Dissertation committee:
Μάναλη Ευφροσύνη, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Παπίρης Σπυρίδων, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Λουκίδης Στυλιανός, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μπακάκος Πέτρος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ροβίνα Νικολέττα, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Παπαϊωάννου Ανδριάνα, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Αϊδίνης Βασίλης, Ερευνητής Α΄, ΕΚΕΒΕ Αλέξανδρος Φλέμινγκ
Summary:
Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology, characterized by the scarring and thickening of the lung tissue. The exact cause of IPF is not fully understood, but it is believed to involve a complex interplay of genetic, environmental, and immune factors. Currently, there is no cure for the disease, and treatment options are limited. Two drugs, pirfenidone and nintedanib, have been approved by the FDA for the treatment of IPF. These drugs have been shown to slow the progression of the disease, and manage symptoms, but they do not fully cure IPF.
Fibroblasts and myofibroblasts in IPF demonstrate a pathologic phenotype characterized by uncontrolled proliferation and survival. Fibroblast accumulation has been suggested to rely on their ability to invade the ECM and/or to migrate along it. Critical for these processes is the proteolysis of the ECM through cell’s protrusions known as podosomes. Podosomes and invadopodia (known as invadosomes) are actin rich structures of pericellular proteolytic activity, resulting in ECM degradation. SH3PXD2A (known asTKS5) is a scaffold protein that localizes to, and is required for invadosomes formation. So far, Τks5 has been found highly related with cancer, since induces cell invasion of cancer cells. It is known that lung fibroblasts shared common characteristics with cancer cells, including ECM invasion. So TKS5 and podosomes may be implicated in the pathogenesis of IPF, and therefore we further examined their role.
Increased expression of TKS5, was detected in the lung tissue of IPF patients and bleomycin (BLM)-treated mice, correlating with increased collagen type I alpha 1 chain (COL1A1) expression. Τhe profibrotic milieu, TGFβ, as well as the acellular fibrotic ECM, were found to induce TKS5 expression and the formation of prominent podosome rosettes in LFs, culminating in increased ECM invasion. Podosomes were retained ex vivo in the absence of any stimulation, indicating that the formation of TKS5-enabled podosomes is an inherent property of IPF LFs. Haploinsufficient Tks5+/- mice were relatively resistant to BLM-induced pulmonary fibrosis. Disease protection was largely attributable to diminished podosome formation in LFs and decreased ECM invasion, thus indicating TKS5-enabled and podosome-mediated ECM invasion as a major pathogenic mechanism in pulmonary fibrosis. Expression profiling revealed an ECM-podosome cross talk, and pharmacologic connectivity map analysis suggested several inhibitors that could prevent podosome formation and thus pulmonary fibrosis. Among them, inhibition of Src kinase was shown to potently attenuate podosome formation in LFs, ECM invasion, as well as pulmonary fibrosis in post BLM precision cut lung slices, suggesting that pharmacological targeting of TKS5-enabled podosome formation is a very promising therapeutic option in pulmonary fibrosis.
In addition, in this dissertation the role of Versican in the pathogenesis of IPF was studied. Versican is a large extracellular matrix (ECM) proteoglycan that is present in various tissues, including the lung. Extracellular matrix is a dynamic structure, consisting of more than 300 proteins, glycosaminoglycans and enzymes, which serves as structural scaffold, and provides the mechanical stability for proper lung function. Dysregulation of ECM composition, structure, stiffness, and abundance contributes to several pathological conditions, including IPF. Due to the numerous indications that ECM is involved in the pathogenesis of IPF, Versican, as an ECM component, could be implicated and therefore we pursued its further study.
In silico studies, in human and mouse datasets, unveil upregulated expression of VCAN in IPF patients and murine lungs upon bleomycin induced pulmonary fibrosis. In IPF lungs VCAN is expressed mainly by fibroblasts, macrophages and dendritic cells, where VCAN-expressing LFs were found to belong to a COL1A1-expressing subpopulation In vitro TGFβ was found to stimulate VCAN mRNA and protein levels in human and mouse lung fibroblasts. Moreover, the stiff fibrotic aECM induces Vcan expression in fibroblasts. In in vivo experiments, Vcan was found increased in lungs of WT mice with bleomycin induced PF. Immunohistochemistry revealed increased levels of Vcan protein in bronchial epithelium, fibrotic areas and macrophages. To further dissect the role of Vcan in IPF, the BLM induced PF model performed in Vcan+/- mice. Heterozygous genetic deletion of Vcan leads to exacerbated pulmonary fibrosis phenotype in mice. Also, isolation of haplo-insufficient Vcan+/- primary LFs revealed differences in basic cell functions, while TGF-β1 stimulation, affects the mRNA expression of fibrogenic genes and induces the formation of podosome rosettes, leading in increased invasion. In conclusion, disease exacerbation in Vcan+/- mice was correlated with disturbed ECM homeostasis, modulation of TGFβ responses, increased fibroblasts adhesion and the formation of podosomes leading in increased ECM migration and invasion.
