Albert K. Y. Tsui

Functional Importance of Dicer Protein in the Adaptive Cellular Response to Hypoxia

Background: Functional relationships between the microRNA and cellular hypoxia response pathways are unknown. Results: Dicer is down-regulated in chronic hypoxia; this mechanism maintains the induction of hypoxia-inducible factor-α subunits and hypoxia-responsive genes. Conclusion: Loss of Dicer-dependent microRNA regulation is important for maintaining the concerted cellular response to hypoxia. Significance: Altogether, we provide a newer perspective into the post-transcriptional pathways that regulate the cellular hypoxic response. The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.

Anemia and Cerebral Outcomes: Many Questions, Fewer Answers

A number of clinical studies have associated acute anemia with cerebral injury in perioperative patients. Evidence of such injury has been observed near the currently accepted transfusion threshold (hemoglobin [Hb] concentration, 7–8 g/dL), and well above the threshold for cerebral tissue hypoxia (Hb 3–4 g/dL). However, hypoxic and nonhypoxic mechanisms of anemia-induced cerebral injury have not been clearly elucidated. In addition, protective mechanisms which may minimize cerebral injury during acute anemia have not been well defined. Vasodilatory mechanisms, including nitric oxide (NO), may help to maintain cerebral oxygen delivery during anemia as all three NO synthase (NOS) isoforms (neuronal, endothelial, and inducible NOS) have been shown to be up-regulated in different experimental models of acute hemodilutional anemia. Recent experimental evidence has also demonstrated an increase in an important transcription factor, hypoxia inducible factor (HIF)-1&agr;, in the cerebral cortex of anemic rodents at clinically relevant Hb concentrations (Hb 6–7 g/dL). This suggests that cerebral oxygen homeostasis may be in jeopardy during acute anemia. Under hypoxic conditions, cytoplasmic HIF-1&agr; degradation is inhibited, thereby allowing it to accumulate, dimerize, and translocate into the nucleus to promote transcription of a number of hypoxic molecules. Many of these molecules, including erythropoietin, vascular endothelial growth factor, and inducible NOS have also been shown to be up-regulated in the anemic brain. In addition, HIF-1&agr; transcription can be increased by nonhypoxic mediators including cytokines and vascular hormones. Furthermore, NOS-derived NO may also stabilize HIF-1&agr; in the absence of tissue hypoxia. Thus, during anemia, HIF-1&agr; has the potential to regulate cerebral cellular responses under both hypoxic and normoxic conditions. Experimental studies have demonstrated that HIF-1&agr; may have either neuroprotective or neurotoxic capacity depending on the cell type in which it is up-regulated. In the current review, we characterize these cellular processes to promote a clearer understanding of anemia-induced cerebral injury and protection. Potential mechanisms of anemia-induced injury include cerebral emboli, tissue hypoxia, inflammation, reactive oxygen species generation, and excitotoxicity. Potential mechanisms of cerebral protection include NOS/NO-dependent optimization of cerebral oxygen delivery and cytoprotective mechanisms including HIF-1&agr;, erythropoietin, and vascular endothelial growth factor. The overall balance of these activated cellular mechanisms may dictate whether or not their up-regulation leads to cytoprotection or cellular injury during anemia. A clearer understanding of these mechanisms may help us target therapies that will minimize anemia-induced cerebral injury in perioperative patients.

Metoprolol Reduces Cerebral Tissue Oxygen Tension after Acute Hemodilution in Rats

Background:Perioperative &bgr;-blockade and anemia are independent predictors of increased stroke and mortality by undefined mechanisms. This study investigated the effect of &bgr;-blockade on cerebral tissue oxygen delivery in an experimental model of blood loss and fluid resuscitation (hemodilution). Methods:Anesthetized rats were treated with metoprolol (3 mg · kg−1) or saline before undergoing hemodilution with pentastarch (1:1 blood volume exchange, 30 ml · kg−1). Outcomes included cardiac output, cerebral blood flow, and brain (PBrO2) and kidney (PKO2) tissue oxygen tension. Hypoxia inducible factor-1&agr; (HIF-1&agr;) protein levels were assessed by Western blot. Systemic catecholamines, erythropoietin, and angiotensin II levels were measured. Results:Hemodilution increased heart rate, stroke volume, cardiac output (60%), and cerebral blood flow (50%), thereby maintaining PBrO2 despite an approximately 50% reduction in blood oxygen content (P < 0.05 for all). By contrast, PKO2 decreased (50%) under the same conditions (P < 0.05). &bgr;-blockade reduced baseline heart rate (20%) and abolished the compensatory increase in cardiac output after hemodilution (P < 0.05). This attenuated the cerebral blood flow response and reduced PBrO2 (50%), without further decreasing PKO2. Cerebral HIF-1&agr; protein levels were increased in &bgr;-blocked hemodiluted rats relative to hemodiluted controls (P < 0.05). Systemic catecholamine and erythropoietin levels increased comparably after hemodilution in both groups, whereas angiotensin II levels increased only after &bgr;-blockade and hemodilution. Conclusions:Cerebral tissue oxygen tension is preferentially maintained during hemodilution, relative to the kidney, despite elevated systemic catecholamines. Acute &bgr;-blockade impaired the compensatory cardiac output response to hemodilution, resulting in a reduction in cerebral tissue oxygen tension and increased expression of HIF-1&agr;.

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)−/− mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS−/− mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS−/− mice, despite comparable reductions in tissue PO2. Paradoxically, nNOS−/− mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Differential HIF and NOS Responses to Acute Anemia: Defining Organ Specific Hemoglobin Thresholds for Tissue Hypoxia

Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide (NO) synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ-specific Hb thresholds for tissue hypoxia, HIF-α (oxygen-dependent degradation domain, ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50 g/l, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and HIF-2α increased earlier (Hb ∼70-90 g/l) in response to anemia. Liver also exhibited an early HIF-α response. Carotid blood flow was increased early (Hb ∼70, g/l), but renal blood flow remained relatively constant, only increased at Hb of 50 g/l. Anemia increased nNOS (brain and kidney) and endothelia NOS (eNOS) (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS independent. HIF-dependent RNA levels increased linearly (∼10-fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (∼100-fold). Plasma EPO levels increased near Hb threshold of 90 g/l, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.

Treatment with a highly selective β1 antagonist causes dose-dependent impairment of cerebral perfusion after hemodilution in rats

BACKGROUND:Acute &bgr;-blockade has been associated with a dose-dependent increase in adverse outcomes, including stroke and mortality. Acute blood loss contributes to the incidence of these adverse events. In an attempt to link the risks of acute blood loss and &bgr;-blockade, animal studies have demonstrated that acute &bgr;-blockade impairs cerebral perfusion after hemodilution. We expanded on these findings by testing the hypothesis that acute &bgr;-blockade with a highly &bgr;1-specific antagonist (nebivolol) causes dose-dependent cerebral hypoxia during hemodilution. METHODS:Anesthetized rats and mice were randomized to receive vehicle or nebivolol (1.25 or 2.5 mg/kg) IV before hemodilution to a hemoglobin concentration near 60 g/L. Drug levels, heart rate (HR), cardiac output (CO), regional cerebral blood flow (rCBF, laser Doppler), and microvascular brain PO2 (PBrO2, G2 Oxyphor) were measured before and after hemodilution. Endothelial nitric oxide synthase (NOS), neuronal NOS (nNOS), inducible NOS, and hypoxia inducible factor (HIF)-1&agr; were assessed by Western blot. HIF-&agr; expression was also assessed using an HIF-(ODD)-luciferase mouse model. Data were analyzed using analysis of variance with significance assigned at P < 0.05, and corrected P values are reported for all post hoc analyses. RESULTS:Nebivolol treatment resulted in dose-specific plasma drug levels. In vehicle-treated rats, hemodilution increased CO and rCBF (P < 0.010) whereas PBrO2 decreased to 45.8 ± 18.7 mm Hg (corrected P < 0.001; 95% CI 29.4–69.7). Both nebivolol doses comparably reduced HR and attenuated the CO response to hemodilution (P < 0.012). Low-dose nebivolol did not impair rCBF or further reduce PBrO2 after hemodilution. High-dose nebivolol attenuated the rCBF response to hemodilution and caused a further reduction in PBrO2 to 28.4 ± 9.6 mm Hg (corrected P = 0.019; 95% CI 17.4–42.7). Both nebivolol doses increased brain endothelial NOS protein levels. Brain HIF-1&agr;, inducible NOS, and nNOS protein levels and brain HIF-luciferase activity were increased in the high-dose nebivolol group after hemodilution (P < 0.032). CONCLUSIONS:Our data demonstrate that nebivolol resulted in a dose-dependent decrease in cerebral oxygen delivery after hemodilution as reflected by a decrease in brain tissue PO2 and an increase in hypoxic protein responses (HIF-1&agr; and nNOS). Low-dose nebivolol treatment did not result in worsened tissue hypoxia after hemodilution, despite comparable effects on HR and CO. These data support the hypothesis that acute &bgr;-blockade with a highly &bgr;1-specific antagonist causes a dose-dependent impairment in cerebral perfusion during hemodilution.

Quantitative assessment of brain microvascular and tissue oxygenation during cardiac arrest and resuscitation in pigs

Cardiac arrest is associated with a very high rate of mortality, in part due to inadequate tissue perfusion during attempts at resuscitation. Parameters such as mean arterial pressure and end‐tidal carbon dioxide may not accurately reflect adequacy of tissue perfusion during cardiac resuscitation. We hypothesised that quantitative measurements of tissue oxygen tension would more accurately reflect adequacy of tissue perfusion during experimental cardiac arrest. Using oxygen‐dependent quenching of phosphorescence, we made measurements of oxygen in the microcirculation and in the interstitial space of the brain and muscle in a porcine model of ventricular fibrillation and cardiopulmonary resuscitation. Measurements were performed at baseline, during untreated ventricular fibrillation, during resuscitation and after return of spontaneous circulation. After achieving stable baseline brain tissue oxygen tension, as measured using an Oxyphor G4‐based phosphorescent microsensor, ventricular fibrillation resulted in an immediate reduction in all measured parameters. During cardiopulmonary resuscitation, brain oxygen tension remained unchanged. After the return of spontaneous circulation, all measured parameters including brain oxygen tension recovered to baseline levels. Muscle tissue oxygen tension followed a similar trend as the brain, but with slower response times. We conclude that measurements of brain tissue oxygen tension, which more accurately reflect adequacy of tissue perfusion during cardiac arrest and resuscitation, may contribute to the development of new strategies to optimise perfusion during cardiac resuscitation and improve patient outcomes after cardiac arrest.

Is methemoglobin an inert bystander, biomarker or a mediator of oxidative stress—The example of anemia?

Acute anemia increases the risk for perioperative morbidity and mortality in critically ill patients who experience blood loss and fluid resuscitation (hemodilution). Animal models of acute anemia suggest that neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) is adaptive and protects against anemia-induced mortality. During acute anemia, we have observed a small but consistent increase in methemoglobin (MetHb) levels that is inversely proportional to the acute reduction in Hb observed during hemodilution in animals and humans. We hypothesize that this increase in MetHb may be a biomarker of anemia-induced tissue hypoxia. The increase in MetHb may occur by at least two mechanisms: (1) direct hemoglobin oxidation by increased nNOS-derived NO within the perivascular tissue and (2) by increased deoxyhemoglobin (DeoxyHb) nitrite reductase activity within the vascular compartment. Both mechanisms reflect a potential increase in NO signaling from the tissue and vascular compartments during anemia. These responses are thought to be adaptive; as deletion of nNOS results in increased mortality in a model of acute anemia. Finally, it is possible that prolonged activation of these mechanisms may lead to maladaptive changes in redox signaling. We hypothesize, increased MetHb in the vascular compartment during acute anemia may reflect activation of adaptive mechanisms which augment NO signaling. Understanding the link between anemia, MetHb and its treatments (transfusion of stored blood) may help us to develop novel treatment strategies to reduce the risk of anemia-induced morbidity and mortality.

Anaemia: can we define haemoglobin thresholds for impaired oxygen homeostasis and suggest new strategies for treatment?

Observational clinical studies in perioperative medicine have defined a progressive increase in mortality that is proportional to both chronic preoperative anaemia and acute interpretative reductions in haemoglobin concentration (Hb). However, this knowledge has not yet helped to define the critical Hb threshold for organ injury and mortality in specific patient populations or in individual patients. Nor has this knowledge enabled us to develop effective treatment strategies for anaemia, as evident from the lack of a demonstrable improvement in survival in patients randomised to higher Hb levels by various treatment strategies including allogeneic red blood cell transfusion, erythropoiesis-stimulating agents (ESAs) and haemoglobin-based oxygen carriers (HBOCs). These findings emphasise the need for a clearer understanding of the mechanism of anaemia-induced mortality. Towards achieving this goal, experimental studies have defined adaptive mechanism by which oxygen homeostasis is maintained during acute anaemia. The mechanisms include: (1) effective sensing of anaemia-induced tissue hypoxia; (2) adaptive cardiovascular responses to maintain adequate tissue oxygen delivery; (3) heterogeneity of organ-specific oxygen delivery to preferentially sustain vital organs which are essential for acute survival (heart and brain); (4) evidence of increased vital organ injury with interruption of cardiovascular responses to anaemia and (5) evidence of activation of adaptive cellular responses to maintain oxygen homeostasis and support survival during acute anaemia. Understanding these mechanisms may allow us to define treatment thresholds and novel treatment strategies for acute anaemia based on biological markers of tissue hypoxia. The overall goal of these approaches is to improve patient outcomes, including event-free perioperative survival.

Metoprolol impairs resistance artery function in mice

Acute β-blockade with metoprolol has been associated with increased mortality by undefined mechanisms. Since metoprolol is a relatively high affinity blocker of β(2)-adrenoreceptors, we hypothesized that some of the increased mortality associated with its use may be due to its abrogation of β(2)-adrenoreceptor-mediated vasodilation of microvessels in different vascular beds. Cardiac output (CO; pressure volume loops), mean arterial pressure (MAP), relative cerebral blood flow (rCBF; laser Doppler), and microvascular brain tissue Po(2) (G2 oxyphor) were measured in anesthetized mice before and after acute treatment with metoprolol (3 mg/kg iv). The vasodilatory dose responses to β-adrenergic agonists (isoproterenol and clenbuterol), and the myogenic response, were assessed in isolated mesenteric resistance arteries (MRAs; ∼200-μm diameter) and posterior cerebral arteries (PCAs ∼150-μm diameter). Data are presented as means ± SE with statistical significance applied at P < 0.05. Metoprolol treatment did not effect MAP but reduced heart rate and stroke volume, CO, rCBF, and brain microvascular Po(2), while concurrently increasing systemic vascular resistance (P < 0.05 for all). In isolated MRAs, metoprolol did not affect basal artery tone or the myogenic response, but it did cause a dose-dependent impairment of isoproterenol- and clenbuterol-induced vasodilation. In isolated PCAs, metoprolol (50 μM) impaired maximal vasodilation in response to isoproterenol. These data support the hypothesis that acute administration of metoprolol can reduce tissue oxygen delivery by impairing the vasodilatory response to β(2)-adrenergic agonists. This mechanism may contribute to the observed increase in mortality associated with acute administration of metoprolol in perioperative patients.