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 PO 2 responses to acute hypoxia among these strains of mice. In Wt mice, hypoxia increased mRNA levels for peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1 ␣), 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 Ϫ/Ϫ mice displayed lower basal levels but maintained hypoxic induction of these transcripts. In contrast, nuclear transcriptional regulation of mitochondrial biogenesis in nNOS Ϫ/Ϫ mice was normal at baseline but did not respond to hypoxia. After hypoxia, subcortical mtDNA content increased in Wt and eNOS Ϫ/Ϫ mice but not in nNOS Ϫ/Ϫ mice. Hypoxia stimulated PGC-1␣ 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 Ϫ/Ϫ mice and not in nNOS Ϫ/Ϫ mice. These findings demonstrate that hypoxic preconditioning elicits subcortical mitochondrial biogenesis by a novel mechanism that requires nNOS regulation of PGC-1␣ and CREB.

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-Pselectin 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-Pselectin aptamer may be useful as a novel therapeutic agent for SCD. (Blood. 2011; 117(2):727-735) IntroductionSickle cell disease (SCD) is caused by a point mutation of the ␤-globin chain, but its pathophysiology is extremely complex and heterogeneous. A salient clinical feature of this disorder is vasoocclusive crisis, which is a major cause of morbidity and mortality in SCD patients; repetitive crises could eventually lead to multiorgan damage in the long term. 1 Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells have been implicated as critical pathologic events for the development of vaso-occlusion. Much attention has been directed to identifying adhesion molecules involved in cell-cell interactions.Endothelial cell P-selectin, a member of the selectin family of cell adhesion molecules, 2 plays a key role in leukocyte recruitment as well as the adhesion of sickle RBCs to the endothelium. 3,4 Presynthesized P-selectin is stored in the Weibel-Palade bodies in endothelial cells and rapidly translocated to the cell surface in response to extracellular stimuli such as hypoxia. 5 Expression levels of P-selectin are elevated in patients with SCD. 6,7 The interactions between P-selectin and its ligands are likely to contribute to cell adhesion between multiple types of cells, which results in the impairment of microvascular circulation presumably involved in the development of painful vaso-occlusive episodes. 4,8 Several antiadhesion compounds have been tested for their ability to inhibit sickle RBC adhesion to endothelial cells, however, no agents are currently used...

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 ¼ 0.75) for rats breathing room air but nearly linear (R 2 ¼ 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. IntroductionThe O 2 content of blood is the mathematical product of hemoglobin concentration and arterial hemoglobin O 2 saturation, the latter being a nonlinear function of arterial oxygen partial pressure (PaO 2 ). The small amount of O 2 dissolved in plasma is usually negligible. However, breathing hyperbaric oxygen (HBO 2 ), that is oxygen at pressures greater than 1 atmosphere absolute (ATA), raises PaO 2 beyond the point at which hemoglobin is fully saturated, so that the dissolved fraction becomes the main source of O 2 available to cells. But this does not assure enhanced O 2 delivery to brain, because tissue PO 2 also depends on regional blood flow.Tissue oxygen tension (PO 2 ) is a dynamic balance between O 2 delivery and consumption. Many authors have reported increased brain PO 2 in HBO 2 (Jamieson and Van Den Brenk, 1962;Bennett, 1965;Bean et al, 1971;Torbati et al, 1978;Hunt et al, 1978), and reviews are available (Jamieson, 1989;Camporesi et al, 1996;Dean et al, 2003). It is also known that total or regional cerebral blood flow (rCBF) decreases in HBO 2 as a function of pressure and time. But the contribution of CBF to brain PO 2 had never been quantified. Cerebral vasoconstriction and decreased total or rCBF have been shown in healthy volunteers and patients breathing O 2 at 3.5 ATA for brief periods (Lambertsen et al, 1953;Visser et al, 1996;Omae et al, 1998). In animals, in which HBO 2 is maintained for longer times and at higher pressures, the rCBF response is biphasic: the initial decrease in rCBF is followed by a secondary rise to www.jcbfm.com control leve...

The retinal pigment epithelium (RPE) is consistently exposed to high levels of pro-oxidant and inflammatory stimuli. As such, under normal conditions the antioxidant machinery in the RPE cell is one of the most efficient in the entire body. However, antioxidant defense mechanisms are often impacted negatively by the process of aging and/or degenerative disease leaving RPE susceptible to damage which contributes to retinal dysfunction. Thus, understanding better the mechanisms governing antioxidant responses in RPE is critically important. Here, we evaluated the role of the redox sensitive microRNA miR-144 in regulation of antioxidant signaling in human and mouse RPE. In cultured human RPE, miR-144-3p and miR-144-5p expression was upregulated in response to pro-oxidant stimuli. Likewise, overexpression of miR-144-3p and -5p using targeted miR mimics was associated with reduced expression of Nrf2 and downstream antioxidant target genes (NQO1 and GCLC), reduced levels of glutathione and increased RPE cell death. Alternately, some protection was conferred against the above when miR-144-3p and miR-144-5p expression was suppressed using antagomirs. Expression analyses revealed a higher conservation of miR-144-3p expression across species and additionally, the presence of two potential Nrf2 binding sites in the 3p sequence compared to only one in the 5p sequence. Thus, we evaluated the impact of miR-144-3p expression in the retinas of mice in which a robust pro-oxidant environment was generated using sodium iodate (SI). Subretinal injection of miR-144-3p antagomir in SI mice preserved retinal integrity and function, decreased oxidative stress, limited apoptosis and enhanced antioxidant gene expression. Collectively, the present work establishes miR-144 as a potential target for preventing and treating degenerative retinal diseases in which oxidative stress is paramount and RPE is prominently affected (e.g., age-related macular degeneration and diabetic retinopathy).

SignificanceThe studies described here are relevant to the cure of diabetic retinopathy, a leading cause of blindness with currently limited therapeutic options. Here we provided evidence showing that agonists of growth hormone-releasing hormone (GHRH) can significantly diminish retinal neurovascular injury characterizing the early stages of diabetic retinopathy through antioxidant and anti-inflammatory effects. The results of the presented studies provide information on the potential therapeutic effects of GHRH agonists and shed light on the role of hypothalamic hormones in retinal physiology and their effect on visual disorders. In addition, our findings suggest protective effects of GHRH analogs in other disease conditions affecting retinal neuronal cells and, possibly, other nonretinal neurons.

Stress-associated premature senescence (SAPS) is involved in retinal microvascular injury and diabetic retinopathy. We have investigated the role and mode of action of miR-34a in retinal endothelial cells senescence in response to glucidic stress. Human retinal microvascular endothelial cells (HuREC) were exposed to glucidic stress (high glucose (HG) = 25 mM d-glucose) and compared to cells exposed to normal glucose (NG = 5 mM) or the osmotic control l-glucose (LG = 25 mM). HG stimulation of HuREC increased the expression of miR-34a and induced cellular senescence. HG also increased the expression of p16ink4a and p21waf1, while decreasing the histone deacetylase SIRT1. These effects were associated with diminished mitochondrial function and loss of mitochondrial biogenesis factors (i.e., PGC-1α, NRF1, and TFAM). Transfection of the cells with miR-34a inhibitor (IB) halted HG-induced mitochondrial dysfunction and up-regulation of senescence-associated markers, whereas miR-34a mimic promoted cellular senescence and mitochondrial dysfunction. Moreover, HG lowered levels of the mitochondrial antioxidants TrxR2 and SOD2, an effect blunted by miR-34a IB, and promoted by miR-34a mimic. 3’-UTR (3’-untranslated region) reporter assay of both genes validated TrxR2 as a direct target of miR-34a, but not SOD2. Our results show that miR-34a is a key player of HG-induced SAPS in retinal endothelial cells via multiple pathways involved in mitochondrial function and biogenesis.

Key Points• Adhesion of sRBCs is synergistically regulated by hypoxia and low NO bioavailability.• P-selectin and p38 kinase pathways play a role in the synergistic adhesion of sRBCs.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. (Blood. 2014; 123(12):1917-1926 IntroductionAlthough sickle cell disease (SCD) arises from a single mutation of b-globin, clinical severity varies significantly.1 A well-known clinical modifier is fetal hemoglobin, which is expressed at variable levels in patients and inhibits the polymerization of sickle hemoglobin. 2 The frequency of vaso-occlusive crisis was also used to evaluate disease severity of SCD patients.3 Vaso-occlusive crisis is likely triggered by multiple physiological insults such as infection and cytokine-mediated inflammation that may cause tissue hypoxia, and mediated by multistep cell adhesion mechanisms involving sickle red blood cells (sRBCs). 4 The degree of sRBC adhesion to endothelial cells was shown to correlate with SCD severity. 1 Nitric oxide (NO) bioavailability is another physiological factor capable of modulating clinical severity.5 NO bioavailability decreases during vaso-occlusive crisis, primarily because of NO scavenging by cell-free hemoglobin 6 as well as other mechanisms including arginase, 7 reactive oxygen species, 8 and NO synthases.9 NO metabolite levels vary among patients even at steady state and may inversely correlate with the frequency of vaso-occlusive crisis. 10 We showed that inhalation of low-dose NO permitted SCD mice to survive hypoxic stress. 11 The gender difference in the clinical severity of SCD may result in part from distinct levels of NO bioavailability.12 Thus, heterogeneity of the clinical expression in SCD appears ...

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 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 in 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 counts 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.