Robert R. Clancy

Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase.

Nitric oxide provokes vasodilation and inhibits platelet aggregation. We examined the effect of nitric oxide on superoxide anion production by three sources: activated intact neutrophils, xanthine oxidase/hypoxanthine, and the NADPH oxidase. Nitric oxide significantly inhibited the generation of superoxide anion by neutrophils exposed to either FMLP (10(-7)M) or PMA (150 ng/ml) (IC50 = 30 microM). To determine whether the effect of nitric oxide on the respiratory burst was due to simple scavenging of O2+, kinetic studies that compared effects on neutrophils and the cell-free xanthine oxidase system were performed. Nitric oxide inhibited O2+ produced by xanthine oxidase only when added simultaneously with substrate, consistent with the short half-life of NO in oxygenated solution. In contrast, the addition of nitric oxide to neutrophils 20 min before FMLP resulted in the inhibition of O2+ production, which suggests formation of a stable intermediate. The effect of nitric oxide on the cell-free NADPH oxidase superoxide-generating system was also examined: The addition of NO before arachidonate activation (t = -6 min) significantly inhibited superoxide anion production. Nitric oxide did not inhibit O2+ when added at NADPH initiation (t = 0). Treatment of the membrane but not cytosolic component of the oxidase was sufficient to inhibit O2+ generation. The data suggest that nitric oxide inhibits neutrophil O2+ production via direct effects on membrane components of the NADPH oxidase. This action must occur before the assembly of the activated complex.

The role of nitric oxide in the pathogenesis of preeclampsia

OBJECTIVE Nitric oxide, a potent vasodilator released by endothelial cells, inhibits platelet aggregation and adhesion to vascular endothelial surfaces. Because endothelial cell damage is considered pivotal in the pathogenesis of preeclampsia, this study was initiated to determine whether nitric oxide production is decreased in patients with preeclampsia. STUDY DESIGN Twenty-six patients with preeclampsia (as defined by a blood pressure > or = 140 mm Hg systolic or 90 mm Hg diastolic plus proteinuria, > or = 300 mg per 24 hours or > or = 2+ by dipstick, both occurring on two occasions > or = 4 hours apart) and 26 normotensive women with singleton gestations in the third trimester were studied. Because nitric oxide is spontaneously oxidized to both nitrite and nitrate, two analytic assays were used serially. Serum nitrite levels were initially determined with the Greiss reagent and subsequently analyzed with Escherichia coli nitrate reductase. RESULTS With the Greiss reagent alone the mean +/- SEM of serum nitrite level in 26 patients with preeclampsia was significantly decreased compared with 26 normotensive patients (3.46 +/- 1.43 mumol/L vs 4.65 +/- 0.85 mumol/L, p = 0.02). With the addition of the nitrate reductase enzyme of Escherichia coli the mean +/- SEM of serum nitrite level in 26 preeclamptic patients was again significantly decreased compared with 26 normotensive patients (20.04 +/- 1.25 mumol/L vs 27.38 +/- 2.23 mumol/L, p = 0.02). One patient with the syndrome of hemolysis, elevated liver enzymes, and low platelets demonstrated a concurrent decrease in serum nitrite over a 2-week period, emphasizing the relationship of nitric oxide to the pathophysiologic features of the syndrome. CONCLUSIONS Circulating levels of nitrite are decreased in patients with preeclampsia. These data support the concept that diminished nitric oxide synthesis contributes to the pathophysiologic changes seen in preeclampsia.

Brain maturation is delayed in infants with complex congenital heart defects.

OBJECTIVE Small head circumferences and white matter injury in the form of periventricular leukomalacia have been observed in populations of infants with severe forms of congenital heart defects. This study tests the hypothesis that congenital heart defects delay in utero structural brain development. METHODS Full-term infants with hypoplastic left heart syndrome or transposition of the great arteries were prospectively evaluated with preoperative brain magnetic resonance imaging. Patients with independent risk factors for abnormal brain development (shock, end-organ injury, or intrauterine growth retardation) were excluded. Outcome measures included head circumferences and the total maturation score on magnetic resonance imaging. Total maturation score is a previously validated semiquantitative anatomic scoring system used to assess whole brain maturity. The total maturation score evaluates 4 parameters of maturity: (1) myelination, (2) cortical infolding, (3) involution of glial cell migration bands, and (4) presence of germinal matrix tissue. RESULTS The study cohort included 29 neonates with hypoplastic left heart syndrome and 13 neonates with transposition of the great arteries at a mean gestational age of 38.9 +/- 1.1 weeks. Mean head circumference was 1 standard deviation below normal. The mean total maturation score for the cohort was 10.15 +/- 0.94, significantly lower than reported normative data in infants without congenital heart defects, corresponding to a delay of 1 month in structural brain development. CONCLUSION Before surgery, term infants with hypoplastic left heart syndrome and transposition of the great arteries have brains that are smaller and structurally less mature than expected. This delay in brain development may foster susceptibility to periventricular leukomalacia in the preoperative, intraoperative, and postoperative periods.

Circulating endothelial cells Biomarker of vascular disease

Recent research has recognised new populations of non-hematopoietic cells in the blood. One of these, circulating endothelial cells (CECs), often defined by the expression of membrane glycoprotein CD146, are rarely found in the blood in health, but raised numbers are present in a wide variety of human conditions, including inflammatory, immune, infectious, neoplastic and cardiovascular disease, and seem likely to be evidence of profound vascular insult. An additional population are endothelial progenitor cells, defined by the co-expression of endothelial and immaturity cell surface molecules and also by the ability to form colonies in vitro. Although increased numbers of CECs correlate with other markers of vascular disease, questions remain regarding the precise definition, cell biology and origin of CECs. For example, they may be damaged, necrotic or apopototic, or alive, and could possess procoagulant and/or proinflammatory properties. However, since these cells seem to be representative of in situ endothelium, their phenotype may provide useful information. Indeed, whatever their phenotype, there is growing evidence that CECs may well be a novel biomarker, the measurement of which will have utility in various clinical settings related to vascular injury. Despite this promise, progress is impeded by the diversity of methodologies used to detect these cells. Accordingly, results are sometimes inconclusive and even conflicting. Nevertheless, increased CECs predict adverse cardiovascular events in acute coronary syndromes, suggesting they may move from being simply a research index to having a role in the clinic. The objective of the present communication is to condense existing data on CECs, briefly compare them with progenitor cells, and summarise possible mechanism(s) by which they may contribute to vascular pathology.

NITRIC OXIDE : A NOVEL MEDIATOR OF INFLAMMATION

Nitric oxide (NO), first identified as an endothelium-derived relaxation factor, is now recognized to regulate the functions of many mammalian cells and tissues. Nitric oxide is synthesized via the oxidation of arginine by a family of nitric oxide synthases (NOS), which are either constitutive and calcium dependent (cNOS) or inducible and calcium independent (iNOS). The endogenous production of nitric oxide plays a vital role in the regulation of physiological processes (e.g., blood vessel tone, neurotransmission) as well as in host defense and immunity. There is increasing evidence that nitric oxide also plays a complex role in the modulation of the inflammatory response. The induction of nitric oxide synthase (iNOS) in tissues can lead to the sustained production of high concentrations of nitric oxide which may exert pro-inflammatory effects including vasodilation, edema, cytotoxicity, and the mediation of cytokine dependent processes. Conversely, the production of NO by endothelial cell cNOS may serve a protective, or anti-inflammatory, function by preventing the adhesion and release of oxidants by activated neutrophils in the microvasculature. In this review we describe the multifaceted role of nitric oxide in inflammation, particularly focusing on the regulation of inflammatory cells and the repertoire of molecular processes targeted by NO that control cellular functions. Biosynthesis of Nitric Oxide Nitric oxide is synthesized via L-arginine oxidation by a family of nitric oxide synthases (NOS). Isomeric forms of nitric oxide synthase, representing at least three distinct gene products, have been cloned in bovine, rat, mice, and human tissues (1-5) (Table I). The three isoforms of the enzyme vary in calcium dependence, kinetics, and regulation. However, all nitric oxide synthases are flavoproteins which require NADPH and tetrahydrobiopterin as co-factors. The amino acid sequences deduced from the cDNA of the endothelial and neuronal cNOSs and iNOS have been reported (1-3, 6).

Inattention, hyperactivity, and school performance in a population of school-age children with complex congenital heart disease.

INTRODUCTION. There is a growing interest in characterizing the neurodevelopmental outcomes of school-age survivors of cardiac surgery. The purpose of this study was to examine a population of 5- to 10-year-old children who underwent newborn cardiac surgery for complex congenital heart disease to characterize and assess risk factors for problems with inattention and hyperactivity, as well as the use of remedial school services. PATIENTS AND METHODS. This study was a cross-sectional analysis of patients who underwent newborn cardiac surgery and were enrolled in a neuroprotection trial conducted at our institution between 1992 and 1997. Parents and teachers completed questionnaires for the school-age child to elicit information pertaining to the childs general health and academic performance. The severity of hyperactivity and inattention were assessed by using 2 standardized questionnaires (Attention-Deficit/Hyperactivity Disorder Rating Scale-IV and Behavior Assessment System for Children). In addition to calculating descriptive estimates of their occurrence, single-covariate logistic regression models were specified and tested by using 3 different outcomes (inattention, hyperactivity, and use of remedial school services) and 14 different covariates representing preoperative, intraoperative, and postoperative factors. RESULTS. Data were obtained from parents and/or teachers for 109 children. Fifty-three (49%) were receiving some form of remedial academic services, and 15% were assigned to a special-education classroom. The number of children receiving clinically significant scores for inattention and hyperactivity on the Behavior Assessment System for Children was 3 to 4 times higher than observed in the general population. On the Attention-Deficit/Hyperactivity Disorder Rating Scale-IV, 30% of the parents reported high-risk scores for inattention and 29% reported high-risk scores for hyperactivity. No perioperative factors were statistically associated with adverse outcomes. CONCLUSION. In this cohort of children with complex congenital heart disease, a significant proportion of the children were at risk for inattention and hyperactivity, and nearly half were using remedial school services. We did not identify any perioperative risk factors, which correlated with high-risk scores or the use of remedial school services. Ongoing neurodevelopmental follow-up and screening are recommended in this vulnerable population.

Occult Neonatal Seizures

Summary: Forty‐one infants with neonatal seizures frequent enough to be captured by randomly recorded routine EEG examinations were studied to determine how often their electrographic seizures were occult (subclinical) and to examine the effects of seizure duration and mental status on their clinical visibility. Seizures were the result of diverse etiologies and most infants had received one or more antiepileptic drug prior to the EEG recordings. The majority of electrographic seizures were occult: only 84 of 393 (21%) were accompanied by distinctive clinical seizure activity; the remaining 79% were occult. There was no significant difference between the duration of EEG seizures with distinctive clinical signs and those without. There was no significant difference in the proportion of occult seizures in neonates with preserved consciousness compared with lethargic or comatose infants. We conclude that unaided visual inspection of infants seriously underestimates true seizure frequency. Long‐term EEG monitoring may be necessary in many infants to determine their real seizure frequency and to judge the adequacy of antiepileptic drug treatment.

Nitric Oxide Synthase/COX Cross-Talk: Nitric Oxide Activates COX-1 But Inhibits COX-2-Derived Prostaglandin Production

It is recognized that there is molecular cross-talk between the inflammatory mediators NO and PGs that may regulate tissue homeostasis and contribute to pathophysiological processes. However, the literature is divided with respect to whether NO activates or inhibits PG production. In this study, we sought to determine whether conflicting observations could be accounted for by divergent effects of NO on the two cyclooxygenase (COX) isoforms. Exposure of resting macrophages to NO (30 μM) enhanced PGE2 release by 4.5-fold. This enhancement was inhibited by indomethacin but not by the COX-2 selective inhibitor NS398. To separate the activation of phospholipase A2 and COX, we performed experiments using fibroblasts derived from COX-1-deficient or COX-2-deficient mice. These cells exhibit increased basal PG production, which is due to a constitutively stimulated cytosolic phospholipase A2 and enhanced basal expression of the remaining COX isozyme. The exposure of COX- 2-deficient cells to exogenous NO (10 μM) resulted in a 2.4-fold increase of PGE2 release above controls. Further studies indicated that NO stimulated PGE2 release in COX-2-deficient cells, without altering COX-1 mRNA or protein expression. In contrast, NO inhibited COX-2-derived PGE2 production in both LPS-stimulated macrophages and COX-1 knockout cells. This inhibition was associated with both decreased expression and nitration of COX-2. Thus, these studies demonstrate divergent effects of NO on the COX isoforms. The regulation of PGE production by NO is therefore complex and will depend on the local environment in which these pleiotropic mediators are produced.

Apolipoprotein E genotype and neurodevelopmental sequelae of infant cardiac surgery

BACKGROUND There has been increasing recognition of adverse neurodevelopmental sequelae in some children after repair of congenital heart defects. Even among children with the same cardiac defect, significant interindividual variation exists in developmental outcome. Polymorphisms of apolipoprotein E have been identified as a risk factor for worse neurologic recovery after central nervous system injury. METHODS A single-institution prospective study of patients <or=6 months of age undergoing cardiopulmonary bypass for repair of congenital heart defects was undertaken to evaluate the association between apolipoprotein E genotype and postoperative neurodevelopmental dysfunction. Developmental outcomes were evaluated at 1 year of age by using the Bayley Scales of Infant Development. RESULTS One-year evaluation was performed in 244 patients. After adjustment for preoperative and postoperative covariates-including gestational age, age at operation, sex, race, socioeconomic status, cardiac defect, and use of deep hypothermic circulatory arrest-the apolipoprotein E epsilon2 allele was associated with a worse neurologic outcome as assessed by the Psychomotor Developmental Index of the Bayley Scales of Infant Development (P =.036). Patients with the apolipoprotein E epsilon2 allele had approximately a 7-point decrease in the Psychomotor Developmental Index. CONCLUSIONS Apolipoprotein E epsilon2 allele carriers had significantly lower Psychomotor Development Index scores at 1 year of age after infant cardiac surgery. The effect was independent of ethnicity, socioeconomic status, cardiac defect, and use of deep hypothermic circulatory arrest. An effect of the apolipoprotein E epsilon4 allele was not detected. Genetic polymorphisms that decrease neuroresiliency and impair neuronal repair after central nervous system injury are important risk factors for neurodevelopmental dysfunction after infant cardiac surgery.

Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of TNF-alpha by macrophages.

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16–24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-α from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.