Pulmonary hypertension is associated with diverse cardiac, pulmonary, and systemic diseases in neonates, infants, and older children and contributes to significant morbidity and mortality. However, ...current approaches to caring for pediatric patients with pulmonary hypertension have been limited by the lack of consensus guidelines from experts in the field. In a joint effort from the American Heart Association and American Thoracic Society, a panel of experienced clinicians and clinician-scientists was assembled to review the current literature and to make recommendations on the diagnosis, evaluation, and treatment of pediatric pulmonary hypertension. This publication presents the results of extensive literature reviews, discussions, and formal scoring of recommendations for the care of children with pulmonary hypertension.
Objective
Recent studies have indicated a role of epigenetic phenomena in pathogenesis of pulmonary hypertension, but in foetal pulmonary artery smooth muscle cell (PASMC) proliferation this is still ...largely unknown. G9a is a key enzyme for histone H3 dimethylation at position lysine‐9. In this study, we have investigated the function of G9a in ovine foetal PASMC proliferation, migration and contractility.
Material and methods
Cell proliferation was measured by cell counting and BrdU incorporation assay and cell cycle analysis was performed by flow cytometry. Expression of cell cycle‐related genes was determined by real‐time PCR and the wound‐healing scratch assay was used to measure cell migration. A gel contraction assay was used to determine contractility of foetal PASMCs. Global DNA methylation was measured by liquid chromatography‐mass spectroscopy.
Results
Inhibition of G9a by its inhibitor BIX‐01294 reduced proliferation of foetal PASMCs and induced cell cycle arrest in G1 phase. This was accompanied by increased p21 expression, but not p53 and other cell cycle‐related genes. Treatment of foetal PASMCs with BIX‐01294 inhibited platelet‐derived growth factor‐induced cell proliferation and migration. Contractility of foetal PASMCs was also markedly inhibited by BIX‐01294. Expression of calponin and ROCK‐II proteins was reduced by BIX‐01294 in a dose‐dependent manner and BIX‐01294 significantly increased global methylation level in the foetal PASMCs.
Conclusion
Our results demonstrate for the first time that histone lysine methylation is involved in cell proliferation, migration, contractility and global DNA methylation in foetal PASMCs. Further understanding of this mechanism may provide insight into proliferative vascular disease in the lungs.
Objective
Pulmonary arterial hypertension, characterized by pulmonary vascular remodelling and vasoconstriction, is associated with excessive proliferative changes in pulmonary vascular walls. ...However, the role of HDACs in the phenotypic alteration of pulmonary arterial smooth muscle cells (PASMC) is largely unknown.
Material and methods
Pulmonary arterial smooth muscle cells were isolated from newborn sheep. Cell cycle analysis was performed by flow cytometry. mRNA and protein expression were measured by real‐time PCR and Western blot analysis. Wound‐healing scratch assay was used to measure cell migration. Contractility of newborn PASMCs was determined by gel contraction assay. Chromatin immunoprecipitation was used to examine histone modifications along the p21 promoter region. Global DNA methylation was measured by liquid chromatography‐mass spectroscopy.
Results
Inhibition of class I and class II HDACs by apicidin and HDACi VIII suppressed proliferation of newborn PASMC and induced cell cycle arrest in G1 phase. Acetyl H3 levels were higher in newborn PASMC treated with apicidin and HDACi VIII. This was accompanied by increased expression of p21 and reduced expression of CCND1 but not p53. HDAC inhibition altered histone codes around the p21 promoter region in NPASMC. Apicidin inhibited serum‐induced cell migration, and modulated profiling of expression of genes encoding pro‐oxidant and antioxidant enzymes. Contractility and global DNA methylation levels of newborn PASMCs were also markedly modulated by apicidin.
Conclusion
Our results demonstrate that class I HDACs are clearly involved in phenotypic alteration of newborn PASMC.
Background and purpose:
Coronary venous activity is modulated by endogenous and exogenous nitrovasodilators. The present study was to determine the role of protein kinase G (PKG) in the regulation of ...the basal tension and nitrovasodilator‐induced relaxation of coronary veins.
Experimental approach:
Effects of a PKG inhibitor on the basal tension and responses induced by nitroglycerin, DETA NONOate, and 8‐Br‐cGMP in isolated porcine coronary veins were determined. Cyclic cGMP was measured with radioimmunoassay. PKG activity was determined by measuring the incorporation of 32P from γ‐32P‐ATP into the specific substrate BPDEtide.
Key results:
Rp‐8‐Br‐PET‐cGMPS, a specific PKG inhibitor, increased the basal tension of porcine coronary veins and decreased PKG activity. The increase in tension was 38% of that caused by nitro‐L‐arginine. Relaxation of the veins induced by nitroglycerin and DETA NONOate was accompanied with increases in cGMP content and PKG activity. These effects were largely eliminated by inhibiting soluble guanylyl cyclase with ODQ. The increase in PKG activity induced by the nitrovasodilators was abolished by Rp‐8‐Br‐PET‐cGMPS. The relaxation caused by these dilators and by 8‐Br‐cGMP at their EC50 was attenuated by the PKG inhibitor by 51–66%.
Conclusions and implications:
These results suggest that PKG is critically involved in nitric oxide‐mediated regulation of the basal tension in porcine coronary veins and that it plays a primary role in relaxation induced by nitrovasodilators. Since nitric oxide plays a key role in modulating coronary venous activity, augmentation of PKG may be a therapeutic target for improving coronary blood flow.
British Journal of Pharmacology (2007) 152, 1060–1069; doi:10.1038/sj.bjp.0707479; published online 24 September 2007
Background and purpose:
The cGMP‐dependent protein kinase (PKG) is a key enzyme for nitrovasodilator‐induced vasodilation. The present study was to determine its role in nitrate tolerance.
...Experimental approach:
isolated porcine coronary arteries were incubated for 24 h with nitroglycerin (NTG) and their relaxant responses were determined. PKG activity was assayed by measuring the incorporation of 32P into BPDEtide. PKG protein was determined by Western blotting and PKG mRNA by real‐time PCR.
Key results:
A 24 h incubation with NTG attenuated relaxation of coronary arteries to NTG, which was associated with decreased PKG activity. The nitrate tolerance induced with NTG at 10−7 M was affected by a scavenger of reactive oxygen species and the tolerance induced with NTG at 10−6 and 10−5 M showed cross‐tolerance to DETA NONOate and 8‐Br‐cGMP (a cell permeable cGMP analogue). PKG protein and mRNA were down‐regulated by a 24 h incubation with NTG at 10−5 M but not at 10−7 M. Acute exposure to exogenous superoxide inhibited PKG activity stimulated by NTG at 10−7 M but not at 10−5 M. Superoxide had no effect on PKG activity stimulated with exogenous cGMP.
Conclusions and implications:
Nitrate tolerance induced by NTG at low concentrations may result from an increased production of reactive oxygen species acting on sites upstream of PKG. The tolerance induced by NTG at higher concentrations may be in part due to suppression of PKG expression resulting from sustained activation of the enzyme. These distinct mechanisms of nitrate tolerance may be of clinical significance.
British Journal of Pharmacology (2008) 153, 497–507; doi:10.1038/sj.bjp.0707600; published online 26 November 2007
Chitosan‐functionalized graphene oxides (FGOCs) were successfully synthesized. FGOCs were found to significantly improve the solubility of the GO in aqueous acidic media. The presence of organic ...groups was confirmed by means of XPS and TGA. Restoration of the sp2 carbon network and exfoliation of graphene sheets were confirmed by Raman spectroscopy, UV‐visible spectroscopy and WAXD. The SEM and AFM investigations of the resultant FGOCs showed that most of the graphene sheets were individual and few were layered. Controlled release behavior of Ibuprofen and 5‐fluorouracil was then investigated. We found that FGOCs are a promising new material for biological and medical applications.
GO is successfully functionalized with chitosan. The excellent exfoliation and dispersion of functionalized graphene sheets in aqueous acetic acid solution are presented. Moreover, IBU and 5‐FU are loaded successfully on FGOCs sheets, despite fewer π electrons. The controlled release behavior and long‐term biocompatibility of FGOCs graphene sheets suggest that they are promising novel materials for biomedical applications.
Objectives
Platelet‐activating factor (PAF) is produced by pulmonary vascular smooth muscle cells (PVSMC). We studied effects of Rho kinase on PAF stimulation of PVSMC proliferation in an attempt to ...understand the role of RhoA/Rho kinase on PAF‐induced ovine foetal pulmonary vascular remodelling. Our hypothesis is that PAF acts through Rho kinase, as one of its downstream signals, to induce arterial (SMC‐PA) and venous (SMC‐PV) cell proliferation in the hypoxic lung environment of the foetus, in utero.
Materials and methods
Rho kinase and MAPK effects on PAF receptor (PAFR)‐mediated cell population expansion, and PAFR expression, were studied by DNA synthesis, western blot analysis and immunocytochemistry. Effects of constructs T19N and G14V on PAF‐induced cell proliferation were also investigated.
Results
Hypoxia increased PVSMC proliferation and Rho kinase inhibitors, Y‐27632 and Fasudil (HA‐1077) as well as MAPK inhibitors PD 98059 and SB 203580 attenuated PAF stimulation of cell proliferation. RhoA T19N and G14V stimulated cell proliferation, but co‐incubation with PAF did not affect proliferative effects of the constructs. PAFR protein expression was significantly downregulated in both cell types by both Y‐27632 and HA‐1077, with comparable profiles. Also, cells treated with Y‐27632 had less PAF receptor fluorescence with significant disruption of cell morphology.
Conclusions
Our results show that Rho kinase non‐specifically modulated PAFR‐mediated responses by a translational modification of PAFR protein, and suggest that, in vivo, activation of Rho kinase by PAF may be a further pathway to sustain PAFR‐mediated PVSMC proliferation.
Neuroblastoma (NB), a childhood neoplasm arising from neural crest cells, is characterized by a diversity of clinical behaviors ranging from spontaneous remission to rapid tumor progression and ...death. In addition to genetic abnormalities, recent studies have indicated that epigenetic aberrations also contribute toward NB pathogenesis. However, the epigenetic mechanisms underlying the pathogenesis of NB are largely unknown. Inhibition of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) was evaluated through the measurement of H3K9Me2 levels. Cell proliferation was examined by cell counting in human NB cell lines (LA1-55n, IMR-5, and NMB). The RNA expression of EHMT2, MYCN, and p21 was measured by real-time PCR. The expression of PCNA, MYCN, p53, cyclinD1, H3, H3K27M2, and H3K9Me2 was examined by western blot analysis. In-vitro invasion and the effects of the EHMT2 inhibitor (BIX-01294) were assessed in the Transwell chamber assay. Caspase 3 and 8 activities were measured using a Caspase-Glo assay kit. The level of overall DNA methylation was measured by liquid chromatography–mass spectroscopy. BIX-01294, a specific inhibitor of EHMT2 (a key enzyme for histone H3 dimethylation at lysine-9), specifically decreases the overall H3K9Me2 level but not H3K27Me2. The inhibition of EHMT2 decreased the proliferation of NB cells and induced apoptosis by increasing caspase 8/caspase 3 activity. BIX-01294 inhibited NB cell mobility and invasion. This was accompanied by a decreased expression of the MYCN oncogene. Inhibition of EHMT2 enhanced a doxorubicin-induced inhibitory effect on cell proliferation. Finally, EHMT2 inhibition modulated overall DNA methylation levels in NB cells. Our results show that histone-lysine methylation is involved in cell proliferation, apoptosis, cell invasion, and overall DNA methylation in human NB cells. Further understanding of this mechanism may provide an insight into the pathogenesis of NB progression and lead to novel treatment strategies.
Rationale: Crosstalk between pulmonary arterial endothelial cells (PAEC) and pulmonary arterial smooth muscle cells (PASMC) plays an important role in the pathogenesis of pulmonary hypertension (PH). ......we injected 8-week old C57BL/6 mice with DMSO control or GW4869, a drug that is known to block the generation of extracellular vesicles, and exposed these mice to normoxia or 10% hypoxia for 3 weeks, followed by measurement of right ventricular systolic pressure (RVSP), RV hypertrophy (RV/(LV+S) ratio), and pulmonary arterial wall thickness.
Department of Pediatrics, Harbor-UCLA Medical Center, School of
Medicine, University of California, Los Angeles, Torrance,
California 90509
Agonist-induced smooth muscle relaxation occurs following
...an increase in intracellular concentrations of cGMP or cAMP. However, the role of protein kinase G (PKG) and/or protein kinase A (PKA) in
cGMP- or cAMP-mediated pulmonary vasodilation is not clearly elucidated. In this study, we examined the relaxation responses of
isolated pulmonary arteries of lambs (age = 10 ± 1 days),
preconstricted with endothelin-1, to increasing concentrations of
8-bromo-cGMP (8-BrcGMP) or 8-BrcAMP (cell-permeable analogs), in the
presence or absence of
Rp -8- -phenyl-1, N 2 -etheno-bromoguanosine
cyclic monosphordthioate ( Rp -8-PET-BrcGMPS) or
KT-5720, selective inhibitors of PKG and PKA, respectively. When
examined for specificity, Rp -8-Br-PET-cGMPS abolished PKG, but
not PKA, activity in pulmonary arterial extracts, whereas KT-5720
inhibited PKA activity only. 8-BrcGMP-induced relaxation was inhibited
by the PKG inhibitor only, whereas 8-BrcAMP-induced relaxation was
inhibited by both inhibitors. A nearly fourfold higher concentration of
cAMP than cGMP was required to relax arteries by 50% and to activate
PKG by 50%. Our results demonstrate that relaxation of pulmonary
arteries is more sensitive to cGMP than cAMP and that PKG plays
an important role in both cGMP- and cAMP-mediated relaxation.
protein kinase A; protein kinase G; lung; smooth muscle; lambs
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