Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

31P magnetic resonance spectroscopy of the Sherpa heart: a phosphocreatine/adenosine triphosphate signature of metabolic defense against hypobaric hypoxia.

Of all humans thus far studied, Sherpas are considered by many high-altitude biomedical scientists as most exquisitely adapted for life under continuous hypobaric hypoxia. However, little is known about how the heart is protected in hypoxia. Hypoxia defense mechanisms in the Sherpa heart were explored by in vivo, noninvasive 31P magnetic resonance spectroscopy. Six Sherpas were examined under two experimental conditions [normoxic (21% FiO2) and hypoxic (11% FiO2) and in two adaptational states--the acclimated state (on arrival at low-altitude study sites) and the deacclimating state (4 weeks of ongoing exposure to low altitude). Four lowland subjects were used for comparison. We found that the concentration ratios of phosphocreatine (PCr)/adenosine triphosphate (ATP) were maintained at steady-state normoxic values (0.96, SEM = 0.22) that were about half those found in normoxic lowlanders (1.76, SEM = 0.03) monitored the same way at the same time. These differences in heart energetic status between Sherpas and lowlanders compared under normoxic conditions remained highly significant (P < 0.02) even after 4 weeks of deacclimation at low altitudes. In Sherpas under acute hypoxia, the heart rate increased by 20 beats per min from resting values of about 70 beats per min, and the percent saturation of hemoglobin decreased to about 75%. However, these perturbations did not alter the PCr/ATP concentration ratios, which remained at about 50% of the values expected in healthy lowlanders. Because the creatine phosphokinase reaction functions close to equilibrium, these steady-state PCr/ATP ratios presumably coincided with about 3-fold higher free adenosine diphosphate (ADP) concentrations. Higher ADP concentrations (i.e., lower [PCr]/[ATP] ratios) were interpreted to correlate with the Km values for ADP-requiring kinases of glycolysis and to reflect elevated carbohydrate contributions to heart energy needs. This metabolic organization is postulated as advantageous in hypobaria because the ATP yield per O2 molecule is 25-60% higher with glucose than with free fatty acids (the usual fuels utilized in the human heart in postfasting conditions).

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis.

Hypoxia/reoxygenation is an important cause of tissue injury in a variety of organs and is classically considered to be a necrotic form of cell death. We examined the role of endonuclease activation, considered a characteristic feature of apoptosis, in hypoxia/reoxygenation injury. We demonstrate that subjecting rat renal proximal tubules to hypoxia/reoxygenation results in DNA strand breaks and DNA fragmentation (both by an in situ technique and by agarose gel electrophoresis), which precedes cell death. Hypoxia/reoxygenation resulted in an increase in DNA-degrading activity with an apparent molecular mass of 15 kDa on a substrate gel. This DNA-degrading activity was entirely calcium dependent and was blocked by the endonuclease inhibitor aurintricarboxylic acid. The protein extract from tubules subjected to hypoxia/reoxygenation cleaved intact nuclear DNA obtained from normal proximal tubules into small fragments, which further supports the presence of endonuclease activity. Despite unequivocal evidence of endonuclease activation, the morphologic features of apoptosis, including chromatin condensation, were not observed by light and electron microscopy. Endonuclease inhibitors, aurintricarboxylic acid and Evans blue, provided complete protection against DNA damage induced by hypoxia/reoxygenation but only partial protection against cell death. Taken together, our data provide strong evidence for a role of endonuclease activation as an early event, which is entirely responsible for the DNA damage and partially responsible for the cell death that occurs during hypoxia/reoxygenation injury. Our data also indicate that in hypoxia/reoxygenation injury endonuclease activation and DNA fragmentation occur without the morphological features of apoptosis.

Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.

Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.

Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments.

BDNF impairs recovery of synaptic transmission after hypoxia even with blockage of adenosine A2A receptors

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Partitioning of respiration between the gills and air-breathing organ in response to aquatic hypoxia and exercise in the Pacific tarpon, Megalops cyprinoides

The evolution of air-breathing organs (ABOs) is associated not only with hypoxic environments but also with activity. This investigation examines the effects of hypoxia and exercise on the partitioning of aquatic and aerial oxygen uptake in the Pacific tarpon. The two-species cosmopolitan genus Megalops is unique among teleosts in using swim bladder ABOs in the pelagic marine environment. Small fish ( 58 - 620 g) were swum at two sustainable speeds in a circulating flume respirometer in which dissolved oxygen was controlled. For fish swimming at 0.11 m s(-1) in normoxia (Po-2 = 21 kPa), there was practically no air breathing, and gill oxygen uptake was 1.53 mL kg(-0.67) min(-1). Air breathing occurred at 0.5 breaths min(-1) in hypoxia ( 8 kPa) at this speed, when the gills and ABOs accounted for 0.71 and 0.57 mL kg(-0.67) min(-1), respectively. At 0.22 m s(-1) in normoxia, breathing occurred at 0.1 breaths min(-1), and gill and ABO oxygen uptake were 2.08 and 0.08 mL kg(-0.67) min(-1), respectively. In hypoxia and 0.22 m s(-1), breathing increased to 0.6 breaths min(-1), and gill and ABO oxygen uptake were 1.39 and 1.28 mL kg(-0.67) min(-1), respectively. Aquatic hypoxia was therefore the primary stimulus for air breathing under the limited conditions of this study, but exercise augmented oxygen uptake by the ABOs, particularly in hypoxic water.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

Intrauterine growth restriction due to uteroplacental vascular insufficiency leads to increased hypoxia-induced cerebral apoptosis in newborn piglets

Uteroplacental vascular insufficiency in humans is a common cause of intrauterine growth restriction (IUGR) and is associated with an increased incidence of perinatal asphyxia and neurodevelopmental disorders compared to normal weight newborns. Experimental models that provide an opportunity to analyze the pathogenesis of these relationships are limited. Here, we used neonatal pigs from large litters in which there were piglets of normal birth weight (for controls) and of low birth weight (for uteroplacental vascular insufficiency). Hypoxia was induced in paired littermates by reducing the fraction of inspired oxygen to 4% for 25 min. Brain tissue was collected 4 h post-hypoxia. Cerebral levels of apoptosis were quantified morphologically and verified with caspase-3 activity and TUNEL. Expression of Bcl-2, BcI-XL and Bax proteins was investigated using immunohistochemistry. Cellular positivity for Bcl-2 was consistently higher in the non-apoptotic white matter of the hypoxic IUGR animals compared with their littermates and reached significance at P < 0.05 in several pairs of littermates. Alterations in Bax showed a trend towards higher expression in the hypoxic IUGR littermates but rarely reached significance. The IUGR piglets showed a significantly greater amount of apoptosis in response to the hypoxia than the normal weight piglets, suggesting an increased vulnerability to apoptosis in the IUGR piglets. (c) 2006 Elsevier B.V. All rights reserved.