Enhancement of vascular stability through ARF6 deactivation reduces inflammatory pathologies

The vasculature infuses nearly every tissue with oxygen and nutrients. It facilitates the transport of cells and chemical messages throughout the body and maintains proper fluid balance in the individual tissues. During angiogenesis and inflammation, vessels that compose the vasculature display enhanced permeability. This leak is regulated by the monolayer of endothelial cells that line each vessel. This thesis aims to further describe the molecular mechanisms within endothelial cells that control vascular leak. I present our research identifying a molecular signaling axis that controls vascular leak induced by inflammatory cues through the small GTPase ADPribosylation factor 6 (ARF6). We show that blockade of ARF6 enhances endothelial barrier function, reduces vascular leak, and prevents inflammatory pathology. We identify a signaling axis composed of Myeloid Differentiation Factor 88 (MYD88), ARF-Nucleotide Binding Site Opener (ARNO), and ARF6 in endothelial cells that is used by the inflammatory cytokine, interlukin-1β (IL-1β), to dissociate cell junctions and enhance vascular leak. We determined that IL-1β enhances permeability rapidly, independently of gene-expression changes, and in a manner dependent on ARF6 activation. We showed that ARF6 activation induced the dissociation of the cell junction protein vascular endothelial cadherin (VE-Cadherin) and when ARF6 was blocked, VE-Cadherin dissociation was prevented and vascular permeability was iv reduced. Inhibiting this signaling axis in mice with a small molecule inhibitor reduced vascular leak and inflammatory pathologies associated with arthritis. MYD88 is a crucial adapter protein for many inflammatory pathways, including the Toll-like-receptors (TLRs). TLRs are the primary receptors that detect pathogens by recognizing danger-associated molecular patterns (DAMPs) such as bacterial lipopolysaccharaides (LPS) and initiating an inflammatory cascade. We determined that the MYD88-ARNO-ARF6 cascade is also responsible for vascular leak induced by LPS. We characterized the peptide MyrARF6 2-13 as an inhibitor of ARF6 activation and showed that peptide treatment is sufficient to reduce vascular leak and enhance survival of mice in experimental sepsis. We suggest that ARF6 controls inflammatory pathology independent of inflammatory factors such as cytokines. Such observations suggest that vascular leak itself could be a key driver of inflammatory pathology and may pave the way for the development of therapeutics that do not leave patients immunocompromised. .