Microglia-derived extracellular vesicles: insights into the immune regulation of angiogenesis under ischemic conditions

Ischemic stroke (IS), accounting for 85% of strokes globally, is among the leading causes of death and life-long disabilities [2]. To date, neuroprotective or neuroregenerative therapies are not available and patients are managed by hyperacute reperfusion treatments, symptomatic measures and neurorehabilitation, which only lead to partial recovery [5,16]. Recent research has identified microglial cells as essential orchestrators of post-ischemic neuroinflammation, contributing to IS progression and resolution [78,82,85]. However, microglial functions are still debated as they can exacerbate ischemic injury or favour tissue repair, depending on the disease stages and on their activation states. Understanding how hypoxia-activated microglia interact with other neurovascular unit (NVU) cells is therefore fundamental to effectively boost their protective activities, while limiting their deleterious ones. Here, I focused on the interactions between microglial cells and the brain vasculature, as growing evidence highlights an important role of microglia in modulating brain angiogenesis [90]. Notably, angiogenesis has been associated with improved stroke outcomes in animal models and in humans and is regarded as an attractive therapeutic target in IS [161-165]. However, the dynamics of stroke-induced angiogenesis are poorly understood. Moreover, the mechanisms through which microglia regulate angiogenesis under ischemic conditions remain unclear. Based on recent evidence highlighting the roles of microglia-released EVs (M-EVs) in shaping the functionality of NVU cells after stroke [221], I hypothesised that M-EVs may be crucially implicated in microglial communication with ECs. In detail, the aim of my PhD thesis was to investigate whether M-EVs released under hypoxic conditions could be involved in modulating the angiogenic process. To investigate this hypothesis, I set up an in vitro model to study human microglial behaviour under stroke-like conditions, reporting microglial activation upon hypoxic stimulation. Morphological characterization of EV populations isolated from the CM of normoxic and hypoxic cells revealed similar size distributions and good sample quality. To assess whether the hypoxic activation would influence M-EVs activities on brain endothelial cells (ECs), I performed various in vitro assays to dissect different steps involved in the angiogenic process. Specifically, I reported a pro-angiogenic activity of EVs released under hypoxic conditions on ECs, highlighting a specific effect of hypoxia on M-EVs biological functions. In vitro data were supported by in vivo observations made in a Zebrafish xenograft model, in which hypEVs similarly stimulated angiogenesis. Furthermore, the analysis of M-EVs protein cargo highlighted the hypoxia-induced over-expression of a set of pro-angiogenic proteins, possibly implicated in the reported hypEVs angiogenic effects. Finally, we showed that hypoxic priming of microglia results in the secretion of M-EVs with a stimulatory action on axonal outgrowth in neuronal cells, indicating a possible neurotrophic role for M-EVs under hypoxia, which will be further evaluated in future experiments. Taken together, these results expand our understanding of the communication between microglial and endothelial cells under ischemic conditions and pose the bases for further studies aimed at defining the roles and mechanisms of action of activated microglia in the neuro-vascular repair process. Further research in this direction will contribute to uncover potential targets for more efficient immunomodulatory therapies for IS.