Extracellular Vesicles as Masters of Endothelial Plasticity. Bridging the Gap Between Health and Disease

Abstract

ABSTRACT: Cells communicate in multiple ways, including the secretion of membrane-bound extracellular vesicles (EVs). These EVs carry and deliver a cargo comprised of regulatory factors which may be proteins, lipids, or nucleic acids. Upon EVs entry in a target cell, the cargo will change the phenotype. In pathologies like cancer, tumor cells secrete EVs into their surroundings to adjust them to their specific needs. In this thesis, we focus on the influence of cancer cell-derived EVs on blood vessel formation, known as angiogenesis. Compared with cancer cells, therapeutic stem cells such as adipose tissue-derived stem cells (ASCs) also secrete EVs which are also known for their so-called pro-angiogenic capacity. Therefore, this thesis addresses the question of which components from tumor cells and ASC-derived EVs can be held responsible for the stimulation of angiogenesis. In particular miRNAs and proteins were studied with their respective ‘-omics’-based technologies. In addition, we investigated the influence of low oxygen levels i.e. hypoxia on the contents and function of EVs because, in vivo, hypoxia is a driver of angiogenesis. Finally, we assessed the influence of extracellular matrix (ECM) i.e. nature’s ‘glue’ to keep cells in tissues together. We showed that EVs from carcinoma cells and ASCs promote sprouting angiogenesis in vitro and ex vivo and stabilize these networks over time. However, the EVs induced a different branching pattern/topology depending on the ECM stiffness, showing a differential behavior of endothelial cells (ECs) in response to the extracellular context (ECM stiffness). Next, the miRNAs and proteins in the EVs were comprehensively characterized. Beyond what we have seen frequently reported, we found that hypoxia for 24h does not greatly influence the miRNA composition of EVs, however, the protein profile does reveal major changes. We initially reasoned that this low influence of hypoxia on the expression of miRNAs was due to the constitutive expression of the hypoxia-inducible factor-1α (HIF-1α) transcription factor in HeLa and SiHa cells. Remarkably, non-tumor but therapeutic cells i.e. ASCs show similar behavior in terms of hypoxia-induced changes in miRNA contents of their secreted EVs non-tumor cells. We found and described a set of angiogenesis regulators in both carcinoma and ASCs-derived EVs and tested the pro-angiogenic effect of miR23b-3p in ECs. Additionally, a comprehensive analysis of the protein profile in carcinoma EVs revealed that although the vesicles are already loaded with pro-angiogenic molecules, in hypoxia, these vesicles are enriched in proteins that are associated with an increase in rigidity, which favor an increase in the efficiency of uptake by ECs. ECs take up dye-labeled EVs during the first hours after treatment, which means EVs elicit a rapid response in ECs. In the same way, those EVs are enriched in molecules that facilitate the characteristic metabolic switch of ECs, when they come out of resting mode to activate and generate tip cells. The similarity between the behavior and partial content of these vesicles from pathological and therapeutic contexts made us re-assess the concept of genetic dose. EVs from tumors and ASCs share 75-94% of their contained miRNAs, and more than 80% of the sequence reads were contributed by only the 40 most enriched miRNAs in all samples. This means that for EV research, we should rethink the way of analyzing the information contained in EVs. Beyond that, the redundant stimulatory role between harbored molecules in EVs can be concluded, and a set of co-regulatory cross-talkers as potential regulators of the angiogenic response in recipient ECs with the ability to move the balance from resting to an activated state was identified. Attempting to elucidate the entire biology of vesicles solely through the lens of a single molecule risks overlooking the broader biological context essential for understanding evolutionary signal transmission mechanisms. The results in this thesis show that on top of the well-known growth factor-governed paracrine signaling pathways of intercellular communication, EVs are more than regulatory particles that fine-tune paracrine signaling. Instead, EVs are potent regulators in their own right with a cargo that carries a redundancy to potentiate e.g. pro-angiogenic stimulation in vitro and in vivo. We also show that pathology and therapy might be on the same side of the river and do not need a bridge as in terms of angiogenesis, their function is largely overlapping. However, other influences (such as pro-fibrotic or pro-inflammatory properties) were not studied, therefore the use of carcinoma-derived EVs for therapy requires crossing yet unknown bridges (Chapter 8, Fig. 5).