Intermittent Reoxygenation Research Articles

Bcl-2/Bcl-xL inhibitor ABT-263 overcomes hypoxia-driven radioresistence and improves radiotherapy

Hypoxia, a characteristic of most human solid tumors, is a major obstacle to successful radiotherapy. While moderate acute hypoxia increases cell survival, chronic cycling hypoxia triggers adaptation processes, leading to the clonal selection of hypoxia-tolerant, apoptosis-resistant cancer cells. Our results demonstrate that exposure to acute and adaptation to chronic cycling hypoxia alters the balance of Bcl-2 family proteins in favor of anti-apoptotic family members, thereby elevating the apoptotic threshold and attenuating the success of radiotherapy. Of note, inhibition of Bcl-2 and Bcl-xL by BH3-mimetic ABT-263 enhanced the sensitivity of HCT116 colon cancer and NCI-H460 lung cancer cells to the cytotoxic action of ionizing radiation. Importantly, we observed this effect not only in normoxia, but also in severe hypoxia to a similar or even higher extent. ABT-263 furthermore enhanced the response of xenograft tumors of control and hypoxia-selected NCI-H460 cells to radiotherapy, thereby confirming the beneficial effect of combined treatment in vivo. Targeting the Bcl-2 rheostat with ABT-263, therefore, is a particularly promising approach to overcome radioresistance of cancer cells exposed to acute or chronic hypoxia with intermittent reoxygenation. Moreover, we found intrinsic as well as ABT-263- and irradiation-induced regulation of Bcl-2 family members to determine therapy sensitivity. In this context, we identified Mcl-1 as a resistance factor that interfered with apoptosis induction by ABT-263, ionizing radiation, and combinatorial treatment. Collectively, our findings provide novel insights into the molecular determinants of hypoxia-mediated resistance to apoptosis and radiotherapy and a rationale for future therapies of hypoxic and hypoxia-selected tumor cell fractions.

Intermittent short-duration reoxygenation protects against simulated high altitude-induced pulmonary hypertension in rats.

High-altitude pulmonary hypertension (HAPH) is a severe and progressive disease caused by chronic hypoxia and subsequent pulmonary vascular remodeling. No cure is currently available owing to an incomplete understanding about vascular remodeling. It is believed that hypoxia-induced diseases can be prevented by treating hypoxia. Thus, this study aimed to determine whether daily short-duration reoxygenation at sea level attenuates pulmonary hypertension under high-altitude hypoxia. To this end, a simulated 5000-m hypoxia rat model and hypoxic cultured human pulmonary artery smooth muscle cells were used to evaluate the effect of short-duration reoxygenation. Results show that intermittent, not continuous, short-duration reoxygenation effectively attenuates hypoxia-induced pulmonary hypertension. The mechanisms underlining the protective effects involved that intermittent, short-duration reoxygenation prevented functional and structural remodeling of pulmonary arteries and proliferation, migration, and phenotypic conversion of pulmonary artery smooth muscle cells under hypoxia. The specific genes or potential molecular pathways responsible for mediating the protective effects were also characterised by RNA sequencing. Further, the frequency and the total time of intermittent reoxygenation affected its preventive effect of HAPH, which was likely attributable to augmented oxidative stress. Hence, daily intermittent, not continuous, short-duration reoxygenation partially prevented pulmonary hypertension induced by 5000-m hypoxia in rats. This study is novel in revealing a new potential method in preventing HAPH. It gives insights into the selection and optimisation of oxygen supply schemes in high-altitude areas.

Targeting SLC25A10 alleviates improved antioxidant capacity and associated radioresistance of cancer cells induced by chronic-cycling hypoxia

High tumor heterogeneity and increased therapy resistance acquired in a hypoxic tumor microenvironment remain major obstacles to successful radiotherapy. Others and we have shown that adaptation of cancer cells to cycling severe hypoxia and intermittent reoxygenation stress (chronic-cycling hypoxia) increases cellular antioxidant capacity thereby supporting resistance to chemotherapy and radiotherapy. Here we explored the involvement of antioxidant-associated mitochondrial transport-systems for maintenance of redox-homeostasis in adaptation to chronic-cycling hypoxia and associated radioresistance.Genetic or pharmacological inhibition of the mitochondrial dicarboxylate carrier (SLC25A10) or the oxoglutarate-carrier (SLC25A11) increased the cytotoxic effects of ionizing radiation (IR). But only targeting of SLC25A10 was effective in overcoming chronic-cycling hypoxia-induced enhanced death resistance in vitro and in vivo by disturbing increased antioxidant capacity. Furthermore, in silico analysis revealed that overexpression of SLC25A10 but not SLC25A11 is associated with reduced overall survival in lung- and breast-cancer patients.Our study reveals a role of SLC25A10 in supporting both, redox- and energy-homeostasis, ensuring radioresistance of cancer cells with tolerance to chronic-cycling hypoxia thereby proposing a novel strategy to overcome a mechanism of hypoxia-induced therapy resistance with potential clinical relevance regarding decreased patient survival.

Attenuation of the hypoxia-induced miR-34a protects cardiomyocytes through maintenance of glucose metabolism

Ischemic injury in the heart is associated with death of cardiomyocytes and even after decades of research there is no appropriate therapeutic intervention to treat ischemic injury. The microRNA miR-34a is known to be induced in cardiomyocytes following ischemic injury. Another hallmark of ischemic injury is impaired glycolysis. The objective of the current study was to investigate the effects of short- and long-term exposure to hypoxia on miR-34a expression on apoptosis and regulation of key glycolysis metabolic enzymes. Both repeated short-term (30 min) burst of hypoxia with intermittent reoxygenation (30 min) as well as long-term (4 h) exposure to hypoxia followed by 6 h of reoxygenation robustly induced miR-34a levels. Hypoxia induced changes in cardiac permeability and localization of the channel protein connexin 34 as well as induced apoptosis as evident by levels of cleaved-caspase 3/7 and impaired cell proliferation. Hypoxia was also associated with decreased expression of key glycolytic enzymes hexokinase-1, hexokinase-2, glucose-6-phosphate-isomerase, and pyruvate dehydrogenase kinase 1. Attenuation of hypoxia-induced miR-34a by anti-miR-34a antagomir, but not a control antagomir, decreased miR-34a levels to those observed under normoxia and also inhibited apoptosis, potentially by rescuing expression of the key glycolytic enzymes. Cumulatively, our results establish that therapeutic targeting of miR-34a via antagomir might be a potent therapeutic mechanism to treat ischemic injury in the heart.

Obstructive Sleep Apnea and Cardiovascular Disease - A New Target for Treatment.

Obstructive sleep apnea (OSA) is characterized by recurrent episodes of partial (hypopnea) or complete interruption (apnea) in breathing during sleep due to airway collapse in the oral or pharyngeal region. Prospective studies have established the adverse cardiovascular consequences of OSA, including an increased risk for developing hypertension, coronary artery disease, stroke, and heart failure. However, more studies are needed to better assess the impact of OSA, and possible benefit of treatment with continuous positive airway pressure (CPAP) on cardiovascular mortality. The leading pathophysiological mechanisms involved in the changes triggered by OSA include intermittent hypoxemia and re-oxygenation, arousals and changes in intrathoracic pressure. Hypertension is strongly related with activation of the sympathetic nervous system, stimulation of the renin-angiotensin-aldosterone system and endothelial dysfunction. OSA should be suspected in hypertensive individuals, particularly in patients with resistant hypertension. CPAP treatment reduces blood pressure, and its effects are more pronounced in patients with high baseline blood pressure and elevated treatment compliance. At present, no clear evidence supports CPAP treatment for primary or secondary cardiovascular disease prevention.

Abstract 11556: Association of Sleep Disordered Breathing and ECG R-Wave to Radial Artery Pulse Delay: The MESA Study

Background: Arterial stiffness is a well-recognized predictor of cardiovascular disease (CVD). ECG R-wave to Radial artery pulse delay (RRD) is a novel hemodynamic index in which arterial stiffness is an important component (shorter delay = Higher arterial stiffness) and is obtainable from a single tonometric measurement at the radial artery with simultaneous ECG. Sleep disordered breathing (SDB) has emerged as a risk factor for CVD. The aim of the study was to determine the association of SDB with RRD. Methods: Multi-Ethnic Study of Atherosclerosis participants in 2010-2012 without overt CVD who underwent a sleep study, radial artery tonometry and cardiac MRI were eligible for this cross-sectional analysis (N = 1173, Mean [SD] age: 67.8 ± 8.8, Women: 55.4%). Independent associations between SDB indices including apnea hypopnea index (AHI) and oxygen (O2) desaturation index (ODI: events with more than 4% O2 desaturation), and RRD (transit time in msec) were examined. Model was constructed to adjust for isovolumetric contraction time, another component of RRD, by including measures of contractility and preload (left ventricular ejection fraction (LVEF) and left ventricular end diastolic volume (LVEDV) respectively). Results: Median [IQR] of AHI and ODI were 7.9/hr [2.9- 18.0] and 7.5/hr [3.0- 17.5] respectively. Adjusting for transit path length, demographic factors, BMI and CVD risk factors, both AHI and ODI were inversely associated with RRD (β= -50.3 msec per SD, p = 0.09 and β= -0.60.2 msec per SD, p = 0.04 respectively). In gender stratified analyses given presence of significant interaction, measures of SDB were predictive of RRD only in men. No significant associations were found with key nocturnal hypoxemia indices including mean O2 saturation (SpO2), percent time with SpO2less than 90 % and minimum SpO2. Men, older age, Asian race, high blood pressure, LVEF and LVEDV were also inversely associated with RRD. Conclusion: SDB was associated with shorter RRD implying higher arterial stiffness in men only. These findings suggest the importance of apnea related dynamic change in SpO2 (intermittent hypoxia and reoxygenation) in its potential link to arterial stiffness and also highlights effect modification by gender in the association between the two.

Abstract 2932: HIF-2 alpha is required for the tumor stemness switch of cancer cells during hypoxia/oxidative stress.

Abstract Tumor is characterized by intermittent hypoxia and re-oxygenation that generates oxidative stress. The effect of acute hypoxia microenvironmental stress on the cancer cell behavior is not clearly known. We hypothesized that hypoxia/oxidative stress could re-program cancer cells to a highly aggressive and metastatic state, we termed "tumor stemness state" (1). To test the hypothesis, we have characterized an in vitro model of acute hypoxia/re-oxygenation (2), and identified a highly tumorigenic and metastatic side-population cells (SP) phenotype from a diverse groups of established cancer cell lines including neuroblastoma, rhabdomyosarcoma and small cell lung cancer (3). These post-hypoxia SP cells (henceforth known as SPhox cells) exhibited high metastatic activity, localization in the hypoxic niche, and expressed several stemness associated genes including Nanog, Oct-4 and Bmi-1 (3). In this present study, we report further characterization of the SPhox cells including the expression of stemness associated transcription factors, expression of inflammatory cytokines and receptors, and the putative interaction of SPhox cells with bone marrow (BM) stem cell niche. First, SPhox cells expressed very high level of Nanog, Lin 28, MYC and HIF-2 proteins. Second, SPhox expressed inflammatory Toll-like receptors. Third, SPhox cells increased tolerance to ROS-inducing agents. Fourth, SPhox cells, when co-cultured with mouse BM Sca-1 cells, the CD271+ fraction, enriched in mesenchymal stem cells (4) was expanded. This expanded CD271+/Sca-1+ cells exhibited high expression of Nanog, Lin28, Oct-4, MYC and HIF-2alpha and ABCG2. Furthermore, the conditioned media obtained from the CD271+/Sca-1+/ABCG2+ cells maintained the stemness of the SPhox cells for two weeks. These results indicated the potential interaction between the SPhox and CD271/Sca-1+ cell. However, these characteristics of SPhox cells were transient, reversible, and lasted only for 3-4 weeks, indicating an underlying transient re-programming mechanism behind the SPhox phenotype. Because, recently we discovered a HIF-2alpha dependent transient reprogramming of human ES cells (5), here we investigated the potential role of HIF-2alpha in the re-programming of SP cells to SPhox cells. We found that the specific siRNA gene silencing of HIF-2alpha reduced the SP to SPhox reprogramming by sixteen-fold. Thus, here we have further characterize the tumor stemness state, including the potential role of HIF-2alpha. We suggest that this tumor stemness state could be the part of the cancer cell defense mechanism persist, and reactivate the aggressive/metastatic phenotype of cancer. 1. Das B. PhD thesis, University of Toronto, 2007 2. Das B et al. Cancer Research, 2005 3. Das B et al. Stem Cells, 2008 4. Das B et al. In Press 5. Das B et al. Stem Cells, 2012 Citation Format: Bikul Das, Reza Bayat-Mokhtari, Rika Tsuchida, Herman Yeger. HIF-2 alpha is required for the tumor stemness switch of cancer cells during hypoxia/oxidative stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2013-2932 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Correction: Phosphodiesterase-5 Inhibition Mimics Intermittent Reoxygenation and Improves Cardioprotection in the Hypoxic Myocardium

Correction: Phosphodiesterase-5 Inhibition Mimics Intermittent Reoxygenation and Improves Cardioprotection in the Hypoxic Myocardium

Phosphodiesterase-5 Inhibition Mimics Intermittent Reoxygenation and Improves Cardioprotection in the Hypoxic Myocardium

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. Aim of the study: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O2) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. Conclusions: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.

Ischemic Preconditioning Augments Survival of Stem Cells via miR-210 Expression by Targeting Caspase-8-associated Protein 2

MicroRNAs (miRs) participate in most cellular functions by posttranscriptional regulation of gene expression albeit with little information regarding their role in ischemic preconditioning (IP) of stem cells. We report that IP of bone marrow-derived mesenchymal stem cells (MSCs) with two cycles of 30-min ischemia/reoxygenation (I/R) supported their survival under subsequent longer exposure to anoxia and following engraftment in the infarcted heart. IP significantly reduced apoptosis in MSCs through activation of Akt (Ser(473)) and ERK1/2 (Thr(202)/Tyr(204)) and nuclear translocation of hypoxia-inducible factor-1alpha (HIF-1alpha). We observed concomitant induction of miR-210 in the preconditioned MSCs ((PC)MSCs). Inhibition of HIF-1alpha or of miR-210 abrogated the cytoprotective effects of preconditioning. Extrapolation of these data to in vivo studies in a rat model of acute myocardial infarction predominantly improved stem cell survival after engraftment with a role for miR-210. Notably, multiple I/R cycles more effectively regulated the miR-210 and hence promoted MSC survival compared with single-cycle hypoxia of an equal duration. Real time PCR array for rat apoptotic genes, computational target gene analyses, and luciferase reporter assay identified FLICE-associated huge protein (FLASH)/caspase-8-associated protein-2 (Casp8ap2) in (PC)MSCs as the target gene of miR-210. Induction of FLASH/CASP8AP2 in miR-210 knocked-down (PC)MSCs resulted in increased cell apoptosis. Taken together, these data demonstrated that cytoprotection afforded by IP was regulated by miR-210 induction via FLASH/Casp8ap2 suppression. These results highlighted that IP by multiple short episodes of I/R is a novel strategy to promote stem cell survival.

Development of Resistance of an Organism under Various Conditions of Hypoxic Preconditioning: Role of the Hypoxic Period and Reoxygenation

Single exposure to moderate (10% O(2)) hypobaric, normobaric, and intermittent hypoxia is followed by a preconditioning response of the organism. The mechanisms for immediate adaptation are activated during the hypoxic period. Intermittent reoxygenation not only delays, but even suppresses this process. However, periods of oxygenation during the course of hypoxic training reduce the effect of hypoxia and prevent the possibility for "overdosage" of the adverse stimulus. Hence, they have a regulatory or normalizing role under these conditions. Our results indicate that hypoxitherapy in intermittent hypoxia mode provides optimum conditions for long-term adaptation.

Hypoxia: Unique myocardial morphology?

Objective The objective of this study was to investigate the effects of chronic and intermittent hypoxia on myocardial morphology. Methods Rats randomly divided into 3 groups (n = 14 per group) were exposed to room air (F io 2 = 0.21), chronic hypoxia (F io 2 = 0.10), and intermittent hypoxia (chronic hypoxia with 1 hour per day of room air) for 2 weeks. Weight, blood gas analysis, hematocrit, hemoglobin, red cells, and right and left ventricular pressures were measured. Hearts excised for morphologic examination were randomly divided into 2 groups (9 per group for gross morphologic measurements and 5 per group for histologic and morphometric analysis). The weight ratio of right to left ventricles plus interventricular septum, myocyte diameter, cross-sectional area, and free wall thickness in right and left ventricles were measured. Results Despite the same polycythemia, the right ventricle pressure ( P < .05) and ratio of right to left ventricle pressures ( P < .02) were higher after chronic hypoxia than intermittent hypoxia. The ratio of heart weight to total body weight and the ratio of right to left ventricles plus interventricular septum was higher ( P < .01) in chronic and intermittent hypoxia than in normoxia. Myocyte diameter was not different between the right and left ventricles in normoxia, whereas right ventricle myocytes were larger than left ventricle myocytes in chronic hypoxia ( P < .05) and intermittent hypoxia ( P < .0005). There was marked dilatation of right ventricle size ( P < .001) and marked reduction of left ventricle ( P < .001) size in chronic and intermittent hypoxia compared with normoxia. The total ventricular area (right ventricle plus left ventricle area) remained the same in all groups. The wall thickness ratio in chronic hypoxia and intermittent hypoxia was increased ( P < .001) compared with normoxia in the right ventricle but not in the left ventricle. Conclusions Intermittent reoxygenation episodes do not induce a lesser ventricular hypertrophic response than observed with chronic hypoxia. The functional myocardial preconditioning consequence of intermittent reoxygenation is not supported by structural differences evident with the available techniques.