Eduardo Villamor

Endotoxin induced chorioamnionitis prevents intestinal development during gestation in fetal sheep.

Chorioamnionitis is the most significant source of prenatal inflammation and preterm delivery. Prematurity and prenatal inflammation are associated with compromised postnatal developmental outcomes, of the intestinal immune defence, gut barrier function and the vascular system. We developed a sheep model to study how the antenatal development of the gut was affected by gestation and/or by endotoxin induced chorioamnionitis. Chorioamnionitis was induced at different gestational ages (GA). Animals were sacrificed at low GA after 2d or 14d exposure to chorioamnionitis. Long term effects of 30d exposure to chorioamnionitis were studied in near term animals after induction of chorioamnionitis. The cellular distribution of tight junction protein ZO-1 was shown to be underdeveloped at low GA whereas endotoxin induced chorioamnionitis prevented the maturation of tight junctions during later gestation. Endotoxin induced chorioamnionitis did not induce an early (2d) inflammatory response in the gut in preterm animals. However, 14d after endotoxin administration preterm animals had increased numbers of T-lymphocytes, myeloperoxidase-positive cells and gammadelta T-cells which lasted till 30d after induction of chorioamnionitis in then near term animals. At early GA, low intestinal TLR-4 and MD-2 mRNA levels were detected which were further down regulated during endotoxin-induced chorioamnionitis. Predisposition to organ injury by ischemia was assessed by the vascular function of third-generation mesenteric arteries. Endotoxin-exposed animals of low GA had increased contractile response to the thromboxane A2 mimetic U46619 and reduced endothelium-dependent relaxation in responses to acetylcholine. The administration of a nitric oxide (NO) donor completely restored endothelial dysfunction suggesting reduced NO bioavailability which was not due to low expression of endothelial nitric oxide synthase. Our results indicate that the distribution of the tight junctional protein ZO-1, the immune defence and vascular function are immature at low GA and are further compromised by endotoxin-induced chorioamnionitis. This study suggests that both prematurity and inflammation in utero disturb fetal gut development, potentially predisposing to postnatal intestinal pathology.

Carbamoyl Phosphate Synthetase Polymorphisms as a Risk Factor for Necrotizing Enterocolitis

A C-to-A nucleotide transversion (T1405N) in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1) has been correlated with low plasma concentrations of l-arginine in neonates. As plasma l-arginine concentrations are decreased in premature infants with necrotizing enterocolitis (NEC), we hypothesized that the CPS1 T1405N polymorphism would correlate with the presence of NEC. We analyzed the CPS1 genotypes for the T1405N polymorphism in 17 preterm infants (≤30 wk and <1500 g) with established NEC, 34 preterm infants without NEC, and 25 healthy term infants. Distribution of genotypes did not differ between the NEC population (CC:AC:AA = 70.6%:23.5%:5.9%) and the preterm control group (CC:AC:AA = 41.2%:35.3%:23.5%; p = 0.110) or the term group (CC:AC:AA = 44%:48%:8%; p = 0.228). The C allele frequency was 82.4% in NEC and 58.8% in preterm control infants (p = 0.018) and analysis for linear trend demonstrated that incidence of NEC increased with the number of C alleles (p = 0.037). The CC genotype was associated with an increased risk of NEC in the preterm infants [odds ratio (OR) = 3.43, 95% confidence interval (CI): 1.01–11.49, p = 0.048), when compared with the grouped together AA/AC genotypes. These data suggest that the CPS1 T1405N polymorphism may be associated with the risk of NEC in preterm infants.

Relaxant Effects of Carbon Monoxide Compared with Nitric Oxide in Pulmonary and Systemic Vessels of Newborn Piglets

Nitric oxide (NO) has been implicated in a number of diverse physiologic processes, including regulation of vascular tone. Carbon monoxide (CO) is another endogenously generated diatomic gas that may play an important physiologic role in vascular smooth muscle homeostasis. The purpose of this study was to compare the responses to exogenous NO and CO in isolated vessels (pulmonary arteries, pulmonary veins, and mesenteric arteries) from 12- to 24-h-old and 2-wk-old piglets. Vessels precontracted with the thromboxane A2 mimetic U46619 (10−7 M) relaxed in response to CO (2 × 10−6 to 2 × 10−4 M) and NO (2 × 10−9 to 2 × 10−7 M); these effects were not affected by endothelium removal but were completely abolished by the soluble guanylate cyclase inhibitor ODQ (10−5 M). In pulmonary arteries, the maximal relaxation to NO increased with postnatal age from 33 ± 4% of the precontraction value to 56 ± 5%, in 12- to 24-h-old and 2-week-old piglets, respectively (p < 0.01), but the response to CO decreased from 25 ± 3% to 12 ± 1%, respectively (p < 0.01). The maximal response to CO was greater in pulmonary veins than in pulmonary or mesenteric arteries for both age groups (p < 0.01). Vasorelaxation induced by endogenous NO (stimulated by acetylcholine) was also greater in pulmonary veins when compared with pulmonary arteries and increased with postnatal age in both vessels. In contrast, no age-related differences were observed in the vasorelaxation induced by the cGMP analog 8-bromo cGMP in pulmonary arteries. When the response to NO was analyzed under three different extracellular O2 concentrations (Po2 4.51 ± 0.03, 19.32 ± 0.17, and 86 ± 0.62, kPa), no significant differences were found. However, in the presence of superoxide dismutase (100 U/mL). the response to CO remained unchanged, and the response to NO improved in pulmonary arteries from 2-week-old but not from newborn piglets. In conclusion, both NO and CO relaxed neonatal vessels through soluble guanylate cyclase activation. However, when compared with NO, CO exhibited a poor vasorelaxant activity. Pulmonary vasorelaxation induced by NO increased with postnatal age, whereas that induced by CO decreased. Changes in extracellular oxygen concentration did not alter the pulmonary vascular response to NO. However, the presence of superoxide dismutase improved the response to NO, indicating that oxidant activity limits the vasorelaxant response to NO but not to CO.

Role of Reactive Oxygen Species in Kv Channel Inhibition and Vasoconstriction Induced by TP Receptor Activation in Rat Pulmonary Arteries

Abstract:  Voltage‐gated potassium channels (Kv) and thromboxane A2 (TXA2) have been involved in several forms of human and experimental pulmonary hypertension. We have reported that the TXA2 analog U46619, via activation of TP receptors and PKCζ, inhibited Kv currents in rat pulmonary artery smooth muscle cells (PASMC), increased cytosolic calcium, and induced a contractile response in isolated rat and piglet pulmonary arteries (PA). Herein, we have analyzed the role of reactive oxygen species (ROS) in this signaling pathway. In rat PA, U46619 increased dichlorofluorescein fluorescence, an indicator of intracellular hydrogen peroxide, and this effect was prevented by the NADPH oxidase inhibitor apocynin and by polyethyleneglycol‐catalase (PEG‐catalase, a membrane‐permeable form of catalase). U46619 inhibited Kv currents in native PASMC and these effects were strongly inhibited by apocynin. The contractile responses to U46619 in isolated PA were inhibited by PEG‐catalase and the NADPH oxidase inhibitors diphenylene iodonium (DPI) and apocynin. A membrane permeable of hydrogen peroxide, t‐butyl hydroperoxide, also inhibited Kv currents and induced a contractile response. Activation of NADPH oxidase and the subsequent production of hydrogen peroxide are involved in the Kv channel inhibition and the contractile response induced by TP receptor activation in rat PA.

Isoprostanes in fetal and neonatal health and disease

Isoprostanes are prostaglandin-like bioactive molecules generated via nonenzymatic peroxidation of lipid membrane-derived arachidonic acid by free radicals and reactive oxygen species. Their cognate receptors, biological actions, and signaling pathways are poorly understood. Aside from being sensitive and specific biomarkers of oxidative stress, E- and F-ring isoprostanes have important biological functions and likely mediate many of the disease-related pathological changes for which they are used as indicators. The biochemical pathways involved in isoprostane formation, their pathogenetic relevance to adult disease states, and their biological function are addressed. Developmentally, plasma and tissue content data show that isoprostane levels are highest during fetal and early neonatal life, when compared with adults. As such, the available data suggesting that isoprostanes play an important biological role, as well as possibly actively participate in the regulation of pulmonary vascular tone and the transition from fetal to postnatal life, are here reviewed. Lastly, the association between isoprostanes and certain neonatal clinical conditions is addressed. Although its existence has been recognized for almost 20 years, little is known about the critical importance of isoprostanes during fetal life and immediate neonatal period. This review is an attempt to bridge this knowledge gap.

Postnatal Maturation of Phosphodiesterase 5 (PDE5) in Piglet Pulmonary Arteries: Activity, Expression, Effects of PDE5 Inhibitors, and Role of the Nitric Oxide/Cyclic GMP Pathway

After birth and during the first days of extrauterine life, pulmonary arterial pressure is progressively reduced to reach the adult values. We hypothesized that changes in PDE5 activity might be involved in the pulmonary postnatal maturation of the nitric oxide (NO)/cGMP pathway. The PDE5 inhibitor sildenafil produced vasorelaxant responses in isolated pulmonary arteries. These effects were similar in newborn (3–18 h) and 2-wk-old piglets, unchanged by endothelium removal, and markedly inhibited by the soluble guanylyl cyclase inhibitor ODQ. The peak of the transient vasorelaxant response to NO gas increased with postnatal age but was unaffected by PDE inhibition. However, the duration of the response to NO was significantly increased. The vasorelaxant response to sodium nitroprusside was potentiated by sildenafil in both age groups. The PDE5 inhibitors dipyridamole and zaprinast, produced qualitatively similar effects but with lower potency. Both total and PDE5-dependent cGMP hydrolytic activity and PDE5 protein expression increased with postnatal age. All these results suggest that PDE5 is a key regulator of NO-induced vasodilation in the postnatal pulmonary arteries. PDE5 inhibition is able to produce pulmonary vasodilation even in the absence of a functional endothelium and potentiates the vasorelaxant response to exogenous NO and nitroprusside. However, PDE5 is not responsible for the maturational increase of NO bioactivity during the first days of extrauterine life.

Neutral Sphingomyelinase, NADPH Oxidase and Reactive Oxygen Species. Role in Acute Hypoxic Pulmonary Vasoconstriction

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium‐denuded PA segments and their inhibition prevented hypoxia‐induced voltage‐gated potassium channel (KV) inhibition and pulmonary vasoconstriction. Consistently, H2O2, or its analogue t‐BHP, decreased KV currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia‐induced ROS production, KV channel inhibition and vasoconstriction. Hypoxia induced p47phox phosphorylation and its interaction with caveolin‐1. Inhibition of nSMase (GW4869) or PKCζ prevented p47phox phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase‐PKCζ‐NADPH oxidase as a necessary step required for ROS production and vasoconstriction. J. Cell. Physiol. 226: 2633–2640, 2011.

Role of superoxide anion on basal and stimulated nitric oxide activity in neonatal piglet pulmonary vessels

The superoxide anion (O2·−) appears to be an important modulator of nitric oxide bioavailability. Enzymatic scavenging of O2·− is carried out by superoxide dismutase (SOD). The present study was designed to characterize the developmental changes on pulmonary vascular reactivity induced by 1) exogenous Cu/Zn SOD, 2) several putative SOD mimetics, and 3) endogenous SOD inhibition. We also analyzed age-related changes on pulmonary SOD activity and vascular O2·− levels. SOD (1–300 U/mL) produced endothelium-dependent relaxation of U46619-contracted intrapulmonary arteries (fourth branch) and veins from 12- to 24-h-old and 2-wk-old piglets. SOD-induced relaxation was greater in pulmonary arteries and was abolished by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester. SOD induced a greater pulmonary artery relaxation in the 2-wk-old than in the 12- to 24-h-old piglet. SOD (100 U/mL) did not modify acetylcholine-induced relaxation in pulmonary arteries. In contrast, endogenous SOD inhibition by diethyldithiocarbamate (3 mM) impaired acetylcholine-induced relaxation in pulmonary arteries from newborn but not from 2-wk-old piglets. Total SOD activity in lung tissue did not change with postnatal age. With the use of dihydroethidium, an oxidant-sensitive fluorescent probe, we did not find significant age- or vessel-related differences in O2·− presence. From the putative SOD mimetics tested, only the metal salts MnCl2 and CuSO4 reproduced the vascular effects of SOD. In summary, SOD produces endothelium-dependent pulmonary vascular relaxation by protecting nitric oxide from destruction by O2·−. This effect was less marked in newborns than in 2-wk-old piglets. In contrast, pulmonary arteries from newborn piglets are more sensitive to the inhibition of endogenous SOD.

Involvement of protein kinase C in reduced relaxant responses to the NO/cyclic GMP pathway in piglet pulmonary arteries contracted by the thromboxane A2-mimetic U46619.

Impairment of nitric oxide (NO)/cyclic GMP production and/or increased activities of thromboxane A2 (TXA2) and endothelin‐1 (ET‐1) have been associated with pulmonary hypertension. We have analysed the interactions of noradrenaline (NA), the TXA2‐mimetic U46619 and ET‐1 with the relaxation induced via cyclic GMP in isolated piglet intrapulmonary arteries. The contractions induced by NA were augmented by endothelium removal or by methylene blue and pre‐contracted rings were fully relaxed by acetylcholine, sodium nitroprusside (SNP), atrial natriuretic peptide and 8‐bromo‐cyclic GMP. In contrast, U46619‐ and ET‐1 induced contractions were endothelium‐independent and only partially relaxed by the latter vasodilators. Whereas the reduced responses to SNP in arteries contracted by U46619 were independent of the U46619‐induced tone, a higher concentration of ET‐1 (tone higher than that induced by NA) was required to reduce the vasodilator responses to SNP. NA, U46619 and ET‐1 had no effect on the SNP‐induced increases in cyclic GMP. The reduced relaxant responses to SNP in arteries pre‐contracted by U46619 were specific for piglet pulmonary arteries since they were not observed in piglet mesenteric or coronary arteries or in rat pulmonary arteries. Furthermore, there were no differences in the relaxant response to the adenylate cyclase activator forskolin in piglet pulmonary arteries pre‐contracted by either NA, U46619 or ET‐1. SNP‐induced relaxation was inhibited by thapsigargin (but not by inhibition of the membrane Na+/K+ ATPase nor K+ channels) indicating a role for Ca2+ sequestration by the Ca2+ ATPase in the effects of SNP. The phorbol ester 12‐myristate, 13‐acetate inhibited the relaxant response to SNP. The inhibitory effect of U46619 on SNP‐induced relaxation was abolished by the protein kinase C inhibitor (PKC) staurosporine suggesting that PKC may be a part of the signal transduction mechanism. In summary, piglet pulmonary arteries when activated by a TXA2‐mimetic show abnormally reduced relaxant responses to the NO/cyclicGMP pathway. This effect appears to be mediated by activation of PKC.

Group B Streptococcus and E. coli LPS-induced NO-dependent hyporesponsiveness to noradrenaline in isolated intrapulmonary arteries of neonatal piglets

1 The effects of endotoxin (E. coli lipopolysaccharide, LPS) and heat inactivated group B Streptococcus (GBS) were studied on the contractile responses to noradrenaline (NA) in isolated pulmonary arteries and on the activity of the constitutive and inducible nitric oxide synthase (NOS) in lung fragments of neonatal piglets. 2 Short‐term (≤ 5 h) incubation with LPS (1 μg ml−1) or GBS (3 × 107 colonies forming units ml−1) did not modify the vascular responsiveness to NA (10−8 M‐10−4 m) in isolated intrapulmonary arteries. However, long‐term incubation (20 h) with LPS or GBS produced a significant reduction in the maximal contractile responses and shifted the concentration‐response curve for NA downwards. 3 Endothelium removal or the cyclo‐oxygenase inhibitor meclofenamate (10−5 m) did not affect the GBS‐ and LPS‐induced hyporesponsiveness to NA. 4 The presence of the nitric oxide (NO) precursor, l‐arginine (10−5 m), 30 min prior to the contractility challenge increased the LPS‐ and GBS‐induced pulmonary vascular hyporesponsiveness to NA. In contrast, the addition, prior to the challenge with NA, of the NOS inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−4m) or coincubation with dexamethasone (3 × 10−6m), a potent inhibitor of the induction of NOS, or with the protein synthesis inhibitor cycloheximide (10−5 m) completely restored the reactivity to NA in LPS‐ and GBS‐treated pulmonary arteries. 5 The incubation for 20 h of lung fragments with LPS and GBS produced a significant increase in the Ca2+‐independent (inducible) NOS activity determined by the conversion of radiolabelled l‐arginine to citrulline, but did not modify the constitutive NOS activity. This NOS induction was abolished by coincubation with dexamethasone (3 × 10−6 m). 6 These results demonstrated that prolonged incubation with GBS and LPS causes an induction of NOS activity which results in a reduced vascular responsiveness to NA in pulmonary arteries of neonatal piglets. Thus, induction of NOS seems to be responsible for the delayed pulmonary vascular hyporesponsiveness induced by GBS (a Gram‐positive) and E. coli (a Gram‐negative), the most common causal agents of neonatal sepsis.