Christine Gross

Reactive Oxygen Species in Pulmonary Vascular Remodeling

The pathogenesis of pulmonary hypertension is a complex multifactorial process that involves the remodeling of pulmonary arteries. This remodeling process encompasses concentric medial thickening of small arterioles, neomuscularization of previously nonmuscular capillary-like vessels, and structural wall changes in larger pulmonary arteries. The pulmonary arterial muscularization is characterized by vascular smooth muscle cell hyperplasia and hypertrophy. In addition, in uncontrolled pulmonary hypertension, the clonal expansion of apoptosis-resistant endothelial cells leads to the formation of plexiform lesions. Based upon a large number of studies in animal models, the three major stimuli that drive the vascular remodeling process are inflammation, shear stress, and hypoxia. Although, the precise mechanisms by which these stimuli impair pulmonary vascular function and structure are unknown, reactive oxygen species (ROS)-mediated oxidative damage appears to play an important role. ROS are highly reactive due to their unpaired valence shell electron. Oxidative damage occurs when the production of ROS exceeds the quenching capacity of the antioxidant mechanisms of the cell. ROS can be produced from complexes in the cell membrane (nicotinamide adenine dinucleotide phosphate-oxidase), cellular organelles (peroxisomes and mitochondria), and in the cytoplasm (xanthine oxidase). Furthermore, low levels of tetrahydrobiopterin (BH4) and L-arginine the rate limiting cofactor and substrate for endothelial nitric oxide synthase (eNOS), can cause the uncoupling of eNOS, resulting in decreased NO production and increased ROS production. This review will focus on the ROS generation systems, scavenger antioxidants, and oxidative stress associated alterations in vascular remodeling in pulmonary hypertension.

PKC-dependent phosphorylation of eNOS at T495 regulates eNOS coupling and endothelial barrier function in response to G+ -toxins.

Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G+-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.

Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: role of protein kinase G nitration.

Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B‐type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO‐cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO−) generation and PKG‐1α nitration, while PKG‐1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1‐methyl‐4‐pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO− donor, 3‐morpholinosydnonimine N‐ethylcarbamide (SIN‐1). Peripheral lung extracts from 4‐week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24 h (with exogenous activation of cGMP) revealed increased ONOO− levels, elevated PKG‐1α nitration, and decreased kinase activity without changes in PKG‐1α protein levels. However, in Shunt lambs treated with L‐arginine or lambs administered polyethylene glycol conjugated‐SOD (PEG‐SOD) during inhaled NO exposure, ONOO− and PKG‐1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG‐1α nitration and subsequent attenuation of activity. J. Cell. Physiol. 226: 3104–3113, 2011.

Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of RhoA

Background: The activation of RhoA is a critical event in acute lung injury (ALI), but the role of nitration in this process is unresolved. Results: The nitration of RhoA at Tyr34 produced GEF-like conformational changes that stimulate RhoA by decreasing GDP binding. Conclusion: We have identified a new mechanism of RhoA activation. Significance: Preventing RhoA nitration may be useful for the management of ALI. Acute lung injury (ALI) is characterized by increased endothelial hyperpermeability. Protein nitration is involved in the endothelial barrier dysfunction in LPS-exposed mice. However, the nitrated proteins involved in this process have not been identified. The activation of the small GTPase RhoA is a critical event in the barrier disruption associated with LPS. Thus, in this study we evaluated the possible role of RhoA nitration in this process. Mass spectroscopy identified a single nitration site, located at Tyr34 in RhoA. Tyr34 is located within the switch I region adjacent to the nucleotide-binding site. Utilizing this structure, we developed a peptide designated NipR1 (nitration inhibitory peptide for RhoA 1) to shield Tyr34 against nitration. TAT-fused NipR1 attenuated RhoA nitration and barrier disruption in LPS-challenged human lung microvascular endothelial cells. Further, treatment of mice with NipR1 attenuated vessel leakage and inflammatory cell infiltration and preserved lung function in a mouse model of ALI. Molecular dynamics simulations suggested that the mechanism by which Tyr34 nitration stimulates RhoA activity was through a decrease in GDP binding to the protein caused by a conformational change within a region of Switch I, mimicking the conformational shift observed when RhoA is bound to a guanine nucleotide exchange factor. Stopped flow kinetic analysis was used to confirm this prediction. Thus, we have identified a new mechanism of nitration-mediated RhoA activation involved in LPS-mediated endothelial barrier dysfunction and show the potential utility of “shielding” peptides to prevent RhoA nitration in the management of ALI.

Asymmetric Dimethylarginine Induces Endothelial Nitric Oxide Synthase Mitochondrial Redistribution through the nitration-mediated activation of Akt1

Background: Asymmetric dimethylarginine (ADMA) can induce endothelial nitric-oxide synthase (eNOS) redistribution from the plasma membrane to the mitochondria. Results: AMDA induces nitration of Akt1 at Tyr350 within the client-binding domain, increasing its activation and enhancing eNOS phosphorylation. Conclusion: Under physiologic conditions, Akt1-mediated redistribution of eNOS to the mitochondria enhances mitochondrial coupling. Significance: Reducing Akt1 nitration may reduce the deleterious effects of Akt1 signaling in various pathologies. We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser617 and Ser1179. Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr350) located within the client-binding domain. Replacement of Tyr350 with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser617 and Ser1179 by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr350.

Endothelin-1 Induces a Glycolytic Switch in Pulmonary Arterial Endothelial Cells via the Mitochondrial Translocation of Endothelial Nitric Oxide Synthase

Recent studies have indicated that, during the development of pulmonary hypertension (PH), there is a switch from oxidative phosphorylation to glycolysis in the pulmonary endothelium. However, the mechanisms underlying this phenomenon have not been elucidated. Endothelin (ET)-1, an endothelial-derived vasoconstrictor peptide, is increased in PH, and has been shown to play an important role in the oxidative stress associated with PH. Thus, in this study, we investigated whether there was a potential link between increases in ET-1 and mitochondrial remodeling. Our data indicate that ET-1 induces the redistribution of endothelial nitric oxide synthase (eNOS) from the plasma membrane to the mitochondria in pulmonary arterial endothelial cells, and that this was dependent on eNOS uncoupling. We also found that ET-1 disturbed carnitine metabolism, resulting in the attenuation of mitochondrial bioenergetics. However, ATP levels were unchanged due to a compensatory increase in glycolysis. Further mechanistic investigations demonstrated that ET-1 mediated the redistribution of eNOS via the phosphorylation of eNOS at Thr495 by protein kinase C δ. In addition, the glycolytic switch appeared to be dependent on mitochondrial-derived reactive oxygen species that led to the activation of hypoxia-inducible factor signaling. Finally, the cell culture data were confirmed in vivo using the monocrotaline rat model of PH. Thus, we conclude that ET-1 induces a glycolytic switch in pulmonary arterial endothelial cells via the redistribution of uncoupled eNOS to the mitochondria, and that preventing this event may be an approach for the treatment of PH.

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.

Nitration Of Tyrosine 247 Inhibits Protein Kinase G-1α Activity By Attenuating Cyclic Guanosine Monophosphate Binding

Background: PKG-1α nitration plays an important role in the development of pulmonary hypertension. Results: We identified Tyr247 as the key residue susceptible to nitration and inhibition of PKG-1α. Conclusion: Nitration attenuates PKG activity by reducing its affinity for cGMP. Significance: Preventing the nitration of PKG-1α could prevent the phenotypic remodeling in the blood vessels during the development of a number of cardiovascular diseases. The cGMP-dependent protein kinase G-1α (PKG-1α) is a downstream mediator of nitric oxide and natriuretic peptide signaling. Alterations in this pathway play a key role in the pathogenesis and progression of vascular diseases associated with increased vascular tone and thickness, such as pulmonary hypertension. Previous studies have shown that tyrosine nitration attenuates PKG-1α activity. However, little is known about the mechanisms involved in this event. Utilizing mass spectrometry, we found that PKG-1α is susceptible to nitration at tyrosine 247 and 425. Tyrosine to phenylalanine mutants, Y247F- and Y425F-PKG-1α, were both less susceptible to nitration than WT PKG-1α, but only Y247F-PKG-1α exhibited preserved activity, suggesting that the nitration of Tyr247 is critical in attenuating PKG-1α activity. The overexpression of WT- or Y247F-PKG-1α decreased the proliferation of pulmonary artery smooth muscle cells (SMC), increased the expression of SMC contractile markers, and decreased the expression of proliferative markers. Nitrosative stress induced a switch from a contractile to a synthetic phenotype in cells expressing WT- but not Y247F-PKG-1α. An antibody generated against 3-NT-Y247 identified increased levels of nitrated PKG-1α in humans with pulmonary hypertension. Finally, to gain a more mechanistic understanding of how nitration attenuates PKG activity, we developed a homology model of PKG-1α. This model predicted that the nitration of Tyr247 would decrease the affinity of PKG-1α for cGMP, which we confirmed using a [3H]cGMP binding assay. Our study shows that the nitration of Tyr247 and the attenuation of cGMP binding is an important mechanism regulating in PKG-1α activity and SMC proliferation/differentiation.

Dimethylarginine Dimethylaminohydrolase II Overexpression Attenuates LPS-Mediated Lung Leak in Acute Lung Injury

Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA), together with exaggerated oxidative/nitrative stress, contributes to the development of ALI in mice exposed to LPS. Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown, and was the focus of this study. In human lung microvascular endothelial cells, DDAH II overexpression prevented the LPS-dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also attenuated the endothelial barrier disruption associated with LPS exposure. Similarly, in vivo, we demonstrated that the targeted overexpression of DDAH II in the pulmonary vasculature significantly inhibited the accumulation of ADMA and the subsequent increase in oxidative/nitrative stress in the lungs of mice exposed to LPS. In addition, augmenting pulmonary DDAH II activity before LPS exposure reduced lung vascular leak and lung injury and restored lung function when DDAH activity was increased after injury. Together, these data suggest that enhancing DDAH II activity may prove a useful adjuvant therapy to treat patients with ALI.

Endothelial cell signaling and ventilator-induced lung injury: molecular mechanisms, genomic analyses, and therapeutic targets

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS). Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The pathobiology of VILI and ARDS shares many inflammatory features including increases in lung vascular permeability due to loss of endothelial cell barrier integrity resulting in alveolar flooding. While there have been advances in the understanding of certain elements of VILI and ARDS pathobiology, such as defining the importance of lung inflammatory leukocyte infiltration and highly induced cytokine expression, a deep understanding of the initiating and regulatory pathways involved in these inflammatory responses remains poorly understood. Prevailing evidence indicates that loss of endothelial barrier function plays a primary role in the development of VILI and ARDS. Thus this review will focus on the latest knowledge related to 1) the key role of the endothelium in the pathogenesis of VILI; 2) the transcription factors that relay the effects of excessive mechanical stress in the endothelium; 3) the mechanical stress-induced posttranslational modifications that influence key signaling pathways involved in VILI responses in the endothelium; 4) the genetic and epigenetic regulation of key target genes in the endothelium that are involved in VILI responses; and 5) the need for novel therapeutic strategies for VILI that can preserve endothelial barrier function.