Christoph Thiemermann

Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo

E. coli lipopolysaccharide (LPS; 15 mg kg-1 i.v.) produced a long-lasting reduction in mean arterial blood pressure (MAP) in the anaesthetized rat. Inhibition of nitric oxide endothelium-derived relaxing factor (EDRF) synthesis with NG-monomethyl-L-arginine (MeArg, 1 mg kg-1 min-1 i.v. for 30 min) produced an increase in MAP and largely attenuated the LPS-induced hypotension; both effects were significantly reversed with L-arginine (6 mg kg-1 min-1 i.v.). When compared to MeArg, phenylephrine (300 mg kg-1 h-1 i.v.) produced a similar pressor response, but much less attenuation of the hypotensive response to LPS. Thus, a stimulation of EDRF release contributes to the LPS-induced hypotension in the anaesthetized rat.

Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock

1 The role of an enhanced formation of nitric oxide (NO) and the relative importance of the constitutive and inducible NO synthase (NOS) for the development of immediate (within 60 min) and delayed (at 180 min) vascular hyporeactivity to noradrenaline was investigated in a model of circulatory shock induced by endotoxin (lipopolysaccharide; LPS) in the rat. 2 Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate. In addition, the calcium‐dependent and calcium‐independent NOS activity was measured ex vivo by the conversion of [3H]‐arginine to [3H]‐citrulline in homogenates from several organs obtained from vehicle‐ and LPS‐treated rats. 3 E. coli LPS (10 mg kg−1, i.v. bolus) caused a rapid (within 5 min) and sustained fall in MAP. At 30 and 60 min after LPS, pressor responses to noradrenaline (0.3, 1 or 3 μg kg−1, i.v.) were significantly reduced. The pressor responses were restored by NG‐nitro‐l‐arginine methyl ester (l‐NAME, 1 mg kg−1, i.v. at 60 min), a potent inhibitor of NO synthesis. In contrast, l‐NAME did not potentiate the noradrenaline‐induced pressor responses in control animals. 4 Dexamethasone (3 mg kg−1, i.v., 60 min prior to LPS), a potent inhibitor of the induction of NOS, did not alter initial MAP or pressor responses to noradrenaline in control rats, but significantly attenuated the LPS‐induced fall in MAP at 15 to 60 min after LPS. Dexamethasone did not influence the development of the LPS‐induced immediate (within 60 min) hyporeactivity to noradrenaline. However, dexamethasone pretreatment prevented the hypotension and vascular hyporeactivity at 180 min. 5 At 60 min after LPS a moderate increase in the activity of a calcium‐independent (inducible) NOS activity was detected in the aorta, but not in any of the other tissues studied. However, at 180 min after LPS, a significant NOS induction was observed in the lung, liver, spleen, mesentery, heart and aorta. This NOS induction was substantially prevented by pretreatment with dexamethasone. 6 These results suggest that the immediate hypotension and vascular hyporeactivity to noradrenaline in endotoxin shock is caused by an enhanced formation of NO due to activation of the constitutive enzyme. The delayed hypotension and vascular hyporeactivity, however, is due to enhanced NO formation by the LPS‐induced enzyme.

Ligands of the peroxisome proliferator-activated receptors (PPAR-γ and PPAR-α) reduce myocardial infarct size

This study was designed to investigate the effects of various chemically distinct activators of PPAR‐γ and PPAR‐α in a rat model of acute myocardial infarction. Using Northern blot analysis and RT‐PCR in samples of rat heart, we document the expression of the mRNA for PPAR‐γ (isoform 1 but not isoform 2) as well as PPAR‐β and PPAR‐α in freshly isolated cardiac myocytes and cardiac fibroblasts and in the left and right ventricles of the heart. Using a rat model of regional myocardial ischemia and reperfusion (in vivo), we have discovered that various chemically distinct ligands of PPAR‐γ (including the TZDs rosiglitazone, ciglitazone, and pioglitazone, as wel as the cyclopentanone prostaglandins 15D‐PGJ2 and PGA1) cause a substantial reduction of myocardial infarct size in the rat. We demonstrate that two distinct ligands of PPAR‐α (including clofibrate and WY 14643) also cause a substantial reduction of myocardial infarct size in the rat. The most pronounced reduction in infarct size was observed with the endogenous PPAR‐γ ligand, 15deoxyΔ12,14‐prostagalndin J2 (15D‐PGJ2). The mechanisms of the cardioprotective effects of 15D‐PGJ2 may include 1) activation of PPAR‐α, 2) activation of PPAR‐γ, 3) expression of HO‐1, and 4) inhibition of the activation of NF‐KB in the ischemic‐reperfused heart. Inhibition by 15D‐PGJ2 of the activation of NF‐κB in turn results in a reduction of the 1) expression of inducible nitric oxide synthase and the nitration of proteins by peroxynitrite, 2) formation of the chemokine MCP‐1, and 3) expression of the adhesion molecule ICAM‐1. We speculate that ligands of PPAR‐γ and PPAR‐α may be useful in the therapy of conditions associated with ischemia‐reperfusion of the heart and other organs. Our findings also imply that TZDs and fibrates may help protect the heart against ischemiareperfusion injury. This beneficial effect of 15D‐PGJ2 was associated with a reduction in the expression of the 1) adhesion molecules ICAM‐1 and P‐selectin, 2) chemokine macrophage chemotactic protein 1, and 3) inducible isoform of nitric oxide synthase. 15D‐PGJ2 reduced the nitration of proteins (immunohistological analysis of nitrotyrosine formation) caused by ischemiareperfusion, likely due to the generation of peroxynitrite. Not all of the effects of 15D‐PGJ2, however, are due to the activation of PPAR‐γ. For instance, exposure of rat cardiac myocytes to 15D‐PGJ2, but not to rosiglitazone, results in an up‐regulation of the expression of the mRNA for heme‐oxygenase‐1 (HO‐1). Taken together, these results provide convincing evidence that several, chemically distinct ligands of PPAR‐γ reduce the tissue necrosis associated with acute myocardial infarction.—Wayman, N. S., Hattori, Y., McDonald, M. C., Mota‐Filipe, H., Cuzzocrea, S., Pisano, B., Chatterjee, P. K., Thiemermann, C. Ligands of the peroxisome proliferator‐activated receptors (PPAR‐γ and PPAR‐α) reduce myocardial infarct size. FASEB J. 16, 1027–1040 (2002)

Erythropoietin Protects the Kidney against the Injury and Dysfunction Caused by Ischemia-Reperfusion

Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X(L) and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.

Nitric oxide and septic shock

1. Nitric oxide (NO) is generated by three different isoforms of NO synthase, two of which are expressed constitutively (in endothelium: eNOS, brain: nNOS), while one is induced by endotoxin (LPS) or cytokines (iNOS). 2. Expression of iNOS in many organs or tissues in septic shock (caused by Gram-negative or Gram-positive bacteria) results in an enhanced formation of NO that contribute to hypotension, vascular hyporeactivity to vasoconstrictors, organ injury, and dysfunction as well as host defense. 3. Inhibition of either the expression of iNOS protein (e.g., with dexamethasone) or of NOS activity (e.g., with selective inhibitors of iNOS activity) exerts beneficial effects in animal models of shock. In contrast, inhibition of eNOS activity may lead to excessive vasoconstriction (adverse effects). 4. There is limited evidence regarding the degree of iNOS induction in human cells or tissues with septic shock. Preliminary data from ongoing clinical trials indicate that nonselective inhibitors of NOS activity (e.g., NG-methyl-L-arginine [L-NMMA]) exert beneficial hemodynamic effects.

Isothioureas: Potent inhibitors of nitric oxide synthases with variable isoform selectivity

1 The induction of a calcium‐independent isoform of nitric oxide (NO) synthase (iNOS) and a subsequent enhanced formation of NO has been implicated in the pathophysiology of a variety of diseases including inflammation and circulatory shock. Here we demonstrate that the S‐substituted isothioureas, S‐methylisothiourea (SMT), S‐(2‐aminoethyl)isothiourea (aminoethyl‐TU), S‐ethylisothiourea (ethyl‐TU) and S‐isopropylisothiourea (isopropyl‐TU) potently inhibit iNOS activity in J774.2 macrophages activated with bacterial endotoxin with EC50 values 8–24 times lower than that of NG‐methyl‐l‐arginine (MeArg) and 200‐times lower than that of NG‐nitro‐l‐arginine (l‐NO2Arg). 2 The inhibition of iNOS activity by these S‐substituted isothioureas is dose‐dependently prevented by excess of l‐arginine suggesting that these isothioureas are competitive inhibitors of iNOS at the l‐arginine binding site. 3 Ethyl‐TU and isopropyl‐TU are 4–6 times more potent than MeArg in inhibiting the constitutive NOS activity in homogenates of bovine aortic endothelial cells (eNOS) and are more potent pressor agents than MeArg in the anaesthetized rat. SMT is equipotent with MeArg, whereas aminoethyl‐TU is 6‐times less potent in inhibiting eNOS activity in vitro. Both SMT and aminoethyl‐TU, however, elicit only weak pressor responses (approximately 15mmHg at 10 mg kg−1, i.v.) in vivo. 4 A comparison of the potencies of ethyl‐, iso‐propyl‐, n‐propyl‐, t‐butyl‐ and n‐butyl‐isothioureas on iNOS activity shows that the inhibitory activity of S‐substituted isothioureas declines sharply if the side chain exceeds 2 carbon atoms in length. Similarly, substitution of the ethylene side chain of ethyl‐TU also results in a diminished potency. Substitution of either one or both nitrogens of SMT with either amino or alkyl groups also substantially reduces its NOS inhibitory potency. 5 In conclusion, isothioureas represent a new class of NOS inhibitors which includes the most potent inhibitors of iNOS activity reported to date. Some members of this class (ethyl‐TU and isopropyl‐TU) are potent inhibitors of eNOS and iNOS with little selectivity towards either isoform, while others (SMT and aminoethyl‐TU) are relatively selective inhibitors of iNOS activity. These latter agents may become useful tools for studying the role of iNOS in various disease models and may be useful in the therapy of diseases that are associated with an enhanced formation of NO due to iNOS induction, such as inflammation, circulatory shock or cancer.

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin.

Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the β common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58–82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia–reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPOs trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPOs three-dimensional structure.

The multiple organ dysfunction syndrome caused by endotoxin in the rat : attenuation of liver dysfunction by inhibitors of nitric oxide synthase

1 We have investigated whether (i) endotoxaemia caused by E. coli lipopolysaccharide in the anaesthetized rat causes a multiple organ dysfunction syndrome (MODS; e.g. circulatory failure, renal failure, liver failure), and (ii) an enhanced formation of nitric oxide (NO) due to induction of inducible NO synthase (iNOS) contributes to the MODS. In addition, this study elucidates the beneficial and adverse effects of aminoethyl‐isothiourea (AE‐ITU), a relatively selective inhibitor of iNOS activity, and NG‐methyl‐l‐arginine (l‐NMMA), a non‐selective inhibitor of NOS activity on the MODS caused by endotoxaemia. 2 In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 117±3mmHg (time 0) to 97±4mmHg at 2 h (P<0.05, n=15) and 84±4mmHg at 6 h (P < 0.05, n= 15). The pressor effect of noradrenaline (NA, 1 μg kg−1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS‐rats with AE‐ITU (1 mg kg−1, i.v. plus 1 mg kg−1 h−1 starting at 2 h after LPS) caused only a transient rise in MAP, but significantly attenuated the delayed vascular hyporeactivity seen in LPS‐rats. Infusion of l‐NMMA (3 mg kg−1, i.v. plus 3 mg kg−1 h−1) caused a rapid and sustained rise in MAP and attenuated the delayed vascular hyporeactivity to NA. Neither AE‐ITU nor l‐NMMA had any effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 3 Endotoxaemia for 6 h was associated with a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT), γ‐glutamyl‐transferase (γGT), and bilirubin, and hence, fiver dysfunction. Treatment of LPS‐rats with AE‐ITU significantly attenuated this liver dysfunction (rise in GOT, GPT, yGT and bilirubin) (P <0.05, n=10). In contrast, l‐NMMA reduced the increase in the serum levels of γGT and bilirubin, but not in GOT and GPT (n = 5). Injection of LPS also caused a time‐dependent, but rapid (almost maximal at 2 h), increase in the serum levels of urea and creatinine, and hence, renal dysfunction. This renal dysfunction was not affected by either AE‐ITU (n= 10) or l‐NMMA (n= 5). In rats infused with saline rather than LPS, neither AE‐ITU (n = 4) nor l‐NMMA (n = 4) had any significant effect on the serum levels of GOT* GPT, yGT, bilirubin, creatinine or urea. 4 Endotoxaemia for 6 h resulted in a 4.5 fold rise in the serum levels of nitrite (9.13 ±0.77 μm, P < 0.01, n= 15), which was significantly reduced by treatment with AE‐ITU (6.32±0.48 μm, P <0.05, n=10) or l‐NMMA (5.10±0.40 μm, P < 0.05, n = 5). In addition, endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver homogenates, which was significantly reduced in lung or liver homogenates obtained from LPS‐rats treated with either AE‐ITU or l‐NMMA. 5 Thus, AE‐ITU or l‐NMMA (i) inhibits iNOS activity in LPS‐rats without causing a significant increase in MAP in rats infused with saline and, hence inhibition of endothelial NOS activity, and (ii) attenuates the delayed circulatory failure as well as the liver dysfunction caused by endotoxaemia in the rat. Thus, an enhanced formation of NO may contribute to the development of fiver failure in endotoxic shock.

Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock.

1 We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine‐ inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2 Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg−1, i.v.) resulted in a fall in MAP from 115 ± 4mmHg (time 0, control) to 79 ± 9mmHg at 180 min (P<0.05, n = 10). The pressor effect of noradrenaline (NA, 1 μg kg−1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg_1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 ± 3mmHg, n= 10, P<0.05) when compared to rats given only LPS (LPS‐rats). Cumulative administration of aminoguanidine (15 mg kg−1 and 45mgkg−1) given 180 min after LPS caused a dose‐related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS‐ rats. 3 Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractile responses elicited by NA (10−9‐10−6 m). Pretreatment with aminoguanidine (15 mg kg−1, i.v., at 20 min prior to LPS) significantly prevented this LPS‐induced hyporeactivity to NA ex vivo. 4 Endotoxaemia for 180 min resulted in a significant increase in iNOS activity in the lung from 0.6 ± 0.2 pmol mg−1 min−1 (control, n = 4) to 4.8 ± 0.3 pmol mg−1 min−1 (P<0.05, n = 6). In LPS‐rats treated with aminoguanidine, iNOS activity in the lung was attenuated by 44 ± 5% (n = 6, P<0.05). Moreover, when added in vitro to lung homogenates obtained from LPS‐rats, aminoguanidine and Nω‐nitro‐L‐arginine methyl ester (l‐NAME; 10−8 to 10−3 m) caused a concentration‐dependent inhibition of iNOS activity (n = 3–6, IC50: 30 ± 12 and 11 ± 6μm, respectively P>0.05). In contrast, aminoguanidine was a less potent inhibitor than L‐NAME of the constitutive nitric oxide synthase in rat brain homogenates (n = 3–6, IC50 is 140 ± 10 and 0.6 ± 0.1 μm, respectively, P<0.05). In addition, the inhibitory effect of aminoguanidine on iNOS activity showed a slower onset than that of L‐NAME (maximal inhibition at 90 min and 30 min, respectively). 5 Treatment of conscious Swiss albino (T/O) mice with a high dose of endotoxin (60 mg kg−1, i.p.) resulted in a survival rate of only 8% at 24 h (n=12). However, therapeutic application of aminoguanidine (15 mg kg−1, i.p. at 2 h and 6 h after LPS) increased the 24 h survival rate to 75% (n = 8), whereas L‐NAME (3 mg kg−1, i.p. at 2h and 6h after LPS) did not affect the survival rate (11%, n = 9). 6 Thus, aminoguanidine inhibits iNOS activity and attenuates the delayed circulatory failure caused by endotoxic shock in the rat and improves survival in a murine model of endotoxaemia. Aminoguanidine, or novel, more potent selective inhibitors of iNOS may be useful in the therapy of septic shock.

Definition and drivers of acute traumatic coagulopathy: clinical and experimental investigations

Summary.  Background: Acute traumatic coagulopathy (ATC) is an impairment of hemostasis that occurs early after injury and is associated with a 4‐fold higher mortality, increased transfusion requirements and organ failure. Objectives: The purpose of the present study was to develop a clinically relevant definition of ATC and understand the etiology of this endogenous coagulopathy. Patients/methods: We conducted a retrospective cohort study of trauma patients admitted to five international trauma centers and corroborated our findings in a novel rat model of ATC. Coagulation status on emergency department arrival was correlated with trauma and shock severity, mortality and transfusion requirements. 3646 complete records were available for analysis. Results: Patients arriving with a prothrombin time ratio (PTr) > 1.2 had significantly higher mortality and transfusion requirements than patients with a normal PTr (mortality: 22.7% vs. 7.0%; P < 0.001. Packed red blood cells: 3.5 vs. 1.2 units; P < 0.001. Fresh frozen plasma: 2.1 vs. 0.8 units; P < 0.001). The severity of ATC correlated strongly with the combined degree of injury and shock. The rat model controlled for exogenously induced coagulopathy and mirrored the clinical findings. Significant coagulopathy developed only in animals subjected to both trauma and hemorrhagic shock (PTr: 1.30. APTTr: 1.36; both P < 0.001 compared with sham controls). Conclusions: ATC develops endogenously in response to a combination of tissue damage and shock. It is associated with increased mortality and transfusion requirements in a dose‐dependent manner. When defined by standard clotting times, a PTr > 1.2 should be adopted as a clinically relevant definition of ATC.