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Vitamin D is a known modulator of inflammation. Native dietary vitamin D3 is thought to be bio-inactive, and beneficial vitamin D3 effects are thought to be largely mediated by the metabolite 1,25(OH)2D3. Reduced serum levels of the most commonly measured precursor metabolite, 25(OH)D3, is linked to an increased risk of multiple inflammatory diseases, including: cardiovascular disease, arthritis, multiple sclerosis, and sepsis. Common to all of these diseases is the disruption of endothelial stability and an enhancement of vascular leak. We previously performed an unbiased chemical suppressor screen on a genetic model of vascular instability, and identified cholecalciferol (D3, dietary Vitamin D3) as a factor that had profound and immediate stabilizing and therapeutic effects in that model. In this manuscript we show that the presumed inactive sterol, D3, is actually a potent and general mediator of endothelial stability at physiologically relevant concentrations. We further demonstrate that this phenomenon is apparent in vitamin D3 metabolites 25(OH)D3 and 1,25(OH)2D3, and that the effects are independent of the canonical transcription-mediated vitamin D pathway. Our data suggests the presence of an alternative signaling modality by which D3 acts directly on endothelial cells to prevent vascular leak. The finding that D3 and its metabolites modulate endothelial stability may help explain the clinical correlations between low serum vitamin D levels and the many human diseases with well-described vascular dysfunction phenotypes. [ABSTRACT FROM AUTHOR]

The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia. [ABSTRACT FROM AUTHOR]