After birth, the full-term ductus arteriosus actively constricts and undergoes extensive histologic changes that prevent subsequent reopening. These changes are thought to occur only if a region of intense hypoxia develops within the ductus wall after the initial active constriction. In preterm infants, indomethacininduced constriction of the ductus is often transient and is followed by reopening. Prostaglandins and nitric oxide both play a role in inhibiting ductus closure in vitro. We hypothesized that combined inhibition of both prostaglandin and nitric oxide production (with indomethacin and N-nitro-L-arginine (L-NA), respectively) may be required to produce the degree of functional closure that is needed to cause intense hypoxia. We used preterm (0.67 gestation) newborn baboons that were mechanically ventilated for 6 d: 6 received indomethacin alone, 7 received indomethacin plus L-NA, and 16 received no treatment (control). Just before necropsy, only 25% of control ductus and 33% of indomethacin-treated ductus were closed on Doppler examination; in contrast, 100% of the indomethacin-plus-L-NA-treated ductus were closed. Control and indomethacin-treated baboons developed negligible-to-mild ductus hypoxia (EF5 technique). Similarly, there was minimal evidence of ductus remodeling. In contrast, indomethacin-plus-L-NA-treated baboons developed intense hypoxia in regions where the ductus was most constricted. The hypoxic muscle strongly expressed vascular endothelial growth factor, and proliferating luminal endothelial cells filled and occluded the lumen. In addition, cells in the most hypoxic regions were undergoing DNA fragmentation. In conclusion, preterm newborns are capable of remodeling their ductus, just like the full-term newborn, if they can reduce their luminal blood flow to a point that produces intense ductus wall hypoxia. Combined prostaglandin and nitric oxide inhibition may be necessary to produce permanent closure of the ductus and prevent reopening in preterm infants. In the full-term infant, closure of the DA occurs in two phases: 1) initial "functional" closure of the DA lumen by smooth muscle constriction, and 2) "anatomic" occlusion of the lumen resulting from endothelial proliferation, neointimal thickening, and loss of smooth muscle cells from the inner muscle media (1, 2). Hypoxia of the DA wall seems to be the required stimulus for irreversible, anatomic closure (3). Anatomic remodeling occurs only in the presence of moderate to intense hypoxia (3).

Solid tumors frequently contain hypoxic subregions due to insufficient blood supply. In these domains, cells can undergo p53-dependent apoptosis. Therefore, hypoxia has been implicated as a physiological stimulus for p53 accumulation and activation. In such an environment, p53 mutant cells exhibit a selective growth advantage. Hypoxic regulation of p53 has been proposed to be hypoxia inducible factor (HIF) dependent; however, controversy remains over whether and to what extent low oxygen (O 2 ) tension by itself enhances p53 protein stability. Here, we examined the p53 response to hypoxia and hypoxia mimetics in several cell lines expressing different HIF-a proteins. Most cells exhibited elevated levels of p53 in response to hypoxia mimetics such as deferoxamine mesylate and CoCl 2 , regardless of their HIF-a protein expression profile. However, over a range of O 2 levels, from 1.5% to less than 0.02%, we failed to observe p53 accumulation or p53 nuclear translocation in any cell lines tested. Only after treatment with a combination of hypoxia and acidosis/nutrient deprivation did some cells exhibit p53 induction. Our results suggest that, although hypoxia induces p53 accumulation in vivo, secondary effects such as acidosis caused by a hypoxic Pasteur effect (instead of low O 2 by itself) are necessary for p53 accumulation. Therefore, the expression of HIF-1a and p53 proteins is not coupled during the cellular hypoxia response.

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O 2 . We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O 2 ), whereas hyperoxia (95% O 2 ) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1␣ (HIF-1␣) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1␣ stabilization under moderate hypoxia. Moreover, the levels of HIF-1␣ that we observed after exposure to moderate hypoxia were comparable between 0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1␣ in response to the non-toxic levels of H 2 O 2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1␣ stability under moderate as well as severe hypoxia.The primary role of the oxidative pentose phosphate cycle (OPPC) 2 in mammalian cells is to maintain the [NADPH/ NADPϩ] ratio, thereby helping to regulate the cellular redox equilibrium (1-3). Glucose-6-phosphate dehydrogenase (G6PD), the initial and rate-limiting enzyme of the OPPC, exists in a dimer-tetramer equilibrium with the tetramer being the catalytically active conformation. Each of four identical G6PD monomers contains a structural NADPϩ binding site (4, 5). When NADPϩ is bound at this site, formation of the active tetramer is favored. In non-stressed cells, the [NADPH]/ [NADPϩ] ratio is very high (approaching 1000) and flux through the OPPC is minimal. However, even a slight increase in [NADPϩ] can increase the number of active G6PD tetramers. Therefore, G6PD activity, by regulating flux through the OPPC, is uniquely sensitive to reactive oxygen species (ROS) as well as other chemical oxidants.The importance of the OPPC for the cellular response to ROS is evident from the elevated incidence of apoptosis that we observed in G6PD Ϫ Chinese hamster ovary cells following exposure to ionizing radiation (2). Likewise, Efferth et al. (6) observed an elevated incidence of oxidant-induced apoptosis in macrophages isolated from patients suffering from G6PD deficiency syndrome, while Fico et al. (42) observed H 2 O 2 -induced apoptosis in G6PD Ϫ mouse embryo fibroblasts. Notably, a 10-fold reduction in cloning efficiency was seen in G6PD Ϫ mouse embryo fibroblasts (MEFs) incubated in a...

Previous work from this laboratory demonstrated that MCF-7 breast carcinoma cells grown in nude mice contained minimal hypoxia but that tamoxifen treatment of these tumors resulted in increased hypoxia (Evans S. et al., Cancer Research, 1997). These findings led to studies exploring the link between estrogen signaling and tumor oxygenation and determining the role of VEGF in this process. The stimulation of estrogen-dependent MCF-7 breast carcinoma cells in vitro with beta-estradiol resulted in a two-fold induction of VEGF mRNA and 1.3-2-fold increase in protein, similar to what was observed when these cells were exposed to 0. 1% oxygen. Furthermore, the two stimuli given together had an additive effect on (increasing) VEGF expression, suggesting that the combination of hypoxia and estrogen may be important in upregulating VEGF in some breast cancers. Estrogen-independent MCF-7-5C cells, developed by growing MCF-7 cells in long-term culture in estrogen-free media, were also studied. Using EF5, a fluorinated 2-nitroimidazole which localizes to hypoxic cells, MCF-7-5C tumors grown in nude mice were found to contain lower pO2 levels and more hypoxic regions than similarly grown MCF-7 tumors. We tested the hypothesis that this might be the result of defective expression of VEGF in MCF-7-5C cells in response to beta-estradiol and/or hypoxia. However, MCF-7-5C and MCF-7 cells showed a similar induction of VEGF in vitro in response to either beta-estradiol or hypoxia. Therefore, although these two cell lines grown as tumors have substantial differences in the presence and patterns of hypoxia, this could not be explained by a difference in VEGF induction.

Since tissue oxygen tension is a balance between delivery and consumption of oxygen, considerable effort has been directed at increasing the former and/or decreasing the latter. Techniques to decrease the rate of cellular oxygen consumption (increasing the distance oxygen can diffuse into tissues) include increasing glycolysis by administering supraphysiologic levels of glucose. We have examined the effect of hyperglycemia produced by intravenous glucose infusion on the tissue oxygenation and radiation response of subcutaneously implanted murine radiation induced fibrosarcomas (RIF-1). A 0.3 M glucose solution was delivered via tail vein injection according to a protocol that maintained glucose at a plasma concentration of 17±1 mM. The effect of this treatment on radiation response (clonogenic and growth delay studies), tumor oxygenation (needle electrode pO 2 and 2-[2-nitro-1Himidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) binding), and tumor bioenergetics and pH (31 P NMR spectroscopy) was examined. Systemic measurements included hematocrit and blood glucose and lactate concentrations. The results of these studies suggest that these subcutaneously implanted RIF-1 tumors are both radiobiologically and metabolically hypoxic and that intravenous glucose infusion is not an effective method of modifying this metabolic state.