title: ROCK induced inflammation of the microcirculation during endotoxemia mediated by nitric oxide synthase.
abstract: Sepsis may be modeled using lipopolysaccharide (LPS), which alters levels of nitric oxide (NO), synthesized via endothelial and inducible nitric oxide synthase (eNOS and iNOS). This study aimed to determine whether the Rho kinase (ROCK) inhibitor fasudil protected against LPS-induced (endotoxemia) macromolecular leak and leukocyte adhesion via NOS pathways. Male Wistar rats (283±8g, n=36) were anaesthetized with thiopental and the mesentery prepared for fluorescent intravital microscopy (IVM). Animals received either (i) LPS alone (150μg kg(-1) h(-1) i.v., n=6); (ii) fasudil (FAS, 3mg kg(-1) i.v., n=6) or (iii) fasudil (10mg kg(-1) i.v., n=6), immediately prior to LPS administration, (iv) fasudil (FAS, 3mg kg(-1) i.v., n=6) alone or (v) fasudil (FAS, 10mg kg(-1) i.v., n=6) alone, or (vi) saline alone (1ml kg(-1) h(-1) i.v, n=6) for 4h (240min). LPS increased macromolecular leak (cumulative normalized grey levels, arbitrary units) from post capillary venules (<40μm) and this was reduced by 3mg kg(-1) fasudil, however, 10mg kg(-1) was less effective (t=240min, control: 3.3±1.7; LPS: 15.1±2.0; LPS+3mg kg(-1) fasudil: 3.3±1.1 (p<0.05), LPS+10mg kg(-1) fasudil: 8.4±3.2 NS). The numbers of leukocytes adhering for >1min/100μm venule were reduced by fasudil (t=240min, control: 1.8±0.7; LPS: 7.0±1.0; LPS+3mg kg(-1) fasudil: 1.75±0.25, p<0.05; LPS+10mg kg(-1) fasudil: 1.8±0.8, p<0.05). Immunohistochemistry demonstrated that fasudil increased endothelial cell expression of eNOS during sepsis, and decreased LPS-induced up-regulation of iNOS. Inhibition of ROCK in rats increases eNOS and decreases iNOS during endotoxemia, concomitantly reducing microvascular inflammation. Thus, targeting the ROCK pathway during sepsis could have therapeutic potential for reducing inflammation via a NO dependent mechanism.
authors.affiliation: Microcirculation Research Group, University of Sheffield, Faculty of Medicine, Dentistry and Health, Beech Hill Road, Sheffield S10 2RX, UK. caroline_mcgown@hotmail.com
authors.forename: Caroline C
authors.initials: CC
authors.lastname: McGown
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Brown
authors.forename: Paul G
authors.initials: PG
authors.lastname: Hellewell
authors.forename: Zoë L S
authors.initials: ZL
authors.lastname: Brookes
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D002199:Capillary Permeability / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D019446:Endotoxemia / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism / Q000503:physiopathology; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008833:Microcirculation / Q000187:drug effects / Q000502:physiology*; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013152:Splanchnic Circulation / Q000187:drug effects / Q000502:physiology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D047428:Protein Kinase Inhibitors; D009569:Nitric Oxide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat; D054460:rho-Associated Kinases; C049347:fasudil