The role of pulmonary fibroblast subtypes in lung development

The aim of the present study was to characterize pulmonary fibroblast subtypes regarding differentiation and localization during alveolarization and to reveal the impact of fibroblast function on alveolarization. A further aim was to validate and characterize a previously generated Cre driver mouse line to target lipofibroblasts and the further usage of this mouse line to target lipofibroblasts in vivo. To address these aims, using in vivo tools of the CreERT2 stop loxP system and reporter genes, the present study demonstrated that platelet-derived growth factor receptor α+ (PDGFRα+) cells of the early postnatal period could give rise to myofibroblasts and lipofibroblasts during alveolarization. Furthermore, markers of mesenchymal stem cells were detected in generated myo- and lipo-fibroblasts, supporting the progenitor cell character of PDGFRα+ cells. Consequently, the depletion of early postnatal PDGFRα+ cells caused a disruption of alveolarization resulting in an abnormal lung structure. This proves the need of PDGFRα+ cells for proper alveolarization. The present study further validated and characterized the inducible function to target lipofibroblasts of a recently generated Plin2tm1.1(Cre/ERT2)Mort mouse line. Using this functional validated mouse line the present study revealed the spatiotemporal mode of differentiation of lipofibroblasts during alveolarization by labeling and characterizing cells of the ADRP cell-lineage. Finally using the Plin2tm1.1(Cre/ERT2)Mort mouse line it was demonstrated for the first time, that the early postnatal ADRP cell-lineage (lipofibroblast lineage) is essential for appropriate alveolarization. Data of the present study give new insights into the differentiation and function of fibroblast subtypes during alveolarization and provide a new tool to target and manipulate lipofibroblasts in vivo by validating the previously generated Cre driver line. The study makes a new contribution to the identification of cellular and molecular targets for the development of new therapeutic strategies for pulmonary structural diseases since the induction of alveolarization in the diseased lung represents a desirable therapeutic approach.Ziel der vorliegenden Arbeit war die Charakterisierung pulmonaler Fibroblasten Subtypen in Bezug auf Differenzierung und Lokalisation während der Alveolarisierung sowie die Aufdeckung des Einflusses der Fibroblastenfunktion auf die Alveolarisierung. Ein weiteres Ziel stellte die Validierung und Charakterisierung einer kürzlich generierten Cre Treiber Mauslinie zur Manipulation von Lipofibroblasten und deren weiterer Gebrauch zur Manipulation von Lipofibroblasten in vivo dar. Um diese Ziele zu erreichen wurde mittels in vivo Methoden des CreERT2 stop loxP Systems und Reporter Genen gezeigt, dass platelet-derived growth factor receptor α+ (PDGFRα+) Zellen der frühen postnatalen Phase Myofibroblasten und Lipofibroblasten in der Alveolarisierung generieren können. Desweitern wurde gezeigt, dass so generierte Myofibroblasten und Lipofibroblasten Marker für mesenchymale Stammzellen exprimieren, als weiteren Hinweis für einen Vorläuferzellcharakter der PDGFRα+ Zellen. Folgerichtig führte die Depletion früh postnataler PDGFRα+ Zellen zu einem Abbruch der Alveolarisierung und abnormaler Lungenstruktur. Dieser Effekt beweist die Notwendigkeit PDGFRα+ Zellen für eine regelrechte Alveolarisierung. Des Weiteren wurde in der vorliegenden Arbeit die induzierbare Funktion einer kürzlich generierten Mauslinie, der Plin2tm1.1(Cre/ERT2)Mort Mauslinie, Lipofibroblasten zu modulieren validiert und charakterisiert. Mit Hilfe dieser funktionell validierten Mauslinie zeigte die vorliegende Arbeit die zeitlich und räumlich abhängige Art der Differenzierung von Lipofibroblasten in der Alveolarisierung durch Markieren und Charakterisieren von Zellen der ADRP Zellabstammungslinie. Schließlich wurde mittels der Plin2tm1.1(Cre/ERT2)Mort Maus Linie erstmalig gezeigt, dass die früh postnatale ADRP Zellabstammunglinie (Lipofibrobalstenabstammungslinie) essentiell für eine adäquate Alveolarisierung ist. Die Daten der vorliegenden Arbeit geben neue Einsichten in die Differenzierung und Funktion von Fibroblasten Subtypen während der Alveolarisierung und liefert durch die Validierung der kürzlich generierten Cre Treiber Mauslinie ein neues Werkzeug, um Lipofibroblasten in vivo zu manipulieren. Die vorliegende Arbeit leistet einen neuen Beitrag zur Identifizierung zellulärer und molekularer Zielkandidaten für die Entwicklung neuer therapeutischer Konzepte für strukturelle Lungenerkrankungen, da das Anregen der Alveolarisierung in der erkrankten Lunge einen wünschenswerten therapeutischen Ansatz darstellt

Cell Autonomous and Non-cell Autonomous Regulation of SMC Progenitors in Pulmonary Hypertension

Summary: Pulmonary hypertension is a devastating disease characterized by excessive vascular muscularization. We previously demonstrated primed platelet-derived growth factor receptor β+ (PDGFR-β+)/smooth muscle cell (SMC) marker+ progenitors at the muscular-unmuscular arteriole border in the normal lung, and in hypoxia-induced pulmonary hypertension, a single primed cell migrates distally and expands clonally, giving rise to most of the pathological smooth muscle coating of small arterioles. Little is known regarding the molecular mechanisms underlying this process. Herein, we show that primed cell expression of Kruppel-like factor 4 and hypoxia-inducible factor 1-α (HIF1-α) are required, respectively, for distal migration and smooth muscle expansion in a sequential manner. In addition, the HIF1-α/PDGF-B axis in endothelial cells non-cell autonomously regulates primed cell induction, proliferation, and differentiation. Finally, myeloid cells transdifferentiate into or fuse with distal arteriole SMCs during hypoxia, and Pdgfb deletion in myeloid cells attenuates pathological muscularization. Thus, primed cell autonomous and non-cell autonomous pathways are attractive therapeutic targets for pulmonary hypertension. : Sheikh et al. demonstrate that hypoxia-induced expression of KLF4 and HIF1-α in specialized lung arteriole SMC progenitors is required for distal migration and smooth muscle expansion, respectively. A HIF1-α/PDGF-B axis in endothelial cells non-cell autonomously regulates progenitor SMC induction, proliferation, and differentiation. The myeloid cell lineage marks SMCs. Keywords: smooth muscle biology, vascular wall, vascular biology, pulmonary artery, pulmonary hypertension, pulmonary vascular disease, vasculoproliferative disease, cardiovascular disease, endothelial-smooth muscle cell interactions, smooth muscle progenitor

Macrophage-derived PDGF-B induces muscularization in murine and human pulmonary hypertension

Excess macrophages and smooth muscle cells (SMCs) characterize many cardiovascular diseases, but crosstalk between these cell types is poorly defined. Pulmonary hypertension (PH) is a lethal disease in which lung arteriole SMCs proliferate and migrate, coating the normally unmuscularized distal arteriole. We hypothesized that increased macrophage platelet-derived growth factor–B (PDGF-B) induces pathological SMC burden in PH. Our results indicate that clodronate attenuates hypoxia-induced macrophage accumulation, distal muscularization, PH, and right ventricle hypertrophy (RVH). With hypoxia exposure, macrophage Pdgfb mRNA was upregulated in mice, and LysM‑Cre mice carrying floxed alleles for hypoxia-inducible factor 1a, hypoxia-inducible factor 2a, or Pdgfb had reduced macrophage Pdgfb and were protected against distal muscularization and PH. Conversely, LysM‑Cre von-Hippel Lindaufl/fl mice had increased macrophage Hifa and Pdgfb and developed distal muscularization, PH, and RVH in normoxia. Similarly, Pdgfb was upregulated in macrophages from human idiopathic or systemic sclerosis–induced pulmonary arterial hypertension patients, and macrophage-conditioned medium from these patients increased SMC proliferation and migration via PDGF-B. Finally, in mice, orotracheal administration of nanoparticles loaded with Pdgfb siRNA specifically reduced lung macrophage Pdgfb and prevented hypoxia-induced distal muscularization, PH, and RVH. Thus, macrophage-derived PDGF-B is critical for pathological SMC expansion in PH, and nanoparticle-mediated inhibition of lung macrophage PDGF-B has profound implications as an interventional strategy for PH