Clinical profiles of post-load glucose subgroups and their association with glycaemic traits over time: An IMI-DIRECT study.

AIM: To examine the hypothesis that, based on their glucose curves during a seven-point oral glucose tolerance test, people at elevated type 2 diabetes risk can be divided into subgroups with different clinical profiles at baseline and different degrees of subsequent glycaemic deterioration. METHODS: We included 2126 participants at elevated type 2 diabetes risk from the Diabetes Research on Patient Stratification (IMI-DIRECT) study. Latent class trajectory analysis was used to identify subgroups from a seven-point oral glucose tolerance test at baseline and follow-up. Linear models quantified the associations between the subgroups with glycaemic traits at baseline and 18 months. RESULTS: At baseline, we identified four glucose curve subgroups, labelled in order of increasing peak levels as 1-4. Participants in Subgroups 2-4, were more likely to have higher insulin resistance (homeostatic model assessment) and a lower Matsuda index, than those in Subgroup 1. Overall, participants in Subgroups 3 and 4, had higher glycaemic trait values, with the exception of the Matsuda and insulinogenic indices. At 18 months, change in homeostatic model assessment of insulin resistance was higher in Subgroup 4 (ß = 0.36, 95% CI 0.13-0.58), Subgroup 3 (ß = 0.30; 95% CI 0.10-0.50) and Subgroup 2 (ß = 0.18; 95% CI 0.04-0.32), compared to Subgroup 1. The same was observed for C-peptide and insulin. Five subgroups were identified at follow-up, and the majority of participants remained in the same subgroup or progressed to higher peak subgroups after 18 months. CONCLUSIONS: Using data from a frequently sampled oral glucose tolerance test, glucose curve patterns associated with different clinical characteristics and different rates of subsequent glycaemic deterioration can be identified.

STAT3 Expression and Activity are Up-Regulated in Diffuse Large B Cell Lymphoma of Dogs.

BACKGROUND: The Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT) pathways play important roles in the pathogenesis of diffuse large B cell lymphoma (DLBCL) in humans, and up-regulated STAT3 expression and activity are associated with worse clinical outcome in humans. No studies have evaluated the JAK-STAT signaling pathway in DLBCL of dogs. HYPOTHESIS: STAT3 pathway is deregulated in DLBCL in dogs. We aim to assess the expression, activation, and cellular localization of STAT3 and mitogen-activated protein kinase ERK1/2 in DLBCL of dogs. ANIMALS: Forty-three client-owned dogs diagnosed with DLBCL by histopathology METHODS: Retrospective analysis of DLBCL in dogs, including patient characteristics and treatment, immunohistochemistry, and protein expressions by Western blot. RESULTS: A higher percentage of STAT3 and p-STAT3 immunolabelled cells were observed in DLBCL of dogs when compared to normal canine lymph nodes. In STAT3 immunolabelled cells, STAT3 has higher nuclear expression in lymphoma samples than in normal or reactive lymph nodes. In addition to up-regulated STAT3 expression and activation, mitogen-activated kinase ERK1/2 activation is up-regulated in DLBCL of dogs. CONCLUSION AND CLINICAL IMPORTANCE: Compared with the normal canine lymph node, DLBCL of dogs has up-regulated STAT3 pathway. Our results support future investigation of JAK inhibitors in the treatment of DLBCL in dogs.

The eNOS enhancer AVE 9488: a novel cardioprotectant against ischemia reperfusion injury.

Nitric oxide (NO) is an important regulator of vascular and myocardial function. Cardiac ischemia/reperfusion injury is reduced in mice overexpressing endothelial NO synthase (eNOS) suggesting cardioprotection by eNOS. Novel pharmacological substances, so called eNOS enhancers, upregulate eNOS expression and thereby increase NO production. We tested the effects of the eNOS enhancer AVE 9488 on cardiac ischemia/reperfusion injury in vivo in mice. After treatment with the eNOS enhancer AVE 9488 (30 mg/kg/day) or placebo for one week mice underwent 30 min of coronary artery ligation and 24 h of reperfusion in vivo. Ischemia-reperfusion damage was significantly reduced in mice treated with the eNOS enhancer when compared to placebo treated mice (infarct/area at risk 65.4 +/- 4.1 vs. 36.9 +/- 4.0%, placebo vs. eNOS enhancer, P = 0.0002). The protective effect was blunted in eNOS knockout mice treated with the eNOS enhancer (infarct/area at risk 64.1 +/- 6.2%, eNOS knockout + eNOS enhancer vs. WT + eNOS enhancer, P = ns). Reactive oxygen species were significantly reduced in mice treated with the eNOS enhancer as indicated by significantly lower malondialdehyde-thiobarbituric acid levels (placebo vs. eNOS enhancer, 3.2 +/- 0.5 vs. 0.8 +/- 0.07 micromol/l, P = 0.0003). Thus pharmacological interventions addressed to increase eNOS-derived NO production constitute a promising therapeutic approach to prevent myocardial ischemia/reperfusion injury.

Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects.

OBJECTIVES: The aim of this study was to investigate whether the caspase-3 inhibitor Ac-DEVD-CHO functionally improves stunned myocardium. BACKGROUND: Degradation of troponin I contributes to the pathogenesis of myocardial stunning, whereas the role of apoptosis is unknown. Caspase-3 is an essential apoptotic protease that is specifically inhibited by Ac-DEVD-CHO. METHODS: Isolated working hearts of rats were exposed to 30 min of low-flow ischemia, followed by 30 min of reperfusion. Ac-DEVD-CHO (0.1 to 1 micromol/l) was added 15 min before ischemia/reperfusion or 5 min before reperfusion. Cardiac output, external heart power, left ventricular (LV) developing pressure and contractility (dp/dt(max)) were measured. Apoptosis was assessed by TUNEL staining and internucleosomal deoxyribonucleic acid fragmentation. Caspase-3 processing and troponin I cleavage were determined by immunoblotting. Caspase-3 activity was measured using a fluorogenic substrate. RESULTS: The addition of Ac-DEVD-CHO before ischemia/reperfusion or before reperfusion dose-dependently and significantly (p < 0.05) improved post-ischemic recovery of cardiac output, external heart power, LV developing pressure and dp/dt(max), compared with the vehicle (0.01% dimethyl sulfoxide). Ac-DEVD-CHO was similarly effective when given before reperfusion. Ac-DEVD-CHO blocked ischemia/reperfusion-induced caspase-3 activation, but cardiomyocyte apoptosis was unaffected. Troponin I cleavage was not inhibited by Ac-DEVD-CHO. CONCLUSIONS: Caspase-3 is activated in stunned myocardium. Inhibition of caspase-3 by Ac-DEVD-CHO significantly improves post-ischemic contractile recovery of stunned myocardium, even when given after the onset of ischemia. The mechanism(s) of protection by Ac-DEVD-CHO appear to be independent of apoptosis. Inhibition of caspase-3 is a novel therapeutic strategy to improve functional recovery of stunned myocardium.

Effects of the cardioselective KATP channel blocker HMR 1098 on cardiac function in isolated perfused working rat hearts and in anesthetized rats during ischemia and reperfusion.

It has been argued that activation of KATP channels in the sarcolemmal membrane of heart muscle cells during ischemia provides an endogenous cardioprotective mechanism. In order to test whether the novel cardioselective KATP channel blocker HMR 1098 affects cardiac function during ischemia, experiments were performed in rat hearts during ischemia and reperfusion. Isolated perfused working rat hearts were subjected to 30 min of low-flow ischemia in which the coronary flow was reduced to 10% of its control value, followed by 30-min reperfusion. In the first set of experiments the hearts were electrically paced at 5 Hz throughout the entire protocol. At the end of the 30-min ischemic period the aortic flow had fallen to 44 +/- 2% (n=8) of its nonischemic value in vehicle-treated hearts, whereas in the presence of 0.3 micromol/l and 3 micromol/l HMR 1098 it had fallen to 29 +/- 7% (n=5, not significant) and 8 +/- 2% (n=12, P<0.05), respectively. Glibenclamide (3 micromol/l) reduced the aortic flow to 9.5 +/- 7% (n=4, P<0.05). In control hearts the QT interval in the electrocardiogram shortened from 63 +/- 6 ms to 36 +/- 4 ms (n=10, P<0.05) within 4-6 min of low-flow ischemia. This shortening was completely prevented by 3 micromol/l HMR 1098 (60 +/- 5 ms before ischemia, 67 +/- 6 ms during ischemia, n=9, not significant). When rat hearts were not paced, the heart rate fell spontaneously during ischemia, and HMR 1,098 (3 micromol/l) caused only a slight, statistically non-significant reduction in aortic flow during the ischemic period. In order to investigate whether HMR 1098 shows cardiodepressant effects in a more pathophysiological model, the left descending coronary artery was occluded for 30 min followed by reperfusion for 60 min in anesthetized rats. Treatment with HMR 1098 (10 mg/kg i.v.) had no statistically significant effects on mean arterial blood pressure and heart rate during the control, ischemia and reperfusion periods. At the end of the reperfusion period, aortic blood flow was slightly reduced by HMR 1098, without reaching statistical significance (two-way analysis of ANOVA, P=0.15). Myocardial infarct size as a percentage of area at risk was not affected by HMR 1098 (vehicle: 75 +/- 3%, HMR 1098: 72 +/- 2%, n=7 in each group). In conclusion, cardiodepressant effects of HMR 1098 were observed only in isolated perfused working rat hearts which were continuously paced during global low-flow ischemia. In the model of anesthetized rats subjected to regional ischemia, HMR 1098 had no significant effect on cardiac function or infarct size.

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice.

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to beta-adrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein-kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein-kinin system could be involved in the development or progression of cardiovascular diseases.

Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway.

OBJECTIVE: Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS: Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

Release of nitric oxide from endothelial cells stimulated by YC-1, an activator of soluble guanylyl cyclase.

1 In this study we examined the endothelium-dependent effect of YC-1 - a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC) - on vascular relaxation and nitric oxide (NO) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. 2 In preconstricted rat aortic rings with intact endothelium, YC-1 produced a concentration-dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC-1, when (i) the aortas were pre-treated with L-NG-nitroarginine methylester (L-NAME) or (ii) the endothelium was removed. 3 Incubation of bovine aortic endothelial cells (BAEC) with YC-1 produced a concentration-dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre-incubating cells with L-NAME or with 8-bromo-cyclic GMP decreased this effect indicating that the YC-1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC-1 on recombinant endothelial constitutive NO synthase activity was observed. 4 The YC-1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. 5 In human umbilical vein endothelial cells (HUVEC), YC-1 stimulated intracellular cyclic GMP production in a concentration- and time-dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC-1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). 6 When HUVEC and RMCEC were pre-treated with L-NG-nitroarginine (L-NOARG), the maximum YC-1 stimulated cyclic GMP increase was reduced by >/=50%. 7 These results indicate, that beside being a direct activator of sGC, YC-1 stimulates a NO-synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.

Ramiprilat prevents the development of acute coronary endothelial dysfunction in the dog.

We investigated the effect of an infusion of ramiprilat on the development of coronary endothelial dysfunction. In anesthetized dogs, the endothelium-dependent vasodilators acetylcholine (ACh, 5 and 10 microg x min(-1) for 1 min) and serotonin (5-HT, 50 and 100 microg x min(-1) for 1 min) and the endothelium-independent vasodilator nitroglycerin (NTG, 50 and 100 microg x min(-1) for 1 min) were given intracoronarily (i.c.) both prior to and after 60 min of ischemia (I) and 180 min of reperfusion (R) of a coronary artery. During I/R the dogs received i.c. either saline (N = 22) or ramiprilat (40 ng/kg x min(-1), N = 14). At the end of the experiment, a biopsy of the most distal coronary bed was processed for scanning electron microscopy (SEM). Prior to I/R all vasodilators induced a similar dose-related increase in coronary flow in both groups. Following I/R, in controls the responses to ACh and 5-HT were significantly blunted (ACh: -39% and -34%; 5-HT: -48% and -49%); those to NTG were unchanged. Ramiprilat significantly prevented the blunting of the responses to ACh (-5%, and -10%) and 5-HT (-11%, and -19%). SEM of control subepicardial arterioles showed adhesion of leukocytes to the endothelium and crater formation. No craters were seen in the ramiprilat-treated dogs. Thus, an acute infusion of ramiprilat significantly prevents the development of coronary endothelial dysfunction. Additionally, the appearance of crater-like changes on the endothelial surface can be taken as a morphological marker of endothelial dysfunction.

Downregulation of soluble guanylyl cyclase in young and aging spontaneously hypertensive rats.

Endothelial dysfunction, as observed in hypertension and atherosclerosis, is associated with a reduction in the bioavailability of endothelium-derived nitric oxide (NO). We tested the hypothesis that alterations in the soluble guanylyl cyclase (sGC) pathway may also contribute to the pathogenesis of hypertension. Therefore, we investigated the expression and activity of sGC in young (6 weeks) and aging (17 months) spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Endothelium-independent relaxation of aortic rings in response to the sGC activator YC-1 was attenuated in SHR, and expression of both alpha(1) and beta(1) subunits of heterodimeric sGC and the basal contents of cGMP were reduced specifically in SHR aorta. Moreover, mRNA expression of the cGMP receptor and effector protein cGMP-dependent protein kinase type Ialpha (cGKIalpha) was also reduced. Interestingly, downregulation of both sGC and cGKIalpha expression was observed in young, ie, normotensive SHR, whereas impairment of the endothelium-independent relaxation was found only in aging SHR. Accordingly, similar cGMP levels were reached in response to YC-1 in young SHR and young WKY, suggesting a compensatory increased sensitivity or effectiveness of the sGC pathway in young SHR. In aging SHR, however, increased sensitivity to YC-1 no longer compensated for the impairment of endothelium-independent relaxation, suggesting that other mechanisms were involved. In fact, endothelium-independent relaxations were partially restored by superoxide dismutase, suggesting a pathophysiological role of superoxide production, particularly at later disease stages. Thus, tissue-specific downregulation of components of the sGC/cGMP pathway is an early event in the pathogenesis of hypertension.

Upregulation of ICAM-1 expression on J774.2 macrophages by endotoxin involves activation of NF-kappaB but not protein tyrosine kinase: comparison to induction of iNOS.

This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule-1 (ICAM-1) expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS) protein and activity caused by endotoxin in cultured J774.2 macrophages. Treatment of J774.2 cells with lipopolysaccharide E. coli (LPS) induced a concentration-dependent increase in the expression of ICAM-1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h. The upregulation of ICAM-1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF-kappaB, such as L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK), pyrrolidine dithiocarbamate (PDTC), rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein. In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS. Thus, the increase in the expression of ICAM-1 on J774.2 macrophages by endotoxin involves the activation of NFkappaB, but not of protein tyrosine kinase.

Reduction of myocardial reperfusion injury by an inhibitor of poly (ADP-ribose) synthetase in the pig.

The effect of the Poly (adenosine 5'-diphosphate ribose) synthetase (PARS) inhibitor 3-aminobenzamide on (i) infarct size caused by regional myocardial ischaemia (60 min) and reperfusion (3 h) in the anaesthetised pig, and (ii) on the cell injury/necrosis of human cardiomyoblasts caused by hydrogen peroxide (3 mM) was investigated. Regional myocardial ischaemia and reperfusion resulted in an infarct size of 66+/-3% of the area at risk, which was reduced by 3-aminobenzamide (to 44+/-2%, n=6), but not 3-aminobenzoic acid (66+/-5%, n=4). 3-aminobenzamide also reduced the postischaemic contractile dysfunction. 3-aminobenzamide, but not 3-aminobenzoic acid, abolished the increase in PARS activity as well as the cell injury/necrosis caused by hydrogen peroxide in the cardiomyoblasts. In conclusion, the PARS inhibitor 3-aminobenzamide reduces myocardial reperfusion injury in the pig, and attenuates the cell injury and death associated with oxidant stress in human cardiomyoblasts. We propose that the activation of PARS plays an important role in the injury associated with oxidant stress of the heart.

Coronary endothelial dysfunction after ischemia and reperfusion in the dog: a functional and morphological investigation.

Coronary endothelial dysfunction is characterized by a lower response to endothelium-dependent vasodilators such as acetylcholine (ACh) and serotonin (5HT), but by an unaltered response to endothelium-independent vasodilators such as nitroglycerin (NTG). In the present study, we investigated the vasoreactivity of the coronary bed in vivo, in a dog model of ischemia and reperfusion (I/R). We also assessed the morphology of the subepicardial arterioles and capillary bed by means of scanning electron microscopy (SEM). Anesthetized, instrumented dogs were divided in two groups. One group (N = 27) was submitted to ischemia (60 min) and reperfusion (180 min) of the left circumflex coronary artery, the second group (N = 8) was sham-operated. Prior to and following I/R, ACh, 5-HT, and NTG were given intracoronarily. At the end of the experiment a 1 cm3 myocardial biopsy was processed for SEM. The sham-operated dogs showed a reduction of basal coronary flow of 11%, but the vasoreactivity to ACh and 5-HT remained constant. In the I/R group, basal coronary flow was reduced by 35% (p < 0.05), and the vasoreactivity to ACh and 5-HT, but not to NTG, was significantly blunted. At SEM the arterioles of the dogs submitted to I/R showed a marked adhesion of leukocytes associated with holes on the endothelial surface, while the capillary bed was free of changes and patient. Thus, following I/R, coronary endothelial dysfunction could be demonstrated in vivo by the blunting of the vasoreactive responses to two different endothelium-dependent vasodilators. The responses to NTG were not affected, probably because the function of the smooth muscle cell was preserved, and the capillary bed was patent.

Effects of tyrphostins and genistein on the circulatory failure and organ dysfunction caused by endotoxin in the rat: a possible role for protein tyrosine kinase.

1 Here we compared the effects of various inhibitors of the activity of protein tyrosine kinase on (i) the expression of the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) caused by endotoxin (lipopolysaccharide, LPS) in cultured macrophages, (ii) the induction of iNOS and cyclooxygenase 2 (COX-2) protein and activity in rats with endotoxaemia, and (iii) the circulatory failure and organ dysfunction caused by LPS in the anesthetized rat. 2 Activation of murine cultured macrophages with LPS (1 microgram ml-1) resulted, within 24 h, in a significant increase in nitrite (an indicator of the formation of NO) in the cell supernatant. This increase in nitrate was attenuated by the tyrphostins AG126, AG556, AG490 or AG1641 or by genistein in a dose-dependent fashion (IC50: approximately 15 microM). In contrast, tyrphostin A1 (an analogue of tyrphostin AG126) or daidzein (an analogue of genistein) had no effect on the rise in nitrite caused by LPS. 3 Administration of LPS (E. coli, 10 mg kg-1, i.v.) caused hypotension and a reduction of the pressor responses elicited by noradrenaline (NA, 1 microgram kg-1, i.v.). Pretreatment of rats with the tyrphostins AG126, AG490, AG556, AG1641 or A1 attenuated the circulatory failure caused by LPS. Although genistein attenuated the vascular hyporeactivity to NA, it did not affect the hypotension caused by LPS. Daidzein did not affect the circulatory failure caused by LPS. 4 Endotoxaemia for 360 min resulted in rises in the serum levels of (i) urea and creatinine (indicators of renal failure), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin and gamma-glutamyl transferase (gamma GT) (indicators of liver injury/dysfunction), lipase (an indicator of pancreatic injury) as well as lactate (an indicator of tissue hypoxia). None of the tyrosine kinase inhibitors tested had a significant effect on the rise i the serum levels of urea, but the tyrphostins AG126, AG556 or A1 significantly attenuated the rises in the serum level of creatinine caused by LPS. In addition, all tyrphostins and genistein attenuated the liver injury/failure, the pancreatic injury, the hypoglycaemia and the lactic acidosis caused by LPS. In contrast, daidzein did not reduce the organ injury/dysfunction or the lactic acidosis caused by LPS. 5 Injection of LPS resulted (within 90 min) in a substantial increase in the serum level of tumor necrosis factor alpha (TNF alpha), which was attenuated by pretreatment of LPS-rats with any of the tyrphostins used. Genistein, but not daidzein, also reduced the rise in the serum levels of TNF alpha caused by LPS. Endotoxaemia for 6 h also resulted in a substantial increase in the expression of iNOS and COX-2 protein and activity in the lung, which was attenuated by pretreatment of LPS-rats with the tyrphostins AG126, AG556 or genistein, but not by daidzein. 6 Thus, tyrphostins (AG126, AG556, AG1641 or A1) and genistein, but not daidzein (inactive analogue of genistein), prevent the (i) circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury lactacidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX-2 protein and activity in rats with endotoxic shock.

Effect of calpain inhibitor I, an inhibitor of the proteolysis of I kappa B, on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat.

1. We compared the effects of calpain inhibitor I (inhibitor of the proteolysis of I kappa B and, hence, of the activation of nuclear factor kappa B (NF kappa B) and dexamethasone on (i) the circulatory failure, (ii) multiple organ dysfunction and (iii) induction of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclo-oxygenase (COX-2) in anaesthetized rats with endotoxic shock. 2. Injection of lipopolysaccharide (LPS, E. coli, 10 mg kg-1, i.v.) resulted in hypotension and a reduction of the pressor responses elicited by noradrenaline. This circulatory dysfunction was attenuated by pretreatment of LPS-rats with calpain inhibitor I (10 mg kg-1, i.v., 2 h before LPS) or dexamethasone (1 mg kg-1, i.v.). 3. Endotoxaemia also caused rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST) (hepatocellular injury), bilirubin and gamma-glutamyl transferase (gamma GT) (liver dysfunction), (iii) lipase (pancreatic injury) and (iv) lactate. Calpain inhibitor I and dexamethasone attenuated the liver injury, the pancreatic injury, the lactic acidosis as well as the hypoglycaemia caused by LPS. Dexamethasone, but not calpain inhibitor I, reduced the renal dysfunction caused by LPS. 4. Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX-2 protein and activity in lung and liver, which was attenuated in LPS-rats pretreated with calpain inhibitor I or dexamethasone. 5. Thus, calpain inhibitor I and dexamethasone attenuate (i) the circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury, lactic acidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX-2 protein and activity in rats with endotoxic shock. We propose that prevention of the activation of NF-kappa B in vivo may be useful in the therapy of circulatory shock or of disorders associated with local or systemic inflammation.

Interleukin-13 is a more potent inhibitor of the expression of inducible nitric oxide synthase in smooth muscle cells than in macrophages: a comparison with interleukin-4 and interleukin-10.

This study compares the effects of interleukin (IL)-13, a cytokine with anti-inflammatory properties, with those of IL-4 or IL-10 on the expression of inducible nitric oxide synthase (iNOS) protein and activity in 1) a murine macrophage cell line (J774.2) activated with lipopolysaccharide (LPS) and 2) rat aortic smooth muscle cells (RASM) activated with LPS plus interferon-gamma. Pretreatment of macrophages with IL-4 or IL-13 caused a similar, concentration-dependent inhibition of the formation of nitrite and the expression of iNOS protein elicited by LPS. In contrast, IL-13 was a much more potent inhibitor of the formation of nitrite and the expression of iNOS protein in activated RASM than IL-4. IL-10 caused only a small, but significant, inhibition of the nitrite formation induced by LPS in macrophages and RASM. Pretreatment of J774.2 macrophages, but not of RASM, with the phosphatidylinositol-3-kinase inhibitor, wortmannin (10-100 nM), attenuated the inhibition by either IL-13 or IL-4 of the LPS-induced increase in nitrite in a dose-related fashion. Thus, IL-13 is more potent than IL-4 in preventing the expression of iNOS protein and activity in activated RASM, whereas IL-13 and IL-4 are equipotent in inhibiting the expression of iNOS protein and activity in J774.2 macrophages.

Combination immunotherapy which neutralises the effects of TNF alpha and IL-1 beta attenuates the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat.

Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF alpha) or interleukin-1 beta (IL-1 beta) are implicated in the pathogenesis of septic shock. Here we investigate the role of endogenous TNF alpha and IL-1 beta on (i) the circulatory failure, (ii) the multiple organ dysfunction syndrome (MODS) and (iii) the induction of the inducible isoform of nitric oxide (NO) synthase (iNOS) caused by endotoxin (LPS) in the anaesthetised rat. Here we demonstrate that (i) a polyclonal antibody against TNF alpha, (ii) a polyclonal antibody against IL-1, (iii) co-administration of polyclonal antibodies against TNF alpha and IL-1 and (iv) neutralisation of the effects of both TNF alpha and IL-1 with one polyclonal antibody directed against both cytokines reduces the circulatory failure, the liver injury/dysfunction, the pancreatic injury (but not the renal dysfunction) caused by endotoxin in the rat. The beneficial effects of these interventions on haemodynamics and organ injury/dysfunction are most likely due to prevention of the induction of iNOS. The two different interventions which neutralised the effects of both TNF alpha and IL-1 were superior in reducing the circulatory failure and the organ injury caused by endotoxin in the rat, than single interventions aimed at neutralising the effects of either cytokine. Thus, we propose that interventions which are able to neutralise the effects of both TNF alpha and IL-1 (combination immunotherapy) may be of benefit in the treatment of patients with septic shock.

Endothelin-1 stimulates the biosynthesis of tumour necrosis factor in macrophages: ET-receptors, signal transduction and inhibition by dexamethasone.

Endothelin-1 (ET-1) enhances the biosynthesis of interleukin-6 (IL-6) in endothelial cells and bone marrow-derived stromal cells of the rat. This study investigates (i) whether ET-1 stimulates the formation of tumour necrosis factor alpha (TNF alpha) or interferon-gamma (IFN gamma) in cultured macrophages or in the anaesthetized rat. Incubation of J774.2 macrophages with ET-1 (0.001-1 microM) caused a concentration- and time-dependent increase in the concentration of TNF alpha, but not of IFN gamma, in the culture medium. The increase in TNF alpha caused by stimulation of J774.2 macrophages was abolished by pretreatment of cells with (i) the protein synthesis inhibitor cycloheximide, (ii) with the selective ETA-receptor antagonists BQ-123 or BQ-485 (but not the selective ETB-receptor antagonist BQ-788), (iii) the tyrosine kinase inhibitors genistein or tyrphostin AG126, or (iv) with the glucocorticoid, dexamethasone. The inhibition by dexamethasone of the formation of TNF alpha by cells activated with ET-1 is not due to the formation of lipocortin-1 (LC1), as it was not reduced by a monoclonal antibody against LC1. Systemic administration (i.v.) of ET-1 (1 nmol.kg-1) to anaesthetized rats caused a rapid and sustained (maximum: 45 min; return to baseline: within 180 min) rise in the plasma levels of TNF alpha. This is the first demonstration that ET-1 can release proinflammatory cytokines in vitro and in vivo. The generation of TNF alpha caused by ET-1 involves (in sequence) the (i) activation of ETA-receptors, (ii) activation of tyrosine kinase resulting in the phosphorylation of intracellular proteins, (iii) the activation of, hitherto, unknown transcription factors, finally resulting in (iv) transcription and translation of the TNF alpha gene. The generation of TNF alpha by cells activated with ET-1 points to a pro-inflammatory role of ET-1 in diseases associated with local (e.g. atherosclerosis, heart failure) or systemic inflammation (circulatory shock), which are associated with high ET-1 plasma levels.

Effect of selective blockade of endothelin ETB receptors on the liver dysfunction and injury caused by endotoxaemia in the rat.

1. We investigated the effects of the selective endothelin (ET)A receptor antagonist BQ-485 and the selective ETB receptor antagonist BQ-788 on circulatory failure, multiple organ dysfunction syndrome (MODS) and the alterations in acid base balance caused by endotoxaemia in the anaesthetized rat. 2. Male Wistar rats were anaesthetized (thiopentone sodium; 120 mg kg-1, i.p.) and received a continuous infusion of vehicle (saline, 0.6 ml kg-1h-1, i.v.), BQ-485 (10 nmol kg-1 min-1, i.v.) or BQ-788 (10 nmol kg-1 min-1, i.v.). Fifteen min later, animals received a bolus injection of either saline (0.9% NaCl, 1 ml kg-1, i.v.) or E. coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.). 3. Injection of LPS resulted in a fall in blood pressure from 115 +/- 4 mmHg (time 0) to 82 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the pressor responses to noradrenaline (NA, 1 microgram kg-1, i.v.). Infusion of BQ-788 attenuated the delayed hypotension (at 360 min: 100 +/- 4 mmHg, n = 7; P < 0.05) and significantly enhanced the pressor responses elicited by NA (at 60 to 240 min). In contrast, treatment of LPS-rats with BQ-485 augmented the hypotension (at 360 min), but did not affect the vascular hyporeactivity elicited by endotoxaemia. 4. Endotoxaemia for 360 min resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), glutamate-oxalate-transferase (GOT) and glutamate-pyruvate-transferase (GPT) (indicators of hepatocellular injury), and bilirubin and gamma-glutamyl transferase (gamma GT) (indicators of liver failure) as well as nitrite (indicator of the induction of nitric oxide synthase; iNOS). Treatment of LPS-rats with BQ-788, but not with BQ-485, attenuated the degree of liver injury and failure, while neither BQ-788 nor BQ-485 affected the acute renal failure or the induction of iNOS caused by endotoxin. 5. Endotoxaemia also caused (within 15 min) an acute metabolic acidosis (falls in pH, HCO3-and base excess) which was compensated by hyperventilation (fall in PaCO2). Treatment of LPS-rats with BQ-788 or BQ-485 did not affect the metabolic acidosis caused by LPS. 6. Thus, the selective ETB receptor antagonist BQ-788 attenuated (i) the delayed hypotension, (ii) the vascular hyporeactivity to NA as well as (iii) the degree of hepatocellular injury and dysfunction caused by endotoxin in the anaesthetized rat. In contrast, the selective ETA receptor antagonist did neither attenuate the circulatory failure nor the liver or renal dysfunction associated with endotoxaemia. We propose that the prevention of the hepatocellular dysfunction and injury caused BQ-788 in endotoxaemia is due to an improvement in oxygen delivery to the liver secondary to (i) inhibition of pre-sinusoidal constriction, (ii) inhibition of sinusoidal constriction, and (iii) improvement in perfusion pressure.

Prevention of the expression of inducible nitric oxide synthase by aminoguanidine or aminoethyl-isothiourea in macrophages and in the rat.

An enhanced formation of nitric oxide (NO) following the induction of the inducible isoform of NOS (iNOS) has been implicated in the pathogenesis of circulatory shock and inflammation. This study elucidates the effects of the NOS inhibitors aminoethyl-isothiourea (AE-ITU), aminoguanidine (AG), NG-methyl-L-arginine (L-NMMA) or N omega-Nitro-L-arginine methyl ester (L-NAME) on the expression of iNOS protein lipopolysaccharide (LPS) in macrophages and the rat (lung homogenates). The expression of iNOS protein was detected by Western blot analysis using a specific iNOS antibody. In the absence of LPS, the iNOS protein was expressed neither in macrophages nor in the lung. LPS (1 microgram.ml-1) resulted in the expression of iNOS protein in macrophages, which was significantly inhibited by pretreatment of cells with AE-ITU or AG, but not by L-NMMA or L-NAME. In addition, LPS (10 mg.kg-1, i.v.) also caused an increase in the expression of iNOS protein in lungs obtained from rats at 6 h after LPS, which was significantly reduced by treatment of LPS-rats with AE-ITU or AG, but not with L-NMMA or L-NAME. Thus, AE-ITU or AG inhibit not only iNOS activity, but also the induction of iNOS protein in vitro and in vivo caused by endotoxin.