Publications (8)32.2 Total impact
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Article: DISRUPTION OF ENDOTHELIAL CELL MITOCHONDRIAL BIOENERGETICS IN LAMBS WITH INCREASED PULMONARY BLOOD FLOW.
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ABSTRACT: AIMS: The mitochondrial dysfunction in our lamb model of congenital heart disease with increased pulmonary blood flow (Shunt) is associated with disrupted carnitine metabolism. Our recent studies have also shown that asymmetric dimethylarginine (ADMA) levels are increased in Shunt lambs and ADMA increases the nitration of mitochondrial proteins in lamb pulmonary arterial endothelial cells (PAEC) in a nitric oxide synthase (NOS)-dependent manner. Thus, we determined if there was a mechanistic link between eNOS, ADMA and the disruption of carnitine homeostasis in PAEC. RESULTS: Exposure of PAEC to ADMA induced the redistribution of eNOS to the mitochondria, resulting in an increase in carnitine acetyl transferase (CrAT) nitration and decreased CrAT activity. The resulting increase in acyl-carnitine levels resulted in mitochondrial dysfunction and the disruption of mitochondrial bioenergetics. As the addition of L-arginine prevented these pathologic changes we examined the affect of L-arginine supplementation on carnitine homeostasis, mitochondrial function and NO signaling in Shunt lambs. We found that the treatment of Shunt lambs with L-arginine prevented the ADMA-mediated mitochondrial redistribution of eNOS, the nitration-mediated inhibition of CrAT, and maintained carnitine homeostasis. In turn ATP levels and eNOS/Hsp90 interactions were preserved and this decreased NOS uncoupling and enhanced NO generation. INNOVATION: Our data link alterations in cellular L-arginine metabolism with the disruption of mitochondrial bioenergetics and implicate altered carnitine homeostasis as a key player in this process. CONCLUSIONS: L-arginine supplementation may be a useful therapy to prevent the mitochondrial dysfunction involved in the pulmonary vascular alterations secondary to increased pulmonary blood flow.Antioxidants & Redox Signaling 12/2012; · 8.20 Impact Factor -
Article: Rosiglitazone preserves pulmonary vascular function in lambs with increased pulmonary blood flow.
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ABSTRACT: Background:Pulmonary vascular function is impaired with increased pulmonary blood flow (PBF). We hypothesized that a peroxisome proliferator-activated receptor-gamma (PPARγ) agonist would mitigate this effect.Methods:An aorta to pulmonary artery shunt was placed in 11 fetal lambs. Lambs received the PPARγ agonist rosiglitazone (RG, 3 mg/kg/day, n=6) or vehicle (n=5) for 4-weeks. Lung tissue from 5 normal 4-week lambs was used for comparisons.Results:At 4-weeks, pulmonary artery pressure (PAP) and vascular resistance (PVR) decreased with inhaled NO in RG and vehicle-treated shunt lambs. PAP and PVR decreased with acetylcholine in RG-treated, not vehicle-treated shunt lambs. In vehicle-treated shunt lambs, NADPH oxidase activity, rac1, superoxide, and 3-nitrotyrosine (3-NT) levels were increased and Ser1177 endothelial NO synthase (eNOS) protein was decreased compared to normal lambs. In RG-treated shunt lambs, NO(X), Ser1177 eNOS protein and eNOS activity were increased, and NADPH activity, rac1, superoxide levels and 3-NT levels were decreased, compared to vehicle-treated shunt lambs. PPARγ protein expression was lower in vehicle-treated shunt lambs than normal and RG-treated shunt lambs.Conclusions:The PPARγ agonist, RG, prevents the loss of agonist induced endothelium-dependent pulmonary vascular relaxation in lambs with increased PBF, in part, due to decreased oxidative stress and/or increased NO production.Pediatric Research (2012); doi:10.1038/pr.2012.149.Pediatric Research 11/2012; · 2.70 Impact Factor -
Article: Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures.
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ABSTRACT: Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.Free radical biology & medicine 06/2012; 53(5):1139-51. · 5.42 Impact Factor -
Article: Glutathione supplementation attenuates lipopolysaccharide-induced mitochondrial dysfunction and apoptosis in a mouse model of acute lung injury.
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ABSTRACT: Acute lung injury (ALI) is a life threatening condition associated with hypoxemia, diffuse alveolar damage, inflammation, and loss of lung function. Lipopolysaccharide (LPS; endotoxin) from the outer membrane of Gram-negative bacteria is a major virulence factor involved in the development of ALI. The depletion of glutathione (GSH), an essential intra- and extra-cellular protective antioxidant, by LPS is an important event that contributes to the elevation in reactive oxygen species. Whether restoring GSH homeostasis can effectively ameliorate mitochondrial dysfunction and cellular apoptosis in ALI is unknown and therefore, was the focus of this study. In peripheral lung tissue of LPS-treated mice, hydrogen peroxide and protein nitration levels were significantly increased. Pre-treatment with GSH-ethyl ester (GSH-EE) prevented this increase in oxidative stress. LPS also increased the lactate/pyruvate ratio, attenuated SOD2 protein levels, and decreased ATP levels in the mouse lung indicative of mitochondrial dysfunction. Again, GSH-EE treatment preserved the mitochondrial function. Finally, our studies showed that LPS induced an increase in the mitochondrial translocation of Bax, caspase 3 activation, and nuclear DNA fragmentation and these parameters were all prevented with GSH-EE. Thus, this study suggests that GSH-EE supplementation may reduce the mitochondrial dysfunction associated with ALI.Frontiers in physiology. 01/2012; 3:161. -
Article: Oxygen glucose deprivation in rat hippocampal slice cultures results in alterations in carnitine homeostasis and mitochondrial dysfunction.
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ABSTRACT: Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neuroprotective. Thus, this study was undertaken to elucidate the molecular mechanisms by which HI alters carnitine metabolism and to begin to elucidate the mechanism underlying the neuroprotective effect of L-carnitine (LCAR) supplementation. Utilizing neonatal rat hippocampal slice cultures we found that oxygen glucose deprivation (OGD) decreased the levels of free carnitines (FC) and increased the acylcarnitine (AC): FC ratio. These changes in carnitine homeostasis correlated with decreases in the protein levels of carnitine palmitoyl transferase (CPT) 1 and 2. LCAR supplementation prevented the decrease in CPT1 and CPT2, enhanced both FC and the AC∶FC ratio and increased slice culture metabolic viability, the mitochondrial membrane potential prior to OGD and prevented the subsequent loss of neurons during later stages of reperfusion through a reduction in apoptotic cell death. Finally, we found that LCAR supplementation preserved the structural integrity and synaptic transmission within the hippocampus after OGD. Thus, we conclude that LCAR supplementation preserves the key enzymes responsible for maintaining carnitine homeostasis and preserves both cell viability and synaptic transmission after OGD.PLoS ONE 01/2012; 7(9):e40881. · 4.09 Impact Factor -
Article: Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures.
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ABSTRACT: The pathological basis of neonatal hypoxia-ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI-induced neuronal cell death. The p38 mitogen-activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase-3 and the appearance of apoptotic neuronal cells. Pre-treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I-1-driven adeno-associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD-mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.European Journal of Neuroscience 09/2011; 34(7):1093-101. · 3.63 Impact Factor -
Article: Delineating the angiogenic gene expression profile before pulmonary vascular remodeling in a lamb model of congenital heart disease.
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ABSTRACT: Disordered angiogenesis is implicated in pulmonary vascular remodeling secondary to congenital heart diseases (CHD). However, the underlying genes are not well delineated. We showed previously that an ovine model of CHD with increased pulmonary blood flow (PBF, Shunt) has an "angiogenesis burst" between 1 and 4 wk of age. Thus we hypothesized that the increased PBF elicited a proangiogenic gene expression profile before onset of vessel growth. To test this we utilized microarray analysis to identify genes that could be responsible for the angiogenic response. Total RNA was isolated from lungs of Shunt and control lambs at 3 days of age and hybridized to Affymetrix gene chips for microarray analyses (n = 8/group). Eighty-nine angiogenesis-related genes were found to be upregulated and 26 angiogenesis-related genes downregulated in Shunt compared with control lungs (cutting at 1.2-fold difference, P < 0.05). We then confirmed upregulation of proangiogenic genes FGF2, Angiopoietin2 (Angpt2), and Birc5 at mRNA and protein levels and upregulation of ccl2 at mRNA level in 3-day Shunt lungs. Furthermore, we found that pulmonary arterial endothelial cells (PAEC) isolated from fetal lambs exhibited increased expression of FGF2, Angpt2, Birc5, and ccl2 and enhanced angiogenesis when exposed to elevated shear stress (35 dyn/cm²) compared with cells exposed to more physiological shear stress (20 dyn/cm²). Finally, we demonstrated that blocking FGF2, Angpt2, Birc5, or ccl2 signaling with neutralizing antibodies or small interfering RNA (siRNA) significantly decreased the angiogenic response induced by shear stress. In conclusion, we have identified a "proangiogenic" gene expression profile in a lamb model of CHD with increased PBF that precedes onset of pulmonary vascular remodeling. Our data indicate that FGF2, Angpt2, Birc5, and ccl2 may play important roles in the angiogenic response.Physiological Genomics 10/2010; 43(2):87-98. · 2.73 Impact Factor -
Article: A novel role for caveolin-1 in regulating endothelial nitric oxide synthase activation in response to H2O2 and shear stress.
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ABSTRACT: Previous studies have shown that acute increases in oxidative stress induced by the addition of hydrogen peroxide (H(2)O(2)) can increase endothelial nitric oxide synthase (eNOS) catalytic activity via an increase in the phosphorylation of eNOS at serine 1177. However, it is unclear how increased H(2)O(2) affects nitric oxide (NO) signaling when endothelial cells are exposed to biomechanical forces. Thus, the purpose of this study was to evaluate the acute effects of H(2)O(2) on NO signaling in the presence or absence of laminar shear stress. We found that acute sustained increases in cellular H(2)O(2) levels in bovine aortic endothelial cells did not alter basal NO generation but the NO produced in response to shear stress was significantly increased. This amplification in NO signaling was found to correlate with an H(2)O(2)-induced increase in eNOS localized to the plasma membrane and an increase in total caveolin-1 protein levels. We further demonstrated that overexpressing caveolin-1 increased eNOS localized to the plasma membrane again without altering total eNOS protein levels. We also found that caveolin-1 overexpression increased NO generation in response to shear stress but only in the presence of H(2)O(2). Conversely, depleting caveolin-1 with an siRNA decreased eNOS localized to the plasma membrane and abolished the enhanced NO generation. Finally, we found that expressing a caveolin-1 binding-site deletion mutant of eNOS in COS-7 cells decreased its plasma membrane localization and resulted in attenuated NO production in response to calcium activation. In conclusion, we have identified a new role for caveolin-1 in enhancing eNOS trafficking to the plasma membrane that seems to be involved in priming eNOS for flow-mediated activation under conditions of oxidative stress. To our knowledge, this is the first report that H(2)O(2) modulates eNOS activity by altering its subcellular location and that caveolin-1 can play a stimulatory role in NO signaling.Free radical biology & medicine 03/2010; 49(2):159-70. · 5.42 Impact Factor
Top co-authors
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Institutions
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2012
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University of Montana
Missoula, MT, USA
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2010–2012
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Georgia Health Sciences University
- Vascular Biology Center
Augusta, GA, USA
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