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- Therapeutic application of Argonaute-2 to enhance neurovascular repair and inflammatory restitution in strokePublication . Pereira, Marta Sofia Machado; Ferreira, Raquel Margarida da Silva; Cristóvão, Ana Clara BrazStroke is the leading cause of death and adult disability in Portugal and the second cause of death worldwide. In the coming years, this health condition will worsen due to the increase in average life expectancy and the increase in the number of individuals with risk factors for stroke. Currently, the recommended treatment for stroke patients is the intravenous thrombolysis therapy (with alteplase or endovascular thrombectomy), although it has limited efficacy due to the narrow therapeutic window where it can be applied without causing hemorrhagic transformations, limiting the benefit to many patients and highlighting the need to develop alternative and safer therapies. The development of strategies aiming to potentiate endogenous neuroprotective mechanisms has emerged as a safe alternative to thrombolysis, however, these approaches have undervalued the importance of the cerebral vasculature in the post-stroke recovery. Previously, it was demonstrated that cerebral vascular remodeling is correlated with higher survival rates and improved long-term neurological function in stroke patients. Accordingly, in this research work, it was proposed the modulation of the Argonaute (Ago)-2 endogenous mechanism to enhance the brain vascular function compromised by cerebral ischemia and, consequently, contributing to full parenchymal recovery after a stroke. Besides being the major coordinator of microRNA traffic and activity, Ago2 protein is pivotal for cell survival and tube network formation in peripheral endothelial cells. However, these mechanisms and the role of Ago2 in the ischemic brain vasculature remain unknown. In this sense, the potential of this protein in the inflammatory process was first assessed. This pathological process is one of the most important post-stroke pathological events that can lead to short-term mortality or promote long-term disability. Accordingly, in vitro (mouse primary brain cultures of endothelial and glial cells stimulated with lipopolysaccharide) and in vivo (adult mice intraperitoneally injected with lipopolysaccharide) data revealed that exogenous Ago2 application (downregulated in brain vasculature in these conditions) normalized endothelial markers associated with inflammatory-induced damage which, in turn, reestablished previously compromised glial and neuronal function. Then, after confirming the potential of Ago2 for cerebral ischemia studies, a strategy to enhance the Ago2 intracellular levels (also downregulated in the ischemic context) and to promote endothelial cell survival was defined. The controlled release of retinoic acid (a signaling molecule with defined anti-inflammatory and pro-angiogenic properties) via polymeric nanoparticles arose as a promising approach. In vitro data (mouse primary brain cultures of endothelial cells subjected to oxygen and glucose deprivation) revealed the protective potential of RA-NP in ischemic conditions, only observed by restoring basal levels of Ago2 protein (an effect mediated by the nitric oxide signaling), highlighting the importance of Ago2 in vascular function. Additionally, in vivo data (adult mice subjected to transient occlusion of the middle cerebral artery) showed that the systemic administration of this formulation promoted tissue survival, normalized inflammatory response, and promoted neurovascular repair (with a robust pro-angiogenic effect) in the peri-infarct region after stroke. However, further studies are needed to establish a correlation between the observed in vivo outcome and the Ago2 protein, and of RA-NP treatment and to decipher interactions between this protein and retinoic acid. Altogether, the experimental research developed throughout this Thesis provided evidence that the modulation of the Ago2 endogenous mechanism can be envisioned as a neuroprotective approach in post-stroke recovery. Furthermore, it revealed a safe nanotechnological tool able to modulate this system, suitable for intravenous administration, and compatible with pre-existing therapies.