochemical methods 72 2.2.4.1. Western Blot analyses 72 2.2.4.2. TGF β activity assay 73 2.2.5. Statistical analyses 74 3\. Results 76 Ltbp4 -/-mice develop severe hypercapnia and Polycytemia 76 Lack of Ltbp4 impairs postnatal alveolarisation in Ltbp4 -/-mice 78 Impaired myofibroblast trans-differentation in lung tissue of Ltbp4 -/-mice 81 Disruption of elastic fibres in lung tissue of Ltbp4 -/-mice 85 Characterisation of underlying molecular mechanisms of myofibroblast transdifferentiation in Ltbp4-/-mice 87 Impaired ability of matrix organisation of LTBP4-/-fibroblasts 87 Ctgf and Pai-1 are negatively regulated in primary murine Ltbp4-/- lung fibroblasts 94 Tgfβ1 activity is altered in primary murine Ltbp4-/-lung fibroblasts 97 Downregulation of Ctgf and Pai-1 is reversible in primary murine Ltbp-4-/- lung fibroblasts 99 4\. Discussion 102 5\. Zusammenfassung 117 6\. Summary 121 7\. References 124 8\. Acknowledgements 137 9\. Erklärung 141The American Journal of Human Genetics published in 2009 four cases of newborn children with a complex disease pattern, the Urban-Rifkin-Davis Syndrome (URDS). All patients had mutations of the Latent TGF¬β binding Protein 4 (LTBP¬4) gene. LTBP¬4 is one of four known LTB proteins. Eponymous for this protein family is the strong interaction with Transforming growth factor β (TGF¬β). In addition LTBP¬4 it is assumed as a distinct structural protein of the ECM. Disruption of LTBP¬4 leads to a variety of disease patterns. Preceding studies have shown that mice lacking the short variant of Ltbp¬4 (Ltbp4s) develop a complex phenotype with cardiomyopathy, colorectal cancer and pulmonary emphysema. Human patients suffering URDS exhibit impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal and dermal development. Despite a huge variety of disease patterns all individuals with mutations in the LTBP¬4 gene share one phenotype; the development of pulmonary emphysema at the stage of postnatal alveolarisation. Aim of this study was the structural and functional analysis of LTBP¬4 as a factor of pathogenesis in the development of pulmonary emphysema. To avoid any splicevariant specific effects, Ltbp4 complete knockout mice (Ltbp4-/-) were analysed via assessment of oxygenation and lung structure. In vitro investigations on myofibroblast transdifferentation, matrix structuration and TGFβ activation were performed on primary murine Ltbp4-/- lung fibroblasts as well as siRNA transfected, LTBP4 deficient human lung fibroblasts. Ltbp4-/- mice die within the first ten days of life with severe hypercapnia and polycythemia. Lung tissue of Ltbp4-/- mice exhibit impaired alveolarisation, disrupted alveolar elastic fibre distribution and reduced myofibroblast transdifferentation. Both primary Ltbp4-/- lung fibroblasts as well as siRNA transfected, LTBP4 deficient human lung fibroblasts revealed impaired matrix structuration in vitro. In summary two key processes of postnatal alveolarisation: myofibroblast transdifferentation and alveolar elastic fibre formation are disrupted in Ltbp4-/- mice, thereby implicating the pivotal role of LTBP4 during alveolarisation. This is the first study investigating the impact of Ltbp¬4 on postnatal alveolarisation by means of a new animal model, the Ltbp4-/- model.Im Jahr 2009 veröffentlichte die Arbeitsgruppe um Davis erstmals vier Fälle von humanen Patienten welche ein komplexes Krankheitsbild zeigten, das Urban- Rifkin-Davis Syndrom (URDS) (Urban, Hucthagowder et al. 2009). Alle beschriebenen Fälle hatten Mutationen im Latent Transforming growth factor β (TGF¬β) bindenden Protein 4 (LTBP¬4). LTBP¬4 ist eines von vier Latent TGF-β bindenden Proteinen (LTBP¬1-4). Namensgebend für die Familie der LTBP Moleküle ist die enge Interaktion mit (TGF¬β). Neben der Beteiligung von LTBP¬4 im TGF-β1 Signalweg gibt es jedoch Hinweise, dass LTBP¬4 zusätzlich eine Funktion als Strukturprotein der extrazellulären Matrix (ECM) erfüllt. Mutationen des LTBP¬4 Genes resultieren in einer Vielzahl von Erkrankungen. Vorangegangene Studien haben gezeigt, dass Knockoutmäuse denen die kurze Variante des Ltbp¬4 (Ltbp¬4s) fehlt ein komplexes Krankheitsbild mit unter anderem Störungen des Herz-Kreislaufsystems und des Atmungstraktes ausbilden. URDS Patienten zeigen Veränderungen im Gastrointestinaltrakt, Urogenitaltrakt, der Haut und der Lunge. Allen Individuen mit Mutationen im LTBP¬4 Gen ist dabei die Entwicklung von Lungenemphysemen im Stadium der postnatalen Alveolarisation gemeinsam. Um durch Splicevarianten verursachte Effekte auszuschließen, wurden in dieser Studie erstmals bis dato noch nicht beschriebene Ltbp4 komplett Knockout - Mäuse (Ltbp¬4-/- Mäuse) untersucht. Ziel war die Analyse der Rolle von LTBP¬4 als Faktor in der Pathogenese von Lungenemphysemen. Ltbp¬4-/- Mäuse wurden mittels Blutgasanalysen und Lungenhistologischen Untersuchungen charakterisiert. Sowohl primäre murine Ltbp¬4-/- Lungenfibroblasten, als auch siRNA transfizierte LTBP4 defiziente humane Lungenfibroblasten wurden im Hinblick auf Myofibroblastentransdifferenzierung, Matrixstrukturierung und TGFβ Aktivität untersucht. Ltbp¬4-/- Mäuse versterben innerhalb der ersten zehn Lebenstage nach schwerer Hyperkapnie und Polyzytämie. Das Lungengewebe zeigt eine verminderte Alveolarisation, fragmentierte elastische Fasern in den Alveolen und reduzierte myofibroblasten Transdifferenzierung. In vitro zeigten die LTBP4 defizienten humanen und murinen Lungenfibroblasten eine gestörte Strukturierung der extrazellulären Matrix. Zusammengefasst sind sowohl die Myofibroblastendifferenzierung als auch weitere Schlüsselmechanismen der postnatalen Alveolarisation, wie die Bildung von alveolären elastischen Fasern bei Ltbp¬4-/- Mäusen gestört. Diese Studie ist die erste, die eine gestörte postnatale Alveolarisation im Zusammenhang mit Mutationen im LTBP¬4 Gen untersucht und gibt neue Einblicke in die Funktion von LTBP-4 als Faktor in der Lungenentwicklung

Identification of Critical Windows of Metabolic Programming of Metabolism and Lung Function in Male Offspring of Obese Dams

Perinatal nutritional determinants known as metabolic programming could be either detrimental or protective. Maternal obesity in the perinatal period determines susceptibility for diseases, such as obesity, metabolic disorders, and lung disease. Although this adverse metabolic programming is well-recognized, the critical developmental window for susceptibility risk remains elusive. Thus, we aimed to define the vulnerable window for impaired lung function after maternal obesity; and to test if dietary intervention protects. First, we studied the impact of high-fat diet (HFD)-induced maternal obesity during intrauterine (HFDiu), postnatal (HFDpost), or perinatal (i.e., intrauterine and postnatal (HFDperi) phase on body weight, white adipose tissue (WAT), glucose tolerance, and airway resistance. Although HFDiu, HFDpost, and HFD(peri)induced overweight in the offspring, only HFD(peri)and HFD(iu)led to increased WAT in the offspring early in life. This early-onset adiposity was linked to impaired glucose tolerance in HFDperi-offspring. Interestingly, these metabolic findings in HFDperi-offspring, but not in HFDiu-offspring and HFDpost-offspring, were linked to persistent adiposity and increased airway resistance later in life. Second, we tested if the withdrawal of a HFD immediately after conception protects from early-onset metabolic changes by maternal obesity. Indeed, we found a protection from early-onset overweight, but not from impaired glucose tolerance and increased airway resistance. Our study identified critical windows for metabolic programming of susceptibility to impaired lung function, highlighting thereby windows of opportunity for prevention

Prevention of Early Postnatal Hyperalimentation Protects against Activation of Transforming Growth Factor-beta/Bone Morphogenetic Protein and Interleukin-6 Signaling in Rat Lungs after Intrauterine Growth Restriction

Background: Intrauterine growth restriction (IUGR) is intimately linked with postnatal catch-up growth, leading to impaired lung structure and function. However, the impact of catch-up growth induced by early postnatal hyperalimentation (HA) on the lung has not been addressed to date. Objective: The aim of this study was to investigate whether prevention of HA subsequent to IUGR protects the lung from 1) deregulation of the transforming growth factor-beta(TGF-beta)/bone morphogenetic protein (BMP) pathway, 2) activation of interleukin (IL)-6 signaling, and 3) profibrotic processes. Methods: IUGR was induced in Wistar rats by isocaloric protein restriction during gestation by feeding a control (Co) or a low-protein diet with 17% or 8% casein, respectively. On postnatal day 1 (P1), litters from both groups were randomly reduced to 6 pups per dam to induce HA or adjusted to 10 pups and fed with standard diet: Co, Co with HA (Co-HA), IUGR, and IUGR with HA (IUGR-HA). Results: Birth weights in rats after IUGR were lower than in Co rats (P < 0.05). HA during lactation led to accelerated body weight gain from P1 to P23 (Co vs. Co-HA, IUGR vs. IUGR-HA; P < 0.05). At P70, prevention of HA after IUGR protected against the following: 1) activation of both TGF-beta [phosphorylated SMAD (pSMAD) 2; plasminogen activator inhibitor 1 (Pai1)] and BMP signaling [pSMAD1; inhibitor of differentiation (Id1)] compared with Co (P < 0.05) and Co or IUGR (P < 0.05) rats, respectively; 2) greater mRNA expression of interleukin (Il) 6 and Il13 (P < 0.05) as well as activation of signal transducer and activator of transcription 3 (STAT3) signaling (P < 0.05) after IUGR-HA; and 3) greater gene expression of collagen I alpha 1 and osteopontin (P < 0.05) and increased deposition of bronchial subepithelial connective tissue in IUGR-HA compared with Co and IUGR rats. Moreover, HA had a significant additive effect (P < 0.05) on the increased enhanced pause (indicator of airway resistance) in the IUGR group (P < 0.05). at P70. Conclusions: This study demonstrates a dual mechanism in IUGR-associated lung disease that is 1) IUGR-dependent and 2) HA-mediated and thereby offers new avenues to develop innovative preventive strategies for perinatal programming of adult lung diseases

Strain-dependent effects on lung structure, matrix remodeling, and Stat3/Smad2 signaling in C57BL/6N and C57BL/6J mice after neonatal hyperoxia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm infants, characterized by lung growth arrest and matrix remodeling. Various animal models provide mechanistic insights in the pathogenesis of BPD. Since there is increasing evidence that genetic susceptibility modifies the response to lung injury, we investigated strain-dependent effects in hyperoxia (HYX)-induced lung injury of newborn mice. To this end, we exposed newborn C57BL/6N and C57BL/6J mice to 85% O-2 (HYX) or normoxia (NOX; 21% O-2) for 28 days, followed by lung excision for histological and molecular measurements. BL/6J-NOX mice exhibited a lower body and lung weight than BL/6N-NOX mice; hyperoxia reduced body weight in both strains and increased lung weight only in BL/6J-HYX mice. Quantitative histomorphometric analyses revealed reduced alveolar formation in lungs of both strains after HYX, but the effect was greater in BL/6J-HYX mice than BL/6N-HYX mice. Septal thickness was lower in BL/6J-NOX mice than BL/6N-NOX mice but increased in both strains after HYX. Elastic fiber density was significantly greater in BL/6J-HYX mice than BL/6N-HYX mice. Lungs of BL/6J-HYX mice were protected from changes in gene expression of fibrillin-1, fibrillin-2, fibulin-4, fibulin-5, and surfactant proteins seen in BL/6N-HYX mice. Finally, Stat3 was activated by HYX in both strains; in contrast, activation of Smad2 was markedly greater in lungs of BL/6N mice than BL/6J mice after HYX. In summary, we demonstrate strain-dependent differences in lung structure and matrix, alveolar epithelial cell markers, and Smad2 (transforming growth factor beta) signaling in neonatal HYX-induced lung injury. Strain-dependent effects and genetic susceptibility need be taken into consideration for reproducibility and reliability of results in animal models

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential