Fatty Acid Composition and Stoichiometry Determine the Angiogenesis Microenvironment.

The current study tested the hypothesis of whether specific lipids may control angiogenic reactions. Using the chorioallantoic membrane assay of the chick embryo, new vessel formation was analyzed quantitatively by gas chromatography and mass spectrometry as well as bioinformatics tools including an angiogenesis analyzer. Our biochemical experiments showed that a specific lipid composition and stoichiometry determine the angiogenesis microenvironment to accelerate or inhibit vessel formation. Specific lipids of angiogenesis determinants in the vessel area and the non-vessel area were identified as nitrooleic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitic acid, oleic acid, linoleic acid, linolenic acid, epoxyoleic acid, lysophosphatidylcholine (LPC), cholesterol, 7-ketocholesterol, and docosahexaenoyl lysophosphatidylcholine (DHA-LPC). Vessel formation happens on the surface area of the hydrophilic membrane of the yolk. Our biochemical data demonstrated that angiogenesis was followed in the white lipid complex area to generate more branches, junctions, segments, and extremities. We analyzed lipid fragments in the vessel, non-vessel, and albumen area to show that each area contains a specific lipid composition and stoichiometry. Mass spectrometry data demonstrated that the vessel area has higher concentrations of nitrooleic acid, palmitic acid, stearic acid, LPC, lysophosphatidylethanolamine, cholesterol, oleic acid, linoleic acid, 7-ketocholesterol, and DHA-LPC; however, DHA and EPA were abundant in the hydrophobic non-vessel area. The purpose of vessel formation is to wrap up the yolk area to transport nutrients including specific fatty acids. Besides, angiogenesis requires aqueous albumen shown by distance-dependent vessel formation from albumen and oxygen. Higher concentrations of fatty acids are required for energy and carbon structure from the carbon-carbon bond, membrane building blocks, and amphiphilic detergent to solubilize a hydrophobic environment in the aqueous blood layer. The current study may guide that the uncovered hydrophobic or zwitterionic molecules such as DHA and DHA-LPC may control angiogenesis as antiangiogenic or proangiogenic molecules as potential drug targets for treating uncontrolled angiogenesis-related diseases, including diabetic retinopathy and age-related macular degeneration.

Dual Switch Mechanism of Erythropoietin as an Antiapoptotic and Pro-Angiogenic Determinant in the Retina.

Constant or intense light degenerates the retina and retinal pigment epithelial cells. Light generates reactive oxygen species and nitric oxide leading to initial reactions of retinal degeneration. Apoptosis is the primary mechanism of abnormal death of photoreceptors, retinal ganglion cells, or retinal pigment epithelium (RPE) in degenerative retinal diseases, including diabetic retinopathy and age-related macular degeneration. The current study evaluated the function of erythropoietin (EPO) on angiogenesis and apoptosis in the retina and RPE under oxidative stress. We determined the pro-angiogenic and antiapoptotic mechanism of EPO under stress conditions using a conditional EPO knockdown model using siRNA, EPO addition, proteomics, immunocytochemistry, and bioinformatic analysis. Our studies verified that EPO protected retinal cells from light-, hypoxia-, hyperoxia-, and hydrogen peroxide-induced apoptosis through caspase inhibition, whereas up-regulated angiogenic reactions through vascular endothelial growth factor (VEGF) and angiotensin pathway. We demonstrated that the EPO expression in the retina and subsequent serine/threonine/tyrosine kinase phosphorylations might be linked to oxidative stress response tightly to determining angiogenesis and apoptosis. Neuroprotective roles of EPO may involve the balance between antiapoptotic and pro-angiogenic signaling molecules, including BCL-xL, c-FOS, caspase-3, nitric oxide, angiotensin, and VEGF receptor. Our data indicate a new therapeutic application of EPO toward retinal degeneration based on the dual roles in apoptosis and angiogenesis at the molecular level under oxidative stress.

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Oleic Acid, Cholesterol, and Linoleic Acid as Angiogenesis Initiators.

The current study determined the natural angiogenic molecules using an unbiased metabolomics approach. A chick chorioallantoic membrane (CAM) model was used to examine pro- and antiangiogenic molecules, followed by gas chromatography-mass spectrometry (GCMS) analysis. Vessel formation was analyzed quantitatively using the angiogenic index (p < 0.05). At embryonic day one, a white streak or circle area was observed when vessel formation begins. GCMS analysis and database search demonstrated that angiogenesis may initiate when oleic, cholesterol, and linoleic acids increased in the area of angiogenic reactions. The gain of function study was conducted by the injection of cholesterol and oleic acid into a chick embryo to determine the role of each lipid in angiogenesis. We propose that oleic acid, cholesterol, and linoleic acid are natural molecules that set the platform for the initiation stage of angiogenesis before other proteins including the vascular endothelial growth factor, angiopoietin, angiotensin, and erythropoietin join as the angiome in sprout extension and vessel maturation.

Inhibition of HDAC6 Attenuates Diabetes-Induced Retinal Redox Imbalance and Microangiopathy.

We investigated the contributing role of the histone deacetylase 6 (HDAC6) to the early stages of diabetic retinopathy (DR). Furthermore, we examined the mechanism of action of HDAC6 in human retinal endothelial cells (HuREC) exposed to glucidic stress. Streptozotocin-induced diabetic rats (STZ-rats), a rat model of type 1 diabetes, were used as model of DR. HDAC6 expression and activity were increased in human diabetic postmortem donors and STZ-rat retinas and were augmented in HuREC exposed to glucidic stress (25 mM glucose). Administration of the HDAC6 specific inhibitor Tubastatin A (TS) (10 mg/kg) prevented retinal microvascular hyperpermeability and up-regulation of inflammatory markers. Furthermore, in STZ-rats, TS decreased the levels of senescence markers and rescued the expression and activity of the histone deacetylase sirtuin 1 (SIRT1), while downregulating the levels of free radicals and of the redox stress markers 4-hydroxynonenal (4-HNE) and nitrotyrosine (NT). The antioxidant effects of TS, consequent to HDAC6 inhibition, were associated with preservation of Nrf2-dependent gene expression and up-regulation of thioredoxin-1 activity. In vitro data, obtained from HuREC, exposed to glucidic stress, largely replicated the in vivo results further confirming the antioxidant effects of HDAC6 inhibition by TS in the diabetic rat retina. In summary, our data implicate HDAC6 activation in mediating hyperglycemia-induced retinal oxidative/nitrative stress leading to retinal microangiopathy and, potentially, DR.

Ursodeoxycholic Acid Halts Pathological Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy.

Retinopathy of prematurity (ROP) is the leading cause of blindness in infants. We have investigated the efficacy of the secondary bile acid ursodeoxycholic acid (UDCA) and its taurine and glycine conjugated derivatives tauroursodeoxycholic acid (TUDCA) and glycoursodeoxycholic acid (GUDCA) in preventing retinal neovascularization (RNV) in an experimental model of ROP. Seven-day-old mice pups (P7) were subjected to oxygen-induced retinopathy (OIR) and were treated with bile acids for various durations. Analysis of retinal vascular growth and distribution revealed that UDCA treatment (50 mg/kg, P7-P17) of OIR mice decreased the extension of neovascular and avascular areas, whereas treatments with TUDCA and GUDCA showed no changes. UDCA also prevented reactive gliosis, preserved ganglion cell survival, and ameliorated OIR-induced blood retinal barrier dysfunction. These effects were associated with decreased levels of oxidative stress markers, inflammatory cytokines, and normalization of the VEGF-STAT3 signaling axis. Furthermore, in vitro tube formation and permeability assays confirmed UDCA inhibitory activity toward VEGF-induced pro-angiogenic and pro-permeability effects on human retinal microvascular endothelial cells. Collectively, our results suggest that UDCA could represent a new effective therapy for ROP.

Mechanistic dissection of diabetic retinopathy using the protein-metabolite interactome.

PURPOSE: The current study aims to determine the molecular mechanisms of diabetic retinopathy (DR) using the protein-protein interactome and metabolome map. We examined the protein network of novel biomarkers of DR for direct (physical) and indirect (functional) interactions using clinical target proteins in different models. METHODS: We used proteomic tools including 2-dimensional gel electrophoresis, mass spectrometry analysis, and database search for biomarker identification using in vivo murine and human model of diabetic retinopathy and in vitro model of oxidative stress. For the protein interactome and metabolome mapping, various bioinformatic tools that include STRING and OmicsNet were used. RESULTS: We uncovered new diabetic biomarkers including prohibitin (PHB), dynamin 1, microtubule-actin crosslinking factor 1, Toll-like receptor (TLR 7), complement activation, as well as hypothetical proteins that include a disintegrin and metalloproteinase (ADAM18), vimentin III, and calcium-binding C2 domain-containing phospholipid-binding switch (CAC2PBS) using a proteomic approach. Proteome networks of protein interactions with diabetic biomarkers were established using known DR-related proteome data. DR metabolites were interconnected to establish the metabolome map. Our results showed that mitochondrial protein interactions were changed during hyperglycemic conditions in the streptozotocin-treated murine model and diabetic human tissue. CONCLUSIONS: Our interactome mapping suggests that mitochondrial dysfunction could be tightly linked to various phases of DR pathogenesis including altered visual cycle, cytoskeletal remodeling, altered lipid concentration, inflammation, PHB depletion, tubulin phosphorylation, and altered energy metabolism. The protein-metabolite interactions in the current network demonstrate the etiology of retinal degeneration and suggest the potential therapeutic approach to treat DR.

Monosodium Urate Contributes to Retinal Inflammation and Progression of Diabetic Retinopathy.

We have investigated the contributing role of monosodium urate (MSU) to the pathological processes associated with the induction of diabetic retinopathy (DR). In human postmortem retinas and vitreous from donors with DR, we have found a significant increase in MSU levels that correlated with the presence of inflammatory markers and enhanced expression of xanthine oxidase. The same elevation in MSU levels was also detected in serum and vitreous of streptozotocin-induced diabetic rats (STZ-rats) analyzed at 8 weeks of hyperglycemia. Furthermore, treatments of STZ-rats with the hypouricemic drugs allopurinol (50 mg/kg) and benzbromarone (10 mg/kg) given every other day resulted in a significant decrease of retinal and plasma levels of inflammatory cytokines and adhesion factors, a marked reduction of hyperglycemia-induced retinal leukostasis, and restoration of retinal blood-barrier function. These results were associated with effects of the hypouricemic drugs on downregulating diabetes-induced levels of oxidative stress markers as well as expression of components of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome such as NLRP3, Toll-like receptor 4, and interleukin-1ß. The outcomes of these studies support a contributing role of MSU in diabetes-induced retinal inflammation and suggest that asymptomatic hyperuricemia should be considered as a risk factor for DR induction and progression.

STAT3-mediated activation of miR-21 is involved in down-regulation of TIMP3 and neovascularization in the ischemic retina.

Retinal neovascularization (RNV) is a sight threatening complication of ischemic retinopathies with limited therapeutic options. The transcription factor signal transducer and activator of transcription 3 (STAT3) has been shown to play a crucial role in promoting RNV. However, manipulating of STAT3 activity can cause significant adverse side effects due to its neurotrophic properties. In this study, we identified microRNA-21 (miR-21) as a downstream effector of STAT3 activity in the ischemic retinas and determined its role in promoting RNV through inhibition of its molecular target, the tissue inhibitor of matrix metalloproteinases 3 (TIMP3). Using human retinal endothelial cells (HREC) exposed to hypoxia and a mouse model of oxygen-induced retinopathy (OIR), we found that TIMP3 expression was significantly decreased at both mRNA and protein levels and this paralleled the activation of STAT3 and up-regulation of miR-21. Moreover, TIMP3 expression was restored by knockdown of STAT3 or blocking of miR-21 in HREC, thus, confirming TIMP3 as a downstream target of STAT3/miR-21 pathway. Finally, in a mouse model of OIR, blockade of miR-21 by a specific antisense (a.miR-21), halted RNV and this effect was associated with rescuing of TIMP3 expression. Our data show that miR-21 mediates STAT3 pro-angiogenic effects in the ischemic retina, thus suggesting its blockade as a potential therapy to prevent/halt RNV.

Protective effects of agonists of growth hormone-releasing hormone (GHRH) in early experimental diabetic retinopathy.

The potential therapeutic effects of agonistic analogs of growth hormone-releasing hormone (GHRH) and their mechanism of action were investigated in diabetic retinopathy (DR). Streptozotocin-induced diabetic rats (STZ-rats) were treated with 15 µg/kg GHRH agonist, MR-409, or GHRH antagonist, MIA-602. At the end of treatment, morphological and biochemical analyses assessed the effects of these compounds on retinal neurovascular injury induced by hyperglycemia. The expression levels of GHRH and its receptor (GHRH-R) measured by qPCR and Western blotting were significantly down-regulated in retinas of STZ-rats and in human diabetic retinas (postmortem) compared with their respective controls. Treatment of STZ-rats with the GHRH agonist, MR-409, prevented retinal morphological alteration induced by hyperglycemia, particularly preserving survival of retinal ganglion cells. The reverse, using the GHRH antagonist, MIA-602, resulted in worsening of retinal morphology and a significant alteration of the outer retinal layer. Explaining these results, we have found that MR-409 exerted antioxidant and anti-inflammatory effects in retinas of the treated rats, as shown by up-regulation of NRF-2-dependent gene expression and down-regulation of proinflammatory cytokines and adhesion molecules. MR-409 also significantly down-regulated the expression of vascular endothelial growth factor while increasing that of pigment epithelium-derived factor in diabetic retinas. These effects correlated with decreased vascular permeability. In summary, our findings suggest a neurovascular protective effect of GHRH analogs during the early stage of diabetic retinopathy through their antioxidant and anti-inflammatory properties.

Melatonin Modulates Prohibitin and Cytoskeleton in the Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) plays imperative roles in normal retinal function by photoreceptor protection from light and phagocytosis of rod and cone outer segments during disc shedding. Melatonin is the free radical scavenger and circadian determinant to protect the RPE and retina from oxidative stress and regulate the circadian clock. The current study tested the hypothesis whether melatonin could affect cytoskeletal structure within RPE. Our Western blot analysis demonstrated that melatonin treatment up-regulated prohibitin 3-fold compared to control. ß-tubulin levels were also up-regulated by melatonin but to a lesser extent. Initial cell shape of ARPE-19 is epitheloid, however, after 30-minute treatment with melatonin, RPE cells undergo a morphological change to a fusiform shape with spindle outgrowth. Cells return to epitheloid shape after 12 hours in untreated medium. Melatonin treated cells showed increased and dissimilar distribution of prohibitin and ß-tubulin compared to non-treated cells, thus altered cytoskeletal and mitochondrial structure in the RPE. Our data implies that melatonin may play a protective role under oxidative stress, which is shown by the marker prohibitin in terms of increased expression and nuclear distribution. During the protective process, cells change their morphology. Our results suggest that melatonin treatment could be beneficial to protect mitochondria under oxidative stress and treat certain ocular diseases, including age-related macular degeneration.

Interactome Mapping Guided by Tissue-Specific Phosphorylation in Age-Related Macular Degeneration.

The current study aims to determine the molecular mechanisms of age-related macular degeneration (AMD) using the phosphorylation network. Specifically, we examined novel biomarkers for oxidative stress by protein interaction mapping using in vitro and in vivo models that mimic the complex and progressive characteristics of AMD. We hypothesized that the early apoptotic reactions could be initiated by protein phosphorylation in region-dependent (peripheral retina vs. macular) and tissue-dependent (retinal pigment epithelium vs. retina) manner under chronic oxidative stress. The analysis of protein interactome and oxidative biomarkers showed the presence of tissue- and region-specific post-translational mechanisms that contribute to AMD progression and suggested new therapeutic targets that include ubiquitin, erythropoietin, vitronectin, MMP2, crystalline, nitric oxide, and prohibitin. Phosphorylation of specific target proteins in RPE cells is a central regulatory mechanism as a survival tool under chronic oxidative imbalance. The current interactome map demonstrates a positive correlation between oxidative stress-mediated phosphorylation and AMD progression and provides a basis for understanding oxidative stress-induced cytoskeletal changes and the mechanism of aggregate formation induced by protein phosphorylation. This information could provide an effective therapeutic approach to treat age-related neurodegeneration.

Disruption of Angiogenesis by Anthocyanin-Rich Extracts of Hibiscus sabdariffa.

Abnormal vessel formations contribute to the progression of specific angiogenic diseases including age-related macular degeneration. Adequate vessel growth and maintenance represent the coordinated process of endothelial cell proliferation, matrix remodeling, and differentiation. However, the molecular mechanism of the proper balance between angiogenic activators and inhibitors remains elusive. In addition, quantitative analysis of vessel formation has been challenging due to complex angiogenic morphology. We hypothesized that conjugated double bond containing-natural products, including anthocyanin extracts from Hibiscus sabdariffa, may control the proper angiogenesis. The current study was designed to determine whether natural molecules from African plant library modulate angiogenesis. Further, we questioned how the proper balance of anti- or pro-angiogenic signaling can be obtained in the vascular microenvironment by treating anthocyanin or fatty acids using chick chorioallantoic membrane angiogenesis model in ovo. The angiogenic morphology was analyzed systematically by measuring twenty one angiogenic indexes using Angiogenic Analyzer software. Chick chorioallantoic model demonstrated that anthocyanin-rich extracts inhibited angiogenesis in time- and concentration-dependent manner. Molecular modeling analysis proposed that hibiscetin as a component in Hibiscus may bind to the active site of vascular endothelial growth factor receptor 2 (VEGFR2) with ΔG= -8.42 kcal/mol of binding energy. Our results provided the evidence that anthocyanin is an angiogenic modulator that can be used to treat uncontrolled neovascular-related diseases, including age-related macular degeneration.

Molecular mechanisms underlying synergistic adhesion of sickle red blood cells by hypoxia and low nitric oxide bioavailability.

The molecular mechanisms by which nitric oxide (NO) bioavailability modulates the clinical expression of sickle cell disease (SCD) remain elusive. We investigated the effect of hypoxia and NO bioavailability on sickle red blood cell (sRBC) adhesion using mice deficient for endothelial NO synthase (eNOS) because their NO metabolite levels are similar to those of SCD mice but without hypoxemia. Whereas sRBC adhesion to endothelial cells in eNOS-deficient mice was synergistically upregulated at the onset of hypoxia, leukocyte adhesion was unaffected. Restoring NO metabolite levels to physiological levels markedly reduced sRBC adhesion to levels seen under normoxia. These results indicate that sRBC adherence to endothelial cells increases in response to hypoxia prior to leukocyte adherence, and that low NO bioavailability synergistically upregulates sRBC adhesion under hypoxia. Although multiple adhesion molecules mediate sRBC adhesion, we found a central role for P-selectin in sRBC adhesion. Hypoxia and low NO bioavailability upregulated P-selectin expression in endothelial cells in an additive manner through p38 kinase pathways. These results demonstrate novel cellular and signaling mechanisms that regulate sRBC adhesion under hypoxia and low NO bioavailability. Importantly, these findings point us toward new molecular targets to inhibit cell adhesion in SCD.

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease.

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.

Inhibition of cell adhesion by anti-P-selectin aptamer: a new potential therapeutic agent for sickle cell disease.

Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-P-selectin aptamer may be useful as a novel therapeutic agent for SCD.

Contributions of nitric oxide synthase isoforms to pulmonary oxygen toxicity, local vs. mediated effects.

Reactive species of oxygen and nitrogen have been collectively implicated in pulmonary oxygen toxicity, but the contributions of specific molecules are unknown. Therefore, we assessed the roles of several reactive species, particularly nitric oxide, in pulmonary injury by exposing wild-type mice and seven groups of genetically altered mice to >98% O2 at 1, 3, or 4 atmospheres absolute. Genetically altered animals included knockouts lacking either neuronal nitric oxide synthase (nNOS(-/-)), endothelial nitric oxide synthase (eNOS(-/-)), inducible nitric oxide synthase (iNOS(-/-)), extracellular superoxide dismutase (SOD3(-/-)), or glutathione peroxidase 1 (GPx1(-/-)), as well as two transgenic variants (S1179A and S1179D) having altered eNOS activities. We confirmed our earlier finding that normobaric hyperoxia (NBO2) and hyperbaric hyperoxia (HBO2) result in at least two distinct but overlapping patterns of pulmonary injury. Our new findings are that the role of nitric oxide in the pulmonary pathophysiology of hyperoxia depends both on the specific NOS isozyme that is its source and on the level of hyperoxia. Thus, iNOS predominates in the etiology of lung injury in NBO2, and SOD3 provides an important defense. But in HBO2, nNOS is a major contributor to pulmonary injury, whereas eNOS is protective. In addition, we demonstrated that nitric oxide derived from nNOS is involved in a neurogenic mechanism of HBO2-induced lung injury that is linked to central nervous system oxygen toxicity through adrenergic/cholinergic pathways.

Transient hypoxia stimulates mitochondrial biogenesis in brain subcortex by a neuronal nitric oxide synthase-dependent mechanism.

The adaptive mechanisms that protect brain metabolism during and after hypoxia, for instance, during hypoxic preconditioning, are coordinated in part by nitric oxide (NO). We tested the hypothesis that acute transient hypoxia stimulates NO synthase (NOS)-activated mechanisms of mitochondrial biogenesis in the hypoxia-sensitive subcortex of wild-type (Wt) and neuronal NOS (nNOS) and endothelial NOS (eNOS)-deficient mice. Mice were exposed to hypobaric hypoxia for 6 h, and changes in immediate hypoxic transcriptional regulation of mitochondrial biogenesis was assessed in relation to mitochondrial DNA (mtDNA) content and mitochondrial density. There were no differences in cerebral blood flow or hippocampal PO2 responses to acute hypoxia among these strains of mice. In Wt mice, hypoxia increased mRNA levels for peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1 alpha), nuclear respiratory factor-1, and mitochondrial transcription factor A. After 24 h, new mitochondria, localized in reporter mice expressing mitochondrial green fluorescence protein, were seen primarily in hippocampal neurons. eNOS-/- mice displayed lower basal levels but maintained hypoxic induction of these transcripts. In contrast, nuclear transcriptional regulation of mitochondrial biogenesis in nNOS-/- mice was normal at baseline but did not respond to hypoxia. After hypoxia, subcortical mtDNA content increased in Wt and eNOS-/- mice but not in nNOS-/- mice. Hypoxia stimulated PGC-1alpha protein expression and phosphorylation of protein kinase A and cAMP response element binding (CREB) protein in Wt mice, but CREB only was activated in eNOS-/- mice and not in nNOS-/- mice. These findings demonstrate that hypoxic preconditioning elicits subcortical mitochondrial biogenesis by a novel mechanism that requires nNOS regulation of PGC-1alpha and CREB.

Cerebral blood flow and brain oxygenation in rats breathing oxygen under pressure.

Hyperbaric oxygen (HBO(2)) increases oxygen tension (PO(2)) in blood but reduces blood flow by means of O(2)-induced vasoconstriction. Here we report the first quantitative evaluation of these opposing effects on tissue PO(2) in brain, using anesthetized rats exposed to HBO(2) at 2 to 6 atmospheres absolute (ATA). We assessed the contribution of regional cerebral blood flow (rCBF) to brain PO(2) as inspired PO(2) (PiO(2)) exceeds 1 ATA. We measured rCBF and local PO(2) simultaneously in striatum using collocated platinum electrodes. Cerebral blood flow was computed from H(2) clearance curves in vivo and PO(2) from electrodes calibrated in vitro, before and after insertion. Arterial PCO(2) was controlled, and body temperature, blood pressure, and EEG were monitored. Scatter plots of rCBF versus PO(2) were nonlinear (R(2)=0.75) for rats breathing room air but nearly linear (R(2)=0.88-0.91) for O(2) at 2 to 6 ATA. The contribution of rCBF to brain PO(2) was estimated at constant inspired PO(2), by increasing rCBF with acetazolamide (AZA) or decreasing it with N-nitro-L-arginine methyl ester (L-NAME). At basal rCBF (78 mL/100 g min), local PO(2) increased 7- to 33-fold at 2 to 6 ATA, compared with room air. A doubling of rCBF increased striatal PO(2) not quite two-fold in rats breathing room air but 13- to 64-fold in those breathing HBO(2) at 2 to 6 ATA. These findings support our hypothesis that HBO(2) increases PO(2) in brain in direct proportion to rCBF.