Intermittent hypoxia: cell to system

This symposium was organized to present research dealing with the effects of intermittent hypoxia on cardiorespiratory systems and cellular mechanisms. The pattern of neural impulse activity has been shown to be critical in the induction of genes in neuronal cells and involves distinct signaling pathways. Mechanisms associated with different patterns of intermittent hypoxia might share similar mechanisms. Chronic intermittent hypoxia selectively augments carotid body sensitivity to hypoxia and causes long-lasting activation of sensory discharge. Intermittent hypoxia also activates hypoxia-inducible factor-1. Reactive oxygen species are critical in altering carotid body function and hypoxia-inducible factor-1 activation caused by intermittent hypoxia. Blockade of serotonin function in the spinal cord prevents long-term facilitation in respiratory motor output elicited by episodic hypoxia and requires de novo protein synthesis. Chronic intermittent hypoxia leads to sustained elevation in arterial blood pressure and is associated with upregulation of catecholaminergic and renin-angiotensin systems and downregulation of nitric oxide synthases.     

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.     

Osteocyte hypoxia: a novel mechanotransduction pathway

Bone is a unique tissue in which to examine mechanotransductiondue to its essential role in weight bearing. Within bone, the osteocyteis an ideal cellular mechanotransducer candidate. Because osteocytesreside distant from the blood supply, their metabolic needs are met bya combination of passive diffusion and enhanced diffusion, arising whenthe tissue is loaded during functional activity. Therefore, wehypothesized that depriving a bone of mechanical loading (and thuseliminating diffusion enhanced by loading) would rapidly induceosteocyte hypoxia. Using the avian ulna model of disuse osteopenia, wefound that 24 h of unloading results in significant osteocyte hypoxia(8.4 ± 1.8%) compared with control levels (1.1 ± 0.5%;P = 0.03). Additionally, we present preliminary data suggesting that a brief loading regimen is sufficient to rescue osteocytes from this fate. The rapid onset of the observed osteocyte hypoxia, the inhibition of hypoxia by brief loading, and thecellular consequences of oxygen deprivation are suggestive of a novelmechanotransduction pathway with implications across organ systems.

     

Nickel carcinogenesis: epigenetics and hypoxia signaling

Both water soluble and insoluble nickel compounds have been implicated in the etiology of human lung and nasal cancers. Water insoluble nickel compounds have been shown to enter cells by phagocytosis and are contained in cytoplasmic vacuoles, which are acidified thus accelerating the dissolution of soluble nickel from the particles. Using Newport Green, a dye that fluoresces when ionic nickel is bound, we have shown that following exposure (48-72 h) of human lung (A549) cells to NiS particles, most of the nickel is contained in the nucleus, while cells exposed to soluble NiCl2 exhibit most of the ions localized in the cytoplasm. This effect is consistent with previously published reports showing that short-term exposure of cells to crystalline nickel particles (1-3 days) is able to epigenetically silence target genes placed near heterochromatin, while similar short-term exposure to soluble nickel compounds are not able to induce silencing of genes placed near heterochromatin. However, a 3 week exposure of cells to soluble NiCl2 is also able to induce gene silencing. A similar effect was found in yeast cells where nickel was able to silence the URA-3 gene placed near (1.3 kb) a telomere silencing element, but not when the gene was placed farther away from the silencing element (2.0 kb). In addition to epigenetic effects, nickel compounds activate hypoxia signaling pathways. The mechanism of this effect involves the ability of either soluble or insoluble nickel compounds to block iron uptake leading to cellular iron depletion, directly affect iron containing enzymes, or both. This results in the inhibition of a variety of iron-dependent enzymes, such as aconitase and the HIF proline hydroxylases (PHD1-3). The inhibition of the HIF proline hydroxylases stabilizes the HIF protein and activates hypoxic signaling. Additional studies have shown that nickel and hypoxia decrease histone acetylation and increase the methylation of H3 lysine 9. These events are involved in gene silencing and hypoxia can also cause these effects in human cells. It is hypothesised that the state of hypoxia either by low oxygen tension or as a result of agents that signal hypoxia under normal oxygen tension (iron chelation, nickel and cobalt) results in low levels of acetyl CoA, which is a substrate for histone and other protein acetylation. This effect may in part be responsible for the gene silencing following nickel exposure and during hypoxia.     

ATIA: a link between inflammation and hypoxia

In this issue of Molecular Cell,Choksi et al. (2011) report the identification of an NF-κB-independent ATIA (anti-TNFα-induced apoptosis)-Thioredoxin 2 axis that inhibits TNFα- and hypoxia-induced apoptosis through elimination of excessive reactive oxygen species directly.     

Angiogenesis: how a tumor adapts to hypoxia

Early atherosclerotic lesions are characterized by increased monocyte adhesion to the overlying endothelium. Oxidized LDL (oxLDL) stimulates the adhesion of human monocytes to endothelial cells, in part, by increasing expression of ICAM-1. However, the cellular role of oxLDL in endothelial adhesiveness is not well understood. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is expressed in vascular endothelial cells. Whether it can be activated by a synthetic ligand, troglitazone, as well as by natural ligands, oxLDL and its lipid components (i.e., 9- and 13-HODE), has not yet been explored. This study was undertaken to determine whether PPARgamma is expressed in ECV304 human vascular endothelial cells and if so to define the biological effects of its activation by these agonists. Our results demonstrate that PPARgamma mRNA is expressed in ECV304 cells, and transfected cells with a PPARE luciferase construct respond to these agonists. In addition, ligand-dependent PPARgamma activation increased ICAM-1 protein expression and enhanced adherence of monocytes to ECV304 cells by two- to threefold. These findings suggest that the PPARgamma signaling pathway might contribute to the atherogenicity of oxLDL in vascular endothelial cells.     

Chronic intermittent hypoxia increases sympathetic responsiveness to hypoxia and hypercapnia

We sought to determine whether chronic exposure tointermittent hypoxia (CIH) increases sympathetic responsiveness tosubsequent chemoreflex stimulation. Sprague-Dawley rats were exposed to30 days of CIH: exposure chamber%O₂ [fractionalconcentration of chamber O₂(Fc_O2)]nadir 6.5-7% with return to 21% each minute for 8 h/day duringthe diurnal sleep period (Exp group). Sham controls (SC group) weresimilarly handled but kept at 21%Fc_O2 andcompared with unhandled controls (UC group). Rats were then anesthetized with urethan, and preganglionic cervical sympathetic activity (CSA), diaphragm electromyogram, arterial pressure, and electrocardiogram were recorded while the rats were spontaneously breathing 100% O₂, room air, 10%O₂, 12%CO₂, and 10%O₂-12%CO₂. CSA and heart rate were alsorecorded during phenylephrine infusion to assess baroreceptor function.Mean arterial pressure was significantly greater in Exp than in SC andUC rats during all conditions (P < 0.05). A vasopressor response to 10%O₂-12%CO₂ was observed only in Exp rats.CSA was greater in Exp than in SC and UC rats during 10%O₂, 12%CO₂, and 10%O₂-12%CO₂ but not during room-air exposure. A significant increase in CSA compared with room air wasnoted during 10% O₂, 12%CO₂, and 10%O₂-12%CO₂ in Exp but not in SC or UCrats. No differences in baroreceptor function were observed amonggroups. We conclude that CIH leads to increased sympatheticresponsiveness to chemoreflex stimulation.

     

Chronic hypoxia attenuates nitric oxide-dependent hemodynamic responses to acute hypoxia

Alterations in the nitric oxide (NO) pathway have been implicated in the pathogenesis of chronic hypoxia-induced pulmonary hypertension. Chronic hypoxia can either suppress the NO pathway, causing pulmonary hypertension, or increase NO release in order to counteract elevated pulmonary arterial pressure. We determined the effect of NO synthase inhibitor on hemodynamic responses to acute hypoxia (10% O(2)) in anesthetized rats following chronic exposure to hypobaric hypoxia (0.5 atm, air). In rats raised under normoxic conditions, acute hypoxia caused profound systemic hypotension and slight pulmonary hypertension without altering cardiac output. The total systemic vascular resistance (SVR) decreased by 41 +/- 5%, whereas the pulmonary vascular resistance (PVR) increased by 25 +/- 6% during acute hypoxia. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME; 25 mg/kg) attenuated systemic vasodilatation and enhanced pulmonary vasoconstriction. In rats with prior exposure to chronic hypobaric hypoxia, the baseline values of mean pulmonary and systemic arterial pressure were significantly higher than those in the normoxic group. Chronic hypoxia caused right ventricular hypertrophy, as evidenced by a greater weight ratio of the right ventricle to the left ventricle and the interventricular septum compared to the normoxic group (46 +/- 4 vs. 28 +/- 3%). In rats which were previously exposed to chronic hypoxia (half room air for 15 days), acute hypoxia reduced SVR by 14 +/- 6% and increased PVR by 17 +/- 4%. Pretreatment with L-NAME further inhibited the systemic vasodilatation effect of acute hypoxia, but did not enhance pulmonary vasoconstriction. Our results suggest that the release of NO counteracts pulmonary vasoconstriction but lowers systemic vasodilatation on exposure to acute hypoxia, and these responses are attenuated following adaptation to chronic hypoxia.