Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.     

The therapeutic value of protein (de)nitrosylation in experimental septic shock

Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier of protein function. We studied whether protein nitrosylation/denitrosylation would impact positively in hemodynamic parameters of septic rats. Polymicrobial sepsis was induced by cecal ligation and puncture. Female Wistar rats were treated with increasing doses of DTNB [5,5′-dithio-bis-(2-nitrobenzoic acid)] 30 min before or 4 or 12 h after sepsis induction. Twenty-four hours after surgery the following data was obtained: aorta response to phenylephrine, mean arterial pressure, vascular reactivity to phenylephrine, biochemical markers of organ damage, survival and aorta protein nitrosylation profile. Sepsis substantially decreases blood pressure and the response of aorta rings and of blood pressure to phenylephrine, as well as increased plasma levels of organ damage markers, mortality of 60% and S-nitrosylation of aorta proteins increased during sepsis. Treatment with DTNB 12 h after septic shock induction reversed the loss of response of aorta rings and blood pressure to vasoconstrictors, reduced organ damage and protein nitrosylation and increased survival to 80%. Increases in protein S-nitrosylation are related to cardiovascular dysfunction and multiple organ injury during sepsis. Treatment of rats with DTNB reduced the excessive protein S-nitrosylation, including that in calcium-dependent potassium channels (BK_Ca), reversed the cardiovascular dysfunction, improved markers of organ dysfunction and glycemic profile and substantially reduced mortality. Since all these beneficial consequences were attained even if DTNB was administered after septic shock onset, protein (de)nitrosylation may be a suitable target for sepsis treatment.     

A selective inducible NOS dimerization inhibitor prevents systemic,cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.     

Relative contribution of hemopoietic and pulmonary parenchymal cells to lung inducible nitric oxide synthase (inos) activity in murine endotoxemia

Acute lung injury is an important feature of sepsis and increased iNOS expression and NO production contribute to the pathogenesis of this syndrome. We generated bone marrow-transplanted chimeric mice with iNOS expression limited to either inflammatory or pulmonary parenchymal cells, and assessed pulmonary iNOS activity and systemic levels of NO metabolites in an endotoxemic model of sepsis. We found that while both pulmonary parenchymal cells and inflammatory cells contribute to the increased lung iNOS activity in endotoxemia, pulmonary parenchymal cells contribute to a significantly greater degree. Using measurement of plasma NO(-)(x), whole body NO production was assessed in this model. We found that the main source of NO(-)(x) was again, parenchymal cells and not inflammatory cells. This is the first study to demonstrate that most of the increased NO production in this model of endotoxemic sepsis derives from parenchymal cells rather than inflammatory cells.     

Release of atrial natriuretic polypeptide by graded right atrial distension in anesthetized dogs

In order to verify the contribution of right atrial pressure to atrial natriuretic polypeptides (ANP) release, we measured plasma levels of immunoreactive (ir)-ANP when graded rise of right atrial pressure was executed in anesthetized dogs. Increasing right atrial pressure (RAP) from 2.7 +/- 0.6 to 9.0 +/- 0.7 mmHg, plasma levels of ir-ANP in aorta tended to increase by 33% but not significantly (p greater than 0.05). However, when RAP was increased from 9.0 +/- 0.7 to 17.0 +/- 1.1 mmHg, ir-ANP levels in aorta were significantly (p less than 0.05) increased by 132% of control within 5 min from the start of RAP elevation. The RAP elevation produced a sustained increase in plasma levels of ir-ANP. There was a positive correlation between right atrial pressure and plasma levels of ir-ANP. The plasma levels of ir-ANP were similar between aorta and pulmonary artery. These results demonstrate that increasing atrial pressure is closely correlated with ANP release and ANP is not greatly metabolized by pulmonary circulation.     

Differential effects of PACAP and VIP on the pulmonary and hindquarters vascular beds of the cat.

Responses to pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide derived from ovine hypothalamus with 68% sequence homology with vasoactive intestinal polypeptide (VIP), were investigated in the pulmonary and hindquarters vascular beds of the anesthetized cat under conditions of controlled blood flow. Injection of the peptide into the perfused lung lobe under elevated tone conditions produced dose-dependent decreases in lobar arterial pressure that were accompanied by biphasic changes in systemic arterial pressure characterized by an initial decrease followed by a secondary increase in pressure. When compared with other vasodilator agents in the pulmonary vascular bed, the relative order of potency was isoproterenol greater than PACAP greater than acetylcholine greater than calcitonin gene-related peptide greater than VIP. In the hindquarters vascular bed, intra-arterial injections of PACAP produced biphasic changes in hindquarters perfusion pressure characterized by initial decreases followed by secondary increases, which were accompanied by biphasic changes in systemic arterial pressure. In terms of relative vasodilator activity in the hindlimb, the order of relative potency was isoproterenol greater than acetylcholine greater than calcitonin gene-related peptide greater than VIP greater than PACAP. PACAP was the only agent that caused a secondary vasoconstrictor response in the hindlimb and produced biphasic changes in systemic arterial pressure. D-Phe2-VIP, a VIP receptor antagonist, blocked the hindquarters vasodilation in response to VIP but had no effect on responses to PACAP. The present investigation shows that PACAP produces pulmonary vasodilation, as well as dilation, and vasoconstriction in the systemic (hindlimb) vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taurine and zoo felids: considerations of dietary and biological tissue concentrations

Taurine (TAU) is an essential amino acid required in the diets of Felidae at concentrations ranging between 0.04 and 0.2% on a dry matter (DM) basis (in purified, highly digestible diets, and canned diets, respectively). Although the domestic cat seems to be an appropriate physiologic model for zoo felids, it is sometimes difficult to assess TAU status in zoo feeding programs owing to scattered information on feed ingredient TAU content as well as a lack of normal ranges for assessment of TAU in biological tissues. Knowing that TAU is required in the formulation of hand‐rearing diets for exotic felids, the TAU content of 38 ingredients or products used in zoo carnivore feeding or hand‐rearing programs was summarized, including 21 new feedstuffs for which TAU data were previously lacking. The kitten milk replacer contained a lower than expected value for TAU. Commercially prepared frozen or canned meat products, seafood products, whole rodent prey, and most strained meat jarred baby foods contained adequate TAU; chunk meats, and some specific types of jarred baby food meats were considerably lower in TAU content (≤0.10% DM) than other foodstuffs. TAU concentrations in plasma and whole blood of eight spp. of zoo felids sampled opportunistically fell within reference ranges for domestic cats (80–120 and 300–600 nmol/ml in plasma and whole blood, respectively). Plasma concentrations are a useful measure of dietary impact, whereas whole blood concentrations seem to reflect tissue storage of this nutrient. Zoo Biol 26:517–531, 2007. © 2007 Wiley‐Liss, Inc.     

Augmented secretion of brain natriuretic peptide in acute myocardial infarction.

In order to elucidate biosynthesis and secretion of natriuretic peptides in the early phase of acute myocardial infarction (AMI), we measured the plasma level of brain natriuretic peptide (BNP), a novel cardiac hormone secreted from the ventricle, in patients with AMI and compared with that of atrial natriuretic peptide (ANP). The plasma level of BNP increased rapidly (within hours from the onset of AMI) and markedly (greater than 100 times the normal level) as compared to that of ANP. The plasma ANP level correlated with pulmonary capillary wedge pressure (PCWP), whereas the plasma BNP level did not correlate with PCWP but highly correlated inversely with cardiac index. These results indicate that BNP is secreted from the heart much more acutely and prominently than ANP in the early phase of AMI, in association with left ventricular dysfunction.     

An Exploratory Analysis of the Utility of Adding Cardiorespiratory Biofeedback in the Standard Care of Pregnancy-Induced Hypertension

This study examined the efficacy of a cardiorespiratory biofeedback intervention compared to bed rest in the treatment of 47 women diagnosed with pregnancy-induced hypertension (PIH). The investigation consisted of a historical control group with 31 PIH subjects receiving treatment as usual (TAU), bed rest and antihypertensive medications, and an experimental group with 16 PIH subjects receiving TAU and instruction on using a portable respiratory sinus arrhythmia (RSA) biofeedback device once daily until delivery. Results indicated that systolic and diastolic blood pressure levels were unchanged for either group. Failing to find the intended main effects, a series of exploratory analyses were performed. Findings of associated hypotheses revealed that the RSA BF group had a 35 % higher birth weight than the TAU group. The gestational age at delivery was 10 % greater in the RSA BF group than in the TAU group. A significant relationship was found between the StressEraser Total and the 1-min Apgar score. Eighty-one percent of the subjects stated that the device was relaxing. Fifty percent of the subjects believed that the device helped them fall asleep. Overall, these results suggest that portable RSA biofeedback may be effective in reducing stress during pregnancy and improving perinatal outcomes.     

Effect of prolonged renal dysfunction on intravascular and extravascular pulmonary fluid volumes during left atrial hypertension

To examine the development of pulmonary edema during experimental renal dysfunction, left atrial pressure was altered in 14 mongrel dogs divided into two groups. Group 1 was composed of seven control animals, and Group 2 was composed of seven animals with surgically induced renal failure (1 week of bilateral ureteral ligation). Data were obtained at two levels of matched transmural pulmonary vascular pressure (defined as mean left atrial pressure less serum protein osmotic pressure). In the animals with renal dysfunction, extravascular lung water (EVLW) (thermal-green dye technique) was higher at moderately (-1 to -2 mm Hg) and severely elevated (11 to 12 mm Hg) vascular driving pressures (11.5 +/- 1.2 cc/kg vs 10.6 +/- 0.8 cc/kg and 14.8 +/- 1.3 cc/kg vs 13.0 +/- 1.9 cc/kg, respectively, both P less than 0.05 vs control). Because protein osmotic pressure was lower in the renal failure group (15.0 +/- 1.8 mm Hg vs 18.4 +/- 1.4 mm Hg, P less than 0.05), greater accumulations of extravascular lung water occurred at lower levels of left atrial pressure (14.2 +/- 1.4 mm Hg vs 17.1 +/- 1.2 mm Hg, P less than 0.05; 26.8 +/- 2.6 mm Hg vs 29.5 +/- 2.3 mm Hg, P less than 0.01). In addition, when the ratio of EVLW/PBV (pulmonary blood volume) was examined in both groups at each stage of the experiment, the ratio was greater in the Group 2 animals at each elevated pressure, suggesting increased permeability with renal dysfunction. In conclusion, pulmonary edema formation occurs at lower left atrial pressures in the setting of sustained renal dysfunction, this phenomenon can be partially explained by lower protein osmotic pressure though altered pulmonary microvascular permeability may contribute to edema formation.     

Role of C5a in multiorgan failure during sepsis

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.     

老年急性呼吸窘迫综合征肺内及肺外源性危险因素分析

目的:探讨老年急性呼吸窘迫综合征肺内及肺外源性危险因素。方法:回顾性分析130例老年ARDS患者,对其中的肺内及肺外源性危险因素进行分析。结果:肺内源性ARDS病因以误吸和肺炎为主,而肺外源性ARDS则以脓毒血症、大手术后等为主;在死亡上均与多器官功能障碍综合征、呼吸衰竭为主要因素,且两组死亡率接近。结论:在老年急性呼吸窘迫综合征中,肺外源性在器官功能衰竭和氧合指数上重于肺内源性,但是在其他因素和死亡结局上均无明显差异性。     

Comparative study of the binding characteristics to and inhibitory potencies towards PARP and in vivo antidiabetogenic potencies of taurine, 3-aminobenzamide and nicotinamide

Background

Poly(ADP-ribose) is a NAD⁺-requiring, DNA-repairing, enzyme playing a central role in pancreatic β-cell death and in the development of endothelial dysfunction in humans and experimental animals. PARP activation is also relevant to the development of complications of diabetes. Hence, agents capable of inhibiting PARP may be useful in preventing the development of diabetes and in slowing down complications of diabetes.

Methods

PARP inhibition was assessed with a colorimetric assay kit. Molecular docking studies on the active site of PARP were conducted using the crystalline structure of the enzyme available as Protein Data Bank Identification No. 1UK1. Type 2 diabetes was induced in male Sprague-Dawley rats with streptozotocin (STZ, 60 mg/kg, i.p.). The test compounds (3-aminobenzamide = 3-AB, nicotinamide = NIC, taurine = TAU) were given by the i.p. route 45 min before STZ at 2.4 mM/kg (all three compounds) or 1.2 and 3.6 mM/kg (only NIC and TAU). Blood samples were collected at 24 hr after STZ and processed for their plasma. The plasma samples were used to measure glucose, insulin, cholesterol, triglycerides, malondialdehyde, nitric oxide, and glutathione levels using reported methods.

Results

3-AB, NIC and TAU were able to inhibit PARP, with the inhibitory potency order being 3-AB>NIC>>TAU. Molecular docking studies at the active site of PARP showed 3-AB and NIC to interact with the binding site for the nicotinamide moiety of NAD⁺ and TAU to interact with the binding site for the adenine moiety of NAD⁺. While STZ-induced diabetes elevated all the experimental parameters examined and lowered the insulin output, a pretreatment with 3-AB, NIC or TAU reversed these trends to a significant extent. At a dose of 2.4 mm/kg, the protective effect decreased in the approximate order 3-AB>NIC≥TAU. The attenuating actions of both NIC and TAU were dose-related except for the plasma lipids since NIC was without a significant effect at all doses tested.

Conclusions

At equal molar doses, 3-AB was generally more potent than either TAU or NIC as an antidiabetogenic agent, but the differences were not as dramatic as would have been predicted from their differences in PARP inhibitory potencies. NIC and TAU demonstrated dose-related effects, which in the case of TAU were only evident at doses ≥2.4 mM/kg. The present results also suggest that in the case of NIC and TAU an increase in dose will enhance the magnitude of their attenuating actions on diabetes-related biochemical alterations to that achieved with a stronger PARP inhibitor such as 3-AB. Hence, dosing will play a critical role in clinical studies assessing the merits of NIC and TAU as diabetes-preventing agents.

     

Superior cervical ganglionectomy effects on median eminence and anterior and mediobasal hypothalamic taurine content in male rats: effects of hyperprolactinemia

The neuroendocrine sequelae of acute or chronic superior cervical ganglionectomy in control or pituitary-grafted rats were studied by analyzing both plasma prolactin, growth hormone (GH) and ACTH levels, and taurine (TAU) content in the hypophysiotropic area of the hypothalamus or the median eminence. As expected, after either acute or chronic ganglionectomy, norepinephrine (NE) content decreased in the brain areas studied, although the values remained higher in hyperprolactinemic rats. TAU content was differentially modified by acute vs. chronic surgeries, thus indicating the possible existence of hypothalamic interactions between TAU and NE to regulate pituitary hormone secretion. Indeed, associated differential changes in plasma prolactin, GH and ACTH levels may be due to the observed TAU changes. As expected, pituitary grafting increased plasma prolactin, GH and ACTH levels, so that the presence of a pituitary graft differentially interferes with the effects of either surgery not only on TAU content but also on the plasma levels of the hormone studied. Globally, ongoing studies confirm the differential effects of acute and chronic superior cervical ganglionectomy on plasma prolactin, GH and ACTH levels, and provide new evidence about its effects on TAU content in the hypophysiotropic area of the hypothalamus and the median eminence that may partially explain the changes observed in the pituitary hormones studied.     

Ibuprofen attenuates plasma tumor necrosis factor activity during sepsis-induced acute lung injury.

Plasma tumor necrosis factor (TNF) activity, cardiac index, extravascular lung water, systemic and pulmonary arterial pressures, pulmonary vascular resistance index, and arterial PO2 were monitored for 300 min in four groups of anesthetized pigs: saline-infused animals (n = 5), saline-infused animals given ibuprofen (12.5 mg/kg iv) at 0 and 120 min (n = 4), animals infused for 60 min with live Pseudomonas aeruginosa (Ps, 5 x 10(8) organisms/ml at 0.3 ml.20 kg-1.min-1, n = 6), and animals infused for 60 min with Ps plus ibuprofen administered at 0 and 120 min (n = 4). Infusion of Ps induced significant elevations (greater than 4-fold increase in units/ml of TNF by 60 min, P less than 0.05) in plasma TNF activity (L929 cytolysis assay) and alterations (P less than 0.05) in all hemodynamic and pulmonary parameters within 30-60 min. Ibuprofen administration in sepsis significantly decreased peak TNF activity by 2 units/ml and attenuated many of the physiological alterations due to sepsis. These results show that ibuprofen attenuates sepsis-induced injury and that alterations of acute septic insult are correlated with reduced plasma TNF activity in septic animals given ibuprofen.     

Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets.

The pulmonary vasoconstrictor, thromboxane, may contribute to the development of pulmonary hypertension. Our objective was to determine whether a combined thromboxane synthase inhibitor-receptor antagonist, terbogrel, prevents pulmonary hypertension and the development of aberrant pulmonary arterial responses in newborn piglets exposed to 3 days of hypoxia. Piglets were maintained in room air (control) or 11% O(2) (hypoxic) for 3 days. Some hypoxic piglets received terbogrel (10 mg/kg po bid). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. A cannulated artery technique was used to measure responses to acetylcholine. Pulmonary vascular resistance for terbogrel-treated hypoxic piglets was almost one-half the value of untreated hypoxic piglets but remained greater than values for control piglets. Dilation to acetylcholine in preconstricted pulmonary arteries was greater for terbogrel-treated hypoxic than for untreated hypoxic piglets, but it was less for pulmonary arteries from both groups of hypoxic piglets than for control piglets. Terbogrel may ameliorate pulmonary artery dysfunction and attenuate the development of chronic hypoxia-induced pulmonary hypertension in newborns.     

感染性疾病内皮活化/功能障碍的生物标记物的研究进展

内皮功能障碍在脓毒症、重症疟疾及登革出血热等多种感染性疾病发病机制中起着重要作用。由于内皮活化常先于内皮功能障碍,血清或/血浆中相应内皮生物标记物常较传统疾病标记物更早被检测到,故可作为评估全身感染性疾病严重程度或预后的指标。本文主要介绍了内皮细胞功能介质(血管生成素-1和血管生成素-2)和可溶性细胞表面黏附分子(可溶性E-选择素、sICAM-1和sVCAM-1)在脓毒症、疟疾及登革出血热等疾病中的表达及临床意义。     

An observational cohort study of the kynurenine to tryptophan ratio in sepsis: association with impaired immune and microvascular function

Both endothelial and immune dysfunction contribute to the high mortality rate in human sepsis, but the underlying mechanisms are unclear. In response to infection, interferon-γ activates indoleamine 2,3-dioxygenase (IDO) which metabolizes the essential amino acid tryptophan to the toxic metabolite kynurenine. IDO can be expressed in endothelial cells, hepatocytes and mononuclear leukocytes, all of which contribute to sepsis pathophysiology. Increased IDO activity (measured by the kynurenine to tryptophan [KT] ratio in plasma) causes T-cell apoptosis, vasodilation and nitric oxide synthase inhibition. We hypothesized that IDO activity in sepsis would be related to plasma interferon-γ, interleukin-10, T cell lymphopenia and impairment of microvascular reactivity, a measure of endothelial nitric oxide bioavailability. In an observational cohort study of 80 sepsis patients (50 severe and 30 non-severe) and 40 hospital controls, we determined the relationship between IDO activity (plasma KT ratio) and selected plasma cytokines, sepsis severity, nitric oxide-dependent microvascular reactivity and lymphocyte subsets in sepsis. Plasma amino acids were measured by high performance liquid chromatography and microvascular reactivity by peripheral arterial tonometry. The plasma KT ratio was increased in sepsis (median 141 [IQR 64–235]) compared to controls (36 [28–52]); p<0.0001), and correlated with plasma interferon-γ and interleukin-10, and inversely with total lymphocyte count, CD8+ and CD4+ T-lymphocytes, systolic blood pressure and microvascular reactivity. In response to treatment of severe sepsis, the median KT ratio decreased from 162 [IQR 100–286] on day 0 to 89 [65–139] by day 7; p = 0.0006) and this decrease in KT ratio correlated with a decrease in the Sequential Organ Failure Assessment score (p<0.0001). IDO-mediated tryptophan catabolism is associated with dysregulated immune responses and impaired microvascular reactivity in sepsis and may link these two fundamental processes in sepsis pathophysiology.     

Disruption of methylarginine metabolism impairs vascular homeostasis

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.     

In vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue.

In vitro evidence suggests that resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation are mediated by changes in vascular smooth muscle concentrations of guanosine 3',5'-cyclic monophosphate (cGMP). We investigated this hypothesis in vivo in 19 mechanically ventilated intact lambs by determining the hemodynamic effects of methylene blue (a guanylate cyclase inhibitor) and then by comparing the hemodynamic response to five vasodilators during pulmonary hypertension induced by the infusion of U-46619 (a thromboxane A2 mimic) or methylene blue. Methylene blue caused a significant time-dependent increase in pulmonary arterial pressure. During U-46619 infusions, acetylcholine, ATP-MgCl2, sodium nitroprusside, isoproterenol, and 8-bromo-cGMP decreased pulmonary arterial pressure. During methylene blue infusions, the decreases in pulmonary arterial pressure caused by acetylcholine and ATP-MgCl2 (endothelium-dependent vasodilators) and sodium nitroprusside (an endothelium-independent guanylate cyclase-dependent vasodilator) were attenuated by greater than 50%. The decreases in pulmonary arterial pressure caused by isoproterenol and 8-bromo-cGMP (endothelium-independent vasodilators) were unchanged. This study in intact lambs supports the in vitro evidence that changes in vascular smooth muscle cell concentrations of cGMP in part mediate resting pulmonary vascular tone and endothelium-dependent pulmonary vasodilation.