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During embryonic development, cells differentiate in a coordinated manner, aligning their fate decisions and differentiation stages with those of surrounding cells. However, little is known about the mechanisms that regulate this synchrony. Here we show that cells in close proximity synchronize their differentiation stages and cellular phenotypes with each other via extracellular vesicle (EV)-mediated cellular communication. We previously established a mouse embryonic stem cell (ESC) line harbouring an inducible constitutively active protein kinase A (CA-PKA) gene and found that the ESCs rapidly differentiated into mesoderm after PKA activation. In the present study, we performed a co-culture of Control-ESCs and PKA-ESCs, finding that both ESC types rapidly differentiated in synchrony even when PKA was activated only in PKA-ESCs, a phenomenon we named ‘Phenotypic Synchrony of Cells (PSyC)’. We further demonstrated PSyC was mediated by EVs containing miR-132. PKA-ESC-derived EVs and miR-132-containing artificial nano-vesicles similarly enhanced mesoderm and cardiomyocyte differentiation in ESCs and ex vivo embryos, respectively. PSyC is a new form of cell-cell communication mediated by the EV regulation of neighbouring cells and could be broadly involved in tissue development and homeostasis.

Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGFC and VEGFD, are key drivers of lymphatic remodelling in cancer, the means by which many signalling pathways in endothelial cells are co-ordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. In this thesis, I seek to understand the broader molecular mechanisms that control cancer metastasis through the analysis of microRNAs which act to co-ordinately regulate signalling pathways involved in complex biological responses, such as lymphatic remodelling, in health and disease. Here, using high-throughput small RNA sequencing, I found that a specific microRNA, miR-132, is up-regulated in expression in lymphatic endothelial cells (LECs) in response to stimulation with VEGFC and VEGFD. Interestingly, inhibiting the effects of miR-132 in LECs in vitro blocked proliferation and tube formation of these cells induced by VEGFC and VEGFD - LEC proliferation and tube formation are key steps in lymphatic remodelling. Moreover, I demonstrated that miR-132 is expressed in the lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGFD. In order to dissect the complexity of molecular regulation by miR-132 in lymphatic biology, my collaborators and I identified miR-132 target mRNAs in LECs, using high-throughput sequencing after RNA-protein cross-linking and immunoprecipitation of the Argonaute protein (Argonaute HITS-CLIP), which led us to define the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different molecular pathways mainly involved in cell proliferation and reg

Alterations in the neuro-immune balance play a major role in the pathophysiology of chronic neuropathic pain. MicroRNAs (miRNA) can regulate both immune and neuronal processes and may function as master switches in chronic pain development and maintenance. We set out to analyze the role of miR-132-3p, first in patients with peripheral neuropathies and second in an animal model of neuropathic pain. We initially determined miR-132-3p expression by measuring its levels in white blood cells (WBC) of 30 patients and 30 healthy controls and next in sural nerve biopsies of 81 patients with painful or painless inflammatory or non-inflammatory neuropathies based on clinical diagnosis. We found a 2.6 fold increase in miR-132-3p expression in WBC of neuropathy patients compared to healthy controls (p<0.001). MiR-132-3p expression was also slightly up-regulated in sural nerve biopsies from neuropathy patients suffering from neuropathic pain compared to those without pain (1.2 fold; p<0.001). These promising findings were investigated further in an animal model of neuropathic pain, the spared nerve injury model (SNI). For this purpose miR-132-3p expression levels were measured in dorsal root ganglia and spinal cord of rats. Subsequently, miR-132-3p expression was pharmacologically modulated with miRNA antagonists or mimetics, and evoked pain and pain aversion were assessed. Spinal miR-132-3p levels were highest 10days after SNI, a time when persistent allodynia was established (p<0.05). Spinal administration of miR-132-3p antagonists via intrathecal (i.t.) catheters dose dependently reversed mechanical allodyina (p<0.001) and eliminated pain behavior in the place escape avoidance paradigm (p<0.001). Intrathecal administration of miR-132-3p mimetic dose-dependently induced pain behavior in naïve rats (p<0.001). Taken together these results indicate a pro-nociceptive effect of miR-132-3p in chronic neuropathic pain.

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