Euoxic Conditions Research Articles

Cerebral and myocardial blood flow responses to hypercapnia and hypoxia in humans

In humans, cerebrovascular responses to alterations in arterial Pco(2) and Po(2) are well documented. However, few studies have investigated human coronary vascular responses to alterations in blood gases. This study investigated the extent to which the cerebral and coronary vasculatures differ in their responses to euoxic hypercapnia and isocapnic hypoxia in healthy volunteers. Participants (n = 15) were tested at rest on two occasions. On the first visit, middle cerebral artery blood velocity (V(P)) was assessed using transcranial Doppler ultrasound. On the second visit, coronary sinus blood flow (CSBF) was measured using cardiac MRI. For comparison with V(P), CSBF was normalized to the rate pressure product [an index of myocardial oxygen consumption; normalized (n)CSBF]. Both testing sessions began with 5 min of euoxic [end-tidal Po(2) (Pet(O(2))) = 88 Torr] isocapnia [end-tidal Pco(2) (Pet(CO(2))) = +1 Torr above resting values]. Pet(O(2)) was next held at 88 Torr, and Pet(CO(2)) was increased to 40 and 45 Torr in 5-min increments. Participants were then returned to euoxic isocapnia for 5 min, after which Pet(O(2)) was decreased from 88 to 60, 52 and 45 Torr in 5-min decrements. Changes in V(P) and nCSBF were normalized to isocapnic euoxic conditions and indexed against Pet(CO(2)) and arterial oxyhemoglobin saturation. The V(P) gain for euoxic hypercapnia (%/Torr) was significantly higher than nCSBF (P = 0.030). Conversely, the V(P) gain for isocapnic hypoxia (%/%desaturation) was not different from nCSBF (P = 0.518). These findings demonstrate, compared with coronary circulation, that the cerebral circulation is more sensitive to hypercapnia but similarly sensitive to hypoxia.

Oxygen Sensing of Mesenchymal Stem and Progenitor Cells Facilitates Neo-Vasculogenesis In Vivo

AbstractAbstract 4313 Background:Vascular homeostasis and regeneration are maintained by proliferating vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs). Despite promising experimental data, regenerative stem cell therapy approaches employing ECFCs have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. We and others have recently shown that ECFC function in vivo requires a stringent interaction with mesenchymal stem and progenitor cells (MSPCs) * [Blood 2009; 113 (26):6716-25]. Co-transplantation of ECFCs and MSPCs is considered to be an advantageous strategy for vascular regenerative medicine.Hypoxia in ischemic tissue is considered to be a key factor influencing pro- and anti-angiogenic treatment by driving the revascularization machinery. In vivo most cells exist under an O2 pressure considerably below air oxygen. In vitro cells are usually expanded under air oxygen and suddenly encounter reduced O2 conditions when re-injected for therapy. Preliminary data suggests that low oxygen conditions differentially regulate stem cell function. We hypothesized that MSPCs act as hypoxia sensors and drive ECFCs to form functioning vessels in vivo. Methods:Adult human ECFCs were isolated and propagated directly from whole venous blood using a novel recovery strategy **[J Vis Exp. 2009;(32) pii: 1524]. MSPCs were isolated from human bone marrow aspirates. During cell culture, pooled human platelet lysate (pHPL) entirely replaced fetal bovine serum. Throughout this study we designated the oxygen level present in vivo in the venous environment as euoxia (41.5±3.4 mmHg). Oxygen levels below euoxia are defined as hypoxia (27.4±7.3 mmHg). Air-oxygen commonly used in standard laboratory practice is above euoxia and is therefore referred to as hyperoxia (139.8±2.9 mmHg). Progenitor cell phenotype, hierarchy, long-term proliferation, wound repair as well as migratory and vasculogenic functions were monitored under euoxia as compared to hypoxic or hyperoxic conditions. Molecular regulation of cellular responses to different oxygen levels was assessed by flow cytometry, immune cytochemistry and proteomic profiling. ECFC and MSPC interactions in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after sub-cutaneous co-implantation in matrigel plugs. Immune histochemistry and TUNEL assays were performed on plugs at day 1, 7 and 14 after transplantation. Results:Compared to hyperoxic standard laboratory conditions in vitro, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced under euoxia, and even more under hypoxic conditions. Hyperoxic conditioning resulted in a shift in progenitor hierarchy with an augmented number of ECFC high proliferative potential (HPP) colonies (60±18% of total colonies) as compared to euoxia (9±6%) and a complete loss of HPP colonies under hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair in scratch assays and matrigel vascular-like network formation in vitro were improved with escalating oxygen supply. The reoxygenation of hypoxic and euoxic ECFCs led to enhanced proliferation and function. Furthermore, MSPCs stabilized hypoxia inducible factor-1α (HIF-1α) under hypoxic as well as euoxic conditions, whereas ECFCs only stabilized HIF-1α when confronted with hypoxia in vitro. In a mouse model, subcutaneously injected ECFCs in matrigel underwent apoptosis after 1 day and attracted mouse leucocytes which infiltrated the matrigel plug. Co-implantation of ECFCs and MSPCs in these matrigel plugs resulted in reduced apoptosis and formation of perfused human vessels as soon as 7 days after transplantation. In this in vivo setting, perivascular cells but not endothelial cells were positive for HIF-1α in immune histochemistry. Background:These data indicate that oxygen levels differentially regulate ECFC and MSPC function during vascular homeostasis and regeneration. While hypoxic ECFCs alone are not able to function in vitro and form patent vessels in vivo, MSPCs react to the low oxygen environment and support ECFCs to perform vessel formation in vivo at least in part by rescuing ECFCs from hypoxia-induced apoptosis. This suggests that oxygen appears to be a key factor in stem cell transplantation and regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Modulation of the genotoxicity of bleomycin by amines through noncovalent DNA interactions and alteration of physiological conditions in yeast

The effects of amines on the induction of mitotic gene conversion by bleomycin (BLM) were studied at the trp5 locus in Saccharomyces cerevisiae strain D7. BLM induces double-strand breaks in DNA and is a potent recombinagen in this assay. The polyamine spermidine causes concentration-dependent protection against the genotoxicity of BLM, reducing the convertant frequency by over 90% under the most protective conditions. Spermine, diethylenetriamine, ethylenediamine, putrescine, and ethylamine were also antigenotoxic in combined treatments with BLM. There was a general correspondence between the protective effect and the number of amino groups, suggesting that more strongly cationic amines tend to be stronger antirecombinagens. Electrostatic association of the amines with DNA probably hinders BLM access to the 4′ position of deoxyribose where it generates a free radical. Other amines interact with BLM differently from these unbranched aliphatic amines. The aminothiol cysteamine inhibits the genotoxicity of BLM under hypoxic conditions but increases it under euoxic conditions. In contrast, pargyline potentiates the genotoxicity of BLM under hypoxic conditions but not under euoxic conditions. The antirecombinagenic effect of cysteamine apparently involves DNA binding and depletion of oxygen needed for BLM activity, whereas its potentiation of BLM entails its serving as an electron source for the activation of BLM. Pargyline may enhance BLM indirectly by preventing the depletion of oxygen by monoamine and polyamine oxidase. The planar 9-aminoacridine weakly induces gene conversion in strain D7, but it is strongly synergistic with BLM. Enhancement of BLM activity by this compound and by the related nitroacridine Entozon is apparently mediated by intercalation of the acridine ring system into DNA. Thus, the influence of amines on the genotoxicity of BLM in yeast encompasses antigenotoxic, potentiating, and synergistic interactions. The underlying mechanisms involve noncovalent association with DNA, altered BLM access to DNA, and modulation of physiological conditions.

Microvessel density, thymidine phosphorylase expression and resistance of head and neck cancer to chemo-radiotherapy.

Cancer growth, invasion and metastasis is strongly dependent on the angiogenic ability. A growing amount of clinicopathological studies confirms that intratumoural microvessel density (MVD) is one of the strongest prognostic variables in human cancer. However, vasculature is also related to the amount of haemoglobin that reaches the tumour, thus oxygenation. Moreover, during chemotherapy, the vessel density strongly defines the drug availability to the tumour. Since euoxic conditions are related to a higher response to radiotherapy (RT) and high drug availability to higher chances for the tumour to respond to chemotherapy, it would be suggested that high microvessel density is a marker of a better response to cytotoxic therapy.KeywordsMicrovessel DensityComplete Response RateThymidine PhosphorylaseDrug AvailabilityThymidine Phosphorylase ExpressionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synthesis and evaluation of a boronated nitroimidazole for boron neutron capture therapy.

We postulated that nitroimidazoles, previously used for radiosensitizing solid tumors, may be interesting templates as carriers of 10B for boron neutron capture therapy. To test this hypothesis, we synthesized a 10B-enriched nitroimidazole, 1-2[(undecahydro-closo-dodecaborato)thio]ethyl]-2- methyl-5-nitroimidazole (imidocaptate), by coupling the Cs salt of BSH (Cs2-10B12H11SH) with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole followed by purification of the adduct. Imidocaptate was taken up by V-79 cells in culture and showed no inherent toxicity under euoxic conditions up to 1.05 mM (126 micrograms of 10B/mL of culture medium). Imidocaptate showed a dose-dependent decrease in D0 when the treated cells were irradiated with a thermal neutron beam. At the highest dose tested (126 micrograms of 10B/mL of culture medium), the ratio of control to sample D0 values was 2.6 for both linear quadratic and single-hit multitarget models. At 33 micrograms of 10B/mL, imidocaptate showed a control/treated D0 ration (1.5) equal to that observed with the disulfide form of BSH at 28 micrograms of 10B/mL. Compared to BSH and its disulfide, the reduced toxicity and equipotency of imidocaptate suggest that this agent may be useful for boron neutron capture therapy of cancer.

Radiation Response of E. Coli After Combined Treatment with Misonidazole and WR-2721 at Various Oxygen Concentrations

It has been reported that the aminothiol compound WR-2721 is a promising radioprotective agent and in combination with misonidazole (MISO) seems to be of therapeutic benefit. Since the radiomodification is oxygen-dependent, the actual oxygen status of cells and the surrounding media is an important factor influencing their effectiveness. Escherichia coli B/r radioresponse was studied either alone or in combination with these compounds at various oxygen concentrations ranging from anoxia to high oxygen content. WR-2721 had a protective effect under anoxic conditions and gave overall protection when oxygen was present. The maximum protection was seen at 3.2% O2 in N2 (PF 2.08). In combination with MISO the hypoxic sensitization of MISO was completely abolished by WR-2721, resulting in radioprotection under hypoxic conditions as well. Under euoxic conditions MISO was able to reduce the protective effect of WR-2721 by about 21%. According to our results MISO and WR-2721 influence each other in their radiomodifying effect in either fixation or repair of the radiation-induced damage.

Carbon dioxide-heart rate responses during hyperoxia, euoxia, and hypoxia.

We studied the steady state responses of heart rate (HR) to carbon dioxide inhalation under hyperoxic, euoxic, and hypoxic conditions in 9 healthy men. With increasing end-tidal PCO2, HR generally increased slightly. On the other hand, distinct increment in VE in response to step increase in end-tidal PCO2 was observed in all three different conditions. Significant positive correlation between hypercapnic VE and HR responsiveness was found in both hyperoxic and hypoxic conditions, whereas no such tendency was seen in euoxic condition. We suggest that the effect of CO2 inhalation on HR is mainly determined by the pulmonary inflation reflex in hyperoxia, the pulmonary inflation reflex plus peripheral chemoreceptor activity in euoxia, and the additional sympathetic and humoral factors in hypoxia, respectively.

Effect of Hypoxia on Recovery from Damage Induced by Heat and Radiation in Plateau-Phase CHO Cells

The effect of hypoxia on the induction of and recovery from damage by radiation alone and in combination with heat has been investigated using plateau-phase Chinese hamster ovary (CHO) cells. Postirradiation hypoxia reduced the potentially lethal damage recovery (PLDR) in cells irradiated under an euoxic state and completely eliminated PLDR in cells irradiated under hypoxia. Cells which were maintained under hypoxia during both irradiation and a 4-hr recovery period and then incubated for a further period of 4 hr under euoxic conditions showed PLDR, suggesting that the inhibition of PLDR by hypoxia is reversible. Oligomycin, an inhibitor of energy metabolism, completely eliminated PLDR when present at a concentration of 1 microM during the postirradiation period. Pre- or postirradiation heat treatment at 42.5 degrees C for 30 min appreciably sensitized the cells to the induction of lethality. Thermal enhancement ratio (TER) was 1.7 for cells irradiated and heat treated under hypoxic conditions. The same heat treatment reduced the oxygen enhancement ratio (OER) associated with gamma radiation from 3.1 to 2.5. Cells subjected to this postirradiation heat treatment showed a small extent of PLDR, whereas the pre-heat-treated cells showed as much recovery as non-heat-treated cells. When hypoxic conditions prevailed during the post-treatment incubation period, PLDR was reduced in preheated cells and completely eliminated in postheated cells. The kinetics of interaction between heat and radiation damage were studied by introducing a time gap of 4 hr between the treatments. Cells maintained under euoxic conditions between the treatments showed an appreciable decrease in interaction, suggesting recovery from damage induced by the first treatment. Hypoxic conditions intervening the two treatments largely inhibited the loss of sensitization. Analysis of the results suggests that cells fail to recover from sublethal heat damage when held for 4 hr under hypoxic conditions. Cells held under hypoxic conditions partly recover from the radiation damage which subsequently interacts with sublethal heat damage, resulting in cell lethality.

Drug resistance of hypoxic tumour cells in vitro

In 1959 Thomlinson and Gray postulated that hypoxic cells might exist in the larger solid tumours ( 13), and some evidence to support this has been obtained in trials of hyperbaric oxygen (3). Hypoxic cells in solid tumours, however, appear to be not only relatively resistant to the cytotoxic effect of ionizing radiation, but also to some chemotherapeutic agents (12). Cytotoxic drugs which may be at least as active under hypoxic as under euoxic conditions are therefore of considerable clinical interest. Lin et al. (8) postulated that bioreductive drugs like Mitomycin C should have a particular toxicity for hypoxic tumours. The decreased oxygen tension in hypoxic tumour cells should create conditions conducive to reductive processes thought to be necessary for activation of Mitomycin C. The reduction of the benzoquinone ring of the Mitomycin molecule to dihydrobenzoquinone has been proposed as an essential step for the biological activity of this drug (8), and it has therefore been classed as a “bioreductive alkylating agent”. Using the murine sarcoma cell lines EMT6 and S180, Kennedy et al. (7) have demonstrated that Mitomycin C is preferentially activated and metabolized by hypoxic murine tumour cells. We have studied the cytotoxic effect ofMitomycin C on human carcinomas under euoxic and hypoxic conditions in the tumour clonogenic assay (HTCA) of Hamburger et al. (6). We also measured the rate of metabolism of Mitomycin C by EMT6 cells and the human colon tumour cell line WIDR, under both euoxic and hypoxic conditions. Additionally we have studied the cytotoxic effect of Bisantrene, a new anthracene bishydrazone derivative, on human carcinomas under euoxic and hypoxic conditions in the HTCA. This drug has shown antitumour activity in phase I trials (1, 10, 14) and in the HTCA (1). In contrast to Mitomycin C or Adriamycin, the drug has no quinone ring and in cardiac studies carried out in beagle dogs, it showed no cardiotoxicity (11). In addition to the clonogenic experiments, the induction of protein associated DNA breaks by Bisantrene has also been investigated under euoxic and hypoxic conditions in the murine L12 10 cell line, using the alkaline elution technique (2). For our experiments, cells were rendered hypoxic by gassing the suspension containing the medium and 10% serum in completely gas tight tubes, designed to obtain and maintain

6-[125I]-iodo-2-methyl-1,4-naphthoquinol bis(diammonium phosphate): a new potential anti-tumour drug

6-[125I]-Iodo-2-methyl-1,4-naphtoquinol bis-(diammonium phosphate) has been prepared as a potential radiochemotherapeutic drug with a specific activity of 1·82mCi/mg; laboratory studies have shown comparable uptake and cytotoxicity for tumour cells cultured in vitro under anoxic and euoxic conditions.

The influence of hypoxia and acidity on the hyperthermic response of malignant cells in vitro.

Colony formation of JB-1-E tumor cells was studied after hyperthermic treatment (42.5 degrees C) at a pH of 6.4 or 7.2 under hypoxic and euoxic conditions. At a pH of 7.2 and normal oxygen tension, there was a moderate decrease in colony formation with increasing duration of hyperthermic treatment (To = 65 min.). This effect was slightly enhanced under hypoxic conditions (To = 36 min.). The hyperthermic effect was enhanced to a considerably greater degree when treatment was performed at a pH of 6.4 (To = 19 min.), with no observable difference between hypoxia and euoxia. These findings indicate that environmental acidity is a determining factor in the hyperthermic effect. The hypoxic effect at a pH of 7.2 is probably due to a slight decrease in the intracellular pH caused by increased production of lactic acid.

Differential radioprotection of euoxic and hypoxic mouse mammary tumors by a thiophosphate compound.

The radioprotective drug S-2-(3-aminopropylamino) ethyl phosphorothioate (WR2721) was evaluated in the EMT-6 mouse mammary tumor. Under euoxic conditions (DMF approximately 2), the drug protected the oxygenated portion of the tumor but not chronically hypoxic cells. Acutely hypoxic cells were protected to a slight degree. Since WR2721 has been shown to protect normal tissues with a DMF of 2–3, it is likely that decreased protection of anoxic tumor cells will be clinically useful.