Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia.

BACKGROUND: Sphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1 -/- ) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1 -/- mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1 -/- neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45. CONCLUSION: Using SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

Clasificación funcional de la hipertensión pulmonar en niños: informe del task force pediátrico del Pulmonary Vascular Research Institute (PVRI), Panamá 2011 / Functional classification of pulmonary hypertension in children: Report from the PVRI pediatric taskforce, Panama 2011

Los miembros del Task Force pediátrico del Pulmonary Vascular Research Institute (PVRI, su sigla en Inglés) fueron conscientes de la necesidad de desarrollar una clasificación funcional de la hipertensión pulmonar en niños. La clasificación que se propone sigue el mismo patrón y utiliza los mismos criterios de la clasificación de la hipertensión pulmonar específica para adultos de Dana Point. Fue necesario incluir modificaciones para los niños, teniendo en cuenta que la edad, el crecimiento físico y la madurez influyen en la expresión funcional de la enfermedad. Es necesario definir el estado clínico del niño, pues ello facilita revisar la evolución del mismo en una forma consistente y objetiva a medida que él/ella crecen. Particularmente en los niños más jóvenes, se trató de incluir indicadores objetivos como el crecimiento, la necesidad de alimentos suplementarios y los registros de asistencia al colegio y a la guardería. Esto ayuda a monitorear la evolución clínica y la respuesta al tratamiento a través de los años y facilita el desarrollo de algoritmos de tratamiento en estos pacientes. Se presenta un artículo de consenso sobre una clasificación aplicable a los niños con hipertensión pulmonar que se discutió en la reunión anual del PVRI que se llevó a cabo en Panamá en febrero de 2011.

The members of the Pediatric Task Force of the Pulmonary Vascular Research Institute (PVRI) were aware of the need to develop a functional classification of pulmonary hypertension in children. The proposed classification follows the same pattern and uses the same criteria as the Dana Point pulmonary hypertension specific classification for adults. Modifications were necessary for children, since age, physical growth and maturation influences the way in which the functional effects of a disease are expressed. It is essential to encapsulate a child's clinical status, to make it possible to review progress with time as he/she grows up, as consistently and as objectively as possible. Particularly in younger children we sought to include objective indicators such as thriving, need for supplemental feeds and the record of school or nursery attendance. This helps monitor the clinical course of events and response to treatment over the years. It also facilitates the development of treatment algorithms for children. We present a consensus paper on a functional classification system for children with pulmonary hypertension, discussed at the Annual Meeting of the PVRI in Panama City, February 2011.

Consenso sobre la clasificación de la enfermedad vascular pulmonar hipertensiva en niños: reporte del task force pediátrico del Pulmonary Vascular Research Institute (PVRI) Panamá 2011 / A consensus approach to the classification of pediatric pulmonary hypertensive vascular disease: Report from the PVRI Pediatric Taskforce, Panama 2011

Las clasificaciones actuales de la hipertensión pulmonar han contribuido significativamente al conocimiento de la enfermedad vascular pulmonar, han facilitado ensayos farmacológicos y han mejorado nuestro conocimiento de las cardiopatías congénitas del adulto; sin embargo estas clasificaciones no son aplicables completamente a la enfermedad en el niño. La clasificación que aquí se propone se basa principalmente en la práctica clínica. Los objetivos específicos de esta nueva clasificación son mejorar las estrategias diagnósticas, promover la investigación clínica, mejorar nuestro conocimiento de la patogénesis, de la fisiología y de la epidemiología de la enfermedad y orientar el desarrollo de modelos de la enfermedad humana en el laboratorio y estudios en animales; también puede servir como un recurso docente. Se hace énfasis en los conceptos de maladaptación perinatal, alteraciones del desarrollo e hipoplasia pulmonar como factores causantes de la hipertensión pulmonar pediátrica; así mismo, en la importancia de los múltiples síndromes malformativos congénitos, genéticos y cromosómicos en la presentación de la hipertensión pulmonar pediátrica. La enfermedad vascular pulmonar hipertensiva en niños se divide en diez grandes categorías.

Current classifications of pulmonary hypertension have contributed a great deal to our understanding of pulmonary vascular disease, facilitated drug trials, and improved our understanding of congenital heart disease in adult survivors. However, these classifications are not applicable readily to pediatric disease. The classification system that we propose is based firmly in clinical practice. The specific aims of this new system are to improve diagnostic strategies, to promote appropriate clinical investigation, to improve our understanding of disease pathogenesis, physiology and epidemiology, and to guide the development of human disease models in laboratory and animal studies. It should be also an educational resource. We emphasize the concepts of perinatal maladaptation, maldevelopment and pulmonary hypoplasia as causative factors in pediatric pulmonary hypertension. We highlight the importance of genetic, chromosomal and multiple congenital malformation syndromes in the presentation of pediatric pulmonary hypertension. We divide pediatric pulmonary hypertensive vascular disease into 10 broad categories.

Role of Rho kinases in PKG-mediated relaxation of pulmonary arteries of fetal lambs exposed to chronic high altitude hypoxia.

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.

Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells.

We determined the effect of oxygen [approximately 100 Torr (normoxia) and approximately 30-40 Torr (hypoxia)] on functions of endothelial nitric oxide (NO) synthase (NOS-3) and its negative regulator caveolin-1 in ovine fetal and neonatal lung microvascular endothelial cells (MVECs). Fetal NOS-3 activity, measured as NO production with 0.5-0.9 microM 4-amino-5-methylamino-2,7-difluorofluorescein, was decreased in hypoxia by 14.4% (P < 0.01), inhibitable by the NOS inhibitor N-nitro-L-arginine, and dependent on extracellular arginine. Caveolar function, assessed as FITC-BSA (160 microg/ml) endocytosis, was decreased in hypoxia by 13.5% in fetal and 22.8% in neonatal MVECs (P < 0.01). NOS-3 and caveolin-1 were physically associated, as demonstrated by coimmunoprecipitation and colocalization, and functionally associated, as shown by cross-activation of endocytosis, by their specific antibodies and activation of NOS by albumin. Caveolin peptide, containing the sequence for the PKC phosphorylation site of caveolin, and caveolin antiserum against the site increased NO production and endocytosis by 12.3% (P < 0.05) and 16% (P < 0.05), respectively, in normoxia and increased endocytosis by 25% (P < 0.001) in hypoxia. PMA decreased NO production in normoxia and hypoxia by 19.32% (P < 0.001) and 11.8% (P < 0.001) and decreased endocytosis in normoxia by 20.35% (P < 0.001). PKC kinase activity was oxygen sensitive, and threonine phosphorylation was enhanced in hypoxia. Pertussis toxin increased caveolar and NOS functions. These data support our hypothesis that increased Po(2) at birth promotes dissociation of caveolin-1 and NOS-3, with an increase in their activities, and that PKC and an oxygen-sensitive cell surface G protein-coupled receptor regulate caveolin-1 and NOS-3 interactions in fetal and neonatal lung MVECs.

Metabolism of platelet activating factor by intrapulmonary vascular smooth muscle cells. Effect of oxygen on phospholipase A2 protein expression and activities of acetyl-CoA acetyltransferase and cholinephosphotransferase.

We have demonstrated that platelet activating factor (PAF) plays an important physiological role in the maintenance of high pulmonary vascular tone in fetal lambs, a role attributable to increased PAF receptor binding (J. Appl. Physiol. 85 (1998) 1079; Am J. Physiol. 278 (2000) H1168). In this study, we examined the possibility that increased PAF synthesis via de novo and remodeling pathways as well as decreased PAF catabolism in hypoxic state of fetal lungs may account for the PAF action in vivo. We investigated effect of oxygen tension on PAF synthesis by ovine fetal intrapulmonary venous (PV) and arterial (PA) smooth muscle cells pulsed with [3H]choline (de novo), or [3H]acetate (remodeling), while PAF catabolism was studied by assay of acetylhydrolase (PAF-Ah) activity. Hypoxia stimulated PAF synthesis by choline incorporation (pmol/10(6)cells) in both PVSMC (1.14+/-0.13 vs 0.53+/-0.05) and PASMC (0.39+/-0.12 vs 0.22+/-0.04). Hypoxia stimulated PAF synthesis via remodeling pathway only in PVSMC (408+/-32 vs 225+/-17) which was 5-fold greater than in PASMC (77+/-15 vs 105+/-24), however, with A23187 in remodeling pathway, PAF synthesis increased 5-fold compared to baseline conditions and then synthesis in hypoxia was greater than in normoxia in both cell types. Phospholipase A2 protein expression was significantly higher in hypoxia in both cells and was approximately 2-fold higher in PVSMC. PAF-Ah activity (nmol lyso-PAF/min/mg protein) was greater in hypoxia vs normoxia in PVSMC (0.81+/-0.24 vs 0.44+/-0.088), but in PASMC activity was less in hypoxia vs normoxia (1.68+/-0.24 vs 3.93+/-0.44). Compared to PVSMC PAF-Ah activity in PASMC was 4-fold higher in hypoxia. Our data demonstrate that (1) PAF synthesis in intrapulmonary SMC of fetal lambs occurs by both de novo studied by choline incorporation and remodeling pathways, the latter being predominant. (2) There is heterogeneity in PAF synthetic and catabolic activities in lung vasculature of fetal lambs. We conclude that increased PAF synthesis in veins by the two synthetic pathways coupled with decreased catabolism will result in a higher venous PAF levels in the hypoxic environment of fetal lungs. We speculate that in vivo, a high PAF level in veins will make more PAF available for binding to its receptors so as to sustain the desired high venous tone in the fetal pulmonary circulation.

Effects of SQ 22536, an adenylyl cyclase inhibitor, on isoproterenol-induced cyclic AMP elevation and relaxation in newborn ovine pulmonary veins.

The effects of inhibition of adenylyl cyclase on isoproterenol-induced relaxation were determined in isolated pulmonary veins of newborn lambs (7-12 days old). In veins constricted with endothelin-1, isoproterenol at concentrations < or = 3 x 10(-9) M had no effect on the cyclic AMP (cAMP) content but caused up to 56% relaxation. At higher concentrations (> or = 10(-8) M), isoproterenol elevated cAMP content and caused further relaxation. In veins constricted with endothelin-1 or U46619 (9,11-dideoxy-11, 9-epoxymethanoprostaglandin prostaglandin F2alpha), the cAMP elevation but not relaxation caused by isoproterenol was abolished by SQ 22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; an adenylyl cyclase inhibitor]. The effects of isoproterenol on vessel tension and cAMP content were inhibited by propranolol. Rp-8-CPT-cAMPS [8-(4-Chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer] and Rp-8-Br-PET-cGMPS [beta-phenyl-1, N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer], inhibitors of cAMP- and guanosine-3',5'-cyclic monophosphate (cGMP)-dependent protein kinases, respectively, attenuated relaxation caused by a cAMP analog but not that by isoproterenol. In the crude membrane preparations of pulmonary veins, an increase in the activity of adenylyl cyclase caused by isoproterenol was abolished by propranolol and SQ 22536. These results suggest that cAMP may not play a critical role in isoproterenol-induced relaxation of pulmonary veins of newborn lambs.

Endothelin effects in isolated, perfused lamb lungs: role of cyclooxygenase inhibition and vasomotor tone.

We have determined the sites of action of endothelin-1 (ET) in the lamb pulmonary circulation. The influence of cyclooxygenase inhibition and baseline vasomotor tone on ET effects was also studied. Lungs of 14 lambs (6-9 wk of age, 12.1 +/- 0.6 kg body wt) were isolated and perfused with blood. Group I lungs (n = 5) were untreated, group II lungs (n = 5) were treated with indomethacin to inhibit cyclooxygenase, and group III lungs (n = 4) were treated with indomethacin and a thromboxane A2 analogue, U-46619, to elevate vasomotor tone. All lungs were perfused with constant flow in zone 3, with left atrial and airway pressures being 8 and 6 cmH2O, respectively. We measured pulmonary arterial pressure and, by the micropuncture servo-null method, pressures in 20- to 50-microns diameter subpleural venules, both before and after each dose of ET was infused (50, 100, 250, and 500 ng/kg). Group I lungs, with high baseline vasomotor tone, exhibited a biphasic response to ET; 50-100 ng/kg of ET dilated both arteries and veins, whereas 500 ng/kg of ET constricted both arteries and veins. In group II lungs with low vasomotor tone, all doses of ET caused constriction of arteries only. In group III lungs (indomethacin treated with elevated vasomotor tone), 50-100 ng/kg of ET caused dilation of arteries and veins, whereas 500 ng/kg of ET induced constriction, this time only in arteries. We conclude that ET has both dilator and constrictor effects in arteries and veins of isolated, perfused lamb lungs. ET-induced arterial and venous dilation is dependent on initial vasomotor tone but not on cyclooxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Segmental vascular resistance during pulsatile and steady perfusion in 3- to 5-wk-old rabbit lungs.

To determine the mechanisms by which pulsatile perfusion reduces vascular resistance in isolated lungs, we compared the effects of pulsatile versus steady perfusion on total and segmental vascular resistance in isolated lungs of 3- to 5-wk-old rabbits. Lungs were perfused alternately with steady and pulsatile flow for 45-min periods at a constant total arteriovenous pressure drop. Blood flow was adjusted to keep mean pulmonary arterial pressure at 20 cmH2O, when left atrial and airway pressures were 8 and 6 cmH2O, respectively. We partitioned the pulmonary circulation into three longitudinal vascular segments, i.e., arteries, microvessels, and veins, by measuring pressures in 20- to 50-microns-diameter subpleural arterioles and venules by the micropipette servo-nulling method. We found that in the isolated, perfused 3- to 5-wk-old rabbit lung, in which arteries and veins are the main sites of resistance, pulsatile flow results in a 20-36% reduction in total vascular resistance, mainly due to a reduction in arterial and venous resistances. The decrease in total vascular resistance was similar in lungs that were untreated or treated with papaverine, indicating that the effect of pulsatile flow was not due to active vasomotion. The reduction in arterial resistance was greater than that in veins (31-55 vs. 19-22%), especially when pulse amplitude was high (5-10 vs. 20-30 cmH2O). Total vascular resistance was also lower after 45 min of pulsatile perfusion with a pulse rate of 200 pulses/min than 80 pulses/min (0.126 +/- 0.04 vs. 0.154 +/- 0.059 cmH2O.min.ml-1.kg).(ABSTRACT TRUNCATED AT 250 WORDS)