The Impact of Hypoxia on Neutrophil Degranulation and Consequences for the Host.

Neutrophils are key effector cells of innate immunity, rapidly recruited to defend the host against invading pathogens. Neutrophils may kill pathogens intracellularly, following phagocytosis, or extracellularly, by degranulation and the release of neutrophil extracellular traps; all of these microbicidal strategies require the deployment of cytotoxic proteins and proteases, packaged during neutrophil development within cytoplasmic granules. Neutrophils operate in infected and inflamed tissues, which can be profoundly hypoxic. Neutrophilic infiltration of hypoxic tissues characterises a myriad of acute and chronic infectious and inflammatory diseases, and as well as potentially protecting the host from pathogens, neutrophil granule products have been implicated in causing collateral tissue damage in these scenarios. This review discusses the evidence for the enhanced secretion of destructive neutrophil granule contents observed in hypoxic environments and the potential mechanisms for this heightened granule exocytosis, highlighting implications for the host. Understanding the dichotomy of the beneficial and detrimental consequences of neutrophil degranulation in hypoxic environments is crucial to inform potential neutrophil-directed therapeutics in order to limit persistent, excessive, or inappropriate inflammation

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart

This is the final version. It was first published by Wiley in The Journal of Physiology at http://onlinelibrary.wiley.com/doi/10.1113/jphysiol.2014.275263/abstract.Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species (ROS) generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n=40) were given water supplemented with 0.7 mmol/L NaCl (as control) or 0.7 mmol/L NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n=10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac L-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics

Targeting HIF2α-ARNT hetero-dimerisation as a novel therapeutic strategy for pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a destructive disease of the pulmonary vasculature often leading to right heart failure and death. Current therapeutic intervention strategies only slow disease progression. The role of aberrant hypoxia-inducible factor (HIF)2α stability and function in the initiation and development of pulmonary hypertension (PH) has been an area of intense interest for nearly two decades. Here we determine the effect of a novel HIF2α inhibitor (PT2567) on PH disease initiation and progression, using two pre-clinical models of PH. Haemodynamic measurements were performed, followed by collection of heart, lung and blood for pathological, gene expression and biochemical analysis. Blood outgrowth endothelial cells from idiopathic PAH patients were used to determine the impact of HIF2α-inhibition on endothelial function. Global inhibition of HIF2a reduced pulmonary vascular haemodynamics and pulmonary vascular remodelling in both su5416/hypoxia prevention and intervention models. PT2567 intervention reduced the expression of PH-associated target genes in both lung and cardiac tissues and restored plasma nitrite concentration. Treatment of monocrotaline-exposed rodents with PT2567 reduced the impact on cardiovascular haemodynamics and promoted a survival advantage. In vitro, loss of HIF2α signalling in human pulmonary arterial endothelial cells suppresses target genes associated with inflammation, and PT2567 reduced the hyperproliferative phenotype and overactive arginase activity in blood outgrowth endothelial cells from idiopathic PAH patients. These data suggest that targeting HIF2α hetero-dimerisation with an orally bioavailable compound could offer a new therapeutic approach for PAH. Future studies are required to determine the role of HIF in the heterogeneous PAH population

Endothelial cell regulation of systemic haemodynamics and metabolism acts through the HIF transcription factors.

BackgroundThe vascular endothelium has important endocrine and paracrine roles, particularly in the regulation of vascular tone and immune function, and it has been implicated in the pathophysiology of a range of cardiovascular and inflammatory conditions. This study uses a series of transgenic murine models to explore for the first time the role of the hypoxia-inducible factors, HIF-1α and HIF-2α in the pulmonary and systemic circulations as potential regulators of systemic vascular function in normoxic or hypoxic conditions and in response to inflammatory stress. We developed a series of transgenic mouse models, the HIF-1α Tie2Cre, deficient in HIF1-α in the systemic and pulmonary vascular endothelium and the L1Cre, a pulmonary endothelium specific knockout of HIF-1α or HIF-2α. In vivo, arterial blood pressure and metabolic activity were monitored continuously in normal atmospheric conditions and following an acute stimulus with hypoxia (10%) or lipopolysaccharide (LPS). Ex vivo, femoral artery reactivity was assessed using wire myography.ResultsUnder normoxia, the HIF-1α Tie2Cre mouse had increased systolic and diastolic arterial pressure compared to litter mate controls over the day-night cycle under normal environmental conditions. VO2 and VCO2 were also increased. Femoral arteries displayed impaired endothelial relaxation in response to acetylcholine mediated by a reduction in the nitric oxide dependent portion of the response. HIF-1α L1Cre mice displayed a similar pattern of increased systemic blood pressure, metabolic rate and impaired vascular relaxation without features of pulmonary hypertension, polycythaemia or renal dysfunction under normal conditions. In response to acute hypoxia, deficiency of HIF-1α was associated with faster resolution of hypoxia-induced haemodynamic and metabolic compromise. In addition, systemic haemodynamics were less compromised by LPS treatment.ConclusionsThese data show that deficiency of HIF-1α in the systemic or pulmonary endothelium is associated with increased systemic blood pressure and metabolic rate, a pattern that persists in both normoxic conditions and in response to acute stress with potential implications for our understanding of the pathophysiology of vascular dysfunction in acute and chronic disease

Cardiovascular adaptation to hypoxia and the role of peripheral resistance.

Systemic vascular pressure in vertebrates is regulated by a range of factors: one key element of control is peripheral resistance in tissue capillary beds. Many aspects of the relationship between central control of vascular flow and peripheral resistance are unclear. An important example of this is the relationship between hypoxic response in individual tissues, and the effect that response has on systemic cardiovascular adaptation to oxygen deprivation. We show here how hypoxic response via the HIF transcription factors in one large vascular bed, that underlying the skin, influences cardiovascular response to hypoxia in mice. We show that the response of the skin to hypoxia feeds back on a wide range of cardiovascular parameters, including heart rate, arterial pressures, and body temperature. These data represent the first demonstration of a dynamic role for oxygen sensing in a peripheral tissue directly modifying cardiovascular response to the challenge of hypoxia

Cutaneous exposure to hypoxia does not affect skin perfusion in humans.

$\textbf{Aim:}$ Experiments have indicated that skin perfusion in mice is sensitive to reductions in environmental O$_2$ availability. Specifically, a reduction in skin-surface PO$_2$ attenuates transcutaneous O$_2$ diffusion, and hence epidermal O$_2$ supply. In response, epidermal HIF-1$\alpha$ expression increases and facilitates initial cutaneous vasoconstriction and subsequent nitric oxide-dependent vasodilation. Here, we investigated whether the same mechanism exists in humans. $\textbf{Methods:}$ In a first experiment, eight males rested twice for 8 h in a hypobaric chamber. Once, barometric pressure was reduced by 50%, while systemic oxygenation was preserved by O$_2$-enriched (42%) breathing gas (Hypoxia$_\text{Skin}$), and once barometric pressure and inspired O$_2$ fraction were normal (Control$_1$). In a second experiment, nine males rested for 8 h with both forearms wrapped in plastic bags. O$_2$ was expelled from one bag by nitrogen flushing (Anoxia$_\text{Skin}$), whereas the other bag was flushed with air (Control$_2$). In both experiments, skin blood flux was assessed by laser Doppler on the dorsal forearm, and HIF-1$\alpha$ expression was determined by immunohistochemical staining in forearm skin biopsies. $\textbf{Results:}$ Skin blood flux during Hypoxia$_\text{Skin}$ and Anoxia$_\text{Skin}$ remained similar to the corresponding Control trial ($P$ = 0.67 and $P$ = 0.81). Immunohistochemically stained epidermal HIF-1$\alpha$ was detected on 8.2 ± 6.1 and 5.3 ± 5.7% of the analysed area during Hypoxia$_\text{Skin}$ and Control$_1$ ($P$ = 0.30) and on 2.3 ± 1.8 and 2.4 ± 1.8% during Anoxia$_\text{Skin}$ and Control$_2$ ($P$ = 0.90) respectively. $\textbf{Conclusion:}$ Reductions in skin-surface PO$_2$ do not affect skin perfusion in humans. The unchanged epidermal HIF-1$\alpha$ expression suggests that epidermal O$_2$ homoeostasis was not disturbed by Hypoxia$_\text{Skin}$/Anoxia$_\text{Skin}$, potentially due to compensatory increases in arterial O$_2$ extraction.Gösta Fraenckel Foundatio

Pulmonary endothelial HIF2α\alpha-arginase axis plays an essential role in the development of hypoxia pulmonary hypertension

This is the author accepted manuscript. The final version is available from the Proceedings of the National Academy of Sciences (PNAS) via https://doi.org/10.1073/pnas.1602978113Hypoxic pulmonary vasoconstriction is correlated with pulmonary vascular remodelling. The hypoxia-inducible transcription factors (HIFs), HIF-1$\alpha$ and HIF-2$\alpha$ are known to contribute to the process of hypoxic pulmonary vascular remodelling; however, the specific role of pulmonary endothelial HIF expression in this process, and in the physiological process of vasoconstriction in response to hypoxia, remains unclear. Here we show that pulmonary endothelial HIF-2$\alpha$ is a critical regulator of hypoxia-induced pulmonary arterial hypertension (PAH). The rise in right ventricular systolic pressure (RVSP) normally observed following chronic hypoxic exposure was absent in mice with pulmonary endothelial HIF-2$\alpha$ deletion. The RVSP of mice lacking HIF-2$\alpha$ in pulmonary endothelium after exposure to hypoxia was not significantly different from normoxic wild type (WT) mice and much lower than the RVSP values seen in WT littermate controls and mice with pulmonary endothelial deletion of HIF-1$\alpha$ exposed to hypoxia. Endothelial HIF-2$\alpha$ deletion also protected mice from hypoxia remodelling. Pulmonary endothelial deletion of arginase-1, a downstream target of HIF-2$\alpha$, likewise attenuated many of the pathophysiological symptoms associated with HPH. We propose a mechanism whereby chronic hypoxia enhances HIF-2$\alpha$ stability, which causes increased arginase expression and dysregulates normal vascular NO homeostasis. These data offer new insight into the role of pulmonary endothelial HIF-2$\alpha$ in regulating the pulmonary vascular response to hypoxia.This study was funded by The Wellcome Trust, Papworth Hospital NIHR Cambridge Biomedical Research Centre

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism.

BACKGROUND: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. RESULTS: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. CONCLUSIONS: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.This work was kindly supported by a British Heart Foundation studentship to TA (FS/09/050). AJMu thanks the Research Councils UK for supporting his academic fellowship. LDR is supported by the Medical Research Council-Human Nutrition Research Elsie Widdowson Fellowship. AJMo thanks the Natural Sciences and Engineering Research Council for supporting her postdoctoral fellowship. MF acknowledges support from the Medical Research Council (G1001536). JLG thanks the Medical Research Council (MC_UP_A090_1006), the Biotechnology and Biological Sciences Research Council (BB/H013539/2) and British Heart Foundation for supporting work in his laboratory

Functional Redundancy of Class I Phosphoinositide 3-Kinase (PI3K) Isoforms in Signaling Growth Factor-Mediated Human Neutrophil Survival

We have investigated the contribution of individual phosphoinositide 3-kinase (PI3K) Class I isoforms to the regulation of neutrophil survival using (i) a panel of commercially available small molecule isoform-selective PI3K Class I inhibitors, (ii) novel inhibitors, which target single or multiple Class I isoforms (PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ), and (iii) transgenic mice lacking functional PI3K isoforms (p110δKOγKO or p110γKO). Our data suggest that there is considerable functional redundancy amongst Class I PI3Ks (both Class IA and Class IB) with regard to GM-CSF-mediated suppression of neutrophil apoptosis. Hence pharmacological inhibition of any 3 or more PI3K isoforms was required to block the GM-CSF survival response in human neutrophils, with inhibition of individual or any two isoforms having little or no effect. Likewise, isolated blood neutrophils derived from double knockout PI3K p110δKOγKO mice underwent normal time-dependent constitutive apoptosis and displayed identical GM-CSF mediated survival to wild type cells, but were sensitized to pharmacological inhibition of the remaining PI3K isoforms. Surprisingly, the pro-survival neutrophil phenotype observed in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD) was resilient to inactivation of the PI3K pathway