Title: BMP and Wnt signaling in mouse models of systemic sclerosis
Other Titles: BMP en Wnt signaalweg in muismodellen van systeemsclerose
Authors: De Langhe, Ellen
Issue Date: 3-Jun-2013
Abstract: Systemic sclerosis (SSc) is a rare, severe chronic autoimmune connective tissuedisease. The triad of vasculopathy, immunological abnormalities and fibrosischaracterizes the clinical and pathogenic picture. Fibrosis can affect the skin, but alsovarious internal organs, leading to architectural damage and organ failure. SSc is adisease with substantial mortality, of which the majority is caused by pulmonaryinvolvement, both pulmonary arterial hypertension and pulmonary interstitial fibrosis.Current therapeutic strategies for scleroderma-associated lung fibrosis are limited.Immunosuppression using cyclophosphamide and corticosteroids remains thecornerstone, but results in only moderate improvement or temporary stabilization.Targeted therapies in SSc are non-existing, since the exact pathogenesis of the diseaseis still obscure. These observations strongly emphasize the need for further researchinto scleroderma pathogenesis, allowing the future development of new treatmentstrategies. The TGFß signaling pathway plays a central role in the process of fibrosis. However,recent data also demonstrate a role for the bone morphogenetic protein (BMP)pathway, also belonging to the TGFß superfamily, in human idiopathic fibrosis (IPF)and mouse models of lung fibrosis, suggesting interaction of these two relatedpathways. Furthermore, cumulating evidence supports a key role of wingless type(Wnt) signaling in both skin and lung fibrosis. However, it is still largely unknown howthese pathways interact and relate to each other in the process of experimentalfibrosis. The main goal of this PhD project was to further unravel the role of BMP signalingin bleomycin-induced skin and lung fibrosis and to further elaborate on its interactionwith the TGFß pathway. Furthermore, we studied the role of two WNT antagonists,SFRP1 and FRZB, in fibrogenesis, both in vivo and in vitro. Finally, we aimed atvalidating micro-computed tomography (μ-CT) imaging as a non-invasive quantitativetool to assess lung fibrosis progression in mice. The work was therefore mainly focusedon molecular signaling pathways influencing the process of fibrosis and theirinteraction with the TGFß pathway. To investigate fibrogenesis in vivo, we used the bleomycin-induced model of skinand lung fibrosis. Severity of fibrosis is currently evaluated by end-stage proceduressuch as histopathology and collagen quantification, requiring sacrifice of the animal, hence precluding dynamic monitoring of the disease process. Non-invasive imagingtechniques, such as μ-CT imaging, could offer the potential of longitudinal follow-upof disease progression and therapeutic interventions in an individual animal, on thecondition that the image-derived data are quantitative and can be validated with thecurrent golden standards. In the first part of this thesis, we present an automated analysis algorithm thatcalculates aerated lung volumes from in vivo μ-CT images. We showed that theimaging technique and subsequent analysis are robust with low inter-observationvariation. Using the bleomycin-induced lung fibrosis model, we demonstrated that theacquired quantitative volume data are valid and correlate to histopathological scoresand collagen content. Finally, we showed that longitudinal follow-up of an individualanimal through weekly scanning is feasible and safe. This work validated a valuabletool for preclinical fibrosis research, allowing true dynamic quantitative monitoring offibrosis progression in vivo. Next, we studied the role of the BMP signaling pathway in bleomycin-induced lungand skin fibrosis, using Nog+/LacZ mice, a model of enhanced BMP signaling. Nog+/LacZmice were partially protected from bleomycin-induced lung fibrosis, but showedidentical fibrotic response in the skin fibrosis model. We demonstrated that normallungs have active BMP signaling in bronchial epithelial cells, but this activity vanishesupon bleomycin instillation, due to reduced BMP ligand expression and an increase inexpression of the BMP antagonist noggin. In vivo, BMP and TGFß activity follow aninverse course, as bleomycin instillation results in activation of the TGFß pathway andrepression of the related BMP pathway. In a series of in vitro experiments, we showedthat BMP ligands counteract the profibrotic effects of TGFß in pulmonary fibroblasts,due to altered complex formation and nuclear shuttling of the receptor-associatedSMAD proteins with the common mediator SMAD4. This work further strengthens theevidence for a counter-regulatory interaction between the related TGFß and BMPpathway. Moreover, we demonstrate that preservation of BMP signaling activity isbiologically relevant and alters fibrotic outcomes in the lung. Finally, we investigated the role of the Wnt antagonists SFRP1 and FRZB inexperimental fibrosis. We confirmed the increased activity of the Wnt signalingpathway in bleomycin-induced lung fibrosis and demonstrated an upregulation of both antagonists. In vitro, we showed that SFRP1 counteracts the profibrotic effect ofTGFß in pulmonary fibroblasts and epithelial cells. We demonstrated that inpulmonary fibroblasts, but not in epithelial cells, TGFß activates canonical Wntsignaling, and this effect can be abrogated by SFRP1. In vivo, we induced lung fibrosisin Sfrp1-/- and Frzb-/- mice, and showed that they have identical fibrotic responsescompared to wild-type mice. We assume that this can be explained by functionalredundancy between the different SFRP family members. This work confirms theinteractionbetween TGFß and Wnt signaling and highlights the complexity of thispathway in vivo. In conclusion, this PhD research supports the notion that BMP signaling has abiologically relevant role in counter-regulating TGFß signaling in bleomycin-inducedlung fibrosis. Furthermore, we show that Wnt signaling and TGFß signaling interact invitro in pulmonary fibroblasts and we highlight the complexity of the Wnt/SFRPpathway. However, more basic research is needed to further unravel the complexinteractions between the different morphogenic pathways. Only true insight in fibrosispathogenesis can lead to targeted therapies in systemic sclerosis.
Table of Contents: 9 Abbreviations
13 CHAPTER 1: General introduction and rationale
43 CHAPTER 2: Aims of the study
47 CHAPTER 3: Quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography
73 CHAPTER 4: TGFβ and BMP signaling in bleomycin-induced lung fibrosis
97 CHAPTER 5: Secreted frizzled related proteins in bleomycin-induced lung fibrosis
117 CHAPTER 6: General discussion and conclusions
129 Summary
133 Samenvatting
137 Reference list
155 Curriculum vitae
163 Dankwoord
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Skeletal Biology and Engineering Research Center (+)

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