Differential expression of inducible nitric oxide synthase in septic shock

During cardiopulmonary bypass (CPB), the septic patient has markedly decreased peripheral vascular resistance as a consequence of endotoxin release from microorganisms. This decrease in peripheral vascular resistance is the result of endotoxin-induced nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). iNOS and eNOS are responsible for the synthesis of NO because of various stimuli, including the bacterial endotoxin, lipopolysaccharide (LPS). We tested the hypothesis that a differential expression of iNOS among human endothelial cells and murine macrophage is dependent upon exposure to endotoxin and various pro-inflammatory cytokines. Using a human endothelial cell line, ECV-304 and murine macrophage cell line, RAW 264.7, we quantified the expression of iNOS with specific FITC-conjugated antibodies using fluorescence activated cell sorter (FACS) and NO production with a Bioxytech nitric oxide spectrophotometric assay. This in vitro septic model utilized LPS supported with species-specific interferon-gamma, interleukin-1 beta, and tumor necrosis factor-alpha. The cell type were stimulated for 8 hours with combinations of the cytokines mentioned. The FACS data demonstrated a significant stimulus-dependent increase in iNOS expression among the macrophage groups; however, the stimulated endothelial cells showed no significant change in iNOS expression. The nitric oxide production data demonstrated significant increases in NO production among macrophage stimulated groups; whereas, endothelial stimulated groups exhibit no significant change. We conclude that NO secreted during septic shock is the result of activated macrophage, not the endothelium. The clinical relevance is that the more severe the infectious process, the lower the PVR may be during CPB because of increased NO production from activated macrophage.     

Fructose induces the inflammatory molecule ICAM-1 in endothelial cells

Epidemiologic studies have linked fructose intake with the metabolic syndrome, and it was recently reported that fructose induces an inflammatory response in the rat kidney. Here, we examined whether fructose directly stimulates endothelial inflammatory processes by upregulating the inflammatory molecule intercellular adhesion molecule-1 (ICAM-1). When human aortic endothelial cells were stimulated with physiologic concentrations of fructose, ICAM-1 mRNA and protein expression increased in a time- and dosage-dependent manner, which was independent of NF-kappaB activation. Fructose reduced endothelial nitric oxide (NO) levels and caused a transient reduction in endothelial NO synthase expression. The administration of an NO donor inhibited fructose-induced ICAM-1 expression, whereas blocking NO synthase enhanced it, suggesting that NO inhibits endothelial ICAM-1 expression. Furthermore, fructose resulted in decreased intracellular ATP; administration of exogenous ATP blocked fructose-induced ICAM-1 expression and increased NO levels. Consistent with the in vitro studies, dietary intake of fructose at physiologic dosages increased both serum ICAM-1 concentration and endothelial ICAM-1 expression in the rat kidney. These data suggest that fructose induces inflammatory changes in vascular cells at physiologic concentrations.     

Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1

BACKGROUND: Splanchnic hypoperfusion is believed to be central in the pathogenesis of hemorrhagic shock-induced acute respiratory distress syndrome and multiple organ failure. Our previous work focused on the portal circulation as the conduit for gut-derived mediators of acute respiratory distress syndrome. Our current focus is the proinflammatory effects of postshock mesenteric lymph. We hypothesize that postshock lymph induces neutrophil (PMN)-mediated endothelial cell damage in an intercellular adhesion molecule-1 (ICAM-1)-dependent fashion, and devised a two-insult model to test this hypothesis. METHODS: Rats (n > or = 5) underwent hemorrhagic shock (mean arterial pressure, 40 mm Hg for 30 minutes) and resuscitation (shed blood plus two times crystalloid) with lymph collection. Human pulmonary microvascular endothelial cells (HMVECs) were divided into three groups and grown to near confluence. Group 1 was incubated for 6 hours in 1% preshock or postshock lymph and ICAM-1 was measured by flow cytometry. Group 2 consisted of coculture of HMVECs and PMNs after endothelial cell activation to determine whether postshock lymph would stimulate PMN adherence. Group 3 was incubated under identical conditions, but PMNs were added for 30 minutes, and then activated with 4.5 micromol/L lysophosphatidylcholine (lyso-PC) for 1 hour to ascertain cytotoxicity. HMVEC density was measured using microscopy and recorded as HMVECs per millimeter squared. ICAM-1-blocking antibody and isotype control were used to assess the effects of ICAM-1 on PMN cytotoxicity. A buffer control was used for comparison using analysis of variance with Tukey's correction. RESULTS: Postshock lymph activated HMVECs for increased surface expression of ICAM-1 and stimulated PMNs to adhere to endothelial cell monolayers. Activation of PMNs with lyso-PC in the presence of postshock lymph resulted in marked HMVEC death. The addition of an ICAM-1-blocking antibody abrogated this effect. Neither postshock lymph alone (758 +/- 35 HMVECs/mm(2)), nor postshock lymph in the presence of quiescent PMNs alone (734 +/- 28 HMVECs/mm(2)), nor lymph plus lyso-PC (834 +/- 21 HMVECs/mm(2)) provoked endothelial cell damage. CONCLUSION: Postshock mesenteric lymph activates endothelial cells for increased ICAM-1 expression and PMN adherence. Furthermore, postshock lymph acts as an inciting event in a two-event in vitro model of PMN-mediated endothelial cell injury. These findings further substantiate the key mechanistic role of mesenteric lymph in hemorrhagic shock-induced acute lung injury and suggest that ICAM-1 expression is pivotal in the two-event model of multiple organ failure.     

Upregulated expression of inducible nitric oxide synthase plays a key role in early apoptosis after anterior cruciate ligament injury.

The reason that the anterior cruciate ligament (ACL) has a very poor healing potential after injury is not well understood. In this study, we investigated the role of nitric oxide (NO) in the apoptotic cell death of ACL cells using a rabbit model and in vitro cell culture. The apoptosis of ACL cells in vivo was analyzed by TUNEL assay and electron microscopy. NO synthase (NOS) expression was observed by immunohistochemical analysis. ACL cells were cultured and the susceptibility to NO-induced apoptosis was tested. Inducible NOS (iNOS) expression after treatment with cytokines was examined by immunohistochemical and RT-PCR analyses. Mitogen-activated protein kinase (MAPK) inhibitors were used for the analysis of downstream signals. A significant number of apoptotic cells were observed on days 1 to 3 after injury; the apoptotic rate returned to the control level by day 7. Upregulation of iNOS in the ACL remnant was observed at day 1. Intraarticular injection of NOS inhibitor suppressed the apoptotic rate. Isolated ACL cells showed much higher susceptibility to NO-induced apoptosis than did medial collateral ligament cells. IL-1beta stimulated ACL cells to upregulate iNOS mRNA and increase NO production. p38 MAPK inhibitor decreased NO-induced apoptosis. Rapid iNOS induction after injury contributes to the high apoptotic rate of ACL cells, and this may partly account for the poor healing capacity of this ligament. iNOS and NO production is suggested to be stimulated by IL-1beta, and NO activates the p38 MAPK pathway and triggers an apoptotic signal in ACL cells.     

Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver.

BACKGROUND: Although inhibition of endothelial nitric oxide synthase (eNOS) has been reported to aggravate hepatic ischemia-reperfusion (I/R) injury, the role of inducible nitric oxide synthase (iNOS) has been still unknown. We investigated the role of NO produced by iNOS, and evaluated the effect of an iNOS inhibitor on prolonged warm I/R injury in the pig liver. METHODS: Pigs were subjected to 120 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in serum and hepatic microdialysate NO2- + NO3- (NOx) and the cellular distribution of eNOS and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of iNOS inhibitor was also investigated. RESULTS: Hepatic I/R induced new nitric oxide production in serum and hepatic microdialysate NOx after reperfusion and severe hepatic damage in the centrilobular region where nitrotyrosine was strongly expressed. Diffuse eNOS expression in sinusoidal endothelium did not differ before and after reperfusion. In contrast, strong iNOS expression in Kupffer cells and neutrophils appeared strongly in the centrilobular region after reperfusion. Pigs with intraportal administration of N(G)-nitro-L-arginine (10 mg/kg) died during the period of ischemia or early in the period of reperfusion with a high mortality rate (80.0%). Intraportal administration of aminoguanidine hemisulfate (10 mg/kg) significantly suppressed nitric oxide production and serum aspartate aminotransferase after reperfusion, inhibited nitrotyrosine expression, and attenuated hepatic damage. CONCLUSIONS: These results indicate that hepatic I/R injury is triggered by centrilobular iNOS expression; and attenuated by inhibition of iNOS.     

Hyperbaric oxygen attenuation of lipopolysaccharide-induced acute lung injury involves heme oxygenase-1

BACKGROUND: Hyperbaric oxygen (HBO) attenuates lipopolysaccharide (LPS)-induced acute lung injury. This beneficial effect of HBO involves inhibition of inducible nitric oxide synthase (iNOS) expression and subsequent nitric oxide (NO) biosynthesis. We sought to investigate the role of heme oxygenase-1 (HO-1) on this HBO inhibition of iNOS induction and acute lung injury in septic rat lungs. METHODS: Before the experiment, 72 rats were randomly allocated to receive HBO or air treatment. With or without HBO pre-treatment, the rats were further divided into the following subgroups (n = 6): (i) LPS injection, (ii) normal saline (N/S) injection, (iii) hemin (a HO-1 inducer) plus LPS, (iv) hemin alone, (v) tin protoporphyrin (SnPP; a HO-1 inhibitor) plus LPS, and (vi) SnPP alone. All rats were maintained for 6 h and then sacrificed with a high-dose pentobarbital injection. Lung injuries and relevant enzymes expression were thus assayed. RESULTS: Histological analysis, PMNs/alveoli ratio, and wet/dry weight ratio measurements demonstrated that LPS caused significant lung injury and HBO and/or hemin significantly attenuated this LPS-induced lung injury. Increased pulmonary iNOS expression and NO production were associated with lung injury. Induction of HO-1, by HBO and/or hemin, significantly attenuated this LPS-induced iNOS expression and acute lung injury. SnPP, on the contrary, offset the effects of HBO and worsened the LPS-induced lung injury. CONCLUSIONS: HBO may act through inhibiting pulmonary iNOS expression to attenuate LPS-induced acute lung injury in septic rats. Furthermore, this HBO attenuation of iNOS expression involves HO-1 induction.     

NO donor ameliorates ischemia-reperfusion injury of the rat liver with iNOS attenuation.

This study investigated the effect of a spontaneous nitric oxide (NO) donor, FK409 (FK), in a rat model of segmental hepatic ischemia. Rats were allocated to four experimental groups. Two of the groups underwent segmental hepatic ischemia of 60 min duration and received FK (0.4 mg/kg, iv) or vehicle alone before inducing ischemia and again 5 min before reperfusion. Sham-FK and sham groups were treated identically, but did not have vascular occlusion. Serum aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) were measured, and the livers were examined for histological evidence of injury, polymorphonuclear neutrophil (PMN) infiltration, and immunohistochemical expression of inducible NO synthase (iNOS) before and 6 h after reperfusion. AST, ALT, and LDH levels were significantly (p < .05) reduced 6 h after reperfusion in the FK-treated group compared with the vehicle-treated control group. FK treatment also reduced the degree of hepatic damage apparent on histopathology and reduced PMN infiltration and iNOS expression. Thus, FK treatment is protective against hepatic ischemia reperfusion injury and attenuates neutrophil infiltration and iNOS expression.     

Role of CO-releasing molecules liberated CO in attenuating leukocytes sequestration and inflammatory responses in the lung of thermally injured mice

BACKGROUND: Acute lung injury and pulmonary inflammatory responses are important complications most frequently encountered in severely burned patients. Polymorphonuclear leukocyte (PMN) sequestration and the subsequent generation of oxidants and inflammatory mediators play the key roles in the pathogenesis of acute lung injury. In this study, we used CO-releasing molecules (CORM-2) to determine whether the CO-releasing molecules-liberated CO could attenuate leukocytes sequestration and the inflammatory response in the lung of thermally injured mice. MATERIALS AND METHODS: Fifty-four mice were assigned to three groups in three respective experiments. In each experiment, mice in sham group (n=6) underwent sham thermal injury, whereas mice in the burn group (n=6) received 15% total body surface area (TBSA) full-thickness thermal injury and mice in CORM-2 group (n=6) underwent the same thermal injury with immediate administration of CORM-2 (8 mg/kg, i.v.). PMN accumulation (MPO assay) in mice lungs and tumor necrosis factor-alpha and interleukin-1beta in BAL fluid, pulmonary edema formation, and wet/dry weight ratios of lung were determined. Activation of NF-kappaB and expression level of ICAM-1 in the lung was assessed. In in vitro experiment, PMN adhesion to experimental mice serum-stimulated mouse lung endothelial cells (MLEC) was assessed. RESULTS: Treatment of thermally injured mice with CORM-2 attenuated PMN accumulation and prevented activation of NF-kappaB in the lung. This was accompanied by a decrease of the expression of ICAM-1. In parallel, PMN adhesion to MLEC stimulated by CORM-2-treated thermally injured mice serum was markedly decreased. Also, CORM-2 markedly decreased the production of inflammatory mediators in BAL fluid without suppressing the permeability of pulmonary microcirculation. CONCLUSIONS: CORM-released CO attenuates the inflammatory response in the lung of thermally injured mice by decreasing leukocyte sequestration and interfering with NF-kappaB activation, protein expression of ICAM-1, and therefore, suppressing endothelial cells' pro-adhesive phenotype.     

High glucose increases inducible NO production in cultured rat mesangial cells. Possible role in fibronectin production.

BACKGROUND/AIM: Increased nitric oxide (NO) generation and action have been suggested to be associated with glomerular hyperfiltration and increased vascular permeability early in diabetes. However, previous studies have primarily focused on the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells, and the role of the inducible NOS (iNOS) pathway in diabetic nephropathy has remained unclear. This study examined whether high glucose modulates NO synthesis by the iNOS pathway in rat mesangial cells. In addition, the effect of inhibition of the iNOS pathway on fibronectin production was determined to examine the role of the iNOS pathway in high glucose-induced extracellular expansion by mesangial cells. METHODS: NO synthesis by the iNOS pathway was evaluated by nitrite and iNOS mRNA and protein productions. The effects of protein kinase C (PKC) inhibitor and aldose reductase inhibitor on the iNOS mRNA expression and aminoguanidine, a relatively specific inhibitor of the iNOS on fibronectin protein production were examined. RESULTS: High 30 mM glucose concentration led to significant increases in nitrite production of rat mesangial cells upon stimulation with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) compared with control 5.6 mM glucose concentration. Mesangial iNOS mRNA expression and protein production also increased significantly in response to high glucose. The addition of calphostin C, a PKC inhibitor, and 6-bromo-1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid, an aldose reductase inhibitor, significantly suppressed the enhancement of iNOS mRNA expression in high glucose concentration. High glucose also significantly increased fibronectin protein production of mesangial cells upon stimulation with LPS plus IFN-gamma compared to control glucose. Aminoguanidine reversed this high glucose-induced fibronectin production at dose inhibiting iNOS mRNA expression. CONCLUSIONS: These results indicate that high glucose enhances cytokine-induced NO production by rat mesangial cells, and that the activation of PKC and aldose reductase pathway may play a role in this enhancement. In addition, high glucose-induced NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under certain condition.     

Inducible nitric oxide synthase expressions in different lung injury models and the protective effect of aminoguanidine

OBJECTIVE: Our aim was to study the expression of inducible nitric oxide synthase (iNOS) in 2 experimental models: (1) ischemia/reperfusion (I/R) of the lung tissues and (2) oleic acid infusion. The protective effect of an iNOS inhibitor, aminoguanidine, was evaluated in these 2 injury models. MATERIALS AND METHODS: Real-time polymerase chain reactions and Western blots were used to assess the mRNA and protein expressions of iNOS in lung tissues after applying 2 injury models. In the I/R model, ischemia was induced by clamping one branch of the pulmonary artery for 60 minutes and then reperfusing for 120 minutes. In the bone fracture model, lung injury was induced by intravenous (IV) infusion of oleic acid (0.1 mL/kg); analysis was performed 6 hours after injury. Blood samples were collected for the assay of 3 inflammatory parameters: tumor necrosis factor alpha, hydroxyl radicals, and nitric oxide (NO). The wet/dry lung weight ratio was used as a parameter reflecting the lung injury level. RESULTS: mRNA and protein expressions of iNOS were significantly increased in these 2 lung injury models compared with the controls. Blood concentrations of TNFalpha, hydroxyl radicals, NO, and wet/dry lung weight ratio were also significantly higher in the 2 experimental groups than in the sham-treated group. The iNOS inhibitor aminoguanidine (20 mg/kg) significantly attenuated the lung injury induced by these challenges. CONCLUSIONS: Reperfusion of the ischemic lung tissues or IV infusion of oleic acid can both induce lung injury by activating systemic inflammatory responses and inducing iNOS expression. Administration of aminoguanidine can significantly attenuate the injury, suggesting that iNOS expression may play a critical role in the lung injury induced in these 2 models.     

NF-kappaB involvement in the induction of high affinity CAT-2 in lipopolysaccharide-stimulated rat lungs

BACKGROUND: Endotoxemia stimulates nitric oxide (NO) biosynthesis through induction of inducible NO synthase (iNOS). Cellular uptake of L-arginine, the sole substrate for iNOS, is an important mechanism regulating NO biosynthesis by iNOS. The isozymes of type-2 cationic amino acid transporters, including CAT-2, CAT-2A, and CAT-2B, constitute the most important pathways responsible for trans-membrane L-arginine transportation. Therefore, regulation of CAT-2 isozymes expression may constitute one of the downstream regulatory pathways that control iNOS activity. We investigated the time course of enzyme induction and the role of nuclear factor-kappaB (NF-kappaB) in CAT-2 isozymes expression in lipopolysaccharide-(LPS) treated rat lungs. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous injections of normal saline (N/S), LPS, LPS plus NF-kappaB inhibitor pre-treatment (PDTC, dexamethasone, or salicylate), or an NF-kappaB inhibitor alone. The rats were sacrificed at different times after injection and enzyme expression and lung injury were examined. Pulmonary and systemic NO production were also measured. RESULTS: LPS co-induced iNOS, CAT-2, and CAT-2B but not CAT-2A expression in the lungs. Furthermore, NF-kappaB actively participated in LPS-induction of iNOS, CAT-2, and CAT-2B. LPS induced pulmonary and systemic NO overproduction and resulted in lung injuries. Attenuation of LPS-induced iNOS, CAT-2, and CAT-2B induction significantly inhibited NO biosynthesis and lessened lung injury. CONCLUSION: NF-kappaB actively participates in the induction of CAT-2 and CAT-2B in intact animals. Our data further support the idea that CAT-2 and CAT-2B are crucial in regulating iNOS activity.     

雌激素在大鼠肝移植缺血再灌注损伤中的保护作用

目的 了解雌激素对大鼠肝移植缺血再灌注损伤是否具有保护作用及其可能的作用机制.方法 将大鼠分为三组:雄性组、雌性组以及给予外源性雌激素的雄性组,分别应用两袖套法实施肝移植手术.术后6 h麻醉后处死,取血及肝组织标本.观察外周血丙氨酸氨基转移酶(ALT)和谷氨酸氨基转移酶(AST)以及一氧化氮(NO)水平,免疫组化染色分析内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)在各组肝组织中的表达.结果 与雄性组和雌性组比较给予外源雌激素的雄性大鼠血清ALT水平和AST水平显著降低(分别为561.69 U/L比730.78 U/L和678.82 U/L,726.44 U/L比914.21 U/L和861.86 U/L);血清NO水平明显升高,平均为63.54 μmol/L;肝组织eNOS表达显著增加,iNOS表达显著降低.结论 雌激素对于大鼠肝移植缺血再灌注损伤具有保护作用,这种保护作用一部分是通过调节eNOS和iNOS表达、提高NO水平实现的.

Abstract：

Objective To investigate the protective effects of 17β-estradiol on ischemia reperfusion injury in rat liver transplantation. Methods The rats were divided into three groups: male to male group(MG), female to female group (FG), and male to male group which were given 17β-estradiol 4000 μg/kg 24 hours before liver transplantation intraperitoneally (M + EG). Then transplantation was performed. At 6 hours after portal vein reperfusion, blood samples were obtained to determine the levels of alanine aminotransferase(ALT), aspartate aminotransferase (AST), and nitric oxide(NO). The expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in liver were observed by immunohistochemistry. Results ALT and AST levels in the M+EG group were lower than those in MG and FG (561. 69 U/L vs 730. 78 U/L and 678. 82 U/L; 726. 44 U/L vs 914. 21 U/L and 861. 86 U/L). The NO level (63. 54 μmol/L) was much higher than those in the MG and FG groups. The expression of eNOS in the M+EG group was higher than those in MG and FG, while the expression of iNOS in M+EG were lower than those in MG. and FG. Conclusion 17β-estradiol can attenuate ischemia reperfusion injury in rat liver transplantation by improving the balance of eNOS and iNOS.     

Nitric oxide does not cause extravasation in endotoxemic rats

BACKGROUND: Nitric oxide (NO) formed from inducible NO synthase (iNOS) is assumed to promote vascular permeability in sepsis and endotoxemia. METHODS: Thirty-seven anesthetized rats were examined for the effects of endotoxin. After randomization, 17 animals had lipopolysaccharide (LPS) administered and 20 rats served as controls and were given the corresponding volume of saline. The observation period was 5 hours after administration of endotoxin. Mean arterial blood pressure, heart rate, and hematocrit were recorded in all animals, and transcapillary exchange of albumin, tissue water content, immunohistochemistry for nitric oxide synthase, and blood gases were investigated in subsets of animals. RESULTS: When anesthetized rats were studied for 5 hours after endotoxin (LPS), the sequestration of albumin decreased in the intestine (double-isotope method) and there was no increased water content (freeze-drying technique) when the elevated tissue plasma volume of the LPS-treated rats was corrected for. Immunohistochemical methods showed a similar distribution and intensity of staining for endothelial NOS and neuronal NOS in LPS and control groups. In the lung of the LPS-treated rats, there was a significantly larger number of infiltrating, inflammatory cells staining for iNOS. There was no iNOS demonstrated in vascular structures or heart. CONCLUSION: At 5 hours after LPS, there was no increased loss of water or albumin from the circulation. This challenges the notion that NO causes vascular damage in endotoxemia and extravasation as an obligatory sequela to endotoxemia.