Development of a novel therapy for ARDS based on the identification of miRNAs from mesenchymal stem cells-derived extracellular vesicles

Abstract

Acute Respiratory Distress Syndrome (ARDS) is an acute hypoxemic respiratory failure caused by diffuse inflammatory lung injury, affecting around 10% of ICU patients worldwide. The pathophysiology involves activation and dysregulation of multiple injury response pathways, which vary depending on the injury’s cause and the individual’s immune response. This variability hinders ARDS diagnosis and treatment. Despite advances, including patient subphenotyping and improved supportive care, no definitive pharmacological treatment exists to restore immune homeostasis and regenerate the alveolocapillary membrane, crucial for lung function. The complex lung structure in ARDS further complicates drug delivery, affecting treatment effectiveness._x000D_ _x000D_ Mesenchymal stem cells (MSCs) possess regenerative, immunomodulatory, and antimicrobial properties, showing promise in preclinical acute lung injury (ALI) models and safety in clinical trials. However, translating these findings into clinical treatments remains challenging. MSCs primarily exert their therapeutic effects through paracrine activity, mediated by extracellular vesicles (EVs). Consequently, MSCs-derived EVs have emerged as a potential alternative to MSCs administration, addressing some clinical application challenges. Nonetheless, MSCs-EVs also face hurdles such as pleiotropic effects, reproducibility issues, and difficulties in large-scale production under GMP conditions._x000D_ _x000D_ This thesis aims to identify the most effective miRNAs within MSCs-derived EVs and characterize a pulmonary delivery system to create a novel therapy that regenerates lung tissue and modulates the immune response in ARDS. The research is presented through three individual publications that tackle specific objectives to achieve this goal._x000D_ _x000D_ The first study shows that MSC-derived EVs improve lung architecture and reduce inflammation in a preclinical acute lung injury (ALI) model. When MSCs are primed with lipopolysaccharide (LPS), the therapeutic effect of their EVs is enhanced. LPS-primed EVs (LPS-EVs) display a greater regenerative effect on alveolar epithelial cells (AECs) and uniquely reduce the M1/M2 macrophage ratio after Pseudomonas aeruginosa infection compared to EVs from naïve MSCs (C-EVs). LPS-EVs also reduce alveolar macrophages' chemoattractant activity, thereby decreasing neutrophil recruitment in the alveolar space._x000D_ _x000D_ LPS-priming alters the miRNA profiles of MSCs-derived EVs, with differentially expressed miRNAs implicated in critical pathways regulating immune response, differentiation, apoptosis, proliferation of AECs, endothelial cells, and barrier integrity—key processes in ARDS. Overexpressed miRNAs, including miR-297, miR-93-5p, and let-7b-5p, are identified as potential mediators of the enhanced effects of LPS-EVs. The individual transfection of these miRNAs into THP-1 cells reduces the expression of proinflammatory cytokines induced by Pseudomonas aeruginosa. Interestingly, when they are administered simultaneously, they exert a synergistic immunomodulatory effect,further promoting macrophage polarization towards an anti-inflammatory M2 phenotype._x000D_ _x000D_ To overcome pulmonary drug delivery challenges, a comparative analysis of two poly(lactic-co-glycolic acid) (PLGA) nanocarriers—one positively and one negatively charged—was conducted. Both types showed excellent colloidal stability and did not show toxic effects to AECs or macrophages, key cellular targets in ARDS therapy. Negatively charged nanocapsules (NCs) demonstrate faster uptake and greater cellular accumulation than cationic NCs. When delivered via intratracheal instillation, anionic NCs distribute uniformly across lung lobes, minimizing systemic spread and confirming effective uptake by AECs and macrophages._x000D_ _x000D_ This thesis, by the identification of miR-297, miR-93-5p, and let-7b-5p as crucial regulators for LPS-EVs' therapeutic effects and the characterization of a drug delivery vehicle suitable for pulmonary administration, lays the groundwork for developing a cell-based, but cell-free ARDS therapy focused on tissue regeneration and restoring immune homeostasis. The proposed therapy has the potential to be a product available at any time at bedside.

Date of Award	12 Dec 2024
Original language	English
Supervisor	Antonio Artigas Raventos (Director) & Daniel Closa Autet (Director)