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Hu, Junjie, Wangxiong Hu, and Yanmei Yang. "Hypoxia Confers Tumor with a Higher Immune Infiltration but Lower Mutation Burden in Gastrointestinal Cancer." Journal of Oncology 2022 (September 12, 2022): 1–9. http://dx.doi.org/10.1155/2022/4965167.
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Background. Hypoxia is one of the driving forces of cancer progression, recurrence, and metastasis. However, the association between the tumor hypoxic tumor microenvironment and the tumor mutation burden (TMB) is poorly understood in gastrointestinal cancer. Methods. Approximately 2,000 samples from colorectal cancer (CRC) and stomach adenocarcinoma (STAD) patients were obtained from the gene expression omnibus database and the cancer genome Atlas databases and were clustered and subtyped by nonnegative matrix factorization. Significant differentially expressed genes that were possibly related to survival differences between the hypoxic and normoxic groups were subjected to multivariate Cox regression. Results. Gastrointestinal cancer patients with CRC and STAD were further divided into two subgroups, namely, the hypoxia group and the normoxia group, and hypoxia was correlated with unfavorable outcomes. Notably, hypoxic tumors had lower TMB but significantly higher levels of immune and stromal infiltration. A signature of HEYL and NRP1 selected by LASSO classified gastrointestinal cancer patients into either a low or high-risk group, allowing for the combination of TMB status with markers of hypoxia in future clinical applications. Conclusions. Hypoxia is an independent prognostic factor and a strong immune infiltration indicator in gastrointestinal tumors of different organs, especially for cancers with low TMB.
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Kim, John S., Ali Azarbarzin, Rui Wang, Ina E. Djonlagic, Naresh M. Punjabi, Phyllis C. Zee, Brian B. Koo, Elsayed Z. Soliman, Magdy Younes, and Susan Redline. "Association of novel measures of sleep disturbances with blood pressure: the Multi-Ethnic Study of Atherosclerosis." Thorax 75, no. 1 (August 22, 2019): 57–63. http://dx.doi.org/10.1136/thoraxjnl-2019-213533.
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BackgroundMechanisms underlying blood pressure (BP) changes in obstructive sleep apnoea (OSA) are incompletely understood. We assessed the associations between BP and selected polysomnography (PSG) traits: sleep depth, airflow limitation measurements and OSA-specific hypoxic burden.MethodsThis cross-sectional analysis included 2055 participants from the Multi-Ethnic Study of Atherosclerosis who underwent PSG and BP measurements in 2010–2013. Sleep depth was assessed using the ‘OR product’, a continuous measure of arousability. Airflow limitation was assessed by duty cycle (Ti/Tt) and % of breaths with flow limitation, and hypoxia by ‘hypoxic burden’. Primary outcomes were medication-adjusted systolic BP (SBP) and diastolic BP (DBP). We used generalised linear models adjusted for age, sex, race/ethnicity, smoking, education, body mass index, alcohol use, periodic limb movements and alternative physiological disturbances.ResultsThe sample had a mean age of 68.4 years and apnoea–hypopnoea index of 14.8 events/hour. Sleep depth was not significantly associated with BP. Every 1 SD increment in log-transformed non-rapid eye movement duty cycle was associated with 0.9% decrease in SBP (95% CI: 0.1% to 1.6%), even after adjusting for sleep depth and hypoxic burden. Every 1 SD increment in log-transformed hypoxic burden was associated with a 1.1% increase in SBP (95% CI: 0.1% to 2.1%) and 1.9% increase in DBP (95% CI: 1.0% to 2.8%) among those not using hypertension medications.ConclusionsHigher duty cycle was associated with lower SBP overall and hypoxic burden with higher SBP and DBP among non-BP medication users. These findings suggest changes in both respiratory effort and oxygenation during sleep influence BP.
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Marallano, Valerie, Anirudh Sattiraju, Hongyan Zou, and Roland Friedel. "TAMI-61. EXAMINING THE ROLE OF HYPOXIA INDUCED GENES CXCR4 AND NXPH4 IN INVASION OF HYPOXIC GLIOBLASTOMA CELLS." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi211. http://dx.doi.org/10.1093/neuonc/noab196.843.
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Abstract Hypoxia (low oxygen) has been associated with adverse effects in tumor biology by exaggerating the capabilities of invasion, proliferation, and survival of tumor cells within the tumor microenvironment. We engineered glioblastoma (GBM) proneural cells with a novel hypoxia reporter, HRE-UnaG, to study areas of tumor hypoxia and the effects that these hypoxic cells have on tumorigenesis. Single cell RNA-seq analysis from a mouse intracranially injected with our HRE dUnaG GBM cells revealed a shift to a mesenchymal state upon hypoxia (detected by expression of UnaG). Two genes, CXCR4 and NXPH4, were identified as being specifically induced in the hypoxic population. Our studies focus on the hypothesis that these two hypoxia induced genes, CXCR4 and NXPH4, are upregulated in hypoxic GBM cells, which may allow tumor cells to become more aggressive and resistant to conventional forms of therapies. GBM cells will be transduced with lentiviral vectors for Dox inducible shRNA knockdown of CXCR4 or NXPH4 to test specific contribution of these genes to the phenotype of the hypoxic population, with particular focus on the change in invasion and overall tumor burden upon gene silencing.
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Khouzam, Raefa Abou, Nagwa Ahmed Zeinelabdin, Mohak Sharda, Husam Nawafleh, Ayda shah Mahmood, Munazza Samar Khan, Goutham Hassan Venkatesh, Salem Chouaib, and Shyama Prasad Rao. "Abstract 5679: Application of a hypoxia metric to investigate tumor mutational burden and cancer cell immunogenicity in vitro." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5679. http://dx.doi.org/10.1158/1538-7445.am2022-5679.
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Abstract Hypoxia afflicts the microenvironment of solid tumors fueling tumor malignancy. Using an 8-gene in vitro hypoxia signature, we have recently shown that pancreatic cancer patients with highly hypoxic tumors experience worse survival and exhibit markers of an immunosuppressed microenvironment. Simultaneously, we obtained evidence of increased mutation count in more hypoxic tumors, which accommodates previous reports associating hypoxia with the regulation of tumor mutational burden (TMB) and genomic instability. These two features could enhance tumor immunogenicity by increasing neoantigen expression, enabling immune recognition and tumor eradication. In this work our aim is to further elucidate the underlying role of hypoxia in generating TMB and genomic instability, and how it contributes to cancer cells immunogenicity. To this end, we applied our recently identified 8-gene hypoxia signature to score the hypoxic response of different cancer cells, considering their median expression levels of the signature genes upon exposure to hypoxia (1% oxygen). The hypoxia score was then applied as a metric to distinguish between the highest and lowest scoring cells, enabling a novel approach for evaluating the intrinsic impact of hypoxia on immunogenicity. After twenty passages under hypoxic conditions (1% oxygen), scored cancer cells were subjected to whole exome sequencing (WES) using the Ion Chef System. Following data processing using appropriate bioinformatic tools, variants were called in the hypoxia-treated cells using their normoxic (21% oxygen) counterparts as controls. We first demonstrated that hypoxia induced an increase in TMB in all cell lines. More interestingly, higher scoring cells were found to display, on average, higher TMB than lower scoring cells, suggesting that cells that have a lower hypoxia score tend to be less mutable. Copy number alterations, mutational signature and neoantigen load predictions generated from the WES data will be discussed. The microarray analysis of transcriptomic data from these hypoxia-treated cells, as well as the relationship between the high TMB, high neoantigen load and increased activation of immune response pathways will also be illustrated. Citation Format: Raefa Abou Khouzam, Nagwa Ahmed Zeinelabdin, Mohak Sharda, Husam Nawafleh, Ayda shah Mahmood, Munazza Samar Khan, Goutham Hassan Venkatesh, Salem Chouaib, Shyama Prasad Rao. Application of a hypoxia metric to investigate tumor mutational burden and cancer cell immunogenicity in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5679.
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Weerackody, Roshan P., David J. Welsh, Roger M. Wadsworth, and Andrew J. Peacock. "Inhibition of p38 MAPK reverses hypoxia-induced pulmonary artery endothelial dysfunction." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 5 (May 2009): H1312—H1320. http://dx.doi.org/10.1152/ajpheart.00977.2008.
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Hypoxia-induced endothelial dysfunction plays a crucial role in the pathogenesis of hypoxic pulmonary hypertension. p38 MAPK expression is increased in the pulmonary artery following hypoxic exposure. Recent evidence suggests that increased p38 MAPK activity is associated with endothelial dysfunction. However, the role of p38 MAPK activation in pulmonary artery endothelial dysfunction is not known. Sprague-Dawley rats were exposed to 2 wk hypobaric hypoxia, which resulted in the development of pulmonary hypertension and vascular remodeling. Endothelium-dependent relaxation of intrapulmonary vessels from hypoxic animals was impaired due to a reduced nitric oxide (NO) generation. This was despite increased endothelial NO synthase immunostaining and protein expression. Hypoxia exposure increased superoxide generation and p38 MAPK expression. The inhibition of p38 MAPK restored endothelium-dependent relaxation, increased bioavailable NO, and reduced superoxide production. In conclusion, the pharmacological inhibition of p38 MAPK was effective in increasing NO generation, reducing superoxide burden, and restoring hypoxia-induced endothelial dysfunction in rats with hypoxia-induced pulmonary hypertension. p38 MAPK may be a novel target for the treatment of pulmonary hypertension.
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Pardo, Andrea C. "Impact of Seizure Burden in Hypoxic Ischemic Encephalopathy." Pediatric Neurology Briefs 29, no. 10 (November 17, 2015): 74. http://dx.doi.org/10.15844/pedneurbriefs-29-10-1.
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Azarbarzin, Ali, Scott A. Sands, Luigi Taranto-Montemurro, Susan Redline, and Andrew Wellman. "Hypoxic burden captures sleep apnoea-specific nocturnal hypoxaemia." European Heart Journal 40, no. 35 (May 9, 2019): 2989–90. http://dx.doi.org/10.1093/eurheartj/ehz274.
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Azab, Abdel Kareem A., Phong Quang, Feda Azab, Brian Thompson, Patricia Maiso, Aldo M. Roccaro, Antonio Sacco, et al. "Dynamic Regulation of the Level of Hypoxia In the Bone Marrow Regulates Cell Dissemination In Multiple Myeloma." Blood 116, no. 21 (November 19, 2010): 4035. http://dx.doi.org/10.1182/blood.v116.21.4035.4035.
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Abstract Abstract 4035 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS: To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS: Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION: Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.
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Wiese, Melanie, Roman G. Gerlach, Isabel Popp, Jasmin Matuszak, Mousumi Mahapatro, Kirstin Castiglione, Dipshikha Chakravortty, et al. "Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages." Infection and Immunity 80, no. 4 (January 17, 2012): 1455–66. http://dx.doi.org/10.1128/iai.05972-11.
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ABSTRACTIn infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (Mϕ) to killEscherichia coliorStaphylococcus aureusin a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient Mϕ revealed that inE. coli- orS. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected Mϕ. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxicE. coli- orS. aureus-infected wild-type Mϕ, respectively. Accordingly, inhibition of the respiratory chain ofS. aureus-infected, normoxic PHOX−/−NOS2−/−Mϕ further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX−/−NOS2−/−Mϕ cultures. Our data demonstrate that the reduced killing ofS. aureusorE. coliduring hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.
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Ramzy, J. A., R. Rengan, M. Mandal, S. Rani, M. E. Vega Sanchez, F. Jaffe, G. D’Alonzo, et al. "0567 Hypoxic Burden and Apnea-Hypopnea Duration in Patients with Positional Obstructive Sleep Apnea." Sleep 43, Supplement_1 (April 2020): A217—A218. http://dx.doi.org/10.1093/sleep/zsaa056.564.
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Abstract Introduction Recently, the measurement of the hypoxic burden and apnea-hypopnea duration has been shown to correlate with mortality in patients with obstructive sleep apnea (OSA). We hypothesized that in patients with mild positional OSA (apnea-hypopnea index [AHI] < 5 events/hr in the non-supine position) the hypoxic burden would be increased and apnea-hypopnea duration shortened and similar to patients with non-positional OSA. Methods Fourteen patients with positional OSA and 24 patients non-positional OSA with similar severity of OSA based on the respiratory event index (REI) were included. All patients had a home sleep apnea test for suspected OSA. The hypoxic burden was calculated by the multiplication of REI and the mean area under the desaturation curves. Results Thirty-eight patients [12 (35%) males, 50±12 yrs, BMI 35±7 kg/m2, Epworth Sleepiness Scale (ESS) 11±8, REI 10±3 events/hr, apnea-hypopnea duration 19±4 sec, mean SaO2 94±2%, lowest SaO2 79±8%, % total sleep time (TST) SaO2 < 90% 11±16%, hypoxic burden 30±17 %min/hr] completed the study. Fourteen patients [9 (64%) males, 46±14 yrs, BMI 31±6 kg/m2, ESS 7±5, REI 9±3 events/hr, mean SaO2 94±2%, lowest SaO2 81±6%, %TST SaO2 < 90% 4±6%] had positional OSA (supine REI 16±7 events/hr, non-supine REI 3±1 events/hr) and 24 patients had non-positional OSA [3 (13%) males, 52±10 yrs, BMI 38±7 kg/m2, ESS 12±9, REI 10±3 events/hr, mean SaO2 94±2%, lowest SaO2 77±9%, %TST SaO2 < 90% 14±19%]. The hypoxic burden was elevated in both the positional and non-positional OSA patients with no difference between the groups (26±19 %min/hr and 32±15 %min/hr, respectively, p=0.13). The apnea-hypopnea duration was similar in positional and non-positional OSA patients (20±3 sec and 18±4 sec, respectively, p=0.08 sec). Conclusion In patients with mild positional OSA the hypoxic burden, which has been associated with cardiovascular mortality, is elevated and similar to patients with non-positional OSA. Support None
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le Feber, Joost, Niels Erkamp, Michel J. A. M. van Putten, and Jeannette Hofmeijer. "Loss and recovery of functional connectivity in cultured cortical networks exposed to hypoxia." Journal of Neurophysiology 118, no. 1 (July 1, 2017): 394–403. http://dx.doi.org/10.1152/jn.00098.2017.
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In the core of a brain infarct, loss of neuronal function is followed by neuronal death within minutes. In an area surrounding the core (penumbra), some perfusion remains. Here, neurons initially remain structurally intact, but massive synaptic failure strongly reduces neural activity. Activity in the penumbra may eventually recover or further deteriorate toward massive cell death. Besides activity recovery, return of brain functioning requires restoration of connectivity. However, low activity has been shown to initiate compensatory mechanisms that affect network connectivity. We investigated the effect of transient hypoxia and compensatory mechanisms on activity and functional connectivity using cultured cortical networks on multielectrode arrays. Networks were exposed to hypoxia of controlled depth (10–90% of normoxia) and duration (6–48 h). First, we determined how hypoxic depth and duration govern activity recovery. Then, we investigated connectivity changes during and after hypoxic incidents, mild enough for activity to recover. Shortly after hypoxia onset, activity and connectivity decreased. Following 4–6 h of ongoing hypoxia, we observed partial recovery. Only if the hypoxic burden was limited did connectivity show further recovery upon return to normoxia. Partial recovery during hypoxia was dominated by restored baseline connections, rather than newly formed ones. Baseline strengths of surviving (persisting or recovered) and lost connections did not differ nor did baseline activity at their “presynaptic” electrodes. However, “postsynaptic” electrodes of surviving connections were significantly more active during baseline than those of lost connections. This implies that recovery during hypoxia reflects an effective mechanism to restore network activity, which does not necessarily conserve prehypoxia connectivity. NEW & NOTEWORTHY Hypoxia reduced the firing rates of cultured neurons. Depending on hypoxic depth and duration, activity recovered during hypoxia and upon return to normoxia. Recovery (partial) during hypoxia was associated with restored baseline connections rather than newly formed ones. Predominantly, baseline connections with most active postsynaptic electrodes recovered, supporting the notion of effective activity homeostasis. This compensatory mechanism remained effective during ~20 h of hypoxia. Beyond 20 h of compensation, loss of activity and connectivity became irreversible.
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Leppänen, Timo, Antti Kulkas, and Juha Töyräs. "The hypoxic burden: also known as the desaturation severity parameter." European Heart Journal 40, no. 35 (May 7, 2019): 2991–93. http://dx.doi.org/10.1093/eurheartj/ehz271.
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Azarbarzin, Ali, Scott A. Sands, Luigi Taranto-Montemurro, Daniel Vena, Tamar Sofer, Sang-Wook Kim, Katie L. Stone, David P. White, Andrew Wellman, and Susan Redline. "The Sleep Apnea-Specific Hypoxic Burden Predicts Incident Heart Failure." Chest 158, no. 2 (August 2020): 739–50. http://dx.doi.org/10.1016/j.chest.2020.03.053.
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Konopleva, Marina, Juliana Benito, Yue-Xi Shi, Sergej Konoplev, Steven M. Kornblau, Olga Frolova, Patrick A. Zweidler-McKay, et al. "Therapeutic Targeting of the Hypoxic Microenvironment in Acute Lymphocytic Leukemia." Blood 114, no. 22 (November 20, 2009): 2040. http://dx.doi.org/10.1182/blood.v114.22.2040.2040.
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Abstract Abstract 2040 Poster Board II-17 The main challenge in the treatment of acute lymphocytic leukemia (ALL) is overcoming resistance to chemotherapy. Recent studies indicate that interactions between leukemia cells and bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to drugs commonly used to treat ALL. We investigated whether hypoxia was a contributing factor in the protective role of the BM microenvironment. We found that the Hypoxia-Inducible-Factor 1a (HIF-1a; a marker normally expressed by only a few hematopoietic cells) was expressed in 68% of BM biopsies from patients with B-lineage ALL (n=53). Expression of HIF-1α detected either histochemically (n=53, p=0.023) or by Reverse Phase Protein Arrays (RPPA, n=116, p=0.0013) inversely correlated with survival of patients with newly diagnosed B-lineage ALL. Silencing of HIF-1α with siRNA, or blockade of mTOR signaling with rapamycin derivatives, reduced expression of the glucose transporter Glut-1, diminished glucose flux, decreased glycolytic rate and ATP production and sensitized leukemic cells to the pro-apoptotic effects of chemotherapeutic agents under hypoxic conditions. In line with this findings, we observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with the ALL cell line Nalm6 or with primary ALL cells, as detected by administration of the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. Altogether, these findings provided a rationale for examining the effects of hypoxia-activated pro-drugs or HIF-1a inhibitors to eliminate ALL progenitor cells within hypoxic niches. To this end, we tested PR-104, a hypoxia-activated dinitrobenzamide mustard currently undergoing Phase II trials in solid tumors. Under hypoxic conditions, this agent is reduced to hydroxylamine and amine metabolites that in turn induce DNA cross-links and cell death (Patterson et al., Clin Can Res 2007). In vitro, 25μM PR-104 induced hypoxia-selective cell death in Nalm6 ALL cells with 80% Annexin V-positivity at 0.1% O2, 46% at 1%O2 compared to 13% at 21%O2. The anti-leukemic efficacy of PR-104 was next examined in the in vivo leukemia models. Administration of PR-104 (250 mg/kg IP TIW for two weeks) prolonged survival of NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML, and decreased leukemia burden as indicated by histopathological analyses of CD45 positive cells in the BM, spleen, lung and liver. Notably, analysis of PIM distribution indicated clearance of the hypoxic leukemic niches. In NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL, PR-104 at 200 mg/kg IP on days 1, 2 and 6 resulted in a dramatic decrease in the percentage of circulating leukemic CD45+ cells on day 15 (control, 92%±6%; treated, 9%±4%; n=7 mice/group). The therapeutic effect of the drug was also tested in a Nalm6-luciferase ALL model where PR-104 administration resulted in decreased tumor burden as determined by luciferase activity and prolonged survival of the PR-104 treated as compared to control mice (p=0.006). Similar to the models of human leukemia, analysis of BM sections of control mice showed extensive areas of hypoxia (PIM-positive) in close proximity to GFP-positive leukemia cells in contrast to the treated mice in which only discrete areas of PIM positivity were detectable. Altogether, these findings strongly suggest that targeting hypoxia is feasible and may increase the sensitivity of ALL cells to chemotherapy. If successful, this approach of targeting hypoxic microenvironment, alone or in combination with other chemotherapeutic or targeted agents, may significantly impact ALL therapy and ultimately improve patient survival. Figure 1. Co-localization of hypoxic PIM(+) areas with GFP positive HALMG tumor areas in bone marrow of control but not of PR-104 treated mice. Figure 1. Co-localization of hypoxic PIM(+) areas with GFP positive HALMG tumor areas in bone marrow of control but not of PR-104 treated mice. Disclosures: No relevant conflicts of interest to declare.
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Azab, Abdel Kareem, Jinsong Hu, Phong Quang, Feda Azab, Costas Pitsillides, Brian Thompson, Patricia Maiso, et al. "Hypoxia Promotes Dissemination of Multiple Myeloma Through Acquisition of Endothelial to Mesenchymal Transition (EMT) Features." Blood 118, no. 21 (November 18, 2011): 471. http://dx.doi.org/10.1182/blood.v118.21.471.471.
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Abstract Abstract 471 Multiple myeloma (MM) is characterized by widespread dissemination of the MM cells at diagnosis associated with multiple focal bone lesions, implying (re)circulation of MM cells into the peripheral blood and (re)entrance or homing into new sites of the BM. However, the driving force for MM cells to leave the BM, egress, and home to new BM niches is still not well understood. Hypoxia (low oxygen) in solid tumors was shown to promote metastasis in solid tumors through activation of proteins involved in the endothelial to mesenchymal Transition (EMT). In this study, we hypothesized that MM tumor progression induces hypoxic conditions, which in turn activates EMT related proteins and promotes metastasis of MM cells. To test this hypothesis, we examined levels of hypoxia in MM cells at different stages of tumor progression in vivo in two animal models: the first by injecting MM1s cell to SCID mice, and the second by injecting 5T33MM cells to C57BL/KaLwRijHsd mice. Hypoxic markers were examined using flow cytometry and immunohistochemistry. We found that tumor progression induced hypoxia in both the MM cells and the tumor microenvironment. Similarly, hypoxia induced genes (HIF1a, HIF1b, HIF2b, CREBBP, HYOU1, VEGF1, HIF1a-inhibitory protein) were increased in MM patients (n=68) compared to plasma cells from healthy donors (n=14). Using flow cytometry we found that the number of circulating MM cells increased with the progression; however, the correlation was observed in late stages of the progression but not in the early stages. A better direct correlation was achieved with the hypoxic state of the MM cells in the BM. Circulating MM cells were more hypoxic that MM cells in the BM (especially at low tumor burden). Moreover, we found that the level of hypoxia in MM cells in the PB did not correlate with the hypoxia in the BM. Next, we tested the mechanism in which hypoxia induces cell egress. We found that MM cells isolated from MM patients have higher gene expression of EMT inducing proteins (E-cadherin, SNAIL, FOXC2, TGFb1) in parallel to a decrease of expression in E-cadherin, and we confirmed the downregulation of E-cadherin expression in correlation with the increase of hypoxia in MM cell and cells in the BM microenvironment in vivo. Culturing MM cells under hypoxic conditions increased the expression of HIF1a and HIF2a. In parallel, hypoxia induced acquisition of EMT related features including downregulation of E-cadherin, upregulation of SNAIL, and inhibition of GSK3b. In addition, hypoxia decreased the adhesion of MM cells to stromal cells. To complete the metastatic process after egress, MM cells need to home to new sites in the BM. Therefore we investigated the effect of hypoxia on expression of CXCR4, chemotaxis and homing of MM cells to the BM. Using flow cytometry we found a direct correlation between hypoxia and the expression of CXCR4 in MM cells in vivo using the SCID-MM1s model. These results were confirmed in vitro, where hypoxia increased the expression of CXCR4 at protein and mRNA levels in MM cells. Moreover, the expression of CXCR4 in MM cells isolated from the PB was higher than cells isolated from the BM especially at low tumor burden, correlating with higher hypoxic state of the circulating tumor cells. Functionally, hypoxia increased the chemotaxis of MM cells towards SDF1a in vitro and, using in vivo confocal microscopy, it was shown to accelerate the homing of MM cells to the BM in vivo. To demonstrate that the chemotaxis and homing were CXCR4 dependent, we treated the hypoxic MM cells with AMD3100 (a CXCR4 inhibitor) and showed that it inhibited chemotaxis in vitro and homing of MM to the BM in vivo. In conclusion, we demonstrate that tumor progression induces hypoxia in the MM cells and in the BM microenvironment. Hypoxia activates EMT-related machinery in MM cells, decreases expression of E-cadherin and consequently decreased the adhesion of MM cells to the BM, and enhance egress of MM cells to the circulation. In parallel, hypoxia increases the expression of CXCR4, and consequently increased the migration and homing of MM cells in from the peripheral blood to the BM. Further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy are warranted. Disclosures: Roccaro: Roche: . Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Slavi, Nefeli, Abduqodir H. Toychiev, Stylianos Kosmidis, Jessica Ackert, Stewart A. Bloomfield, Heike Wulff, Suresh Viswanathan, Paul D. Lampe, and Miduturu Srinivas. "Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy." Proceedings of the National Academy of Sciences 115, no. 26 (June 11, 2018): E5934—E5943. http://dx.doi.org/10.1073/pnas.1803907115.
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Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.
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Park, Hun-young, Sang-seok Nam, Hirofumi Tanaka, and Dong-jun Lee. "Hemodynamic, Hematological, and Hormonal Responses to Submaximal Exercise in Normobaric Hypoxia in Pubescent Girls." Pediatric Exercise Science 28, no. 3 (August 2016): 417–22. http://dx.doi.org/10.1123/pes.2015-0176.
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Purpose:The aim of this study was to investigate hemodynamic, hematological, and immunological responses to prolonged submaximal cycle ergometer exercise at a simulated altitude of 3000 m in pubescent girls.Methods:Ten girls, 12.8 ± 1.0 years old, exercised on a cycle ergometer for 60 min at a work rate corresponding to 50% maximal oxygen consumption measured at sea level, under two environmental conditions; sea level (normoxia) and a simulated 3000 m altitude (normobaric hypoxia).Results:There were no significant differences in tidal volume, ventilation, oxygen consumption, cardiac output, stroke volume, and heart rate between the two exercise conditions. However, reticulocyte, adrenocorticotropic hormone, and cortisol concentrations increased significantly from pre- to postexercise in the hypoxic environment. Leukocyte and T-cell count increased and B-cell count decreased after exercise under both conditions. There were no significant changes in natural killer cell count.Conclusion:Our simulated hypoxic environment provided a mild environmental stressor that did not impose a heavy burden on the cardiovascular, hematological, or immunological functions during submaximal exercise in pubescent girls.
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Kharoshankaya, Liudmila, Nathan J. Stevenson, Vicki Livingstone, Deirdre M. Murray, Brendan P. Murphy, Caroline E. Ahearne, and Geraldine B. Boylan. "Seizure burden and neurodevelopmental outcome in neonates with hypoxic–ischemic encephalopathy." Developmental Medicine & Child Neurology 58, no. 12 (September 6, 2016): 1242–48. http://dx.doi.org/10.1111/dmcn.13215.
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Azarbarzin, Ali, Scott A. Sands, David P. White, Susan Redline, and Andrew Wellman. "The hypoxic burden: a novel sleep apnoea severity metric and a predictor of cardiovascular mortality—Reply to ‘The hypoxic burden: also known as the desaturation severity parameter’." European Heart Journal 40, no. 35 (May 7, 2019): 2994–95. http://dx.doi.org/10.1093/eurheartj/ehz273.
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Eunson, Paul. "The long-term health, social, and financial burden of hypoxic-ischaemic encephalopathy." Developmental Medicine & Child Neurology 57 (March 19, 2015): 48–50. http://dx.doi.org/10.1111/dmcn.12727.
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Boo, NY, and Cheah IG. "The burden of hypoxic-ischaemic encephalopathy in Malaysian neonatal intensive care units." Singapore Medical Journal 57, no. 8 (August 2016): 456–63. http://dx.doi.org/10.11622/smedj.2016137.
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Lynch, Niamh E., Nathan J. Stevenson, Vicki Livingstone, Brendan P. Murphy, Janet M. Rennie, and Geraldine B. Boylan. "The temporal evolution of electrographic seizure burden in neonatal hypoxic ischemic encephalopathy." Epilepsia 53, no. 3 (February 6, 2012): 549–57. http://dx.doi.org/10.1111/j.1528-1167.2011.03401.x.
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Kuwahira, Ichiro, Uguri Kamiya, Tokuzen Iwamoto, Yoshihiro Moue, Tetsuya Urano, Yasuyo Ohta, and Norberto C. Gonzalez. "Splenic contraction-induced reversible increase in hemoglobin concentration in intermittent hypoxia." Journal of Applied Physiology 86, no. 1 (January 1, 1999): 181–87. http://dx.doi.org/10.1152/jappl.1999.86.1.181.
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The effect of intermittent hypoxia (IHx) on blood hemoglobin concentration ([Hb]) and the underlying mechanisms were studied in rats exposed to 10% O2, 1 h/day, for up to 5 wk. IHx protocols with longer daily hypoxic exposure show persistent polycythemia; however, it is unknown whether [Hb] increases transiently during hypoxia in protocols without polycythemia. Hypoxia produced a reversible [Hb] increase after 4 days of IHx but not in normoxic controls (NxC) or after shorter period of IHx. Splenectomy abolished the phenomenon. Plasma epinephrine and norepinephrine levels during hypoxia were comparable in IHx and NxC groups, but the epinephrine-induced [Hb] increase was larger in IHx. The α1- and α2-adrenoreceptor blockade (phentolamine) and α2-blockade (yohimbine) abolished the [Hb] increase of IHx rats. Conversely, α2-receptor stimulation (oxymetazoline) increased [Hb] during normoxia in IHx but not in NxC. In conclusion, this IHx protocol results in reversible [Hb] increases during hypoxia via splenic contraction mediated by increased α2-adrenoreceptor response. This may protect O2supply during hypoxia without the cardiovascular burden of polycythemia during normoxia.
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Azlia, Demara, Yedhi, Laily Annisa Kusumastuti, and Daffa Abhista Wicaksana. "GLYCOLYTIC 2-DEOXY-D-GLUCOSE PERIOCULAR INHIBITOR SEBAGAI TERAPI ADJUVANT PADA RETINOBLASTOMA." Al-Iqra Medical Journal : Jurnal Berkala Ilmiah Kedokteran 1, no. 2 (August 30, 2018): 48–54. http://dx.doi.org/10.26618/aimj.v1i2.2750.
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Retinoblastoma is the most common intraocular tumor in children. Retinoblastoma tumors consist of areas with high angiogenic activity and other areas with low oxygen pressure conditions. Tumor cells that survive in this hypoxic region have been shown to be resistant to chemotherapy and radiation, namely standard retinoblastoma therapy which selectively targets rapidly dividing cell populations, so that therapy is needed that can reach up to the hypoxic cells. 2-deoxy-D-glucose (2-DG) has been shown to be effective in reducing hypoxic areas in tumors, reducing tumor buds, and inhibiting angiogenesis. to discuss the glycolytic 2-DG inhibitors as adjuvant therapy in retinoblastoma. The authors searched for medical articles in the PubMed and ResearchGate literature databases with the keywords treatment, 2-deoxy-D-glucose, 2-DG, retinoblastoma, and cancer. The words are combined with OR or AND using Boolean logic. Based on the selection according to the inclusion and exclusion criteria, 4 articles were used as the main reference for writing this review literature. 2-DG will disregulate (inhibit) the process of angiogenesis through upregulation of Estrogen Receptor 1 Nuclear (ESR 1), ubiquitin, jun activation domain binding protein 1 (JAB1), G-protein alpha S, and CAPER in the apical part of the tumor and B3GNT1 upregulation , CEPT1, PABPC1, myotrophin, HAX1 in the postero-lateral part of the tumor. H3, DTX1, RPL12, and bone morphogenic (BMP) histones are also upregulated so that hypoxia can be inhibited. With this mechanism, the tumor burden can be suppressed. Glycolytic 2-DG inhibitors with anti-angiogenesis and anti-hypoxic abilities can be developed as adjuvant therapy for advanced retinoblastoma patients.
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Benito, Juliana, Yuexi Shi, Barbara Szymanska, Hernan Carol, Ingrid Bohem, Hongbo Lu, Sergej Konoplev, et al. "Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104." Blood 116, no. 21 (November 19, 2010): 868. http://dx.doi.org/10.1182/blood.v116.21.868.868.
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Abstract Abstract 868 Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups. Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. Disclosures: Wilson: PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.
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Sekhon, Mypinder S., Peter Gooderham, David K. Menon, Penelope M. A. Brasher, Denise Foster, Danilo Cardim, Marek Czosnyka, et al. "The Burden of Brain Hypoxia and Optimal Mean Arterial Pressure in Patients With Hypoxic Ischemic Brain Injury After Cardiac Arrest*." Critical Care Medicine 47, no. 7 (July 2019): 960–69. http://dx.doi.org/10.1097/ccm.0000000000003745.
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Dykstra, Kaitlyn M., Hannah R. S. Fay, Ashish C. Massey, Neng Yang, Matthew Johnson, Scott Portwood, Monica L. Guzman, and Eunice S. Wang. "Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis." Blood Advances 5, no. 8 (April 20, 2021): 2087–100. http://dx.doi.org/10.1182/bloodadvances.2020002666.
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Abstract Leukemia stem cells (LSCs) and therapy-resistant acute myeloid leukemia (AML) blasts contribute to the reinitiation of leukemia after remission, necessitating therapeutic interventions that target these populations. Autophagy is a prosurvival process that allows for cells to adapt to a variety of stressors. Blocking autophagy pharmacologically by using mechanistically distinct inhibitors induced apoptosis and prevented colony formation in primary human AML cells. The most effective inhibitor, bafilomycin A1 (Baf A1), also prevented the in vivo maintenance of AML LSCs in NSG mice. To understand why Baf A1 exerted the most dramatic effects on LSC survival, we evaluated mitochondrial function. Baf A1 reduced mitochondrial respiration and stabilized PTEN-induced kinase-1 (PINK-1), which initiates autophagy of mitochondria (mitophagy). Interestingly, with the autophagy inhibitor chloroquine, levels of enhanced cell death and reduced mitochondrial respiration phenocopied the effects of Baf A1 only when cultured in hypoxic conditions that mimic the marrow microenvironment (1% O2). This indicates that increased efficacy of autophagy inhibitors in inducing AML cell death can be achieved by concurrently inducing mitochondrial damage and mitophagy (pharmacologically or by hypoxic induction) and blocking mitochondrial degradation. In addition, prolonged exposure of AML cells to hypoxia induced autophagic flux and reduced chemosensitivity to cytarabine (Ara-C), which was reversed by autophagy inhibition. The combination of Ara-C with Baf A1 also decreased tumor burden in vivo. These findings demonstrate that autophagy is critical for mitochondrial homeostasis and survival of AML cells in hypoxia and support the development of autophagy inhibitors as novel therapeutic agents for AML.
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Coy, Kevin M., Andrii Maryniak, Thomas Blankespoor, and Adam Stys. "Approach to high intracoronary thrombus burden in the era of COVID-19." BMJ Case Reports 14, no. 12 (December 2021): e246223. http://dx.doi.org/10.1136/bcr-2021-246223.
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Since the start of the COVID-19 pandemic, several cases have reported extensive multivessel coronary thrombosis as a cardiovascular manifestation of SARS-CoV-2 infection. This case describes a patient who developed non-ST elevation myocardial infarction during hospitalization for acute hypoxic respiratory failure due to COVID-19. We review the immediate and delayed revascularisation strategies of culprit and non-culprit lesions in the setting of high intracoronary thrombus burden induced by SARS-CoV-2. Successful percutaneous intervention and stenting of a culprit lesion and resolution of an intracoronary thrombus using a delayed strategy of lesion passivation with adjuvant pharmacotherapy are demonstrated on index and follow-up angiography.
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Portwood, Scott, Deepika Lal, Yung-Chun Hsu, Rodrigo Vargas, Meir Wetzler, and Eunice S. Wang. "Activity of the Hypoxia-Activated Prodrug, TH-302, in Human Acute Myeloid Leukemia Models." Blood 120, no. 21 (November 16, 2012): 3611. http://dx.doi.org/10.1182/blood.v120.21.3611.3611.
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Abstract Abstract 3611 Recent evidence has demonstrated the bone marrow (BM) microenvironment, the principal site of acute myeloid leukemia (AML) initiation and expansion, is characterized by intrinsically low oxygen tension. Theoretically, such microenvironmental changes may lead to the selective outgrowth of AML clones which are “better adapted” to survive within a severely hypoxic microenvironment and/or may confer resistance to chemotherapeutic agents, similar to solid tumor cells. We report here that human AML cells (HL60, ML-2) cultured under chronic hypoxic conditions mimicking the marrow microenvironment (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic cells, as determined by flow cytometric analysis, western blot analysis, and cell viability assays. Similar results were noted in primary AML samples treated with cytarabine under normoxic and hypoxic conditions in colony formation assays (n=3 samples, p=0.01). In order to improve upon chemotherapy outcomes, we investigated the effects of TH-302, a hypoxia-activated bromo-isophosphoramidate mustard prodrug, which is currently undergoing clinical trial evaluation in multiple tumor types. Treatment of AML cell lines (HL60, HEL) and primary AML samples with TH-302 (at doses ranging from 0.1– 5 mM, p values ranging from <0.05–0.0001) resulted in dose- and hypoxic-dependent inhibition of AML proliferation and apoptosis. In vivo TH-302 treatment significantly decreased disease burden, as measured by total animal bioluminescence, and prolonged overall survival in two systemic human AML xenograft models (HEL-luciferase, HL60-luciferase) (Figure 1). Immunohistochemical studies demonstrated that TH-302 treatment reduced numbers of hypoxic (pimonidazole-positive) cells within the leukemic marrow microenvironment. Because prior data in animal models has shown that AML progression within the marrow is associated with expansion of hypoxic BM areas, we examined the effects of TH-302 treatment on systemic AML growth when initiated early (prior to AML inoculation) or late (several days following AML engraftment) in the disease process. TH-302 was equally effective at both time points. Although anti-vascular therapy has been shown to enhance tumor hypoxia in other cancer types, we noted no synergistic or additive in vivo effects when TH-302 therapy was combined with sorafenib, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in our models. TH-302 therapy administered for two weeks in non-leukemic and leukemia-engrafted mice was not associated with hematologic toxicities. In summary, our results demonstrate the anti-leukemic activity of TH-302 in preclinical AML models and suggest that the efficacy of this and other drugs for AML therapy may be uniquely affected by the BM microenvironment. Further clinical development of TH-302 and other hypoxia-targeted drugs for AML therapy are warranted. Based on our data, higher TH-302 doses and/or chronic drug administration may be needed for optimal in vivo anti-leukemic activity. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Disclosures: No relevant conflicts of interest to declare.
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Hedlund, McDonald, Nemirovsky, Awrey, Jensen, and Dedhar. "Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts." Cancers 11, no. 7 (July 17, 2019): 1002. http://dx.doi.org/10.3390/cancers11071002.
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Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.
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Zheng, Zhuangzhuang, Chenbin Bian, Huanhuan Wang, Jing Su, Lingbin Meng, Ying Xin, and Xin Jiang. "Prediction of immunotherapy efficacy and immunomodulatory role of hypoxia in colorectal cancer." Therapeutic Advances in Medical Oncology 14 (January 2022): 175883592211383. http://dx.doi.org/10.1177/17588359221138383.
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Immunotherapy has been used in the clinical treatment of colorectal cancer (CRC); however, most patients fail to achieve satisfactory survival benefits. Biomarkers with high specificity and sensitivity are being increasingly developed to predict the efficacy of CRC immunotherapy. In addition to DNA alteration markers, such as microsatellite instability/mismatch repair and tumor mutational burden, immune cell infiltration and immune checkpoints (ICs), epigenetic changes and no-coding RNA, and gut microbiomes all show potential predictive ability. Recently, the hypoxic tumor microenvironment (TME) has been identified as a key factor mediating CRC immune evasion and resistance to treatment. Hypoxia-inducible factor-1α is the central transcription factor in the hypoxia response that drives the expression of a vast number of survival genes by binding to the hypoxia response element in cancer and immune cells in the TME. Hypoxia regulates angiogenesis, immune cell infiltration and activation, expression of ICs, and secretion of various immune molecules in the TME and is closely associated with the immunotherapeutic efficacy of CRC. Currently, various agents targeting hypoxia have been found to improve the TME and enhance the efficacy of immunotherapy. We reviewed current markers commonly used in CRC to predict therapeutic efficacy and the mechanisms underlying hypoxia-induced angiogenesis and tumor immune evasion. Exploring the mechanisms by which hypoxia affects the TME will assist the discovery of new immunotherapeutic predictive biomarkers and development of more effective combinations of agents targeting hypoxia and immunotherapy.
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McLeod, Laurie L., and Abdu I. Alayash. "Detection of a ferrylhemoglobin intermediate in an endothelial cell model after hypoxia-reoxygenation." American Journal of Physiology-Heart and Circulatory Physiology 277, no. 1 (July 1, 1999): H92—H99. http://dx.doi.org/10.1152/ajpheart.1999.277.1.h92.
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A cell culture model of bovine aortic endothelial cells attached to microcarrier beads was used to study the interaction of diaspirin cross-linked hemoglobin (an oxygen-carrying blood substitute) with hypoxia-reoxygenation. Hemoglobin (200 μM) and hypoxia-volume restriction (3–5 h), together and separately, caused toxicity in this model, as measured by decreased cellular replating efficiency. Hemoglobin (60 μM) caused a reduction in hydrogen peroxide concentration and an increase in lipid peroxidation above that induced by hypoxia alone. Incubation of hemoglobin with endothelial cells caused transient oxidation of hemoglobin to its highly reactive and toxic ferryl species after ≥3 h of hypoxia, followed by 1 h of reoxygenation. Lipid peroxidation, which may occur in the presence of ferrylhemoglobin, also occurred after 1 h of reoxygenation. Hemoglobin caused a dose-dependent decrease in intracellular glutathione concentration, suggesting that it caused an oxidative stress to the cells. However, addition of ascorbate, α-tocopherol, or trolox did not decrease hemoglobin oxidation in the presence of normal or hypoxic cells. It is concluded that diaspirin cross-linked hemoglobin forms a ferryl intermediate in the absence of any exogenously added oxidant and contributes to the oxidative burden experienced by endothelial cells after hypoxia-reoxygenation, a condition that is likely to be encountered during trauma and surgery when hemoglobin solutions are used as perfusion agents.
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Hawkins-Daarud, Andrea, Russell Rockne, David Corwin, Alexander R. A. Anderson, Paul Kinahan, and Kristin R. Swanson. "In silico analysis suggests differential response to bevacizumab and radiation combination therapy in newly diagnosed glioblastoma." Journal of The Royal Society Interface 12, no. 109 (August 2015): 20150388. http://dx.doi.org/10.1098/rsif.2015.0388.
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Recently, two phase III studies of bevacizumab, an anti-angiogenic, for newly diagnosed glioblastoma (GBM) patients were released. While they were unable to statistically significantly demonstrate that bevacizumab in combination with other therapies increases the overall survival of GBM patients, there remains a question of potential benefits for subpopulations of patients. We use a mathematical model of GBM growth to investigate differential benefits of combining surgical resection, radiation and bevacizumab across observed tumour growth kinetics. The differential hypoxic burden after gross total resection (GTR) was assessed along with the change in radiation cell kill from bevacizumab-induced tissue re-normalization when starting therapy for tumours at different diagnostic sizes. Depending on the tumour size at the time of treatment, our model predicted that GTR would remove a variable portion of the hypoxic burden ranging from 11% to 99.99%. Further, our model predicted that the combination of bevacizumab with radiation resulted in an additional cell kill ranging from 2.6×10 7 to 1.1×10 10 cells. By considering the outcomes given individual tumour kinetics, our results indicate that the subpopulation of patients who would receive the greatest benefit from bevacizumab and radiation combination therapy are those with large, aggressive tumours and who are not eligible for GTR.
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Cavazos, Antonio, Helen Ma, Juliana Maria Benito, Mark J. Levis, Naval Daver, Amir Ashoorzadeh, Robert Anderson, Adam Patterson, Jeff Smaill, and Marina Konopleva. "Pre-Clinical Activity of Novel Hypoxia-Activated FLT3 Inhibitors in FLT3-Mutated AML." Blood 128, no. 22 (December 2, 2016): 5210. http://dx.doi.org/10.1182/blood.v128.22.5210.5210.
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Abstract Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are most frequently observed molecular abnormalities in AML, which lead to constitutive activation of the tyrosine kinase. While several mechanisms of resistance to FLT3 tyrosine kinase inhibitors (TKIs) have been identified, commonly failure is associated with lack of complete and sustained inhibition of FLT3 tyrosine kinase activity. FLT3 inhibition is particularly challenging in blasts within the hypoxic bone marrow (BM) microenvironment, which plays a crucial role in AML survival. Widespread hypoxia has been shown by us and others to be prevalent within the confines of the leukemic BM space, and to limit the efficacy of standard chemotherapy (Benito et al., PLoS One 6(8):e23108, 2011). With the goal of exploiting hypoxia as a physiological target and increasing selectivity to FLT3-mutant AML blasts, we have developed a series of hypoxia-activated prodrugs of FLT3 TKIs (HA-TKIs) based on the known clinical compounds sunitinib, AC220, MLN-518 and crenolanib. Bioreductive trigger conjugation was performed at the final step of the synthesis to provide the prodrugs as previously described (Lu et al., Tetrahedron, 69, p9130-9138, 2013). Under hypoxia, these compounds can undergo enzymatic one-electron reduction and then fragment selectively to release the cell permeable TKI, generating higher local concentrations of TKIs and limiting normal tissue exposures. Pulse radiolysis was used to determine the electron affinity (E(1)) and rate of fragmentation of each prodrug. The E(1) values ranged from -430 mV to -497 mV, while the rate of fragmentation of the prodrugs following one-electron reduction under hypoxia ranged from 28 s-1 to 400 s-1. SN37156 and SN37169 in particular demonstrated E(1) and kfrag parameters ideal for hypoxia-selective cellular metabolism (-449 mV, 85 s-1; -449 mV, 62 s-1, respectively). With the purpose of identifying the HA-TKIs with the most selective and dose-potent in vitro efficacy, we first performed an Oxic/Hypoxic screening of the hypoxia-dependent activity of HA-TKIs against FLT3-mutant AML cell lines. From the 10 compounds originally developed, SN37156 and SN37169 showed hypoxia-dependent apoptosis and growth arrest. SN37156 demonstrated the best activity against Molm13, Molm14 and MV4-11 cells under hypoxia with hypoxic cytotoxicity ratios (oxic/hypoxic IC50 ratios) of 4.9, 11 and 17.6, respectively. In our secondary screening we compared the activity of SN37156, SN37169 and their parental inhibitors on target modulation. Both HA-TKIs selectively inhibited FLT3 and ERK auto phosphorylation in MV4-11 cell line under hypoxic but not oxic conditions, with similar potency to the parental inhibitor under normal oxygen culture conditions. We next investigated the effects of both HA-TKIs on inhibition of FLT3 downstream targets, including AKT, ERK, pS6 and STAT5 using time-of-flight mass cytometry (CyTOF). The data demonstrated distinct patterns of signaling inhibition, with a dose-dependent p-ERK and p-S6 inhibition in MV4-11 and Molm-14 cells upon treatment with both HA-TKIs. In turn, both compounds at 100nM inhibited p-AKT and p-STAT5. Single dose tolerability studies were conducted in tumor-free C57BL6 mice. Prodrugs were administered by IP injection in 20% β-hydroxypropylcyclodextrin solution starting at 17.8 µmol/kg and escalating in 1.33-fold increments until lethality or severe mortality (mean body weight loss of >10%) was observed. The maximal tolerated dose for both, SN37156 and SN37169 was established at 56.1 µmol/kg. To test efficacy of HA-TKIs in vivo, we injected C57BL6 mice with genetically engineered Baf3-ITD/luc/GFP cells. Mice were randomized to treatment groups on day 8 when engraftment was documented by bioluminescent imaging (BLI), and treated with either vehicle or 56.1 µmol/kg of SN37156 or SN37169 I.P. every 3 days. Multiple doses (up to 10 so far; study is ongoing) were well tolerated. BLI demonstrated reduced leukemia burden in both treatment groups compared to control. In summary, our data indicate that HA-TKIs SN37156 and SN37169 inhibit mutant FLT3 and its selected downstream signaling targets under conditions resembling marrow hypoxia, and induce hypoxia-dependent growth arrest and apoptosis in FLT3-mutant AML cells. Planned studies in FLT3-mutant AML PDX will evaluate in vivo efficacy to eliminate primary human AML cells from hypoxic BM microenvironment. Disclosures Daver: Kiromic: Research Funding; Ariad: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding. Konopleva:Cellectis: Research Funding; Calithera: Research Funding.
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Lowe, E., S. Mathieson, N. Stevenson, D. Murray, J. Rennie, and G. Boylan. "The effect of moderate hypothermia on the electrographic seizure burden in neonatal hypoxic ischaemic encephalopathy." Archives of Disease in Childhood - Fetal and Neonatal Edition 96, Supplement 1 (June 1, 2011): Fa27. http://dx.doi.org/10.1136/archdischild.2011.300164.38.
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Lüscher, Thomas F. "Forgotten cardiovascular risk factors: pregnancy complications and preterm birth, bullying, periodontal disease, and hypoxic burden." European Heart Journal 40, no. 14 (April 7, 2019): 1093–96. http://dx.doi.org/10.1093/eurheartj/ehz171.
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Xue, Xiang, Bryce X. Bredell, Erik R. Anderson, Angelical Martin, Christopher Mays, Hiroko Nagao-Kitamoto, Sha Huang, et al. "Quantitative proteomics identifies STEAP4 as a critical regulator of mitochondrial dysfunction linking inflammation and colon cancer." Proceedings of the National Academy of Sciences 114, no. 45 (October 23, 2017): E9608—E9617. http://dx.doi.org/10.1073/pnas.1712946114.
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Inflammatory bowel disease (IBD) is a chronic inflammatory disorder and is a major risk factor for colorectal cancer (CRC). Hypoxia is a feature of IBD and modulates cellular and mitochondrial metabolism. However, the role of hypoxic metabolism in IBD is unclear. Because mitochondrial dysfunction is an early hallmark of hypoxia and inflammation, an unbiased proteomics approach was used to assess the mitochondria in a mouse model of colitis. Through this analysis, we identified a ferrireductase: six-transmembrane epithelial antigen of prostate 4 (STEAP4) was highly induced in mouse models of colitis and in IBD patients. STEAP4 was regulated in a hypoxia-dependent manner that led to a dysregulation in mitochondrial iron balance, enhanced reactive oxygen species production, and increased susceptibility to mouse models of colitis. Mitochondrial iron chelation therapy improved colitis and demonstrated an essential role of mitochondrial iron dysregulation in the pathogenesis of IBD. To address if mitochondrial iron dysregulation is a key mechanism by which inflammation impacts colon tumorigenesis, STEAP4 expression, function, and mitochondrial iron chelation were assessed in a colitis-associated colon cancer model (CAC). STEAP4 was increased in human CRC and predicted poor prognosis. STEAP4 and mitochondrial iron increased tumor number and burden in a CAC model. These studies demonstrate the importance of mitochondrial iron homeostasis in IBD and CRC.
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Kawarai, Yoshimasa, Hirokazu Tanaka, Tatsuya Kobayashi, and Makio Shozu. "Progesterone as a Postnatal Prophylactic Agent for Encephalopathy Caused by Prenatal Hypoxic Ischemic Insult." Endocrinology 159, no. 6 (April 10, 2018): 2264–74. http://dx.doi.org/10.1210/en.2018-00148.
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Abstract Brain damage caused by hypoxic ischemic insult during the perinatal period causes hypoxic ischemic encephalopathies (HIEs). Therapeutic hypothermia is indicated for HIE, but because the therapeutic burden is large for its limited therapeutic effectiveness, another strategy is needed. Progesterone (P4) plays a neuroprotective role through the actions of its metabolite, allopregnanolone (Allo), on P4 receptor, γ-aminobutyric acid type A receptors or both. We examined the therapeutic potential of P4 using a newborn rat model of HIE. Fetal rats were exposed to transient ischemic hypoxia by 30-minute bilateral uterine artery clamping on gestational day 18. After spontaneous birth, newborn pups were subcutaneously injected with P4 (0.10 or 0.01 mg), medroxyprogesterone acetate (MPA; 0.12 mg), or Allo (0.10 mg) through postnatal days (PDs) 1 to 9. Brain damage in the rats was assessed using the rotarod test at PD50. The HIE insult reduced the rats’ ability in the rotarod task, which was completely reversed by P4 and Allo, but not by MPA. Histological examination revealed that the HIE insult decreased neuronal (the cortex and the hippocampal CA1 region) and oligodendroglial cell density (the corpus callosum) through PD0 to PD50. The axon fiber density and myelin sheath thickness in the corpus callosum were also reduced at PD50. The time-course study revealed that P4 restored oligodendroglial cells by PD5, which was followed by neuroprotective action of P4 that lasted long over the injection period. These results suggest that P4 protects the neonatal brain from HIE insult via restoration of oligodendroglial cells.
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Koehn, Liam M., Xiaodi Chen, Aric F. Logsdon, Yow-Pin Lim, and Barbara S. Stonestreet. "Novel Neuroprotective Agents to Treat Neonatal Hypoxic-Ischemic Encephalopathy: Inter-Alpha Inhibitor Proteins." International Journal of Molecular Sciences 21, no. 23 (December 2, 2020): 9193. http://dx.doi.org/10.3390/ijms21239193.
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Perinatal hypoxia-ischemia (HI) is a major cause of brain injury and mortality in neonates. Hypoxic-ischemic encephalopathy (HIE) predisposes infants to long-term cognitive deficits that influence their quality of life and place a large burden on society. The only approved treatment to protect the brain after HI is therapeutic hypothermia, which has limited effectiveness, a narrow therapeutic time window, and is not considered safe for treatment of premature infants. Alternative or adjunctive therapies are needed to improve outcomes of full-term and premature infants after exposure to HI. Inter-alpha inhibitor proteins (IAIPs) are immunomodulatory molecules that are proposed to limit the progression of neonatal inflammatory conditions, such as sepsis. Inflammation exacerbates neonatal HIE and suggests that IAIPs could attenuate HI-related brain injury and improve cognitive outcomes associated with HIE. Recent studies have shown that intraperitoneal treatment with IAIPs can decrease neuronal and non-neuronal cell death, attenuate glial responses and leukocyte invasion, and provide long-term behavioral benefits in neonatal rat models of HI-related brain injury. The present review summarizes these findings and outlines the remaining experimental analyses necessary to determine the clinical applicability of this promising neuroprotective treatment for neonatal HI-related brain injury.
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Oudaert, Inge, Hatice Satilmis, Philip Vlummens, Anke Maes, Elke De Bruyne, Karin Vanderkerken, Sarah-Maria Fendt, Kim De Veirman, and Eline Menu. "Pyrroline-5-Carboxylate Reductase 1: A Novel Target for Sensitizing Myeloma to Cytotoxic Agents By Inhibition of PRAS40-Mediated Protein Synthesis." Blood 138, Supplement 1 (November 5, 2021): 1574. http://dx.doi.org/10.1182/blood-2021-149156.
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Abstract Introduction Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both cancer cell survival and drug resistance. In this study, we aimed to identify which metabolic changes and downstream pathways are involved in myeloma cell growth and persistence. Methods Correlation of pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) with overall survival was investigated in the gene-expression data of MM patients (MMRF CoMMpass trial). To perform a tracer study, RPMI-8226 cells were supplemented with 13C-glutamine for 48h in both normoxia and hypoxia (<1% O 2, by chamber). For further in vitro investigation, 2 human MM cell lines (OPM-2 and RPMI-8226) were used. Proline concentrations in cell lysates were measured by ELISA-based proline assay kit. We used siRNA to establish a knockdown of PYCR1 and/or PYCR2. Levels of apoptosis were measured using AnnexinV and 7-AAD positivity on flow cytometry. Differential protein expression was evaluated with western blot. Proliferation was measured by assessing BrdU incorporation through flow cytometry. Pargyline was used as a PYCR1 inhibitor. All in vitro experiments were performed in hypoxic conditions. For the in vivo murine experiment, C57BL/KalwRij mice were inoculated with 1 million of eGFP+ 5TGM1 cells, and treated with vehicle, bortezomib (0.6 mpk, 2x/week, starting day 14), pargyline (100 mpk, 5x/week, starting day 1) or combination of both. Tumor burden was measured by flow cytometry when vehicle mice reached end-stage. Results Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are 2 mitochondrial enzymes that facilitate the last step in the enzymatic conversion of glutamine to proline. High expression of both enzymes correlated with a lower overall survival in the CoMMpass trial. Moreover, MM cells from relapse/refractory patients expressed significant higher levels of PYCR1. We performed a tracer study with RPMI-8226 cells, revealing an increased conversion of 13C-glutamine to proline in hypoxia compared to normoxia. We confirmed these results by increased proline production after 48h of hypoxic culture. SiRNA-mediated knockdown of PYCR1 or both PYCR1/2 combined with bortezomib increased apoptotic cell death in OPM-2 and RPMI-8226, which we confirmed by detecting upregulation of cleaved PARP and cleaved CASPASE 3 levels. In contrast, PYCR2 knockdown combined with bortezomib did not significantly alter apoptosis. Further investigation revealed that PYCR1 knockdown reduced proliferation, and led to a decrease in p-AKT, p-p42/44 MAPK and c-MYC levels. Mechanistically, we found that PYCR1 silencing affected protein synthesis, as shown by a downregulation of p-PRAS40, p-MTOR, p-p70, p-S6, p-4EBP1 and p-EIF4e levels. Next, we evaluated whether the clinically relevant anti-hypertensive agent and PYCR1 inhibitor, pargyline, was capable of inducing myeloma cell death. In vitro, pargyline reduced proline production, MM viability and increased apoptotic cell death. Pargyline was also capable of reducing viability in CD138+ cells of primary patient samples . Finally, in vivo combination of pargyline with bortezomib significantly reduced tumor burden in the 5TGM1 model. On protein level, we also observed a significant decrease in p-4EBP1 and p-EIF4e in the freshly isolated 5TGM1 cells for the combination therapy. Conclusion Hypoxia increased glutamine-to-proline conversion in myeloma cells by stimulating PYCR activity. Knockdown of PYCR1 and PYCR1/2 increased bortezomib efficacy and inhibited proliferation. Mechanistically, PYCR1 interference reduced PRAS40-mediated protein synthesis. Pargyline, a PYCR1 inhibitor, also reduced MM viability and increased apoptosis. In vivo, pargyline combined with bortezomib significantly reduced tumor burden in the 5TGM1 model compared to both single agents. In conclusion, this study identifies PYCR1 as a novel target in MM therapy. Disclosures De Veirman: Active Biotech AB: Research Funding. OffLabel Disclosure: Pargyline is a antihypertensive agent and irreversible MAO B inhibitor that also inhibits PYCR1. Pargyline is not approved by the FDA as a PYCR1 inhibitor.
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Bolzoni, Marina, Paola Storti, Gaetano Donofrio, Irma Airoldi, Daniela Guasco, Denise Toscani, Marco Peronaci, et al. "Hypoxia-Inducible Factor (HIF)-1a Is A Therapeutic Target in Myeloma-Induced Angiogenesis and Bone Destruction in Vivo." Blood 120, no. 21 (November 16, 2012): 2947. http://dx.doi.org/10.1182/blood.v120.21.2947.2947.
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Abstract Abstract 2947 It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that hypoxia inducible factor (HIF)-1α is overexpressed by MM cells. However, the potential role of HIF-1α as a therapeutic target in MM is not known and is currently under investigation. In this study we explored the effect of persistent HIF-1α inhibition by expression of a lentivirus shRNA pool in MM cells on proliferation, survival and transcriptional and pro-angiogenic profiles of MM cells either in vitro or in vivo in mouse models. A HIF-1α Lentivirus shRNA pool was used for HIF-1α stable knock-down in human myeloma cell lines (HMCL)s and the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs were infected and then selected with puromycin. Selected clones were screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. The transcriptional profiles were evaluated in the HMCL JJN3 cells transduced with shRNA forHIF-1α (JJN3-anti-HIF-1α) and on those infected with the control vector pKLO.1 (JJN3-pKLO.1) by U133 Plus2.0 Arrays (Affymetrix®) either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR and by ELISA assays for protein levels. Finally the effect of HIF-1α inhibition in MM cells was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models. Together with tumor volume and weight, microvascular density was evaluated by CD34 immunostaining. Cortical bone thickness was determined by microQcT in the intratibial mouse model. Among the genes significantly modulated by HIF-1α inhibition (327 and 361 genes in hypoxic and normoxic condition, respectively), we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated by HIF-1α inhibition. Interestingly some pro-osteoclastogenic cytokines were also inhibited including IL-7 and CCL3/MIP-1α. In the in vivo mouse models, we found that mice, injected either subcutaneously or intratibially with JJN3-anti-HIF-1α, showed a dramatic reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-pKLO.1. A significant reduction in the number of vessels X field and VEGF immunostaining were observed in both mouse models. Moreover in the intratibial experiments HIF-1α inhibition significantly blocked MM-induced bone destruction. Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenesis and reduces the MM tumor burden and bone destruction in vivo suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.
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Trzepizur, Wojciech, Margaux Blanchard, Timothée Ganem, Frédéric Balusson, Mathieu Feuilloy, Jean-Marc Girault, Nicole Meslier, et al. "Sleep Apnea–Specific Hypoxic Burden, Symptom Subtypes, and Risk of Cardiovascular Events and All-Cause Mortality." American Journal of Respiratory and Critical Care Medicine 205, no. 1 (January 1, 2022): 108–17. http://dx.doi.org/10.1164/rccm.202105-1274oc.
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Guidotti, Isotta, Licia Lugli, Maria Pina Guerra, Luca Ori, Claudio Gallo, Francesca Cavalleri, Andrea Ranzi, Rossella Frassoldati, Alberto Berardi, and Fabrizio Ferrari. "Hypothermia reduces seizure burden and improves neurological outcome in severe hypoxic-ischemic encephalopathy: an observational study." Developmental Medicine & Child Neurology 58, no. 12 (July 22, 2016): 1235–41. http://dx.doi.org/10.1111/dmcn.13195.
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Murray, D. M., G. B. Boylan, A. P. Fitzgerald, C. A. Ryan, B. P. Murphy, and S. Connolly. "Persistent lactic acidosis in neonatal hypoxic-ischaemic encephalopathy correlates with EEG grade and electrographic seizure burden." Archives of Disease in Childhood - Fetal and Neonatal Edition 93, no. 3 (May 1, 2008): F183—F186. http://dx.doi.org/10.1136/adc.2006.100800.
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Fay, Hannah R. S., Kaitlyn M. Dykstra, Matthew Johnson, Tara L. Cronin, Linda Lutgen-Dunckley, Brandon L. Martens, Joseph R. Moberg, Monica L. Guzman, and Eunice S. Wang. "Mitophagy Plays a Key Role in the Anti-Leukemic Activity of Autophagy Inhibitors Under Hypoxia in Acute Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 1278. http://dx.doi.org/10.1182/blood-2019-127024.
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Background: Leukemia stem cells (LSCs) and acute myeloid leukemia (AML) blasts persisting in the bone marrow (BM) after chemotherapy are key drivers of AML relapse and chemotherapy refractoriness. The hypoxic microenvironment of the BM is known to protect these cells, however the mechanisms behind this chemoresistance are not well understood. We have previously shown that AML cell lines cultured under hypoxia upregulate autophagy. Blocking autophagy with lysosome-based autophagy inhibitor Bafilomycin A1 (Baf A1) was shown to target LSCs in colony formation (CF) assays and in vivo serial transplants in NSG mice. Another autophagy inhibitor chloroquine (CQ) also demonstrated anti-LSC effects in CF assays. The clinical development of Baf A1 and CQ has been limited due to poor pharmacokinetics and toxicity. Lys05 is a promising CQ derivative with increased potency and therapeutic potential. Recent studies have shown that LSCs have an increased reliance on oxidative phosphorylation (OXPHOS) as well as mitophagy, the autophagic degradation of damaged mitochondria. Hypoxia has also been demonstrated to increase mitophagy in certain cell types through upregulation of BNIP3. We hypothesized that AML cells cultured under hypoxia would have an increased reliance on mitophagy for mitochondrial homeostasis and survival. Treatment with autophagy inhibitors would be expected to block mitophagy and induce AML cell death specifically under hypoxia. Methods: Human AML cells (MOLM13) were cultured under hypoxia (1% O2) or normoxia (21% O2). NSG mice were inoculated with luciferase expressing MOLM13-BLIV & treated with vehicle (DMSO) or Lys05 (40 mg/kg) IP for 18 days. Tumor burden was assessed by bioluminescence. CF assays were established with AML patient cells in the presence of vehicle or Lys05 in MethoCult under normoxia/hypoxia and counted on day 13. Annexin V-FITC/PI flow cytometry was used to measure apoptosis. OXPHOS was assessed with a Seahorse XFe96 using the Mitochondrial Stress Test. Mitochondrial mass was measured by flow cytometry using MITO-ID Green. BNIP3 & PINK1 protein expression was visualized via western blot. Results: Treatment with Lys05 decreased in vivo tumor burden significantly in NSG mice systemically engrafted with human AML cells. We also saw a marked decrease in the number of CF-units in primary AML patient samples treated with Lys05 which was further enhanced under hypoxia. Treatment of AML cell lines with CQ and Lys05 also enhanced apoptosis under hypoxia as compared to normoxia. Baf A1, however, showed equal amounts of apoptosis in normoxia and hypoxia (Fig. 1A). While Baf A1, CQ, and Lys05 all inhibit autophagy through deacidification of the lysosome, Baf A1 has been shown to induce additional effects on mitochondria, inducing uncoupling of OXPHOS and depolarization. We therefore examined the autophagy inhibitors' effect on mitochondrial function. At 24 hours, Baf A1 caused a significant decrease in basal and maximal respiration under both hypoxia and normoxia. CQ and Lys05, however, only showed a significant decrease in OXPHOS under hypoxia, with no effect on mitochondrial function under normoxia. We postulated that this effect arose from increased mitophagy under hypoxia. BNIP3 expression levels were enhanced under hypoxia in MOLM13 cells at 24- 48 hours. If mitophagy is constitutively occurring, blocking this process would cause an increase in the number of mitochondria. As expected, the number of mitochondria increased when treated with Lys05 or CQ under hypoxia, but not under normoxia, suggesting that mitophagy is occurring only under hypoxic conditions. Baf A1 caused an increase in mitochondria under both hypoxia and normoxia, suggesting that Baf A1 can both induce mitophagy and block mitochondrial degradation. We further confirmed this was due to mitophagy by assessing expression of the mitophagy protein PINK1. In normoxia, Baf A1 showed an almost 2-fold increase in PINK1 compared to the vehicle whereas CQ did not (Fig. 1B). Conclusion: We have identified a class of autophagy inhibitors that displays enhanced efficacy in AML cells under hypoxic conditions that reflect the BM microenvironment. This is due in part to their ability to target AML cellular reliance on mitophagy. These results provide the rationale for the further clinical development of Lys05 or other lysosome-based autophagy inhibitors as a novel means of targeting minimal residual disease in AML therapy. Disclosures Guzman: Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; SeqRx: Consultancy; Cellectis: Research Funding. Wang:Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role.
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Mazzotti, D. R., T. Leppänen, S. Sands, J. Töyräs, A. Wellman, A. Kulkas, S. Redline, T. Karhu, and A. Azarbarzin. "0593 Hypoxemia During Sleep Disordered Breathing and Cardiovascular Disease: A Comparison of Different Oxygen Desaturation Measures." Sleep 43, Supplement_1 (April 2020): A227. http://dx.doi.org/10.1093/sleep/zsaa056.590.
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Abstract Introduction The apnea-hypopnea index has been used to characterize obstructive sleep apnea (OSA) severity. However, this metric is limited in providing information about cardiovascular disease (CVD) risk. Recent studies proposed alternative metrics that capture frequency, duration, depth, and combinations of duration and depth of hypoxemia. This study provides a systematic evaluation of the association between conventional or novel nocturnal hypoxemia metrics and the incidence of CVD and CV mortality in the Sleep Heart Health Study (SHHS). Methods We used data from 5,042 participants of the SHHS. Over 10.7 years, there were 1,312 (26.0%) incident CVD events and 359 (7.1%) CV deaths. We calculated standardized (z-scored) values of eight nocturnal hypoxemia indices, including conventional (e.g., oxygen desaturation index) and novel metrics (e.g., hypoxic burden, respiratory event-related area under desaturation curve and desaturation severity, corresponding to alternative quantitative measurements looking at the shape of each desaturation event). The association between each metric and incidence of CVD or CV mortality was evaluated using Cox proportional hazards models. Age, sex, body mass index, race, ethnicity, smoking, total sleep time, number of respiratory events, and prevalent CVD at baseline were used as covariates. Hazard ratios (HR) are presented as the effect of one standard deviation increase in each correponding metric. Results In unadjusted models, all nocturnal hypoxemia indices were associated with increased incidence of CVD and CV mortality. In adjusted models, longer average desaturation duration was associated with lower CVD incidence (HR[95%CI]=0.93[0.86-0.99];p=0.034), higher hypoxic burden with increased CV mortality (HR[95%CI]=1.22[1.04-1.43];p=0.017), and higher % sleep time with oxygen saturation less than 90% (Tlt90%) with increased CV mortality (HR[95%CI]=1.12[1.00-1.26];p=0.040). Conclusion Different metrics of nocturnal hypoxemia derived from polysomnography were associated with CV risk in the SHHS. However, after covariate adjustment, only shorter average desaturation duration, and higher hypoxic burden and Tlt90% were independent CV risk factors. Support AASM Foundation (194-SR-18,188-SR-17); American Heart Association (19CDA34660137); NIH (U01HL53940,U01HL53941,U01HL63463,U01HL53937, U01HL53938,U01HL53916,U01HL53934,U01HL63429,U01HL53931,HL114473, P01HL094307,HL134015,R35HL135818,1R21HL145492-01,R01HL102321,R01HL128658); The State Research Funding (KUH: 5041767, 5041768; TUH: VTR3242, VTR3228, EVO2089), Academy of Finland (313697, 323536), Business Finland (5133/31/2018), Respiratory Foundation of Kuopio Region, Tampere Tuberculosis Foundation, Research Foundation of Pulmonary Diseases, Foundation of Finnish Anti-Tuberculosis Association.
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Häkli, Martta, Joose Kreutzer, Antti-Juhana Mäki, Hannu Välimäki, Reeja Maria Cherian, Pasi Kallio, Katriina Aalto-Setälä, and Mari Pekkanen-Mattila. "Electrophysiological Changes of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes during Acute Hypoxia and Reoxygenation." Stem Cells International 2022 (December 19, 2022): 1–15. http://dx.doi.org/10.1155/2022/9438281.
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Ischemic heart disease is the most common cardiovascular disease and a major burden for healthcare worldwide. However, its pathophysiology is still not fully understood, and human-based models for disease mechanisms and treatments are needed. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to model acute ischemia-reperfusion in our novel cell culture assembly. The assembly enables exchange of oxygen partial pressure for the cells within minutes, mimicking acute ischemic event. In this study, hypoxia was induced using 0% O2 gas for three hours and reoxygenation with 19% O2 gas for 24 hours in serum- and glucose-free medium. According to electrophysiological recordings, hypoxia decreased the hiPSC-CM-beating frequency and field potential (FP) amplitude. Furthermore, FP depolarization time and propagation slowed down. Most of the electrophysiological changes reverted during reoxygenation. However, immunocytochemical staining of the hypoxic and reoxygenation samples showed that morphological changes and changes in the sarcomere structure did not revert during reoxygenation but further deteriorated. qPCR results showed no significant differences in apoptosis or stress-related genes or in the expression of glycolytic genes. In conclusion, the hiPSC-CMs reproduced many characteristic changes of adult CMs during ischemia and reperfusion, indicating their usefulness as a human-based model of acute cardiac ischemia-reperfusion.
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Yoshida, Tatsuro, Angelo D'Alessandro, Xiaoyun Fu, Travis Nemkov, Davide Stefanoni, Michael Dioguardi, Neeta Rugg, et al. "Differential Metabolomics and Lipidomics of Hypoxic/Hypocapnic Long-Term Stored Red Cells Associate with Improved 24-Hour Recovery after Transfusion." Blood 134, Supplement_1 (November 13, 2019): 4994. http://dx.doi.org/10.1182/blood-2019-125961.
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Storage lesion accumulates during refrigerated storage of RBC, and development of novel methods and/or additive solutions are needed for more efficacious RBC with reduced burden of harmful byproducts. Autologous 24-hr post-transfusion recovery (PTR24) at the end of storage measured in healthy volunteers is the only in vivo practical metrics to evaluate consequences of RBC storage. PTR24 studies are logistically and economically demanding and as such, the identification of easier biomarkers of stored blood quality can expedite the development and testing of novel storage strategies. We performed a prospective, randomized, dual‐arm, crossover, two‐center radio labeled 24-hr PTR24 study was conducted as a part of the pivotal study to evaluate Hemanext® hypoxic/hypocapnic processing system for meeting standard FDA acceptability criteria for RBC storage. Subjects donated CP2D whole blood (500 ± 50 mL). The Test arm consisted of whole blood‐derived RBC, leukoreduced, and processed to achieve hypoxic/hypocapnic state in AS-3 additive within 12 hours of phlebotomy then stored for 42 days at 1‐6°C under hypoxic conditions (Test). Unprocessed normal atmosphere stored units (Control) were stored within 8 hours under conventional storage condition. PTR24 data was obtained from 19 test subjects and 21 control subjects (14 paired) by infusion of 51‐Cr/99‐Tc(m) radiolabeled RBCs*. At day 0, 21 and 42, in vitro metrics, including conventional quality parameters (%hemolysis, ATP, 2,3-DPG, morphology, microparticles and deformability), as well as a large panel of metabolites and lipids were analyzed for correlations with PTR24. Bioactive lipids were quantified by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM). Pearson's correlation coefficients for PTR24 and analytes were examined; correlation pattern differed significantly between normal air Control and hypoxic Test RBCs and thus analyzed separately. Among the conventional RBC quality metrics, ATP as well as ability to rejuvenate ATP and 2,3-DPG at end of storage correlated with PTR24. RBC deformability correlated only with Control, while reduced oxidative damage in Test removed its dependency. Test showed stronger correlations with ATP and other metabolic parameters. 302 metabolites and lipids were analyzed by qualitative metabolomics workflow, and significant correlations were observed with |R| ≥ 0.5 for 37 compounds in normal air stored RBCs (15 with p ≤ 0.05), and 41 compounds in hypoxic RBCs (19 with p ≤ 0.05). The top 5 correlating and inversely correlating compounds for each condition are reported along with p-values in Table 1. For RBC stored in normal air, strong correlations were primarily seen with membrane lipids and associated fatty acids, amino acids, and the Pentose Phosphate Pathway intermediates. Hypoxic storage PTR24 values primarily correlated with alternative membrane components, amino acids, and Pentose Phosphate Pathway intermediates (Table 2). Among 67 bio-reactive lipids analyzed quantitatively, negative correlations of oxylipins (9- or 13-HODEs, 5-, 8-, 11-, 12-HETEs) to PTR24 were observed under normal conditions, but not under hypoxic condition (Table 3). In summary, RBC deformability correlated only with PTR24 of normal air stored RBC, while reduced oxidative damage in hypoxic/hypocapnic conditions removed this dependency. Although normal air and hypoxic stored RBCs show differing metabolite correlates with PTR24, significant associations tend to occur within similar overall pathways. Comparing the PTR24-metabolite correlations between normal air and hypoxic/hypocapnic RBC storage reveals that intermediates of oxidative stress management and membrane homeostasis are potential predictors of post-transfusion recovery. Detailed analysis of lipids supports that hypoxic/hypocapnic storage may reduce membrane lipid degradation and generates the hypothesis that the metabolism of bioactive lipids under hypoxia/hypocapnia may be different from the one found in RBC stored under normal air storage conditions. * Radio-labeling for 14 subjects occurred without prior RDRC approval; one site did not meet GMP standards. Disclosures Yoshida: New Health Sciences Inc.: Employment, Equity Ownership. D'Alessandro:Hemanext, Inc.: Membership on an entity's Board of Directors or advisory committees; Omix Technologies, Inc.: Other: Founder. Nemkov:Omix Technologies, Inc.: Equity Ownership, Other: Founder. Dioguardi:Hemanext: Employment. Iselin:Hemanext: Employment. Dunham:Hemanext: Employment. Cancelas:Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding; Velico: Consultancy, Research Funding.
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Herbath, Melinda, Jeffrey S. Harding, Sarah Marcus, George Hasko, Andras Nagy, Zsuzsanna Fabry, and Matyas Sandor. "Regulators of mycobacterial granuloma formation – CCL2 and VEGF-A." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 42.7. http://dx.doi.org/10.4049/jimmunol.200.supp.42.7.
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Abstract Granulomas are macrophage dominated lesions and are a central feature of mycobacterial infections. These cell aggregates are dynamically changing structures as the life span of the granuloma recruited effector cells is relatively short and the cells have to be replaced. CCL2 and VEGF-A are required for granuloma maintenance as blocking the action of these chemokines results in reduction of granuloma size and number after BCG or Mtb infections. We seek to elucidate the relevance of these two cytokines in the timeline of mycobacterial infections and their relative contribution to granuloma formation. CD11c+ cell immigration is impaired, costimulatory molecule expression is lower and granulomas are smaller while the bacterial burden is higher in the liver when CCR2KO mice are infected i.p. with M. bovis BCG. Similarly, in animals that are selectively deficient in macrophage VEGF-A production the granulomas are smaller. Granuloma cells produce VEGF-A. Caseating granulomas, like the ones induced in C3HeB/FeJ (Kramnik) mice are hypoxic and hypoxia is a strong inducer of VEGF-A production. Sarcoid lesions induced by BCG or Mtb in B6 mice are not hypoxic but we show that ATP released from dead cells induces VEGF-A in a subpopulation of macrophages in the granulomas. Additionally, VEGF production proved to be dependent on granuloma size: bigger granulomas produce larger quantities of VEGF-A while smaller lesions produce less or none. This finding suggests that unlike other chemokines such as CCL2, which is described to be involved in both granuloma initiation and maintenance, VEGF-A may support an increased lesion size in the late acute phase of infection. Regulating cellular recruitment may provide new therapies for granulomatous diseases.
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Muz, Barbara, Feda Azab, Pilar De La Puente, Justin King, Micah John Luderer, Ravi Vij, and Abdel Kareem Azab. "Novel Method to Detect Minimal Residual Disease in Multiple Myeloma Predicts Recurrence Better Than CD138-Based Models." Blood 126, no. 23 (December 3, 2015): 2985. http://dx.doi.org/10.1182/blood.v126.23.2985.2985.
Full textAbstract:
Abstract Introduction: Diagnosis, responses to treatment, minimal residual disease (MRD) and circulating tumor cells (CTC) in multiple myeloma (MM) are all detected by the gold-standard marker CD138 flow cytometry-based method. However, the presence of clonogenic CD138-negative MM cells and hypoxia-driven CD138 downregulation were shown previously. In this study, we found that CD138 is significantly downregulated in MRD and CTC populations in MM, thus we developed a novel two-color flow cytometry-based set of biomarkers (independent of CD138) to detect MRD and CTC in MM. Methods: We created a MRD mouse model by treating MM-bearing mice with a high dose of bortezomib. We then tested the effect of hypoxia and drug treatment on the expression of different markers, including CD138. Therefore, to detect MM cells we utilized CD38-APC antibody, followed by the exclusion of non-MM CD38-expressing cells such as T cells, monocytes, NK cells, B cells and dendritic cells using (CD3, CD14, CD16, CD19 and CD123)-V450 antibodies, respectively. To verify the ability of the new method to selectively detect MM cells, mononuclear cells from peripheral blood (PB) from healthy patients and MM cell lines (hypoxic and normoxic) were stained by the antibody cocktail and analyzed by flow cytometry. Next, we compared the sensitivity of the traditional CD138 method to the new method in detecting (a) normoxic or hypoxic MM cells spiked into bone marrow (BM) cells in vitro, (b) normoxic or hypoxic MM cells spiked into PB in vitro, (c) MM1s-GFP+ cells in mice with different tumor burden in the BM in vivo, (d) circulating MM1s-GFP+ in the PB in vivo, (e) MRD cells in the BM samples from 16 patients with complete remission (CR) or very good partial response (VGPR), and (F) CTC in 12 progressive MM patients. We further aimed to predict time to progression (TTP) in 16 patients with complete remission based on the detection of MRD in these patients using the new method and compared with flow cytometry-based CD138 or histology. Results: In vivo, we found that bortezomib-treated MRD cells were hypoxic, compared to a progressive vehicle-treated cells. CD138 expression in these cells was significantly decreased, but CD38 expression was unchanged. In vitro expression of CD138 was decreased due to hypoxia and bortezomib treatment, whereas CD38 expression was unchanged. Furthermore, we developed a new method using an antibody cocktail, where the MM cell population is defined as CD38+/CD3-/CD14-/CD16-/CD19-/CD123- (APC+ and V450-). In vitro, the new method detected 100% of hypoxic and normoxic MM cells, and less than 0.5% of mononuclear cells from the PB or BM of healthy donors. In contrast, CD138+ cells failed to detect 50% of hypoxic MM cells and 10-25% of normoxic cells. In vivo, the amount of cells detected by the new method directly correlated with the number of MM1s-GFP+ cells detected in the BM with a range between 0-60%, with a correlation coefficient (slope) of 0.99 and R2 of 0.999. The CD138 detected only a fraction of the MM1s-GFP+ population (<10%) even in mice with a high BM tumor burden. In addition, the new method detected close to a 100% of the circulating MM1-GFP+ cells in vivo, while the CD138 marker detected less than 1% of the circulating MM1s-GFP+ cells. In patients, the new method detected 0.5-8% MRD cells in the BM of 16 patients with CR or VGPR (which were defined as CD138-negative), and 0.1-1.8% CTC in the PB of 12 progressive MM patients. In contrast, CD138 marker detected less than 0.5% of MRD cells in the BM of the CR and VGPR patients, and less than 0.1% of CTCs in the PB of the progressive patients. Furthermore, we found that, while CD138 and histology failed to predict recurrence in CR patients, the new method successfully detected CD138-negative MM population whose prevalence in the BM inversely correlated with TTP in MM patients defined as CR based on CD138 and histology. Conclusions: We confirmed that CD138 expression is variable on MM cells, and that it is downregulated in MRD and CTC populations, and that it was not effective in detecting these particular populations in MM. Furthermore, we developed a novel two-color flow cytometry-based biomarker-set to detect MM cells independent of CD138. The new methods detected close to a 100% of all MM cells in vitro and in vivo, including MRD and CTC. Moreover, the new method detected a CD138-negative MRD and CTC in MM patients, and the prevalence of this population inversely correlated with TTP in MM patients. Disclosures Vij: Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.