Beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide  C.C. McGown, Z.L.S. Brookes  British Journal of Anaesthesia  Volume 98, Issue 2, Pages 163-175 (February 2007) DOI: 10.1093/bja/ael358 Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 1 The mevalonate pathway. Statin-mediated inhibition of HMG-CoA reductase blocks the conversion of HMG-CoA to mevalonate and thus prevents downstream cholesterol-dependent and cholesterol- independent steps. Activity of the small GTPase Rho depends upon geranylation by the isoprenoid geranylgeranyl-PP and therefore is blocked by statins. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 2 Schematic representation of Rho activation via GGPP. GGPP is involved in the post-translational modifications of small GTPase proteins including Rho. Geranylgeranylation by GGPP causes Rho to cycle between its inactive GDP-bound state and its active GTP-bound state. This shift in activity allows Rho to translocate within the cell from the cytoplasm, where it is inactive, to the membrane where it interacts with the cytoskeleton and other intracellular proteins to regulate processes including endothelial integrity and gene expression. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 3 Intravital microscopy (IVM) images of rat mesenteric vessels, demonstrating macromolecular leak. The fluorescent molecule fluoroscein isothiocyanate (FITC) was conjugated to bovine serum albumin (BSA) to form FITC–BSA and administered i.v. Blue light (495 nm) was then used to activate FITC–BSA, causing it to fluoresce. It can be observed that ‘healthy’ vessels maintain FITC–BSA within the vasculature (a). However, when the integrity of the endothelium is compromised FITC–BSA leaks from gaps forming between the endothelial cells, appearing as a white flare in the interstitium (b). Macromolecular leak occurs from post-capillary venules only. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 4 Schematic representation of tight junctional proteins. Occludin is an integral membrane protein that interacts with the intracellular protein ZO, establishing a link between the cytoskeleton and tight junctions. Signalling molecules such as GTPase Rho act at the cytoskeleton to modulate tight junctions and thus endothelial permeability. Claudin has been proposed as the main tight junctional protein. There is evidence to suggest that occludin and claudin interact to regulate tight junctional permeability. JAM proteins localize close to tight junctions and may mediate the relationship between ZO and occludin. They also dimerize with JAM proteins of adjacent cells but whether this relationship has an influence on endothelial cell permeability is uncertain. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 5 Schematic representation of gap junction structure. Connexin monomers are comprised of four transmembrane subunits and have intracellular termini. Six connexin monomers form a ‘connexon’ hexamer, surrounding a pore that allows the passage of molecules between adjacent cells. The ‘gap’ is the space between the cells, formed because of the presence of gap junctions in the plasma membranes. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 6 Schematic representation of interactions between gap junctions and adherens junctions. Gap junction formation is mediated by adherens junctions cell adhesion molecules including VE-cadherin (Cad), a homodimerizing transmembrane protein and catenin (Cat), an intracellular protein linking cadherin to the cytoskeleton. Cat interacts directly with the cytoskeleton or via ZO. Disruption of Cat expression has an inhibitory effect on gap junction formation and thus increases vascular permeability. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 7 IVM images of a rat mesenteric venule, demonstrating a leucocyte rolling along the endothelial cell surface, as demonstrated by arrows. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 8 IVM image of a rat mesenteric venule, demonstrating leucocyte adhesion (thin arrows) and migration (thick arrows) through the endothelial cell layer. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions

Fig 9 Schematic diagram proposing effects of statins in microvascular endothelial cells. Statins reduce iNOS expression by blocking the transcription factors NFκB and STAT-1. Inhibition of these transcription factors also inhibits cytokine production and thus prevents up-regulation of cell adhesion molecules on both endothelial cells and leucocytes. Statins increase eNOS activity by activation of the eNOS phosphorylator Akt; inhibition of Rho, an eNOS inhibitor, via blockade of the mevalonate pathway; and inhibition of caveolin-1, which down-regulates NO synthesis via sequestering eNOS into caveolae. British Journal of Anaesthesia 2007 98, 163-175DOI: (10.1093/bja/ael358) Copyright © 2007 British Journal of Anaesthesia Terms and Conditions