Inhaled nitric oxide modulates leukocyte kinetics in the mesenteric venules of endotoxemic rats

OBJECTIVE: to determine whether inhaled nitric oxide (NO) would alter leukocyte kinetics in the septic microvasculature. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats. INTERVENTIONS: Rats were treated with either saline or endotoxin (10 mg/kg, iv) and then allowed to breathe either air or air plus NO (10 ppm). MEASUREMENTS AND MAIN RESULTS: After a 4-hr period, rolling, firm adhesion, and emigration of leukocytes and endothelial dysfunction were monitored in mesenteric venules by using intravital videomicroscopy. Compared with controls, endotoxemic rats exhibited a profound influx in mesenteric venule rolling leukocytes (55+/-17 vs. 70+/-19 leukocytes/min; p < .05), associated with a reduction of leukocyte rolling velocity (83+/-14 vs. 34+/-3 microm/sec; p < .05). In endotoxemic rats, venular endothelium leukocyte firm adhesion (1.15+/-0.32 vs. 4.08+/-0.96 leukocytes/ 100 microm; p < .05) and emigration (0.84+/-0.47 vs. 4.23+/-1.2 leukocytes/100 microm; p < .05) increased compared with controls. Inhaled NO had no effect on leukocyte kinetics in control rats. Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08+/-0.96 vs. 1.86+/-0.76 leukocytes/100 microm; p < .05) and emigration (4.23+/-1.2 vs. 1.68+/-0.72 leukocytes/100 microm; p < .05). Compared with control rats, macromolecular (FITC-dextran) vascular leakage, expressed as the perivenular/intravenular fluorescence intensity ratio, increased in endotoxemic rats (0.56+/-0.02 vs. 0.81+/-0.05; p < .01). Endotoxin-induced macromolecular vascular leakage increases were partially prevented by inhaled NO (0.66+/-0.01 vs. 0.56+/-0.02; p < .05). CONCLUSION: These observations suggest that inhaled NO reduces leukocyte adhesion and the degree of vascular permeability dysfunction in mesenteric venule of endotoxemic rats.     

Leukocyte-independent plasma extravasation during endotoxemia

OBJECTIVES: To determine the meaning of leukocyte-endothelial interactions for the development of endotoxin-induced vascular leakage. DESIGN: Randomized, blinded, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Twenty-four male Wistar rats. INTERVENTIONS: After application of fucoidin to prevent leukocyte rolling and adherence (25 mg/kg; n = 8; fucoidin/LPS group) or saline 0.9% (n = 8; LPS group), animals were given an intravenous infusion of endotoxin (Escherichia coli lipopolysaccharide 026:B6; 2 mg/kg/hr) over 120 mins. Animals in the control group (n = 8) received an equivalent volume of saline 0.9%. MEASUREMENTS AND MAIN RESULTS: Leukocyte rolling and leukocyte adherence, red cell velocity, vessel diameters, venular wall shear rate, volumetric blood flow, and macromolecular leakage were determined in mesenteric postcapillary venules using in vivo videomicroscopy at baseline, 60 mins, and 120 mins after start of a continuous endotoxin infusion. Fucoidin prevented leukocyte rolling (baseline, 3+/-2 rollers; 120 mins, 3+/-1 rollers; not significant vs. baseline; p < .01 vs. LPS group) and reduced the adherence of leukocytes at baseline and during endotoxemia and showed only a slight increase in adherent leukocytes (baseline, 100+/-38 cells/mm2; 120 mins, 244+/-68 cells/mm2; p < .05 vs. baseline; p < .01 vs. LPS group). In the LPS group, endotoxin exposure induced a marked increase in adherent leukocytes (baseline, 248+/-24 cells/mm2; 120 mins, 560+/-57 cells/mm2; p < .01). Leukocyte adherence in control animals (control group) did not increase significantly. Macromolecular leakage, expressed as the ratio of perivenular to intravenular fluorescence intensity after injection of fluorescence-labeled albumin, increased from 0.16+/-0.03 to 0.49+/-0.04 (p < .01 vs. baseline; p < .05 vs. control) during the infusion of endotoxin in the LPS group. Fucoidin application did not diminish the extravasation of albumin (baseline, 0.09+/-0.03; 120 mins, 0.61+/-0.10; p < .01 vs. baseline; p < .01 vs. control). CONCLUSIONS: These results demonstrate that despite a significant reduction of adherent leukocytes to the endothelium by fucoidin, there is no reduction in macromolecular leakage, indicating that leukocyte-endothelial interactions only play a minor role for the development of macromolecular leakage and microvascular damage in the early phase of endotoxemia.     

Effects of inhaled nitric oxide in a rat model of Pseudomonas aeruginosa pneumonia

OBJECTIVE: Antimicrobial effects of nitric oxide (NO) have been demonstrated in vitro against a variety of infectious pathogens, yet in vivo evidence of a potential therapeutic role for exogenous NO as an antimicrobial agent is limited. Thus, we assessed the effects of inhaled NO on pulmonary infection, leukocyte infiltration, and NO synthase (NOS) activity in a rat model of Pseudomonas aeruginosa pneumonia. DESIGN: Controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: After intratracheal instillation of either P. aeruginosa or saline (sham), rats were randomly exposed to either 40 ppm of inhaled NO or room air (RA) for 24 hrs before they were killed. MEASUREMENTS AND MAIN RESULTS: Inhaled NO in pneumonia rats markedly reduced pulmonary bacterial load (0.02+/-0.01% vs. 0.99+/-0.59% of bacterial input in pneumonia with room air, p < .05) and pulmonary myeloperoxidase activity, a marker of leukocyte infiltration (21.7+/-3.8 vs. 55.0+/-8.1 units in pneumonia with room air, p < .05), but had no effect on systemic hemodynamics or gas exchange. Pneumonia was associated with enhanced pulmonary NOS activity (8.8+/-2.4 vs. 0.2+/-0.1 pmol citrulline/min/mg protein in sham, p < .01) and increased plasma levels of nitrites/nitrates (NOx-; 45+/-7 vs. 16+/-3 micromol/L in sham, p < .01). Inhaled NO therapy attenuated the pneumonia-induced increase in pulmonary calcium-independent NOS activity (p < .05) and markedly increased plasma NOx- levels. Exposure of P. aeruginosa in culture to 40 ppm of ambient NO confirmed a delayed antibacterial effect of NO in vitro. CONCLUSIONS: Inhaled NO has an important antibacterial effect both in vitro and in vivo against P. aeruginosa and is associated with reduced pulmonary leukocyte infiltration in vivo. These results in a rat model of P. aeruginosa pneumonia suggest that future studies should address the possible clinical effects of inhaled NO therapy in pneumonia.     

Diminished L-arginine bioavailability in hypertension

L-Arginine is the precursor of NO (nitric oxide), a key endogenous mediator involved in endothelium-dependent vascular relaxation and platelet function. Although the concentration of intracellular L-arginine is well above the Km for NO synthesis, in many cells and pathological conditions the transport of L-arginine is essential for NO production (L-arginine paradox). The present study was designed to investigate the modulation of L-arginine/NO pathway in systemic arterial hypertension. Transport of L-arginine into RBCs (red blood cells) and platelets, NOS (NO synthase) activity and amino acid profiles in plasma were analysed in hypertensive patients and in an animal model of hypertension. Influx of L-arginine into RBCs was mediated by the cationic amino acid transport systems y+ and y+L, whereas, in platelets, influx was mediated only via system y+L. Chromatographic analyses revealed higher plasma levels of L-arginine in hypertensive patients (175+/-19 micromol/l) compared with control subjects (137+/-8 micromol/l). L-Arginine transport via system y+L, but not y+, was significantly reduced in RBCs from hypertensive patients (60+/-7 micromol.l(-1).cells(-1).h(-1); n=16) compared with controls (90+/-17 micromol.l(-1).cells(-1).h(-1); n=18). In human platelets, the Vmax for L-arginine transport via system y+L was 86+/-17 pmol.10(9) cells(-1).min(-1) in controls compared with 36+/-9 pmol.10(9) cells(-1).min(-1) in hypertensive patients (n=10; P<0.05). Basal NOS activity was decreased in platelets from hypertensive patients (0.12+/-0.02 pmol/10(8) cells; n=8) compared with controls (0.22+/-0.01 pmol/10(8) cells; n=8; P<0.05). Studies with spontaneously hypertensive rats demonstrated that transport of L-arginine via system y+L was also inhibited in RBCs. Our findings provide the first evidence that hypertension is associated with an inhibition of L-arginine transport via system y+L in both humans and animals, with reduced availability of L-arginine limiting NO synthesis in blood cells.     

Effects of nitric oxide synthase inhibition on microvascular reactivity in septic mice.

Persistent vasodilation refractory to vasopressor agents is characteristic of septic shock. Induction of nitric oxide synthase (NOS) by sepsis-induced cytokines within the vasculature is one of the primary mediators of this refractory vasodilation. To evaluate the mechanism of vasodilation in sepsis, we used in vivo videomicroscopy to measure microvascular vasoconstrictive responses to topical suffusion of norepinephrine in mice made septic by cecal ligation and puncture, and contrasted the effects of topical superfusion of the nonselective NOS inhibitor N(G)-methyl-L-arginine (L-NMMA) and the selective inducible NOS (iNOS) inhibitor S-methyl-isothiourea (SMT). Mice with sepsis were less sensitive to the vasoconstrictive effects of norepinephrine than controls (EC50, the concentration that produces half-maximal response 2.0+/-0.6 x 10(-6) M vs. 7.9+/-2.2 x 10(-8) M, P=0.01). Selective inhibition of inducible iNOS with topical SMT (100 microM) markedly increased catecholamine reactivity in mice with sepsis but did not affect reactivity in controls (P=0.0007 for sepsis, P=0.24 for controls). Nonselective NOS inhibition with topical L-NMMA produced a similar increase in catecholamine reactivity in mice with sepsis but not controls (P=0.001 for sepsis, P=0.56 for controls). When excess (1 mM) L-arginine, the substrate for NOS, was added to the superfusion buffer along with both SMT and L-NMMA, arteriolar responsiveness to norepinephrine was decreased to the original values. These experiments demonstrate that iNOS inhibition is as effective as nonselective NOS inhibition in reversing decreased catecholamine reactivity in sepsis. This suggests a crucial role for microvascular activation of iNOS in the pathophysiology of hypotension and decreased vasopressor responsiveness in sepsis.     

L-arginine and endothelin receptor antagonist bosentan counteract hemodynamic effects of modified hemoglobin

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a nitric oxide scavenger, causes systemic and pulmonary vasoconstriction in normal and septic sheep. We studied the effect of L-arginine and the endothelin-1 (ET-1) antagonist bosentan on the PHP response to determine whether the PHP-induced vasoconstriction resulted predominantly from the action of ET-1 or solely from removal of NO. After 24 h of carrier solution (nonseptic sheep), sheep received PHP (20 mg/kg/h; n = 5), PHP plus L-arginine (at 28 h, 100 mg/kg bolus and 500 mg/kg for 1 h) plus bosentan (at 32 h, 10 mg/kg; n = 6), and only L-arginine and bosentan (n = 5). These protocols were repeated after 24 h of Pseudomonas aeruginosa (S, 6x10(6) colony-forming units/kg/h). PHP induced vasoconstriction in septic and nonseptic sheep for the duration of its infusion. In nonseptic sheep, neither L-arginine nor bosentan significantly lowered systemic (SVRI) and pulmonary (PVRI) vascular resistance and did not antagonize the PHP-induced vasoconstriction. During sepsis, SVRI fell and cardiac index (CI) rose. L-arginine and bosentan further decreased SVRI (L-arginine: 34+/-2%*, p<.05; bosentan: 35+/-5%*, p<.05) and PVRI (L-arginine: 28+/-2%*, p<.05; bosentan: 33+/-7%*, p<.05) and increased CI (L-arginine: 29+/-4%*, p<.05; bosentan: 11+/-5%, NS). Both agents antagonized the PHP-induced vasoconstriction lowering SVRI (L-arginine: 29+/-3%*, p<.05; bosentan: 26+/-5%*, p<.05) and PVRI (L-arginine: 27+/-4%*, p<.05; bosentan: 32+/-4%*, p<.05) to levels before PHP administration. Plasma ET-1 levels increased during sepsis (from 9.8+/-.2 to 15.6+/-.7* pg/mL, p<.05) and fell during PHP infusion (to 9.7+/-1.6* pg/mL, p<.05). In nonseptic sheep, ET-1 levels decreased during PHP (from 8.5+/-.6 pg/mL to 5.9+/-.6*, p<.05). Bosentan increased ET-1 levels 2.7 times higher in septic than in nonseptic sheep. We conclude that during sepsis, the NO scavenger PHP unmasks an underlying ET-1 mediated vasoconstriction, and its effect is antagonized by L-arginine and bosentan.     

Endocan, a new endothelial marker in human sepsis

OBJECTIVES: a) To evaluate in septic patients the blood levels of endocan, a circulating proteoglycan, which regulates leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interactions in vitro; b) to determine whether endocan could be used as a diagnostic and prognostic marker in sepsis in the intensive care unit; and c) to study kinetics of endocan secretion by endothelial cells in vitro after stimulation by soluble mediators involved in sepsis. DESIGN: Prospective observational study. SETTING: Intensive care unit of the University Hospitals of Lille, France, and Geneva, Switzerland. PATIENTS: All patients admitted to the intensive care unit over a 6-month period with clinical evidence of severe sepsis or septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In vitro, we showed a sustained endocan secretion by endothelial cells after stimulation by lipopolysaccharide and tumor necrosis factor-alpha. Circulating levels of endocan measured in 63 patients admitted to the intensive care unit with sepsis were significantly elevated compared with 20 healthy donors and seven patients with systemic inflammatory response syndrome: 2.71 +/- 4.88 ng/mL vs. 0.77 +/- 0.44 ng/mL vs. 0.68 +/- 1.03 ng/mL (median +/- interquartile range, p < .001). Endocan levels were higher in patients with septic shock (6.11 +/- 12.99 ng/mL, n = 22) than in patients with severe sepsis (1.97 +/- 7.8 ng/mL, n = 12) or sepsis (1.95 +/- 1.63 ng/mL, n = 29). Measurement of endocan at intensive care unit admission revealed higher levels in nonsurvivors (n = 12) than in patients still alive 10 days later (n = 51, 6.98 +/- 13.8 vs. 2.45 +/- 4.09, p < .01). CONCLUSIONS: These results suggest that in septic patients, endocan blood level is related to the severity of illness and the outcome of the patient and may represent a novel endothelial cell dysfunction marker.     

Reduction in intestinal leukocyte adherence in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G

OBJECTIVES: To investigate leukocyte adherence in intestinal venules in experimental endotoxemia after treatment with the 21-aminosteroid U-74389G. DESIGN AND SETTING: Prospective, randomized, controlled animal study in an experimental laboratory. SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g. INTERVENTIONS: The rats were divided equally into three groups: (a) control group, (b) endotoxemia (5 mg/kg lipopolysacharide from Escherichia coli O55:B5), and (c) endotoxemia and U-74389G administration 30 min before (3 mg/kg) and 60 min after endotoxin challenge (1.5 mg/ kg). MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined using intravital fluorescence videomicroscopy 2 h after endotoxin challenge. Leukocytes were stained in vivo by means of rhodamine 6G. In the endotoxemic animals we observed a fourfold increase in the count of firmly adherent leukocytes in submucosal post-capillary and collecting venules. Treatment with the 21-aminosteroid U-74389G significantly attenuated the count of sticking leukocytes in the collecting venules (control, 61 +/- 10 cells/mm2; lipopolysaccharide, 237 +/- 42 cells/mm2; U-74389G 125 +/- 9 cells/mm2; p < 0.05). In these venules leukocyte rolling behavior was comparable to that in the control group without endotoxin challenge. CONCLUSIONS: Administration of U-74389G, which has radical scavenging properties, attenuates leukocyte adherence in selected populations of intestinal venules which is found increased during endotoxemia. Thus, 21-aminosteroids may have an impact in the treatment of endotoxin-induced intestinal injury.     

Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion.

Leukocyte rolling has been postulated to be mandatory for subsequent leukocyte adhesion and tissue injury observed during ischemia/reperfusion. The objective of this study was to systematically assess this hypothesis at the microvascular level by examining the effects of various concentrations of a selectin-binding carbohydrate (fucoidin) on the increased rolling and adhesion of leukocytes in postischemic venules. The contribution of L-selectin and/or P-selectin to leukocyte rolling were also assessed in this model. Using intravital microscopy we observed that 60 min of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. A high dose of fucoidin (25 mg/kg) reduced leukocyte rolling by > 90% and significantly reduced leukocyte adhesion, whereas a lower dose of fucoidin still reduced leukocyte rolling by 60% but had no effect on leukocyte adhesion. Moreover, despite the profound reduction in leukocyte rolling with fucoidin, the remaining rolling cells were able to firmly adhere via a CD18-dependent mechanism, particularly in those postcapillary venules with reduced (30-50%) shear rates. The increased rolling was also reduced 60% by either an anti-P-selectin antibody, an anti-L-selectin antibody, or a combination of the two antibodies, but this reduction in rolling cells did not translate into significantly reduced leukocyte adhesion. Our data suggest that L-selectin, P-selectin, and a fucoidin-sensitive pathway contribute to the significant increase in reperfusion-induced leukocyte rolling. However, targeting leukocyte rolling as a form of therapy requires very significant efficacy (> 90%) to achieve reasonable (approximately 50%) attenuation in leukocyte adhesion in postischemic venules.     

Calpain inhibitors improve myocardial dysfunction and inflammation induced by endotoxin in rats

Excessive activation of calpains has been implicated in the pathophysiology of inflammation, trauma, and ischemia reperfusion injury. Here, we investigated the effects of calpain inhibition on myocardial dysfunction and inflammation induced by endotoxin in rats. Rats were treated i.v. with endotoxin (10 mg/kg) or endotoxin plus calpain inhibitors and were then prepared after 4 h for myocardial contractility assessment, detection of endothelium leukocyte interactions, and plasma TNF-alpha, nitrite/nitrate, and endocan levels. Compared with vehicle-treated rats, hearts from endotoxin-treated rats had reduced systolic performance that was partially prevented by calpain inhibitors, i.e., acetyl-leucyl-leucyl-arginal (leupeptin), carbobenzoxy-valyl-phenylalanial (calpain inhibitor III), and N-acetyl-leucinyl-leucinyl-norleucinal (ALLN). Leupeptin and calpain inhibitor III reduced plasma TNF-alpha levels in endotoxin-treated rats. ALLN reduced plasma TNF-alpha and nitrite/nitrate levels in endotoxin-treated rats. Endotoxin treatment increased mesenteric venule leukocyte rolling (10 +/- 3 leukocytes/min vs. 44 +/- 10 leukocytes/min; P < 0.01) and adhesion (2 +/- 2 leukocytes/min vs. 15 +/- 3 leukocytes/min; P < 0.01), which was reduced by calpain inhibitors. Attenuation of leukocyte endothelium interactions observed in calpain inhibitor-treated rats with sepsis was associated with increases in plasma anti-adhesion molecule endocan. In conclusion, calpain inhibitors improved endotoxin-induced cardiac dysfunction, which may be attributed to the modulation of endothelium leukocyte interactions in the inflamed vasculature.     

Fluids reverse the early lipopolysaccharide-induced albumin leakage in rodent mesenteric venules

Objective Volume resuscitation is clinically beneficial in patients with sepsis, but few data exist concerning the effects of fluid administration on early events in the inflammatory process. Vascular permeability, leukocyte rolling and leukocyte adhesion in the rodent mesenteric microcirculation were assessed in vivo using intravital microscopy, and the effect of fluid administration on lipopolysaccharide (LPS)-induced changes recorded.Design Prospective, repeated measures study.Setting University hospital laboratory.Subjects Male Wistar rats in six groups.Interventions All animals underwent intravital microscopic examination of mesenteric post-capillary venules. LPS or vehicle was applied topically. Animals received either no additional fluids, 0.9% saline (16 ml/kg per h) or 5% human albumin (16 ml/kg per h) commencing 30 min prior to LPS/vehicle administration.Measurements and main results Leukocyte rolling, firm adhesion and blood velocity were observed directly. Vascular permeability was assessed using the flux of fluorescently labelled albumin into the interstitium. LPS significantly increased the median (IQR) number of leukocytes rolling and firmly adherent relative to baseline (at 60 min rolling increased from 12.0 (10.3–13.8) to 40.3 (36.0–47.5) cells/min; adhesion increased from 1 (1–2) to 17 (12–26) cells/100 m; n=5, p<0.01). Transvascular albumin flux was significantly increased 45 min after LPS application (p<0.01), but not after vehicle. Administration of either 0.9% saline (n=5) or 5% human albumin (n=6), significantly attenuated LPS-induced increases in albumin flux (p<0.05), leukocyte rolling (p<0.01) and adhesion (p<0.01). Fluid administration did not appear to alter shear rates.Conclusions Pre-emptive volume administration with either saline or albumin prevented early LPS-induced microcirculatory changes by an undefined effect that is unrelated to changes in microvascular flow.Drs. Anning and Finney are supported by grants from the British Heart Foundation.     

Transit time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo.

Leukocyte recruitment requires leukocyte rolling, activation, firm adhesion, and transmigration. Injection of the proinflammatory cytokine TNF-alpha induces expression of E-selectin, interleukin-8, and other adhesion molecules and chemoattractants on the endothelial surface. TNF-alpha- treated CD18 null mouse cremaster muscle venules show increased leukocyte rolling velocity and reduced leukocyte recruitment efficiency. Leukocyte recruitment in CD18 null but not wild-type mice is significantly blocked by an mAb to E-selectin. To understand this overlap between adhesion events previously considered separate, we introduce a quantitative analysis of the efficiency of induction of rolling, conversion of rolling to adhesion, and of adhesion to transmigration. We find that CD18 and E-selectin cooperate to control the time a leukocyte needs to roll through an inflamed area and to convert rolling to firm adhesion. Leukocyte rolling time, defined as the time it takes for a rolling leukocyte to pass through a defined length of a vessel segment, emerges as a unifying parameter determining the efficiency of inducing firm adhesion, which is a rate-limiting step controlling leukocyte recruitment in inflammation. We conclude that leukocytes integrate chemoattractant signals while rolling along the endothelial surface until they reach a critical level of activation and become firmly adherent.     

Antithrombin reduces mesenteric venular leukocyte interactions and small intestine injury in endotoxemic rats

We examined the hypothesis that recombinant human antithrombin would reduce mesenteric venule leukocyte adhesion and small intestine injury in endotoxemic rats. Endotoxemic (endotoxin 10 mg/kg, intravenously) rats were treated either with saline or recombinant human antithrombin (250 and 500 U/kg). In some rats, indomethacin (100 mg/kg, intraperitoneally) was injected 60 min prior to endotoxin and recominant human antithrombin (500 U/kg) treatment. Compared to controls, intravital videomicroscopy of the mesentric venule showed an increase of leukocyte rolling (55+/-17 versus 70+/-19 leukocytes/min; P < 0.05) and firm adhesion (1.1+/-0.3 versus 5.8+/-0.8 leukocytes/100 microm; P < 0.05) in endotoxemic rats. Recombinant human antithrombin attenuated endotoxin-induced venular endothelium leukocyte adhesive cascade. The beneficial effects of recombinant human antithrombin on leukocyte adhesion were inhibited by indomethacin (100 mg/kg, intraperitoneally) in endotoxemic rats. Endotoxin treatment increased fluorescein isothiocyanate (FITC)-labeled dextran 4,000 (FD4) gut lumen to plasma ratio and wet weight/dry weight ratio. Recombinant human antithrombin (500 U/kg) attenuated endotoxin-induced gut injury. These observations suggest that recombinant human antithrombin reduces endothelium-leukocyte interactions in endotoxemic rats by interacting with local prostacyclin production.     

Presence of nitrotyrosine with minimal inducible nitric oxide synthase induction in lipopolysaccharide-treated pigs

The production of large amounts of nitric oxide (NO) by the inducible form of nitric oxide synthase (iNOS) and the subsequent production of peroxynitrite (OONO-) are believed to be major factors in the hemodynamic abnormalities of sepsis. This finding is based on data from rats and mice but has not been established in other species. Therefore, we examined the role of iNOS in lipopolysaccharide (LPS)-treated pigs, which have a hemodynamic pattern with sepsis that is more similar to humans than rats. Pigs were anesthetized, ventilated, and given LPS (n = 12), 20 microg/kg over 2 h, or saline (n = 7). They were killed after 2 (n = 8 LPS, 7 control) or 4 h (4 LPS). We measured cardiac output (CO), mean arterial (Part), and pulmonary and central venous pressures. We evaluated NO production by measuring expired NO, and plasma nitrate/nitrite concentration, NOS activity (in lung tissue), and iNOS protein by Western analysis, and immunohistochemistry (lung and liver), as well as iNOS mRNA by Northern analysis (liver and lung). We also measured nitrotyrosine as evidence of OONO- production by slot blot, Western analysis, and immunohistochemistry. By 2 h, Part fell and CO did not change so that systemic vascular resistance decreased from 21.5+/-2.9 to 12.7+/-3.1 mmHg x L(-1) x min (P < 0.05) and remained at 11.3+/-1.7 mmHg x L(-1) x min in the animals observed for 4 h. Plasma nitrate/nitrite, expired NO, and NOS activity did not change. We found no iNOS in tissues by Western analysis with 5 different antibodies but detected a small amount of iNOS by immunohistochemistry in inflammatory cells and small vessels. There was a small increase in iNOS mRNA in liver and lung. Despite the minimal increase in iNOS, nitrotyrosine was increased in small vessels and in inflammatory cells. In conclusion, caution should be used when extrapolating the septic response in rodents to other species, for the pattern of iNOS induction is very different.     

Microvascular response in patients with cardiogenic shock

OBJECTIVE: To examine the mechanisms contributing to decreased microvascular blood flow in cardiogenic shock by comparing patients with cardiogenic shock with critically ill controls and with patients with septic shock. DESIGN: Prospective, consecutive entry of patients meeting the criteria for septic shock, cardiogenic shock, and critical illness without coexisting infection or shock. SETTING: University hospital, medical intensive care unit, coronary care unit, and respiratory care unit. PATIENTS: Eight patients with cardiogenic shock secondary to acute myocardial infarction, six critically ill controls, and six patients with septic shock. MEASUREMENTS AND MAIN RESULTS: Forearm blood flow was measured at rest and during reactive hyperemia by venous air plethysmography. Red cell deformability was determined by filtration. Leukocyte aggregation was detected by the leukergy test. Neutrophil CD11b/CD18 expression and soluble intercellular adhesion molecule-1 levels were also measured. In cardiogenic shock, forearm arterial resistance was significantly increased at rest and during reactive hyperemia compared with controls and patients with septic shock. The response to reactive hyperemia was attenuated in cardiogenic and septic shock patients, as measured by the absolute change in forearm blood flow from baseline, which was significantly less as compared with controls (p < .01). The percent change in forearm blood flow during reactive hyperemia compared with forearm blood flow at rest was significantly lower in cardiogenic shock (60+/-10) and in septic shock (50+/-11) compared with controls at baseline (145+/-20; p < .01). Red cell deformability was significantly decreased in cardiogenic shock (1.2+/-0.2 mL/min; p < .05) and septic shock (1.1+/-0.2 mL/min; p < .05), compared with controls (1.8+/-0.1 mL/min). Neutrophil CD11b/CD18 expression, leukergy, and serum intercellular adhesion molecule-1 levels in cardiogenic shock patients were not significantly different from controls. CONCLUSION: These data suggest that the response to reactive hyperemia is attenuated in cardiogenic shock. This appears to reflect increased vasoconstriction and an impaired capacity for vasodilation. Decreased erythrocyte deformability may also be important in limiting systemic microvascular flow. However, evidence supporting a role for neutrophil-endothelial cell interactions was not observed.     

Absence of trauma-induced leukocyte rolling in mice deficient in both P- selectin and intercellular adhesion molecule 1

Leukocyte recruitment during inflammation is achieved through a multistep paradigm that includes margination, selectin-mediated rolling, beta 2 integrin-mediated firm adhesion, emigration, and migration into the site of inflammation. We have used the mouse cremaster muscle as a model of trauma- and cytokine-induced inflammation to study the possible role of intercellular adhesion molecule (ICAM) 1 in leukocyte rolling using gene-targeted mice deficient in ICAM-1, P-selectin, and a combination of P-selectin and ICAM-1. Rolling flux and average leukocyte rolling velocity in ICAM-1- deficient mice was not different from wild-type mice, but P- selectin/ICAM-1-deficient mice showed a total absence of rolling for at least 2 h after surgical trauma. Rolling in both wild-type and ICAM-1- deficient mice 60-120 min after trauma was significantly inhibited by a P-selectin monoclonal antibody (mAb) (RB40.34). In contrast, an mAb (KAT-1) blocking ICAM-1 binding to leukocyte function-associated antigen 1 did not block residual rolling in P-selectin-deficient mice. TNF-alpha induced leukocyte rolling in P-selectin/ICAM-1-deficient mice, but the rolling flux fraction was significantly lower than in TNF- alpha-treated ICAM-1-deficient mice. Leukocyte rolling in P- selectin/ICAM-1-deficient mice treated with TNF-alpha for 3 h was completely blocked by an E-selectin mAb (9A9E3), and partially by an L- selectin mAb (MEL-14). This clearly demonstrates E-selectin-dependent rolling in vivo. Leukocyte rolling velocities were significantly reduced after TNF-alpha treatment and were similar in wild-type and gene-targeted strains. We conclude that the residual trauma-induced leukocyte rolling seen in P-selectin-deficient mice is completely abolished by concomitant ICAM-1 deficiency. This severe defect in leukocyte rolling may explain the absence of leukocyte recruitment into the inflamed peritoneal cavity of P-selectin/ICAM-1-deficient mice at early time points (< or = 4 h).     

Rosiglitazone protects against ischemia/reperfusion-induced leukocyte adhesion in the zucker diabetic fatty rat

Increased susceptibility to atherosclerosis increases the risk of mortality in type 2 diabetic patients. Leukocyte adhesion to the endothelium is a critical step in atherogenesis. In addition to its insulin-sensitizing effects, rosiglitazone (RSG) possesses anti-inflammatory properties. However, the effects of RSG on the initial phase of leukocyte recruitment (rolling, adhesion) have not been studied in vivo. This study tested the hypothesis that RSG treatment of Zucker diabetic fatty (ZDF) rats inhibits ischemia/reperfusion-induced leukocyte adhesion to the endothelium. Male ZDF rats (16 weeks) were treated with RSG (3 mg/kg/day, p.o.) 7 days before experimentation. Leukocyte-endothelial interactions in cremaster venules were recorded using intravital microscopy prior to 30 min of ischemia and during a 90-min reperfusion period. Although blood pressure, plasma glucose, and insulin were not different between treatment groups, RSG treatment was associated with reduced leukocyte rolling and inhibition of leukocyte adhesion throughout the reperfusion period (P < 0.01). Cremaster mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) was reduced by 35% in RSG-treated animals (P < 0.01), whereas P- and E-selectin and intercellular adhesion molecule-1 (ICAM-1) were unchanged. Immunostaining for P-selectin, E-selectin, and VCAM-1 was reduced by 21, 61, and 50%, respectively (for all, P < 0.05), in RSG-treated animals. Inhibition of ischemia/reperfusion-induced leukocyte adhesion might contribute to the utility of RSG as a therapy for vascular disease.     

Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion.

We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 microg/ml), or hyperglycemic (174-316 mg/dl) sera from patients with diabetes and abnormal hemoglobin A1c (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dyn/cm2). Rolling and adherent cells were evaluated by digital image processing. Results showed that 30 mM glucose significantly (P < 0. 01) increased the number of adherent leukocytes to endothelial cells in respect to control (5 mM glucose; 151+/-19 versus 33+/-8 cells/mm2). A similar response was induced by endothelial stimulation with IL-1beta, here used as positive control (195+/-20 cells/mm2). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1beta-treated endothelial cells, was significantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 microm). Functional blocking of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion (67+/-16, 83+/-12, 62+/-8 versus 144+/-21 cells/ mm2). Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation. Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate (100 microM) and tosyl-phe-chloromethylketone (25 microM) significantly reduced (P < 0.05) leukocyte adhesion in respect to HUVEC treated with glucose alone. A significant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine (5 nM). When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte adhesion as compared with controls, despite normal levels of IL-1beta and TNFalpha in these sera. These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins, possibly depending on NF-kB activation.