Investigation of molecular mechanisms underlying the pathogenesis of lung fibrosis: from patients to in vitro model

The thesis investigates the molecular mechanisms underlying lung fibrosis, with a particular emphasis on COVID-19. It comprises four chapters, each addressing distinct yet interrelated aspects of the disease. Initially, clinical parameters indicative of pulmonary damage, assessed via CALIPER CT scans, were evaluated to identify simple biomarkers that could mirror fibrotic lung involvement. Among these, serum lactate dehydrogenase (LDH) emerged as a valuable surrogate marker, correlating with both disease severity and imaging findings. Next, the study explored alterations in oxygen homeostasis, focusing on 2,3-bisphosphoglycerate (2,3-BPG) levels and acid-base imbalances in COVID-19 patients. These findings highlighted specific compensatory mechanisms engaged in response to reduced oxygenation and lung damage, thereby elucidating the role of 2,3-BPG in disease progression. Subsequently, a novel pathological mechanism involving angiotensin-converting enzyme 2 (ACE2) cleavage was investigated in lung epithelial cells. Beyond its established enzymatic function, ACE2 underwent proteolytic cleavage in response to pharmacological inhibition and viral spike protein interactions. This process involved both extracellular domain shedding and a regulated intramembrane proteolysis event, leading to nuclear signalling through HIF-1α activation, independently of Ang II stimulation. These results reveal a non-canonical pathway by which ACE2 cleavage may exacerbate lung injury. Finally, the thesis proposes a therapeutic strategy aimed at inhibiting the TGF-β–Smad3 fibrotic pathway, a central driver of fibrosis. The work details the design and development of specific RNA aptamers against Smad3, leveraging robotic-assisted selection and intramer technology for enhanced stability. Collectively, these findings deepen our understanding of COVID-19–related fibrotic lung injury and identify novel diagnostic and therapeutic targets, underscoring the importance of multifaceted approaches in managing pulmonary fibrosis.