Raymond Stidwill

Pulmonary Hypertension in a GTP-Cyclohydrolase 1–Deficient Mouse

Background—GTP-cyclohydrolase 1 (GTP-CH1) catalyzes the first step for the de novo production of tetrahydrobiopterin (BH4), a cofactor for nitric oxide synthase (NOS). The hyperphenylalaninemic mutant mouse (hph-1) displays a 90% reduction in GTP-CH1 activity. Reduced BH4 decreases NOS activity and may lead to endothelial dysfunction, and there is increasing evidence that a dysfunction of the NOS pathway may be implicated in pulmonary hypertension. The aim of the study was to investigate whether reduced BH4 in the hph-1 mouse results in a pulmonary hypertensive phenotype. Methods and Results—Morphological characterization of the heart, lung, and kidney and measurements of systemic and right ventricular blood pressures were performed in both hph-1 and wild-type mice. BH4 and NOx levels were also measured. Hph-1 mice had significantly lower NOx and BH4 levels, consistent with previous findings. Both morphological and in vivo data were indicative of a pulmonary but not systemic hypertensive phenotype. We observed increased right ventricle–left ventricle plus septum ratios attributable only to an increase in right ventricular mass, increased smooth muscle medial area in pulmonary resistance vessels, and significantly higher right ventricular pressures in vivo. There were no significant differences between left ventricular masses and systemic pressures, and there was no observed evidence of systemic hypertension in kidney sections between wild-type and hph-1. Conclusions—This study demonstrates that mice deficient in GTP-CH1/BH4 display a pulmonary hypertensive but not systemic hypertensive phenotype.

Succinate recovers mitochondrial oxygen consumption in septic rat skeletal muscle

Objective:Mitochondrial dysfunction, particularly affecting complex I of the respiratory chain, could play a fundamental role in the development of multiple organ failure during sepsis. Increasing electron flow through complex II by addition of succinate may improve mitochondrial oxygen utilization and thus adenosine triphosphate production. Design:Ex vivo animal study. Setting:University research laboratory. Subjects:Male adult Wistar rats. Interventions:Fecal peritonitis was induced in conscious, fluid-resuscitated, hemodynamically-monitored rats. Sham-operation and naïve animals acted as controls. At 48 hrs, clinical severity was graded. Soleus muscle was taken for measurement of mitochondrial complex activities and oxygen consumption. The effect of glutamate plus malate (complex I substrates) and succinate (complex II substrate) on mitochondrial respiration was assessed. Measurements and Main Results:In the presence of glutamate plus malate, mitochondrial oxygen consumption was abnormally low in skeletal muscle tissue from moderately-to-severely septic animals as compared with naïve and sham-operation controls (both p < .01). On addition of succinate, mitochondrial respiration was augmented in all groups, particularly in moderately-to-severely septic animals (39% ± 6% increase) as compared with naïve (11% ± 5%; p < .01) and sham-operation controls (10% ± 5%; p < .01). In the presence of succinate, mitochondrial oxygen consumption was similar between the groups. Conclusions:Succinate increases mitochondrial oxygen consumption in ex vivo skeletal muscle taken from septic animals, bypassing the predominant inhibition occurring at complex I. This warrants further exploration in vivo as a putative therapeutic modality.

Differential effects of vasopressin and norepinephrine on vascular reactivity in a long-term rodent model of sepsis.

Objective:There is escalating interest in the therapeutic use of vasopressin in septic shock. However, little attention has focused on mechanisms underlying its pressor hypersensitivity, which contrasts with the vascular hyporesponsiveness to catecholamines. We investigated whether a long-term rodent model of sepsis would produce changes in endogenous levels and pressor reactivity to exogenous norepinephrine and vasopressin comparable with those seen in septic patients. Design:In vivo and ex vivo animal study. Setting:University research laboratory. Subjects:Male adult Wistar rats. Interventions and Measurements:Fecal peritonitis was induced in conscious, fluid-resuscitated rats. Biochemical and hormonal profiles were measured at time points up to 48 hrs. Pressor responses to intravenous norepinephrine, vasopressin, and F-180, a selective V1 receptor agonist, were measured at 24 hrs. Contractile responses to these drugs were assessed in mesenteric arteries taken from animals at 24 hrs using wire myography. Comparisons were made against sham operation controls. Main Results:Septic rats became unwell and hypotensive, with a mortality of 64% at 48 hrs (0% in controls). Plasma norepinephrine levels were elevated in septic animals at 24 hrs (1968 ± 490 vs. 492 ± 90 pg/mL in controls, p = .003), whereas vasopressin levels were similar in the two groups (4.5 ± 0.8 vs. 3.0 ± 0.5 pg/mL, p = not significant). In vivo, the pressor response to norepinephrine was markedly reduced in the septic animals, but responses to vasopressin and F-180 were relatively preserved. In arteries from septic animals, norepinephrine contractions were decreased (efficacy as measured by maximum contractile response, Emax: 3.0 ± 0.3 vs. 4.7 ± 0.2 mN, p < .001). In contrast, the potency of vasopressin (expressed as the negative log of the concentration required to produce 50% of the maximum tension, pD2: 9.1 ± 0.04 vs. 8.7 ± 0.05, p < .001) and F-180 (pD2 8.2 ± 0.04 vs. 7.6 ± 0.02, p < .001) was enhanced (n ≥ 6 for all groups). Conclusions:This long-term animal model demonstrates changes in circulating vasoactive hormones similar to prolonged human sepsis, and decreased pressor sensitivity to norepinephrine. Ex vivo sensitivity to vasopressin agonists was heightened. This model is therefore appropriate for the further investigation of mechanisms underlying vasopressin hypersensitivity, which may include receptor or calcium-handling alterations within the vasculature.

Genetic and Pharmacological Inhibition of Dimethylarginine Dimethylaminohydrolase 1 Is Protective in Endotoxic Shock

Objective—The overproduction of vascular NO contributes toward the circulatory collapse observed in patients with septic shock. Dimethylarginine dimethylaminohydrolase (DDAH), which has 2 isoforms, metabolizes asymmetrically methylated arginines (asymmetric mono- or di-methylarginine), endogenously produced NO synthase inhibitors. We wished to investigate whether reducing DDAH1 activity, using genetic and pharmacological approaches, is protective during lipopolysaccharide-induced endotoxic shock. Methods and Results—Experiments were conducted in DDAH1 heterozygous knockout mice (DDAH1+/−) or naive rats treated with a synthetic pharmacological DDAH inhibitor (L-257). We demonstrate for the first time that L-257 is DDAH1 selective using recombinant human DDAH proteins. DDAH1 mRNA was expressed in aortic but not macrophage cDNA, and consistent with this expression profile, L-257 selectively inhibited NO production from lipopolysaccharide-treated aorta but not macrophages, in culture. Conscious and anesthetized cardiovascular hemodynamics were monitored using implanted radiotelemetry devices or invasive catheters, respectively. Lipopolysaccharide was administered intravenously to model endotoxemia, and all animals presented with circulatory shock. DDAH1+/− mice or L-257–treated rats displayed attenuation in the rate of developed hypotension compared with wild-type littermates or vehicle control animals, respectively. Conclusion—Pharmacological and genetic reduction of DDAH1 activity is protective against the vascular changes observed during endotoxic shock.

Variable effects of inhibiting iNOS and closing the vascular ATP-sensitive potassium channel (via its pore-forming and sulfonylurea receptor subunits) in endotoxic shock.

Excess production of NO and activation of vascular ATP-sensitive potassium (KATP) channels are implicated in the hypotension and vascular hyporeactivity associated with endotoxic shock. Using a fluid-resuscitated endotoxic rat model, we compared the cardiovascular effects of an iNOS inhibitor and two distinct inhibitors of the KATP channel. Endotoxin (LPS) was administered to anesthetized, spontaneously breathing, fluid-resuscitated adult male Wistar rats, in which MAP, aortic and renal blood flow, and hepatic microvascular oxygenation were monitored continuously. At 120 min, the iNOS inhibitor, GW273629, and the KATP-channel inhibitors, PNU-37883A and glyburide, were administered separately, and their effects on hemodynamics and oxygenation were examined. We found that GW273629 increased MAP over and above the pressor effect achieved in sham animals. Inhibiting KATP channels via the pore-forming subunit (PNU-37883A and high-dose glyburide) produced significant pressor effects, whereas inhibiting the sulfonylurea receptor with low-dose glyburide was ineffective. No agent reversed the fall in aortic or renal blood flow, the fall in hepatic microvascular oxygenation, or the metabolic acidosis that occurred in LPS-treated animals. We conclude that inhibition of the KATP channel via the pore-forming, but not the sulfonylurea receptor subunit, increases blood pressure in a short-term endotoxic model. However, this was not accompanied by any improvement in macrocirculatory or microcirculatory organ blood flow nor reversal of metabolic acidosis. It therefore remains uncertain whether the iNOS pathway or the KATP channel represents a potential target for drug development in the treatment of endotoxic shock.

Inhibition of vascular adenosine triphosphate-sensitive potassium channels by sympathetic tone during sepsis.

Objective:Excessive opening of the adenosine triphosphate-sensitive potassium channel in vascular smooth muscle is implicated in the vasodilation and vascular hyporeactivity underlying septic shock. Therapeutic channel inhibition using sulfonylurea agents has proved disappointing, although agents acting on its pore appear more promising. We thus investigated the hemodynamic effects of adenosine triphosphate-sensitive potassium channel pore inhibition in awake, fluid-resuscitated septic rats, and the extent to which these responses are modulated by the high sympathetic tone present in sepsis. Temporal changes in ex-vivo channel activity and subunit gene expression were also investigated. Design:In vivo and ex vivo animal study. Setting:University research laboratory. Subjects:Male adult Wistar rats. Interventions and Measurements:Fecal peritonitis was induced in conscious, fluid-resuscitated rats. Pressor responses to norepinephrine and PNU-37883A (a vascular adenosine triphosphate-sensitive potassium channel inhibitor acting on the Kir6.1 pore-forming subunit) were measured at 6 or 24 hrs, in the absence or presence of the autonomic ganglion blocker, pentolinium. The aorta and mesenteric artery were examined ex vivo for 86rubidium efflux as a marker of adenosine triphosphate-sensitive potassium channel activity, and for adenosine triphosphate-sensitive potassium channel subunit gene expression using quantitative reverse transcription-polymerase chain reaction. Main Results:A total of 120 rats (50 sham-operated controls, 70 septic) were included. Septic rats became hypotensive after 12 hrs, with a 24-hr mortality of 51.7% (0% in controls). At 6 hrs, there was an attenuated pressor response to norepinephrine (p < .01) despite blood pressure being elevated (p < .01). PNU-37883A had no pressor effect, except in the presence of pentolinium (p < .01). Kir6.1 subunit mRNA increased significantly in the mesenteric artery while 86rubidium efflux was increased in both the aorta and mesenteric artery at 24 hrs. Conclusions:Despite evidence of increased adenosine triphosphate-sensitive potassium channel activity in sepsis, it appears to be inhibited in vivo by high sympathetic tone. This may explain, at least in part, the reduced efficacy of adenosine triphosphate-sensitive potassium channel blockers in human septic shock. (Crit Care Med 2012; 40:–1268)

A novel DDAH-1 inhibitor improved sepsis-induced impairment in vasoreactivity to noradrenaline in a rat endotoxaemia model

In septic shock, iNOS activation and nitric oxide (NO) overproduction contribute to vascular hyporeactivity to adrenergic vasopressors. The consequent hypotension often necessitates high doses of catecholamine administration. However, this may lead to detrimental effects on tissue perfusion, immune function and myocardial function. Asymmetric dimethlyarginine (ADMA), an endogenous inhibitor of NO synthase, is extensively metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Competitive inhibition of the DDAH-1 isoform should thus reverse hypotension but, as this isoform is absent in immune cells, it should not compromise the immune effects of NO. Hence, we investigated whether L257, a novel DDAH-1 inhibitor, could spare norepinephrine dosing in a rat endotoxic shock model.

A novel DDAH-1 inhibitor improved cardiovascular function in a short-term anesthetized rat model of sepsis

Excessive NOS activity and NO overproduction are believed to play an important role in sepsis-induced macrocirculatory and microcirculatory dysfunction. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, is extensively metabolised by dimethylarginine dimethylaminohydrolase (DDAH). The DDAH-1 isoform is present in vascular smooth muscle so its inhibition should theoretically reverse sepsis-induced hypotension. We thus investigated the dose-dependent cardiovascular effects of a novel DDAH-1 competitive inhibitor, L-257, in experimental sepsis.