Ru-Jeng Teng

Superoxide Reacts with Nitric Oxide to Nitrate Tyrosine at Physiological pH via Peroxynitrite

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO−) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280–27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkalinecis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.

Role of autophagy in angiogenesis in aortic endothelial cells.

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with impaired pulmonary vasodilation at birth. Previous studies demonstrated that a decrease in angiogenesis contributes to this failure of postnatal adaptation. We investigated the hypothesis that oxidative stress from NADPH oxidase (Nox) contributes to impaired angiogenesis in PPHN. PPHN was induced in fetal lambs by ductus arteriosus ligation at 85% of term gestation. Pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were compared for their angiogenic activities and superoxide production. HTFL-PAEC had decreased tube formation, cell proliferation, scratch recovery, and cell invasion and increased cell apoptosis. Superoxide (O(2)(-)) production, measured by dihydroethidium epifluorescence and HPLC, were increased in HTFL-PAEC compared with NFL-PAEC. The mRNA levels for Nox2, Rac1, p47(phox), and Nox4, protein levels of p67(phox) and Rac1, and NADPH oxidase activity were increased in HTFL-PAEC. NADPH oxidase inhibitor, apocynin (Apo), and antioxidant, N-acetyl-cysteine (NAC), improved angiogenic measures in HTFL-PAEC. Apo and NAC also reduced apoptosis in HTFL-PAEC. Our data suggest that PPHN is associated with increased O(2)(-) production from NADPH oxidase in PAEC. Increased oxidative stress from NADPH oxidase contributes to the impaired angiogenesis of PAEC in PPHN.

The human tail

The human tail is a congenital anomaly with a protruding lesion from the lumbosacrococcygeal region. A newborn with a tail-like structure over the coccygeal area observed since birth is presented. Lipoma accompanied by tethered spinal cord were found. In reviewing the literature from 1960 to 1997, 59 cases were described. Higher incidences of spinal dysraphism (49.15%) and tethered spinal cord (20.34%) compared with previous reports were evident. This fact plays an important role in understanding the disturbance of development and regression of human tails. A new classification according to whether the anomaly appears in combination with spinal dysraphism is proposed for clinical usage. Preoperative detailed image studies are needed to clarify the possibility of tethered spinal cord syndrome developing in the future and thus prevent it. Magnetic resonance imaging is the modality of choice if available. Long-term follow-up for possible sequelae after operation, especially in cases with spinal dysraphism, is necessary.

Cross talk between NADPH oxidase and autophagy in pulmonary artery endothelial cells with intrauterine persistent pulmonary hypertension.

Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91(phox) and p47(phox), NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis.

Decreases in manganese superoxide dismutase expression and activity contribute to oxidative stress in persistent pulmonary hypertension of the newborn

A rapid increase in the synthesis and release of nitric oxide (NO) facilitates the pulmonary vasodilation that occurs during birth-related transition. Alteration of this transition in persistent pulmonary hypertension of the newborn (PPHN) is associated with impaired function of endothelial nitric oxide synthase (eNOS) and an increase in oxidative stress. We investigated the hypothesis that a decrease in expression and activity of mitochondrial localized manganese superoxide dismutase (MnSOD) in pulmonary artery endothelial cells (PAEC) increases oxidative stress and impairs eNOS function in PPHN. We isolated PAEC and pulmonary arteries from fetal lambs with PPHN induced by prenatal ductus arteriosus ligation or sham ligation (control). We investigated MnSOD expression and activity, tyrosine nitration of MnSOD, and mitochondrial O(2)(-) levels in PAEC from control and PPHN lambs. We introduced exogenous MnSOD via an adenoviral vector (ad-MnSOD) transduction into PAEC and pulmonary arteries of PPHN lambs. The effect of ad-MnSOD was investigated on: mitochondrial O(2)(-) levels, MnSOD and eNOS expression and activity, intracellular hydrogen peroxide (H(2)O(2)) levels, and catalase expression in PAEC. MnSOD mRNA and protein levels and activity were decreased and MnSOD tyrosine nitration was increased in PPHN-PAEC. ad-MnSOD transduction of PPHN-PAEC increased its activity two- to threefold, decreased mitochondrial O(2)(-) levels, and increased H(2)O(2) levels and catalase expression. ad-MnSOD transduction improved eNOS expression and function and the relaxation response of PPHN pulmonary arteries. Our observations suggest that decreased MnSOD expression and activity contribute to the endothelial dysfunction observed in PPHN.

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.

Granulocyte colony-stimulating factor in the cord blood of premature neonates born to mothers with pregnancy-induced hypertension☆☆☆

OBJECTIVES To estimate the cord blood levels of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in preterm infants and to study the relationship of these levels to pregnancy-induced hypertension (PIH) and absolute neutrophil counts. STUDY DESIGN G-CSF and GM-CSF levels in the cord blood of preterm neonates (n = 74) either with or without maternal PIH were estimated by enzyme-linked immunosorbent assay. RESULTS Infants in the PIH group had lower white blood cell, absolute neutrophil, absolute lymphocyte, and monocyte counts. The levels of G-CSF in cord blood were significantly lower in infants whose mothers had PIH (P =.04) and in infants with neutropenia (P =. 01). G-CSF levels were positively correlated with both absolute neutrophil count (P =.02) and total white blood cell count (P =.01). GM-CSF was undetectable in all subjects. According to logistic regression with neutropenia as the dependent variable, only maternal PIH (P <.001), gestational age (P <.001), and G-CSF (P =.01) were independently related. CONCLUSION In this study maternal PIH and low gestational age were significantly associated with neutropenia in premature infants. Low G-CSF levels may contribute to the neutropenia that is commonly seen in infants born to mothers with PIH.

AMP kinase activation improves angiogenesis in pulmonary artery endothelial cells with in utero pulmonary hypertension.

Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with in utero pulmonary hypertension (IPH) have phenotypical changes that lead to increased reactive oxygen species (ROS) formation and impaired angiogenesis. AMP-activated protein kinase (AMPK) is known to be activated by ROS, which is expected to help angiogenesis in IPH-PAEC. The objectives of this study were to investigate AMPK responses in IPH and its role in angiogenesis. We observed that, compared with control PAEC, IPH-PAEC have decreased phosphorylation of AMPKα catalytic subunit and AMPK downstream enzymes, indicating a decrease in AMPK activity. In addition, the expression of AMPK kinases is decreased, and protein phosphatase 2 is increased in IPH-PAEC, potentially contributing to the decreased AMPK activation. Metformin, an AMPK activator, improved IPH-PAEC angiogenesis while increasing endothelial NO synthase (eNOS) serine(1179) phosphorylation and decreasing the eNOS-caveolin-1 association. Metformin also increased MnSOD activity and the expression of both eNOS and MnSOD. The increase in angiogenesis by Metformin is abolished by pretreatment with AMPK inhibitor, Compound C. Expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor β (PDGFβ) are decreased in IPH-PAEC compared with control PAEC and were not altered by Metformin. These data indicate that Metformin improves angiogenesis through mechanisms independent of these angiogenic factors. In conclusion, activation of AMPK restores angiogenesis and increases the bioavailability of nitric oxide in IPH. Whether Metformin is beneficial in the management of pulmonary hypertension requires further investigation.

Inducible HSP70 regulates superoxide dismutase-2 and mitochondrial oxidative stress in the endothelial cells from developing lungs

Superoxide dismutase 2 (SOD-2) is synthesized in the cytosol and imported into the mitochondrial matrix, where it is activated and functions as the primary antioxidant for cellular respiration. The specific mechanisms that target SOD-2 to the mitochondria remain unclear. We hypothesize that inducible heat shock protein 70 (iHSP70) targets SOD-2 to the mitochondria via a mechanism facilitated by ATP, and this process is impaired in persistent pulmonary hypertension of the newborn (PPHN). We observed that iHSP70 interacts with SOD-2 and targets SOD-2 to the mitochondria. Interruption of iHSP70-SOD-2 interaction with 2-phenylethylenesulfonamide-μ (PFT-μ, a specific inhibitor of substrate binding to iHSP70 COOH terminus) and siRNA-mediated knockdown of iHSP70 expression disrupted SOD-2 transport to mitochondria. Increasing intracellular ATP levels by stimulation of respiration with CaCl2 facilitated the mitochondrial import of SOD-2, increased SOD-2 activity, and decreased the mitochondrial superoxide (O2(·-)) levels in PPHN pulmonary artery endothelial cells (PAEC) by promoting iHSP70-SOD-2 dissociation at the outer mitochondrial membrane. In contrast, oligomycin, an inhibitor of mitochondrial ATPase, decreased SOD-2 expression and activity and increased O2(·-) levels in the mitochondria of control PAEC. The basal ATP levels and degree of iHSP70-SOD-2 dissociation were lower in PPHN PAEC and lead to increased SOD-2 degradation in cytosol. In normal pulmonary arteries (PA), PFT-μ impaired the relaxation response of PA rings in response to nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine. Pretreatment with Mito-Q, a mitochondrial targeted O2(·-) scavenger, restored the relaxation response in PA rings pretreated with PFT-μ. Our observations suggest that iHSP70 chaperones SOD-2 to the mitochondria. Impaired SOD-2-iHSP70 dissociation decreases SOD-2 import and contributes to mitochondrial oxidative stress in PPHN.