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1
Locke, Frederick L., Justin Chou, Saran Vardhanabhuti, Regis Perbost, Peter Dreger, Brian T. Hill, Catherine Lee, et al. "Association of pretreatment (preTx) tumor characteristics and clinical outcomes following second-line (2L) axicabtagene ciloleucel (axi-cel) versus standard of care (SOC) in patients (pts) with relapsed/refractory (R/R) large B-cell lymphoma (LBCL)." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 7565. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.7565.
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7565 Background: The Phase 3 randomized ZUMA-7 trial in 2L R/R LBCL showed axi-cel superiority to SOC (salvage chemotherapy and HDT-ASCT) in event-free survival (EFS; hazard ratio [HR], 0.398; P<.0001; Locke et al. N Eng J Med. 2021). We report results of exploratory analyses of tumor characteristics, including preTx tumor burden (TB), tissue hypoxia-related lactate dehydrogenase (LDH) level, and tumor microenvironment (TME). Methods: TB was calculated as the sum of product diameters of ≤6 reference lesions (Locke et al. Blood Adv. 2020). Serum LDH was assessed. PreTx tumor samples were assessed for RNA expression by the NanoString IO 360™ panel and prespecified immune contexture indexes related to T-cell involvement (Immunosign 15 [IS15] and 21 [IS21]; Galon et al. ASCO 2020. #3022). ZUMA-1 data were used for comparison to 3L R/R LBCL. CD19 protein expression was assessed by immunohistochemistry (H-score). Associations between biomarkers and clinical outcomes were assessed using descriptive statistics ( P<.05 was significant). Results: Axi-cel EFS was superior to SOC for both high and low TB (HR, 0.29 and 0.49, respectively; P<.001 for both) and elevated and nonelevated LDH (HR, 0.32 and 0.5, respectively; P<.001 for both). EFS in axi-cel pts was not associated with preTx TB (HR, 1.01 [95% CI, 0.88-1.16]; P=.89) or LDH (HR, 0.98 [95% CI, 0.74-1.29]; P=.86), but was worse in SOC pts with higher preTx TB (HR, 1.17 [95% CI, 1.03-1.32]; P=.01) or higher LDH (HR, 1.29 [95% CI, 1.02-1.63], P =.03). PreTx TB was lower in SOC pts with ongoing response vs nonresponders and pts who relapsed ( P=.07), but not in axi-cel pts ( P=.99). Non-germinal center B-cell (GCB) cell-of-origin subtypes is a poor prognostic factor for EFS in SOC (EFS was significantly worse in SOC pts with non-GCB vs GCB; HR, 1.82 [95% CI, 1.12-2.96]; P=.02) but not in axi-cel. In IO360 analysis, gene expression of B-cell lineage antigens (CD19, CD20, BCMA) and markers highly expressed by tumor cells (CD45RA, IRF8, BTLA) positively associated with objective and durable responses to axi-cel. While axi-cel remained superior to SOC with high ( > median) or low CD19 expression level, the probability of an ongoing response increased with a higher CD19 H-score. PreTx TME IS15 and IS21 scores were generally higher in 2L vs 3L. Conclusions: Axi-cel was superior to SOC in all subgroup analyses, including higher TB and LDH. Durable responses with axi-cel were greatest in tumors with prominent B-cell features, but were superior to SOC regardless of these features. Axi-cel intervention in 2L is supported by durable response rates not impacted by high TB, as seen in 3L axi-cel or 2L SOC. Higher preTx immune involvement in 2L vs 3L tumors suggests high TB may be overcome with axi-cel in patients with a more favorable immune contexture. Clinical trial information: NCT03391466.
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Locke, Frederick L., Justin Chou, Saran Vardhanabhuti, Regis Perbost, Peter Dreger, Brian T. Hill, Catherine Lee, et al. "Association of pretreatment (preTx) tumor characteristics and clinical outcomes following second-line (2L) axicabtagene ciloleucel (axi-cel) versus standard of care (SOC) in patients (pts) with relapsed/refractory (R/R) large B-cell lymphoma (LBCL)." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 7565. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.7565.
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7565 Background: The Phase 3 randomized ZUMA-7 trial in 2L R/R LBCL showed axi-cel superiority to SOC (salvage chemotherapy and HDT-ASCT) in event-free survival (EFS; hazard ratio [HR], 0.398; P<.0001; Locke et al. N Eng J Med. 2021). We report results of exploratory analyses of tumor characteristics, including preTx tumor burden (TB), tissue hypoxia-related lactate dehydrogenase (LDH) level, and tumor microenvironment (TME). Methods: TB was calculated as the sum of product diameters of ≤6 reference lesions (Locke et al. Blood Adv. 2020). Serum LDH was assessed. PreTx tumor samples were assessed for RNA expression by the NanoString IO 360™ panel and prespecified immune contexture indexes related to T-cell involvement (Immunosign 15 [IS15] and 21 [IS21]; Galon et al. ASCO 2020. #3022). ZUMA-1 data were used for comparison to 3L R/R LBCL. CD19 protein expression was assessed by immunohistochemistry (H-score). Associations between biomarkers and clinical outcomes were assessed using descriptive statistics ( P<.05 was significant). Results: Axi-cel EFS was superior to SOC for both high and low TB (HR, 0.29 and 0.49, respectively; P<.001 for both) and elevated and nonelevated LDH (HR, 0.32 and 0.5, respectively; P<.001 for both). EFS in axi-cel pts was not associated with preTx TB (HR, 1.01 [95% CI, 0.88-1.16]; P=.89) or LDH (HR, 0.98 [95% CI, 0.74-1.29]; P=.86), but was worse in SOC pts with higher preTx TB (HR, 1.17 [95% CI, 1.03-1.32]; P=.01) or higher LDH (HR, 1.29 [95% CI, 1.02-1.63], P =.03). PreTx TB was lower in SOC pts with ongoing response vs nonresponders and pts who relapsed ( P=.07), but not in axi-cel pts ( P=.99). Non-germinal center B-cell (GCB) cell-of-origin subtypes is a poor prognostic factor for EFS in SOC (EFS was significantly worse in SOC pts with non-GCB vs GCB; HR, 1.82 [95% CI, 1.12-2.96]; P=.02) but not in axi-cel. In IO360 analysis, gene expression of B-cell lineage antigens (CD19, CD20, BCMA) and markers highly expressed by tumor cells (CD45RA, IRF8, BTLA) positively associated with objective and durable responses to axi-cel. While axi-cel remained superior to SOC with high ( > median) or low CD19 expression level, the probability of an ongoing response increased with a higher CD19 H-score. PreTx TME IS15 and IS21 scores were generally higher in 2L vs 3L. Conclusions: Axi-cel was superior to SOC in all subgroup analyses, including higher TB and LDH. Durable responses with axi-cel were greatest in tumors with prominent B-cell features, but were superior to SOC regardless of these features. Axi-cel intervention in 2L is supported by durable response rates not impacted by high TB, as seen in 3L axi-cel or 2L SOC. Higher preTx immune involvement in 2L vs 3L tumors suggests high TB may be overcome with axi-cel in patients with a more favorable immune contexture. Clinical trial information: NCT03391466.
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Ho, A. S., X. Huang, H. Cao, A. C. Koong, and Q. T. Le. "Detection of circulating hypoxia-regulated miR-210 in pancreatic adenocarcinoma patients." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 4624. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.4624.
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4624 Background: MicroRNAs (miRs) are small non-coding transcripts involved in many cellular mechanisms, including tumorigenesis. miR-210, in particular, has been shown to be induced by hypoxia, over-expressed in several different cancers, and correlated with adverse outcomes in breast cancer. Moreover, since pancreatic adenocarcinomas have been previously shown to be extremely hypoxic, we hypothesized that miR-210 may be elevated in the plasma of these patients compared to non-cancer controls. Here, we compared the circulating plasma levels of miR-210 in pancreatic cancer patients and controls using a novel miRNA extraction approach and quantitative PCR. Methods: Pretreatment EDTA plasma samples were obtained from pancreatic cancer patients and age-matched non-cancer controls. miRNA was extracted from 40ul of plasma and reverse transcribed to cDNA. A known quantity of c. elegans miR-54 was added to the sample as a normalization control. miR-210 and cel-miR-54 were then quantified using TaqMan MicroRNA Assays. The procedure was performed on the initial 11 pairs of age-matched pancreatic cancer patients and non- cancer controls, then validated with a second cohort of 12 pancreatic cancer patients and 11 controls. Results: miR-210 was reliably detected and quantified in small amounts of plasma using the approach developed in our study. There is a statistically significant four-fold increase of mir-210 expression in pancreatic cancer patients compared to normal controls (Student's t-test, p <0.0001). This difference was confirmed in the validation group (Student's t-test, p<0.05). Conclusions: Circulating miR-210 levels can be readily measured from a small quantity of plasma using a novel extraction method. Its expression is significantly higher in the blood of pancreatic cancer patients compared to controls and may potentially serve as a useful biomarker for pancreatic cancer diagnosis. No significant financial relationships to disclose.
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Douiev, Liza, Chaya Miller, Shmuel Ruppo, Hadar Benyamini, Bassam Abu-Libdeh, and Ann Saada. "Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency." Cells 10, no. 2 (February 20, 2021): 452. http://dx.doi.org/10.3390/cells10020452.
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Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient’s cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.
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Storti, Paola, Irma Airoldi, Marina Bolzoni, Mirca Lazzaretti, Daniela Guasco, Luca Agnelli, Eugenia Martella, et al. "Hypoxia-Inducible Factor (HIF)-1α Is a Therapeutic Target in Myeloma-Induced Angiogenesis." Blood 118, no. 21 (November 18, 2011): 3927. http://dx.doi.org/10.1182/blood.v118.21.3927.3927.
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Abstract Abstract 3927 Hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of tumor associated angiogenesis. It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that HIF-1α is overexpressed by CD138+ MM cells and modulates the transcriptional and pro-angiogenic profiles of MM cells. The potential role of HIF-1α as a therapeutic target in MM is under investigation. To deepen this issue, in this study we explored the effect of a stable HIF-1α inhibition in MM cells on cell proliferation, survival and on MM-induced angiogenesis either in vitro or in vivo using a plasmocytoma model. Human myeloma cell lines (HMCL)s were screened for HIF-1α protein expression in normoxia condition finding that 2 out 5 HMCLs were positive (JJN3 and RPMI-8226). Anti-HIF-1α Lentivirus sh RNA pool was used for HIF-1α stable knock-out in human myeloma cell lines (HMCL)s whereas the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs have been infected and selected with 0.4 ug/ml puromycin for 21 days and the selected clones screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. A selective inhibition on HIF-1α mRNA and protein expression was observed both in normoxic and hypoxic conditions. HMCLs proliferation and apoptosis were not affected by HIF-1α inhibition neither in hypoxia nor normoxia. Thereafter the transcriptional profiles on the HMCL JJN3 transduced with shRNA anti-HIF-1α (JJN3-anti-HIF-1α), as compared to those infected with the control vector pKLO.1 (JJN3-pKLO.1) have been analyzed by U133 Plus2.0 Arrays (Affymetrix ®) either in hypoxic or normoxic conditions. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. Absolute fold change higher than 1.5 was used to select differentially expressed genes. Among the genes significantly modulated (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 in JJN3-anti-HIF-1α as compared to JJN3-pKLO.1 either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR. In line with this observation, we found that the pro-angiogenic properties of JJN3, evaluated by an in vitro angiogenesis assay (Angio-Kit, TCS), were significantly inhibited by the HIF-1α knock-out. Finally the potential involvement of HIF-1α in MM-induced angiogenesis was assessed in vivo in NOD/SCID mice in a subcutaneous model. We found that the mice which were injected subcutaneously with JJN3-anti-HIF-1α, showed a dramatic reduction of the weight and volume of the plasmocytoma mass than mice inoculated with the JJN3-pKLO.1 (P=0.0007 and P=0.012; respectively). Moreover we observed a significant reduction of the number of vessels X field stained by CD31 antigen (anti-HIF-1α vs. pKLO.1: −76%; P=0.003) and VEGF immunostaining, whereas the Ki67 index of the plasmocytoma mass only showed a slight reduction (anti-HIF-1α vs. pKLO.1: −14%, P=NS). Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenic switch both in vitro and in vivo and consequently reduces the MM tumoral burden suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures: Bolzoni: Celgene Italy: Research Funding. Giuliani:Celgene: Research Funding; Novartis: Research Funding.
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Wei, Guijie, Jianhua Chen, Ziqi Jing, Yanyi Li, Zhihui Li, Wei Zheng, Xiurui Sun, et al. "Glucose transporter 1 (GLUT1)-targeting and hypoxia-activated mitochondria-specific chemo-thermal therapy via a glycosylated poly(amido amine)/celastrol (PAMAM/Cel) complex." Journal of Colloid and Interface Science 608 (February 2022): 1355–65. http://dx.doi.org/10.1016/j.jcis.2021.10.129.
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Lee, Sohyeon, Yoonyoung Kim, and Eun Seong Lee. "Hypoxia-Responsive Azobenzene-Linked Hyaluronate Dot Particles for Photodynamic Tumor Therapy." Pharmaceutics 14, no. 5 (April 24, 2022): 928. http://dx.doi.org/10.3390/pharmaceutics14050928.
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In this study, we developed ultra-small hyaluronate dot particles that selectively release phototoxic drugs into a hypoxic tumor microenvironment. Here, the water-soluble hyaluronate dot (dHA) was covalently conjugated with 4,4′-azodianiline (Azo, as a hypoxia-sensitive linker) and Ce6 (as a photodynamic antitumor agent), producing dHA particles with cleavable Azo bond and Ce6 (dHA-Azo-Ce6). Importantly, the inactive Ce6 (self-quenched state) in the dHA-Azo-Ce6 particles was switched to the active Ce6 (dequenched state) via the Azo linker (–N=N–) cleavage in a hypoxic environment. In vitro studies using hypoxia-induced HeLa cells (treated with CoCl2) revealed that the dHA-Azo-Ce6 particle enhanced photodynamic antitumor inhibition, suggesting its potential as an antitumor drug candidate in response to tumor hypoxia.
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Shahzad, Moazzam, Muhammad Salman Faisal, Ernie Shippey, Qamar Iqbal, Laila Hashim, Clint Divine, Zahra Mahmoudjafari, et al. "Evolution in Resource Utilization for Unique Toxicities Related to Chimeric Antigen Receptor T Cell Therapy from 2017 to 2020: A Database Review." Blood 138, Supplement 1 (November 5, 2021): 4844. http://dx.doi.org/10.1182/blood-2021-154341.
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Abstract Introduction: Chimeric antigen receptor T cell therapy (CAR-T) is a novel treatment that utilizes T cells by augmenting them using vector viruses to add antigens to target cancer cells. In 2017, FDA approved CD-19 CAR-T for relapsed/refractory diffuse large B-cell lymphoma and acute lymphoblastic leukemia patients ≤ 26yr old. Unique toxicities associated with CAR-T therapy include cytokine release syndrome (CRS) and immune effector cell-related neurotoxicity (ICANS). Lower-grade CRS and ICANS are managed with tocilizumab, an interleukin-6 antagonist, and steroids. Management of higher-grade CRS and ICANS requires intensive care unit (ICU) admission. Our understanding and management of CRS and ICANS continue to evolve. In this analysis, we conducted a retrospective review using the Vizient database® to investigate toxicity incidence and resource utilization among patients admitted for CAR-T therapy between 2017 and 2020. Methods: We used The Vizient® CDB database to analyze admissions for CAR-T infusion for patients over 18 years of age receiving FDA approved CD19 CAR-T axicabtagene ciloleucel (axi-cel) and tisangenlecleucel (tisa-cel) between 2017 to 2020. We compared patients who received CAR-T between October 2017 and March 2018 (group 1) to those who received CAR-T therapy between October 2019 and March 2020 (group 2). Due to the lack of diagnosis code for CRS or ICANS until 2021, surrogates billing codes such as fever, sepsis, dyspnea were used for CRS. In regards to ICANS, we used codes for febrile seizure, febrile convulsions, altered mental status, somnolence, and stupor. In addition, other adverse events such as weakness and nausea were also collected. Results: Eighty-one institutions had performed CAR-T in the period 2017 through 2020. The 2017-2018 period (group 1) included 215 patients, with a median age of 59 (49-68) years, while the CAR-T recipients in 2019-2020 (group 2) had 655 patients with a median age of 62 (52-69) years. Tisa-cel and Axi-cel was administered to 31% (n= 67) and 69% (n= 148) in group 1 and 26% (182) and 74% (n= 517) of group 2 patients respectively. The incidence of sepsis in group 1 was 18% vs. 13% in group 2, with an absolute difference of -5.8% (P value=0.04). Fever and dyspnea were the most common presentations of CRS present in 44.2% and 49% in group 1 and 35% and 28% in group 2, respectively. The incidence of fever decreased by 8.2% (p=0.02) in group 2 compared to group 1. The incidence of hypoxia was 24.7% vs. 20.5%, and the incidence of hypotension was 32.1% and 33.8% in groups 1 and 2, with no statistically significant difference between the two groups (p=0.64 and 0.19). The incidence of neurotoxicity decreased slightly in group 2 compared to group 1, but it was not statistically significant (P= 2723). Overall ICU utilization was 24.7 and 24.6% in both groups (p=0.9). The 30 days mortality in groups 1 and 2 was 6% vs. 3.7%. Tocilizumab utilization decreased by 20%, and dexamethasone or equivalent steroid usage decreased by 70% in group 2 compared to group 1. (Table 1) Conclusions: The incidence of CRS and ICANS among recipients of CAR-T remains high, with up to one-fourth of the patients requiring ICU, which has remained static. However, the general use of tocilizumab and steroids has decreased by 20% and 70%, respectively, possibly due to the implementation of consensus grading and operation protocols that may have increased awareness and judicious early interventions. Figure 1 Figure 1. Disclosures Mahmoudjafari: Incyte: Membership on an entity's Board of Directors or advisory committees; Omeros: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees.
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Thompson, Alexis A., Janet L. Kwiatkowski, John B. Porter, Suradej Hongeng, Evangelia Yannaki, Andreas E. Kulozik, Martin G. Sauer, et al. "Favorable Outcomes in Pediatric Patients in the Phase 3 Hgb-207 (Northstar-2) and Hgb-212 (Northstar-3) Studies of Betibeglogene Autotemcel Gene Therapy for the Treatment of Transfusion-Dependent β-Thalassemia." Blood 136, Supplement 1 (November 5, 2020): 52–54. http://dx.doi.org/10.1182/blood-2020-135857.
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Introduction Betibeglogene autotemcel (beti-cel; LentiGlobin for β-thalassemia) gene therapy is being evaluated for the treatment of transfusion-dependent β-thalassemia (TDT). Initial positive results of beti-cel in the phase 3 studies, HGB-207 (NCT02906202; non-β0/β0 genotypes) and HGB-212 (NCT03207009; β0/β0, β0/β+ IVS-I-110 and β+ IVS-I-110/β+ IVS-I-110 genotypes), showed 10/12 adult patients achieved transfusion independence. The studies expanded enrollment to include adolescents and children. We present interim results from pediatric patients <18 yrs who received beti-cel in HGB-207 and HGB-212 as of 3 March 2020. Methods After mobilization and apheresis, autologous CD34+ cells were transduced ex vivo with BB305 lentiviral vector, containing a modified human β-globin gene to produce beti-cel drug product (DP). Patients underwent busulfan myeloablation and infusion with beti-cel and were then followed longitudinally. Transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without transfusions for ≥12 mo) was the primary endpoint in HGB-207 and a secondary endpoint in HGB-212. Transfusion reduction (≥60% reduction in transfusion volume between Month 12 to 24 versus baseline) is the primary endpoint in HGB-212. Hb levels, TI characteristics, and quality of life were secondary endpoints. Assessments of ineffective erythropoiesis were exploratory. Data presented as median (min-max). Results Twenty-four pediatric patients were treated including 13 patients <12 yrs old (207: n=8; 212: n=5) and 11 patients ≥12 to <18 yrs old (207: n=6; 212: n=5). Patient, DP, and engraftment characteristics are described in the Table. All patients achieved neutrophil engraftment. Twenty-one patients with >3 mo follow-up achieved platelet engraftment and had platelets ≥100 x109/L by Month 12; one 17-yr old patient did not have platelets ≥100 x109/L until Month 15. In HGB-207, 6/7 (86%) patients <12 yrs and 6/6 (100%) patients ≥12 to <18 yrs with >3 mo follow-up have stopped transfusions for ≥6 mo. TI was achieved in 3/4 (75%) evaluable patients <12 yrs with weighted average Hb during TI of 10.0 (9.4-10.3) g/dL. At last visit, gene therapy-derived HbAT87Q in these patients was 5.1-7.1 g/dL. In patients ≥12 to <18 yrs, all 6 patients achieved TI with weighted average Hb during TI of 11.6 (11.3-12.3) g/dL. These patients had 8.4-10.5 g/dL HbAT87Q at last visit. Myeloid:erythroid (M:E) ratio in patients <12 yrs with TI improved from 1:3.7-1:1.1 at baseline to 1:2-1.5:1 at Month 12; in patients ≥12 to <18 yrs with TI, M:E ratio improved from 1:7.3-1.6:1 at baseline to 1:2.7-1.9:1 at Month 12. Patients <18 yrs who achieved TI in HGB-207 had an improved health state today score as assessed using EQ-5D-Y from 67 (50-96) at baseline (n=7) to 92.5 (85-95) at Month 12 (n=6). In HGB-212, 3/5 (60%) patients <12 yrs and 4/4 (100%) patients ≥12 to <18 yrs with >3mo follow-up have stopped transfusions for ≥6 mo. TI was achieved in 1/2 evaluable patients <12 yrs. Weighted average Hb during TI was 10.3 g/dL and HbAT87Q was 9.2 g/dL at last visit in this patient. All 3 evaluable patients ≥12 to <18 yrs achieved TI with weighted average Hb of 9.6 (9.5-12.8) g/dL. HbAT87Q in these patients was 8.0-10.9 g/dL at last visit. The M:E ratio in the patient <12 yrs with TI improved from 1:4.7 at baseline to 1.2:1 at Month 12. In the 2 patients ≥12 to <18 yrs with baseline and Month 12 assessments, M:E ratios improved from 1:5.1 to 1:1.4 and 1:3.3 to 1:1.8. Post-infusion non-hematologic grade ≥3 adverse events (AEs) in ≥3 patients aged <12 yrs in either study were febrile neutropenia (n=9), stomatitis (n=6), decreased appetite (n=4), increased alanine aminotransferase (n=3), and epistaxis (n=3); in patients ≥12 to <18 yrs, these were stomatitis (n=8), febrile neutropenia (n=3) and hypoxia (n=3). Grade 4 veno-occlusive disease occurred in 2 patients ≥12 to <18 yrs and one grade 2 event occurred in a patient <12 yrs; all cases were successfully treated with defibrotide. Neither replication-competent lentivirus, insertional oncogenesis, nor clonal dominance were observed. Summary Interim results in HGB-207 and HGB-212 show that after treatment with beti-cel, pediatric patients <18 yrs achieved transfusion independence with comparable rates as in adults, suggesting that beti-cel gene therapy represents an effective treatment option across ages. The safety profile of gene therapy with beti-cel was consistent with busulfan myeloablation. Disclosures Thompson: CRISPR/Vertex: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Baxalta: Research Funding; BMS: Consultancy, Research Funding; Biomarin: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding. Kwiatkowski:Novartis: Research Funding; Agios: Consultancy; Sangamo: Research Funding; bluebird bio,Inc.: Consultancy, Research Funding; Apopharma: Research Funding; Imara: Consultancy; BMS: Consultancy; Terumo Co: Research Funding; Celgene: Consultancy. Porter:Vifor Pharmaceuticals: Honoraria; bluebird bio, Inc.: Consultancy, Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; La Jolla Pharmaceuticals: Honoraria; Silence Therapeutics: Honoraria; Protagonist Therapeutics: Honoraria; BMS: Consultancy, Honoraria. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees. Thuret:Novartis pharma: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials. Lal:Insight Magnetics: Research Funding; Celgene, BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Terumo Corporation: Research Funding; Agios Pharmaceuticals: Consultancy; Chiesi USA: Consultancy; La Jolla Pharmaceutical Company: Research Funding; bluebird bio, Inc.: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Guo:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Liu:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Colvin:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Walters:Veevo Biomedicine: Consultancy; AllCells, Inc: Consultancy; Editas: Consultancy. Locatelli:Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.
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Yang, B. C., and J. L. Mehta. "Alterations in pulmonary artery tone during repeated episodes of hypoxia." American Journal of Physiology-Lung Cellular and Molecular Physiology 269, no. 3 (September 1, 1995): L293—L298. http://dx.doi.org/10.1152/ajplung.1995.269.3.l293.
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To examine the basis of pulmonary constriction during chronic hypoxia, rat pulmonary artery rings were precontracted and exposed to multiple episodes of hypoxia. The first hypoxic episode resulted in a transient contraction, followed by potent relaxation, and then a slow sustained contraction. Repeated hypoxic exposure resulted in stronger initial contraction and attenuated relaxation. Prolongation of the normoxic interval between hypoxic episodes reversed the attenuation of hypoxic relaxation. Pulmonary artery rings that were deendothelialized or treated with the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester or oxyhemoglobin displayed only hypoxic relaxation without initial or late contractions and no attenuation of relaxation during repeated hypoxia. Pretreatment of rings with indomethacin or adenosine or endothelin receptor antagonists had no effect on the hypoxia-mediated alterations. Thus repetitive exposure to hypoxia results in an increase in initial contraction and a decrease in relaxation of pulmonary artery rings. Frequency of hypoxic episodes and endothelial integrity determine pulmonary tone during repeated hypoxia. However, cyclooxygenase products, adenosine, or endothelin do not play a role in hypoxia-mediated changes in rat pulmonary artery tone.
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Gong, Yanqing, and Faton H. Agani. "Oligomycin inhibits HIF-1α expression in hypoxic tumor cells." American Journal of Physiology-Cell Physiology 288, no. 5 (May 2005): C1023—C1029. http://dx.doi.org/10.1152/ajpcell.00443.2004.
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Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1α in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1α accumulation in different tumor cell lines. Hypoxia-induced HIF-1α accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F0F1-ATPase, prevented hypoxia-induced HIF-1α protein accumulation and had no effect on HIF-1α induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1α content was pronounced in cells exposed to hypoxia (1.5% O2) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1α regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1α accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function.
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Zhang, Hanying, Miyako Okamoto, Evgeniy Panzhinskiy, W. Michael Zawada, and Mita Das. "PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells." American Journal of Physiology-Cell Physiology 306, no. 7 (April 1, 2014): C648—C658. http://dx.doi.org/10.1152/ajpcell.00351.2013.
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Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.
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Guo, Ying, Jin Tan, Yuyang Miao, Zuoming Sun, and Qiang Zhang. "Effects of Microvesicles on Cell Apoptosis under Hypoxia." Oxidative Medicine and Cellular Longevity 2019 (April 17, 2019): 1–11. http://dx.doi.org/10.1155/2019/5972152.
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Hypoxia, as one of the severe cellular stresses, can cause cellular injury and even cell death. Apoptosis is the main mechanism of regulating cell death and is closely related to the cell death caused by hypoxia. However, hypoxia-induced apoptosis is not entirely the result of direct hypoxic stimulus of cells. In recent years, it has been found that cells injured by hypoxia can shed a kind of membranous vesicles, which are called microvesicles (MVs). MVs can carry bioactive molecules from injured mother cells and appear in blood, cerebrospinal fluid, and other body fluids. MVs can induce normal cell apoptosis by transferring bioactive molecules into adjacent cells and amplifying the hypoxic injury in an organism. This review summarizes the characteristic changes of MVs derived from hypoxic cells and the mechanism of normal cell apoptosis mediated by hypoxic cell-derived MVs. Finally, we introduce the significance of this apoptosis-apoptosis cascade reaction in hypoxic diseases.
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Wodopia, Ralf, Hyun Soo Ko, Javiera Billian, Rudolf Wiesner, Peter Bärtsch, and Heimo Mairbäurl. "Hypoxia decreases proteins involved in epithelial electrolyte transport in A549 cells and rat lung." American Journal of Physiology-Lung Cellular and Molecular Physiology 279, no. 6 (December 1, 2000): L1110—L1119. http://dx.doi.org/10.1152/ajplung.2000.279.6.l1110.
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Fluid reabsorption from alveolar space is driven by active Na reabsorption via epithelial Na channels (ENaCs) and Na-K-ATPase. Both are inhibited by hypoxia. Here we tested whether hypoxia decreases Na transport by decreasing the number of copies of transporters in alveolar epithelial cells and in lungs of hypoxic rats. Membrane fractions were prepared from A549 cells exposed to hypoxia (3% O2) as well as from whole lung tissue and alveolar type II cells from rats exposed to hypoxia. Transport proteins were measured by Western blot analysis. In A549 cells, α1- and β1-Na-K-ATPase, Na/K/2Cl cotransport, and ENaC proteins decreased during hypoxia. In whole lung tissue, α1-Na-K-ATPase and Na/K/2Cl cotransport decreased. α- and β-ENaC mRNAs also decreased in hypoxic lungs. Similar results were seen in alveolar type II cells from hypoxic rats. These results indicate a slow decrease in the amount of Na-transporting proteins in alveolar epithelial cells during exposure to hypoxia that also occurs in vivo in lungs from hypoxic animals. The reduced number of transporters might account for the decreased transport activity and impaired edema clearance in hypoxic lungs.
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Zhou, Shiyu, Yu Lan, Yuqun Li, Zhenxing Li, Jinding Pu, and Liping Wei. "Hypoxic Tumor-Derived Exosomes Induce M2 Macrophage Polarization via PKM2/AMPK to Promote Lung Cancer Progression." Cell Transplantation 31 (January 2022): 096368972211069. http://dx.doi.org/10.1177/09636897221106998.
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Hypoxia is a major regulator of tumor aggressiveness and metastasis in cancer progression. Exosomes (exos) play an important role in the communication between lung cancer and hypoxic microenvironment. However, the underlying mechanisms are largely undefined. Exos were isolated from A549 cells under hypoxia conditions. Transmission electron microscopy and nanoparticle tracking analysis were carried out to characterize exos. CCK-8 assay, flow cytometry, Western blot, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of A549 cells, respectively. The M2 polarization of macrophages was evaluated by RT-qPCR and Western blot analysis. In vivo nude mice model was established to determine the regulatory effect of hypoxia/exos on the progression of lung cancer. Hypoxic A549 cell-derived exos (hypoxia/exos) promoted the proliferation and migration, and inhibited the apoptosis in A549 cells. The expression of PKM2 was significantly upregulated in hypoxia/exos. Hypoxic exosomal PKM2 induced M2 polarization of macrophages by activating AMPK pathway. Co-culture with hypoxia/exos-treated macrophages enhanced the migration, invasion, and epithelial-mesenchymal transition (EMT) in A549 cells. Moreover, treatment with hypoxia/exos facilitated the tumor growth and lung metastasis of A549 cells. Our findings reveal that hypoxic exosomal PKM2 induces M2 macrophage polarization via AMPK pathway, and thus exerts a simulative effect on the growth and metastasis of lung carcinoma.
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Schmaltz, Cornelius, Patricia Harrigan Hardenbergh, Audrey Wells, and David E. Fisher. "Regulation of Proliferation-Survival Decisions during Tumor Cell Hypoxia." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2845–54. http://dx.doi.org/10.1128/mcb.18.5.2845.
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ABSTRACT Hypoxia may influence tumor biology in paradoxically opposing ways: it is lethal as a direct stress trigger, yet hypoxic zones in solid tumors harbor viable cells which are particularly resistant to treatment and contribute importantly to disease relapse. To examine mechanisms underlying growth-survival decisions during hypoxia, we have compared genetically related transformed and untransformed fibroblast cells in vitro for proliferation, survival, clonogenicity, cell cycle, and p53 expression. Hypoxia induces G0/G1 arrest in primary fibroblasts but triggers apoptosis in oncogene-transformed derivatives. Unexpectedly, the mechanism of apoptosis is seen to require accumulated acidosis and is rescued by enhanced buffering. The direct effect of hypoxia under nonacidotic conditions is unique to transformed cells in that they override the hypoxic G0/G1 arrest of primary cells. Moreover, when uncoupled from acidosis, hypoxia enhances tumor cell viability and clonogenicity relative to normoxia. p53 is correspondingly upregulated in response to hypoxia-induced acidosis but downregulated during hypoxia without acidosis. Hypoxia may thus produce both treatment resistance and a growth advantage. Given strong evidence that hypoxic regions in solid tumors are often nonacidotic (G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, Nat. Med. 3:177–182, 1997), this behavior may influence relapse and implicates such cells as potentially important therapeutic targets.
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Gallego-Martin, Teresa, Jesus Prieto-Lloret, Philip Aaronson, Asuncion Rocher, and Ana Obeso. "Hydroxycobalamin Reveals the Involvement of Hydrogen Sulfide in the Hypoxic Responses of Rat Carotid Body Chemoreceptor Cells." Antioxidants 8, no. 3 (March 13, 2019): 62. http://dx.doi.org/10.3390/antiox8030062.
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Carotid body (CB) chemoreceptor cells sense arterial blood PO2, generating a neurosecretory response proportional to the intensity of hypoxia. Hydrogen sulfide (H2S) is a physiological gaseous messenger that is proposed to act as an oxygen sensor in CBs, although this concept remains controversial. In the present study we have used the H2S scavenger and vitamin B12 analog hydroxycobalamin (Cbl) as a new tool to investigate the involvement of endogenous H2S in CB oxygen sensing. We observed that the slow-release sulfide donor GYY4137 elicited catecholamine release from isolated whole carotid bodies, and that Cbl prevented this response. Cbl also abolished the rise in [Ca2+]i evoked by 50 µM NaHS in enzymatically dispersed CB glomus cells. Moreover, Cbl markedly inhibited the catecholamine release and [Ca2+]i rise caused by hypoxia in isolated CBs and dispersed glomus cells, respectively, whereas it did not alter these responses when they were evoked by high [K+]e. The L-type Ca2+ channel blocker nifedipine slightly inhibited the rise in CB chemoreceptor cells [Ca2+]i elicited by sulfide, whilst causing a somewhat larger attenuation of the hypoxia-induced Ca2+ signal. We conclude that Cbl is a useful and specific tool for studying the function of H2S in cells. Based on its effects on the CB chemoreceptor cells we propose that endogenous H2S is an amplifier of the hypoxic transduction cascade which acts mainly by stimulating non-L-type Ca2+ channels.
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Shahzad, Moazzam, Muhammad Salman Faisal, Ernie Shippey, Ali Hussain, Clint Divine, Zahra Mahmoudjafari, Allison Appenfeller, et al. "Safety and Cost Effectiveness of Chimeric Antigen Receptor T Cell Therapy in the Outpatient Setting." Blood 138, Supplement 1 (November 5, 2021): 2823. http://dx.doi.org/10.1182/blood-2021-151872.
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Abstract Introduction Since the commercial approval of chimeric antigen receptor T cell (CAR-T) therapies, administration and toxicity monitoring have largely been in an inpatient setting due to the risk of significant toxicities such as cytokine release syndrome (CRS) and neurotoxicity in the first 30 days. Administration in the outpatient setting can be safe and cost-effective. Here we report the cost savings and adverse events of CAR-T in an outpatient setting as compared to the inpatient setting. Methods Cost differences of the commercial CD19 CAR-T axicabtagene ciloleucel (axi-cel) and tisangenlecleucel (tisa-cel) among inpatient and outpatient settings in 2020 were investigated using the Vizient Database®. Cost analysis for both settings was done for the initial 30 days post-CAR-T infusion. There were no billing codes for CRS and neurotoxicity till 2021. Clinical surrogates such as fever, hypotension, hypoxia, sepsis were used for CRS while febrile convulsion not otherwise specified (NOS), febrile seizure NOS, altered mental status, somnolence, stupor, and coma were used for neurotoxicity. ICD 10 codes for adverse effects were also used. Results In 2017-2020, there were 81 organizations in the database that performed CAR-T procedures. In 2020, there were 1369 inpatient and 71 outpatient encounters, which were analyzed for cost and adverse events. (Table 1) The incidence of CRS was 43.2% (n=592) and 40.8% (n=29) in inpatient and outpatient groups, while that of neurotoxicity was 37.3% (n=511) and 29.6% (n=21) respectively. For cost analysis, we included the 16 centers (22% of all centers) that offered both inpatient and outpatient administration in 2020. Median inpatient cost was $397,610 ($346,550-$650,749) and median outpatient group cost was $243,050 ($204,344-$408,467). An analysis of variance (ANOVA) was run between inpatient and outpatient cases was found to be significant (P<0.0001). (Table 2) (Figure 1) (Figure 2) Conclusion As the field of CAR-T therapy continues to grow, outpatient programs are likely to increase. Incidence of adverse effects was lower in the outpatient group, likely patient selection effect. This data suggests that outpatient CAR-T therapy is feasible cost-effective and has the potential to grow and improve value. While it appears to be an attractive option, there is a need for more studies on patient selection and creating a robust outpatient infrastructure is needed. Figure 1 Figure 1. Disclosures Mahmoudjafari: Incyte: Membership on an entity's Board of Directors or advisory committees; Omeros: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. McGuirk: EcoR1 Capital: Consultancy; Novartis: Research Funding; Fresenius Biotech: Research Funding; Novartis: Research Funding; Astelllas Pharma: Research Funding; Bellicum Pharmaceuticals: Research Funding; Kite/ Gilead: Consultancy, Honoraria, Other: travel accommodations, expense, Kite a Gilead company, Research Funding, Speakers Bureau; Gamida Cell: Research Funding; Magenta Therapeutics: Consultancy, Honoraria, Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Allovir: Consultancy, Honoraria, Research Funding; Pluristem Therapeutics: Research Funding.
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O’Leary, Andrew J., Sarah E. Drummond, Deirdre Edge, and Ken D. O’Halloran. "Diaphragm Muscle Weakness Following Acute Sustained Hypoxic Stress in the Mouse Is Prevented by Pretreatment with N-Acetyl Cysteine." Oxidative Medicine and Cellular Longevity 2018 (2018): 1–19. http://dx.doi.org/10.1155/2018/4805493.
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Oxygen deficit (hypoxia) is a major feature of cardiorespiratory diseases characterized by diaphragm dysfunction, yet the putative role of hypoxic stress as a driver of diaphragm dysfunction is understudied. We explored the cellular and functional consequences of sustained hypoxic stress in a mouse model. Adult male mice were exposed to 8 hours of normoxia, or hypoxia (FiO2 = 0.10) with or without antioxidant pretreatment (N-acetyl cysteine, 200 mg/kg i.p.). Ventilation and metabolism were measured. Diaphragm muscle contractile function, myofibre size and distribution, gene expression, protein signalling cascades, and oxidative stress (TBARS) were determined. Hypoxia caused pronounced diaphragm muscle weakness, unrelated to increased respiratory muscle work. Hypoxia increased diaphragm HIF-1α protein content and activated MAPK, mTOR, Akt, and FoxO3a signalling pathways, largely favouring protein synthesis. Hypoxia increased diaphragm lipid peroxidation, indicative of oxidative stress. FoxO3 and MuRF-1 gene expression were increased. Diaphragm 20S proteasome activity and muscle fibre size and distribution were unaffected by acute hypoxia. Pretreatment with N-acetyl cysteine substantially enhanced cell survival signalling, prevented hypoxia-induced diaphragm oxidative stress, and prevented hypoxia-induced diaphragm dysfunction. Hypoxia is a potent driver of diaphragm weakness, causing myofibre dysfunction without attendant atrophy. N-acetyl cysteine protects the hypoxic diaphragm and may have application as a potential adjunctive therapy.
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Itoh, Mai, Yusuke Takahashi, Yuki Okuhashi, and Shuji Tohda. "Effects of Hypoxia on HIF, Notch, Akt, and NF-κB Signaling in Leukemia Cell Lines." Blood 122, no. 21 (November 15, 2013): 3874. http://dx.doi.org/10.1182/blood.v122.21.3874.3874.
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Abstract Background Leukemia stem cells reside in the bone marrow niche under hypoxic conditions. The activity of various signaling pathways in the hypoxic environment should be known to understand the pathophysiology of leukemia stem cells. Hypoxia is known to stabilize HIF1α, which transactivates various genes allowing the cell to adapt to hypoxic conditions. Two theories have been reported regarding crosstalk between HIF and Notch signaling; one suggests that HIF1α binds to cleaved Notch1, which results in stabilization of Notch signaling, and the second suggests that HIF1α represses a negative feedback loop of the Notch1-Hes1 system by inhibiting Hes1 binding to the HES1 promoter and thus enhancing Notch signaling. In this study, we examined the activity of various signaling pathways in cells cultured under normoxia and hypoxia. We discovered novel processes for HIF, Notch, and their down-stream signaling. Methods Two T-ALL cell lines (KOPT-K1 and DND-41), 3 AML cell lines (NB4, THP-1, and TMD7), and 1 CML cell line (K562) were used in this study. Cells were cultured under normoxia or hypoxia (1% O2). The effect of hypoxia on cell growth was examined using a colorimetric WST-1 assay. The effect of hypoxia on replication of clonogenic cells was studied by colony assay after suspension culture under normoxia or hypoxia. The expression and activation of signaling proteins were examined by immunoblotting using lysates from cells cultured under normoxia or hypoxia for 4, 8, and 24 hours. In some experiments, we used lysates from cells cultured with recombinant Notch ligand Delta1 protein. Results Hypoxic conditions reduced cell growth and amplification of clonogenic cells when compared to the normoxic condition. Hypoxia enhanced HIF1α levels in all 6 cell lines and increased HIF2α levels in 4 cell lines. HIF2α was not expressed in NB4 and THP-1 cells. Hypoxia reduced the levels of Notch1 and cleaved Notch1 in KOPT-K1, DND-41, and NB4 cells. Notch1 expression was not affected by hypoxia in THP-1, TMD7, and K562 cells. In KOPT-K1, DND-41, and NB4 cells, hypoxia reduced Hes1 expression. In KOPT-K1 cells, Hes1 levels decreased after 4–8 hours under hypoxic conditions; however, the levels increased after 24 hours. Hypoxia suppressed Myc expression in 4 cell lines. Hypoxia also promoted Akt phosphorylation in KOPT-K1, NB4, and K562 cells but suppressed Akt phosphorylation in DND-41 cells without affecting total Akt levels. Hypoxia suppressed NF-κB phosphorylation without affecting total NF-κB protein levels in all cell lines. Delta1 enhanced cleaved Notch1 fragment levels in 4 myeloid cell lines under normoxia and hypoxia. The enhanced Hes1 expression was not significantly different between normoxia and hypoxia. Discussion We discovered a novel relationship between hypoxia and Notch signaling. Specifically, hypoxia suppressed Notch1 expression and activation, while hypoxia increased HIF1α and HIF2α levels. We found that Hes1 levels decreased and subsequently increased in KOPT-K1 cells cultured under hypoxia. This could be due to the decrease of cleaved Notch1 as shown here, and subsequent enhancement of Notch signaling owing to increased HIF1α as mentioned in Background section. Hypoxia also suppressed Myc expression and NF-κB phosphorylation, which was likely due to Notch1 suppression. The effect of hypoxia on Akt phosphorylation varied depending on cell lines; however, determining the underlying cause requires further study. In conclusion, we determined that the activities in various signaling pathways under hypoxia differ from those under normoxia. These findings would contribute to the development of various molecular-targeted therapies against leukemic cells, especially leukemia stem cells under hypoxic conditions. Disclosures: No relevant conflicts of interest to declare.
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Sikarwar, A. S., M. Hinton, K. T. Santhosh, P. Dhanaraj, M. Talabis, P. Chelikani, and S. Dakshinamurti. "Hypoxia inhibits adenylyl cyclase catalytic activity in a porcine model of persistent pulmonary hypertension of the newborn." American Journal of Physiology-Lung Cellular and Molecular Physiology 315, no. 6 (December 1, 2018): L933—L944. http://dx.doi.org/10.1152/ajplung.00130.2018.
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Persistent pulmonary hypertension of the newborn (PPHN) features hypoxemia, pulmonary vasoconstriction, and impaired cardiac inotropy. We previously reported low basal and stimulated cAMP in hypoxic pulmonary artery smooth muscle cells (PASMCs). We now examine pulmonary arterial adenylyl cyclase (AC) activity and regulation in hypoxic PPHN. PPHN was induced in newborn swine by normobaric hypoxia (fraction of inspired oxygen 0.10) for 72 h and compared with age-matched normoxic controls. We studied relaxation of pulmonary arterial (PA) rings to AC activator forskolin and cGMP activator sodium nitroprusside (SNP) by isometric myography, ATP content, phosphodiesterase activity, AC content, isoform expression, and catalytic activity in presence or absence of Gαs-coupled receptor agonists, forskolin, or transnitrosylating agents in human and neonatal porcine PASMCs and HEK293T stably expressing AC isoform 6, after 72 h hypoxia (10% O2) or normoxia (21% O2). Relaxation to forskolin and SNP were equally impaired in PPHN PA. AC-specific activity decreased in hypoxia. PASMC from PPHN swine had reduced AC activity despite exposure to normoxia in culture; transient hypoxia in vitro further decreased AC activity. Prostacyclin receptor ligand affinity decreased, but its association with Gαs increased in hypoxia. Total AC content was unchanged by hypoxia, but AC6 increased in hypoxic cells and PPHN pulmonary arteries. Impairment of AC6 activity in hypoxia was associated with nitrosylation. PPHN PA relaxation is impaired because of loss of AC activity. Hypoxic AC is inhibited because of S-nitrosylation; inhibition persists after removal from hypoxia. Downregulation of AC-mediated relaxation in hypoxic PA has implications for utility of Gαs-coupled receptor agonists in PPHN treatment.
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Agani, Faton H., Michelle Puchowicz, Juan Carlos Chavez, Paola Pichiule, and Joseph LaManna. "Role of nitric oxide in the regulation of HIF-1α expression during hypoxia." American Journal of Physiology-Cell Physiology 283, no. 1 (July 1, 2002): C178—C186. http://dx.doi.org/10.1152/ajpcell.00381.2001.
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Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1α and HIF-1β subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1α, is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1α hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1α accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1α hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1α accumulation during hypoxia. Regulation of HIF-1α by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.
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Collard, Charles D., Cuneyt Bukusoglu, Azin Agah, Sean P. Colgan, Wende R. Reenstra, B. Paul Morgan, and Gregory L. Stahl. "Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells." American Journal of Physiology-Cell Physiology 276, no. 2 (February 1, 1999): C450—C458. http://dx.doi.org/10.1152/ajpcell.1999.276.2.c450.
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Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 ± 0.6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-α also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.
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Muz, Barbara, Feda Azab, Pilar De La Puente, Ravi Vij, and Abdel Kareem Azab. "Tumor Hypoxia Promotes Dissemination and Tumor Colonization In Waldenström Macroglobulinemia." Blood 122, no. 21 (November 15, 2013): 3011. http://dx.doi.org/10.1182/blood.v122.21.3011.3011.
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Abstract Introduction Waldenström Macroglobulinemia (WM) is a rare, low-grade B-cell lymphoma characterized by lymphoplasmacytic cells spread widely in the bone marrow (BM) and overproduction of monoclonal immunoglobulins M (IgM). Previous studies showed that tumor hypoxia develops in the BM of other hematologic malignancies and promotes dissemination. In this study, we tested the effect of hypoxia on cell proliferation, cell cycle and apoptosis; on egress and homing of WM cells from and into the BM; and on recovery and tumor colonization in the new BM niche. Methods We characterized the effect of tumor progression on generation of hypoxic conditions in the BM in vivo, by injecting BCWM1-mCherry cells to SCID mice, letting them grow for two weeks, analyzing the hypoxic state of the WM cells in the BM using pimonidazole, and testing the number of circulating cells. Moreover, we tested the effect of hypoxia on the homing of WM cells to the BM by injecting normoxic and hypoxic cells to mice and monitoring the number of the circulating WM cells in the blood at different time points by flow cytometry. Cancer cell colonization was assessed 1 and 3 days post IV injection of normoxic and hypoxic cells to mice; mononuclear cells were isolated from the BM, fixed, permeabilized and stained with antibodies for p-Rb and cyclin-E. The percentile of WM cells in the BM and the expression of cell cycle proteins were analyzed by flow cytometry. BCWM1 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) in vitro for 24hrs, and n some cases reoxygenated for 24hrs. The expression of E-cadherin, VLA-4 and CXCR4 was analyzed by western blot or flow cytometry. We tested the effect of hypoxia on adhesion of WM cells to BM stroma and fibronectin. We further tested the effect of hypoxia on chemotactic properties of WM cells towards SDF-1 using a transwell migration chamber. In addition, we tested the effect of hypoxia on WM cell survival (by MTT assay), apoptosis and cell cycle (by using AnnexinV-PI and PI, respectively), and signaling pathways associated with survival, apoptosis and cell cycle (by western blotting). Results Tumor progression was shown to increase hypoxic conditions in the BM in vivo. We found a direct correlation between the percent of WM cells in the BM to the level of hypoxia. The level of hypoxia was in a direct correlation with the number of circulating WM cells in vivo. Then we mimicked the hypoxic conditions in vitro and found that cell progression (MTT) and cell cycle (PI staining) were decreased, but apoptosis of WM cells was not affected (AnnexinV-PI staining). These results were confirmed by decreased activation of the PI3K signaling pathway (p-PI3K, p-AKT, p-GSK) and decreased expression of cell cycle proteins (p-Rb, CDK2, CDK4, cyclin-D1 and p-cyclin-E); however, no change was observed in apoptosis-related proteins (PARP, cleaved caspase-3, -8 and -9). Moreover, hypoxia decreased the expression of E-cadherin which contributed to reduction of adhesion of WM cells to the BM stromal cells. At the same time, hypoxic WM cells exhibited increased CXCR4 surface expression and augmented migratory abilities in the presence of SDF-1. Neither the expression of integrins (VLA-4) nor the adhesion of WM cells to fibronectin was affected by hypoxia. This data indicates the conservation of the homing machinery of the WM to the BM despite the hypoxic conditions accompanied by increased chemotactic ability. When hypoxic and normoxic cells were injected to naïve mice, hypoxic cells showed enhanced homing to the BM and tumor colonization. Similarly, hypoxic cells which were reoxygenated in vitro showed more proliferation, cell cycle and activation of proliferative signaling pathways compared to normoxic cells. Conclusions We report that WM tumor growth in the BM increases hypoxia, and that hypoxia induces cell cycle arrest, and less proliferation of cells with no apoptosis. At the same time, hypoxia induces egress of WM cells from the BM through reduction of E-cadherin expression and decreased adhesion. When in the circulation, previously hypoxic cells home more efficiently to the BM through increased expression of CXCR4 and chemotaxis, and through maintaining expression of integrins and adhesion to fibronectin. When in the new oxygenated BM niche, hypoxic WM cells recover and colonize the new niche better than normoxic cells, and reoxygenated hypoxic cells have faster cell cycle and proliferation rate. Disclosures: No relevant conflicts of interest to declare.
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Liu, Xin Hua, Alexander Kirschenbaum, Min Lu, Shen Yao, Amy Dosoretz, James F. Holland, and Alice C. Levine. "Prostaglandin E2Induces Hypoxia-inducible Factor-1α Stabilization and Nuclear Localization in a Human Prostate Cancer Cell Line." Journal of Biological Chemistry 277, no. 51 (October 24, 2002): 50081–86. http://dx.doi.org/10.1074/jbc.m201095200.
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Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1α (HIF-1α), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E2(PGE2) restores hypoxic effects on VEGF. We hypothesized that PGE2mediates hypoxic effects on VEGF by modulating HIF-1α expression. Addition of PGE2to PC-3ML human prostate cancer cells had no effect on HIF-1α mRNA levels. However, PGE2significantly increased HIF-1α protein levels, particularly in the nucleus. This effect of PGE2largely results from the promotion of HIF-1α translocation from the cytosol to the nucleus. PGE2addition to PC-3 ML cells transfected with a GFP-HIF-1α vector induced a time-dependent nuclear accumulation of the HIF-1α protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1α levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE2reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE2effects on HIF-1α were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE2production via COX-2-catalyzed pathway plays a critical role in HIF-1α regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.
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Hong, Sang-Eun, Jong Hun An, Seong-Lan Yu, Jaeku Kang, Chang Gyo Park, Hoi Young Lee, Dong Chul Lee, et al. "Ceria-Zirconia Antioxidant Nanoparticles Attenuate Hypoxia-Induced Acute Kidney Injury by Restoring Autophagy Flux and Alleviating Mitochondrial Damage." Journal of Biomedical Nanotechnology 16, no. 7 (July 1, 2020): 1144–59. http://dx.doi.org/10.1166/jbn.2020.2948.
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Oxidative stress is one of the principal causes of hypoxia-induced kidney injury. The ceria nanoparticle (CNP) is known to exhibit free radical scavenger and catalytic activities. When zirconia is attached to CNPs (CZNPs), the ceria atom tends to remain in a Ce3+ form and its efficacy as a free radical scavenger thus increases. We determined the effectiveness of CNP and CZNP antioxidant activities against hypoxia-induced acute kidney injury (AKI) and observed that these nanoparticles suppress the apoptosis of hypoxic HK-2 cells by restoring autophagy flux and alleviating mitochondrial damage. In vivo experiments revealed that CZNPs effectively attenuate hypoxia-induced AKI by preserving renal structures and glomerulus function. These nanoparticles can successfully diffuse into HK-2 cells and effectively counteract reactive oxygen species (ROS) to block hypoxia-induced AKI. This suggests that these particles represent a novel approach to controlling this condition.
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Rico, A. J., J. Prieto-Lloret, C. Gonzalez, and R. Rigual. "Hypoxia and acidosis increase the secretion of catecholamines in the neonatal rat adrenal medulla: an in vitro study." American Journal of Physiology-Cell Physiology 289, no. 6 (December 2005): C1417—C1425. http://dx.doi.org/10.1152/ajpcell.00023.2005.
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Hypoxia elicits catecholamine (CA) secretion from the adrenal medulla (AM) in perinatal animals by acting directly on chromaffin cells. However, whether innervation of the AM, which in the rat occurs in the second postnatal week, suppresses this direct hypoxic response is the subject of debate. Opioid peptides have been proposed as mediators of this suppression. To resolve these controversies, we have compared CA-secretory responses with high external concentrations of K+ ([K+]e) and hypoxia in the AM of neonatal (1- to 2-day-old) and juvenile (14- or 15- and 30-day-old) rats subjected to superfusion in vitro. In addition, we studied the effect of hypercapnic acidosis on the CA-secretory responses in the AM during postnatal development and the possible interaction between acidic and hypoxic stimuli. Responses to high [K+]e were comparable at all ages, but responses to hypoxia and hypercapnic acidosis were maximal in neonatal animals. Suppression of the hypoxic response in the rat AM was not mediated by opioids, because their agonists did not affect the hypoxic CA response. The association of hypercapnic acidosis and hypoxia, mimicking the episodes of asphyxia occurring during delivery, generates a more than additive secretory response in the neonatal rat AM. Our data confirm the loss of the direct sensitivity to hypoxia of the AM in the initial weeks of life and demonstrate a direct response of neonatal AM to hypercapnic acidosis. The synergistic effect of hypoxia and acidosis would explain the CA outburst crucial for adaptation to extrauterine life observed in naturally delivered mammals.
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Eliasson, Pernilla M., and Jan-Ingvar Jönsson. "A Hypoxic Niche in the Mouse Bone Marrow Diminishes Proliferation and Differentiation of Hematopoietic Stem Cells." Blood 112, no. 11 (November 16, 2008): 4777. http://dx.doi.org/10.1182/blood.v112.11.4777.4777.
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Abstract In the bone marrow hematopoietic stem cells (HSCs) reside in specialized niches in close contact with stromal cells and endosteal osteoblasts. It is thought that this environment is hypoxic in nature, where HSCs are maintained in a quiescent state to prevent their depletion. Hypoxia stabilizes the transcription factor HIF-1α which triggers angiogenesis as well as genes slowering the cell cycle, promoting cell survival, and leading to a decrease in cellular metabolism. In this study, hypoxic effects of the maintenance of Lin−Sca1+c-kit+* (LSK) cells derived from mouse bone marrow and the involvement of the transcription factor hypoxia inducible factor 1 α (HIF-1α) were investigated. Hypoxic culture conditions led to an increase in numbers of primitive colony-forming progenitor cells and a preferential expansion of immature blast-like appearing cells. Concurrently, the immature c-kit Sca-1 phenotype was better maintained in hypoxia compared to ambient oxygen levels. Moreover, hypoxia decreased the proliferation of HSCs as measured by CFSE or PKH26 staining. This was confirmed by cell cycle analysis, and hypoxic cultivation decreased the percentage of cells in S-phase whereas cells in G0/G1 phase increased. Cells infected with a constitutively active form of HIF-1α showed the same pattern as cells cultured in hypoxia. To verify that the effect is HIF-1α mediated, we silenced HIF-1α in LSK cells with shRNA. The decrease in proliferation in hypoxic cultivation of cells infected with shRNA against HIF-1α was markedly diminished, indicating that HIF-1α play an important role in controlling proliferation of hematopoietic stem cells. These results suggest that a major function of hypoxia is to counteract proliferation and possibly differentiation, thereby sustaining maintenance. Furthermore, hypoxic culture conditions may have beneficial clinical implications for ex vivo purposes and may improve the yields of stem cells. In our ongoing-studies, we are investigating whether HIF-1α and hypoxia is an absolute prerequisite for the proper maintenance of HSCs in the bone marrow.
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Kugeratski, Fernanda G., Samuel J. Atkinson, Lisa J. Neilson, Sergio Lilla, John R. P. Knight, Jens Serneels, Amelie Juin, et al. "Hypoxic cancer–associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling." Science Signaling 12, no. 567 (February 5, 2019): eaan8247. http://dx.doi.org/10.1126/scisignal.aan8247.
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Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry–based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs.
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Sedivy, Vojtech, Shreena Joshi, Youssef Ghaly, Roman Mizera, Marie Zaloudikova, Sean Brennan, Jana Novotna, Jan Herget, and Alison M. Gurney. "Role of Kv7 channels in responses of the pulmonary circulation to hypoxia." American Journal of Physiology-Lung Cellular and Molecular Physiology 308, no. 1 (January 1, 2015): L48—L57. http://dx.doi.org/10.1152/ajplung.00362.2013.
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Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K+channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3–5 days in an isobaric hypoxic chamber (FiO2= 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease.
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Kroon, Marielle E., Pieter Koolwijk, Bea van der Vecht, and Victor W. M. van Hinsbergh. "Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix." Blood 96, no. 8 (October 15, 2000): 2775–83. http://dx.doi.org/10.1182/blood.v96.8.2775.
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Abstract Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor–2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor–α (TNF-α) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-α was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the αv-integrin subunit was enhanced by hypoxia, blocking antibodies against αvβ3- and αvβ5-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process.
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Kroon, Marielle E., Pieter Koolwijk, Bea van der Vecht, and Victor W. M. van Hinsbergh. "Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix." Blood 96, no. 8 (October 15, 2000): 2775–83. http://dx.doi.org/10.1182/blood.v96.8.2775.h8002775_2775_2783.
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Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor–2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor–α (TNF-α) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-α was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the αv-integrin subunit was enhanced by hypoxia, blocking antibodies against αvβ3- and αvβ5-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process.
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Rehn, Matilda, André Olsson, Kristian Reckzeh, Eva Diffner, Peter Carmeliet, Göran Landberg, and Jörg Cammenga. "Hypoxic induction of vascular endothelial growth factor regulates murine hematopoietic stem cell function in the low-oxygenic niche." Blood 118, no. 6 (August 11, 2011): 1534–43. http://dx.doi.org/10.1182/blood-2011-01-332890.
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Abstract Hypoxia is emerging as an important characteristic of the hematopoietic stem cell (HSC) niche, but the molecular mechanisms contributing to quiescence, self-renewal, and survival remain elusive. Vascular endothelial growth factor A (VEGFA) is a key regulator of angiogenesis and hematopoiesis. Its expression is commonly regulated by hypoxia-inducible factors (HIF) that are functionally induced in low-oxygen conditions and that activate transcription by binding to hypoxia-response elements (HRE). Vegfa is indispensable for HSC survival, mediated by a cell-intrinsic, autocrine mechanism. We hypothesized that a hypoxic HSC microenvironment is required for maintenance or up-regulation of Vegfa expression in HSCs and therefore crucial for HSC survival. We have tested this hypothesis in the mouse model Vegfaδ/δ, where the HRE in the Vegfa promoter is mutated, preventing HIF binding. Vegfa expression was reduced in highly purified HSCs from Vegfaδ/δ mice, showing that HSCs reside in hypoxic areas. Loss of hypoxia-regulated Vegfa expression increases the numbers of phenotypically defined hematopoietic stem and progenitor cells. However, HSC function was clearly impaired when assessed in competitive transplantation assays. Our data provide further evidence that HSCs reside in a hypoxic microenvironment and demonstrate a novel way in which the hypoxic niche affects HSC fate, via the hypoxia-VEGFA axis.
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Sheares, Karen K. K., Trina K. Jeffery, Lu Long, Xudong Yang, and Nicholas W. Morrell. "Differential effects of TGF-β1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 287, no. 5 (November 2004): L919—L927. http://dx.doi.org/10.1152/ajplung.00012.2004.
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Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Previously, we demonstrated that hypoxia modulates the proliferation of human peripheral pulmonary artery smooth muscle cells (PASMCs) by induction of cyclooxygenase-2 (COX-2) and production of antiproliferative prostaglandins ( 55 ). The transforming growth factor (TGF)-β superfamily plays a critical role in the regulation of pulmonary vascular remodeling, although to date an interaction with hypoxia has not been examined. We therefore investigated the pathways involved in the hypoxic induction of COX-2 in peripheral PASMCs and the contribution of TGF-β1 and bone morphogenetic protein (BMP)-4 in this response. In the present study, we demonstrate that hypoxia induces activation of p38MAPK, ERK1/2, and Akt in PASMCs and that these pathways are involved in the hypoxic regulation of COX-2. Whereas inhibition of p38MAPK or ERK1/2 activity suppressed hypoxic induction of COX-2, inhibition of the phosphoinositide 3-kinase pathway enhanced hypoxic induction of COX-2. Furthermore, exogenous TGF-β1 induced COX-2 mRNA and protein expression, and our findings demonstrate that release of TGF-β1 by PASMCs during hypoxia contributes to the hypoxic induction of COX-2 via the p38MAPK pathway. In contrast, BMP-4 inhibited the hypoxic induction of COX-2 by an MAPK-independent pathway. Together, these findings suggest that the TGF-β superfamily is part of an autocrine/paracrine system involved in the regulation of COX-2 expression in the distal pulmonary circulation, and this modulates hypoxia-induced pulmonary vascular cell proliferation.
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Emerling, Brooke M., Leonidas C. Platanias, Emma Black, Angel R. Nebreda, Roger J. Davis, and Navdeep S. Chandel. "Mitochondrial Reactive Oxygen Species Activation of p38 Mitogen-Activated Protein Kinase Is Required for Hypoxia Signaling." Molecular and Cellular Biology 25, no. 12 (June 15, 2005): 4853–62. http://dx.doi.org/10.1128/mcb.25.12.4853-4862.2005.
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ABSTRACT Mammalian cells have the ability to sense low oxygen levels (hypoxia). An adaptive response to hypoxia involves the induction of the transcription factor hypoxia-inducible factor 1 (HIF-1). The intracellular signaling pathways that regulate HIF-1 activation during hypoxia remain unknown. Here, we demonstrate that p38α − / − cells fail to activate HIF-1 under hypoxic conditions. Cells deficient in Mkk3 and Mkk6, the upstream regulators of p38α, also fail to activate HIF-1 under hypoxic conditions. The p38α − / − cells are able to activate HIF-1 in response to anoxia or iron chelators during normoxia. Furthermore, the hypoxic activation of p38α and HIF-1 was abolished by myxothiazol, a mitochondrial complex III inhibitor, and glutathione peroxidase 1 (GPX1), a scavenger of hydrogen peroxide. Thus, the activation of p38α and HIF-1 is dependent on the generation of mitochondrial reactive oxygen species. These results provide genetic evidence that p38 mitogen-activated protein kinase signaling is essential for HIF-1 activation.
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Kenneth, Niall Steven, and Sonia Rocha. "Regulation of gene expression by hypoxia." Biochemical Journal 414, no. 1 (July 29, 2008): 19–29. http://dx.doi.org/10.1042/bj20081055.
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Hypoxia induces profound changes in the cellular gene expression profile. The discovery of a major transcription factor family activated by hypoxia, HIF (hypoxia-inducible factor), and the factors that contribute to HIF regulation have greatly enhanced our knowledge of the molecular aspects of the hypoxic response. However, in addition to HIF, other transcription factors and cellular pathways are activated by exposure to reduced oxygen. In the present review, we summarize the current knowledge of how additional hypoxia-responsive transcription factors integrate with HIF and how other cellular pathways such as chromatin remodelling, translation regulation and microRNA induction, contribute to the co-ordinated cellular response observed following hypoxic stress.
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O’Reilly, Susan M., Martin O. Leonard, Niamh Kieran, Katrina M. Comerford, Eoin Cummins, Marc Pouliot, Sean B. Lee, and Cormac T. Taylor. "Hypoxia induces epithelial amphiregulin gene expression in a CREB-dependent manner." American Journal of Physiology-Cell Physiology 290, no. 2 (February 2006): C592—C600. http://dx.doi.org/10.1152/ajpcell.00278.2005.
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Hypoxia occurs during a number of conditions in which altered epithelial proliferation is critical, including tumor development. Microarray analysis of colon-derived epithelial cells revealed a hypoxia-dependent increase in the expression of amphiregulin, an EGF receptor (EGFR) ligand that activates epithelial proliferation and has been associated with the development of colonic tumors. Amphiregulin expression was also induced in tissues from mice exposed to whole animal hypoxia. The hypoxic upregulation of amphiregulin was independent of the classic transcriptional response mediated via hypoxia-inducible factor (HIF)-1α. Transfection of HeLa cells with truncated amphiregulin promoter reporter constructs revealed that a 37-bp segment upstream from the TATA box retained hypoxic sensitivity. This sequence contains an evolutionarily conserved cAMP response element (CRE) that constitutively binds the CRE binding protein (CREB). Deletion of the CRE abolished sensitivity to hypoxia. Thus hypoxia promotes intestinal epithelial amphiregulin expression in a CRE-dependent manner, an event that may contribute to increased proliferation. These data also further support a role for CREB as an HIF-independent hypoxia-responsive transcription factor in the regulation of intestinal epithelial gene expression.
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Gleadle, Jonathan M., and Peter J. Ratcliffe. "Induction of Hypoxia-Inducible Factor-1, Erythropoietin, Vascular Endothelial Growth Factor, and Glucose Transporter-1 by Hypoxia: Evidence Against a Regulatory Role for Src Kinase." Blood 89, no. 2 (January 15, 1997): 503–9. http://dx.doi.org/10.1182/blood.v89.2.503.
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Abstract The induction by hypoxia of genes such as erythropoietin, vascular endothelial growth factor (VEGF ), and glucose transporter-1 (Glut-1) is mediated in part by a transcriptional complex termed hypoxia-inducible factor-1 (HIF-1). Several lines of evidence have implicated protein phosphorylation in the mechanism of activation of HIF-1 by hypoxia. Recent reports have described the activation of the tyrosine kinase src by severe hypoxia, and a role in the induction of VEGF by severe hypoxia has been proposed. This led us to examine whether src and related kinases operated more widely in the hypoxic induction of HIF-1 and HIF-1–dependent genes regulated by hypoxia. Measurements of src kinase activity in cells exposed to varying severities of hypoxia showed activation by severe hypoxia (0.1% oxygen or catalyst induced anoxia), but not 1% oxygen. This contrasted with the marked induction of HIF-1 by exposure to 1% oxygen. Manipulations of src activity were produced by transient and stable transfection of Hep3B cells. Despite substantial changes in src activity, no alteration was seen in the normoxic or hypoxic expression of erythropoietin, VEGF, or Glut-1, or in the regulation of HIF-1–dependent reporter genes inducible by hypoxia. Similarly, we found that the expression of these genes in src- or c-src kinase-deficient cells did not differ from wild-type cells at either 1% oxygen or more severe hypoxia. These results indicate that src is not critical for the hypoxic induction of HIF-1, erythropoietin, VEGF, or Glut-1.
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Zhang, Junhui, Geoffrey Thomas Gibney, Peng Zhao, and Ying Xia. "Neuroprotective role of δ-opioid receptors in cortical neurons." American Journal of Physiology-Cell Physiology 282, no. 6 (June 1, 2002): C1225—C1234. http://dx.doi.org/10.1152/ajpcell.00226.2001.
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We recently demonstrated that δ-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons ( days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([d-Ala2,d-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) μ- or κ-opioid receptor inhibition (β-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.
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Deng, Fan, Shuang Wang, Riping Xu, Wenqian Yu, Xianyu Wang, and Liangqing Zhang. "Endothelial microvesicles in hypoxic hypoxia diseases." Journal of Cellular and Molecular Medicine 22, no. 8 (May 29, 2018): 3708–18. http://dx.doi.org/10.1111/jcmm.13671.
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Lei, Yan, Xiaoyong Peng, Yi Hu, Mingying Xue, Tao Li, Liangming Liu, and Guangming Yang. "The Calcilytic Drug Calhex-231 Ameliorates Vascular Hyporesponsiveness in Traumatic Hemorrhagic Shock by Inhibiting Oxidative Stress and miR-208a-Mediated Mitochondrial Fission." Oxidative Medicine and Cellular Longevity 2020 (December 3, 2020): 1–13. http://dx.doi.org/10.1155/2020/4132785.
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Background. The calcium-sensing receptor (CaSR) plays a fundamental role in extracellular calcium homeostasis in humans. Surprisingly, CaSR is also expressed in nonhomeostatic tissues and is involved in regulating diverse cellular functions. The objective of this study was to determine if Calhex-231 (Cal), a negative modulator of CaSR, may be beneficial in the treatment of traumatic hemorrhagic shock (THS) by improving cardiovascular function and investigated the mechanisms. Methods. Rats that had been subjected to THS and hypoxia-treated vascular smooth muscle cells (VSMCs) were used in this study. The effects of Cal on cardiovascular function, animal survival, hemodynamics, and vital organ function in THS rats and the relationship to oxidative stress, mitochondrial fusion-fission, and microRNA (miR-208a) were investigated. Results. Cal significantly improved hemodynamics, elevated blood pressure, increased vital organ blood perfusion and local oxygen supply, and markedly improved the survival outcomes of THS rats. Furthermore, Cal significantly improved vascular reactivity after THS in vivo and in vitro. Cal also restored the THS-induced decrease in myosin light chain (MLC) phosphorylation (the key element for VSMC contraction). Inhibition of MLC phosphorylation antagonized the Cal-induced restoration of vascular reactivity following THS. Cal suppressed oxidative stress in THS rats and hypoxic-VSMCs. Meanwhile, THS induced expression of mitochondrial fission proteins Drp1 and Fis1 and decreased expression of mitochondrial fusion protein Mfn1 in vascular tissues. Cal reduced expression of Drp1 and Fis1. In hypoxic-VSMCs, Cal inhibited mitochondrial fragmentation and preserved mitochondrial morphology. In addition, miR-208a mimic decreased Fis1 expression, and miR-208a inhibitor prevented Cal-induced Fis1 downregulation in hypoxic-VSMCs. Conclusion. Calhex-231 exhibits outstanding potential for effective therapy of traumatic hemorrhagic shock, and the beneficial effects result from its protection of vascular function via inhibition of oxidative stress and miR-208a-mediated mitochondrial fission.
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Minamino, T., S. A. Mitsialis, and S. Kourembanas. "Hypoxia Extends the Life Span of Vascular Smooth Muscle Cells through Telomerase Activation." Molecular and Cellular Biology 21, no. 10 (May 15, 2001): 3336–42. http://dx.doi.org/10.1128/mcb.21.10.3336-3342.2001.
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ABSTRACT Chronic hypoxia induces smooth muscle cell proliferation and vessel wall remodeling in the vasculature of the lung. One well-characterized component of the hypoxic response is transcriptional activation of genes encoding vascular smooth muscle cell (VSMC) mitogens. We report here that chronic hypoxia can also prolong the growth of human VSMC by inducing telomerase activity and telomere stabilization. We demonstrate that hypoxia induced phosphorylation of the telomerase catalytic component (TERT) and sustained high levels of TERT protein expression in VSMC compared to normoxia. Furthermore, inhibition of telomerase shortened cell life span in hypoxic cultures, whereas constitutive expression of TERT extended the life span of cells under normoxic conditions. Our data indicate that hypoxic induction of telomerase activity could be involved in long-term growth of VSMC and may thus contribute to human vascular disorders.
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Asosingh, Kewal, Hendrik De Raeve, Mark de Ridder, Guy A. Storme, Angelo Willems, Ivan Van Riet, Benjamin Van Camp, and Karin Vanderkerken. "Role of the Hypoxic Bone Marrow Microenvironment in Multiple Myeloma Tumor Progression." Blood 104, no. 11 (November 16, 2004): 2348. http://dx.doi.org/10.1182/blood.v104.11.2348.2348.
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Abstract Recently we reported that pre-clinical myeloma disease progression in the 5T2MM mouse model is characterized by predominant CD45+ MM-cells in the early, pre-angiogenic stage stage of slow tumor progression, followed by expansion of CD45− MM-cells during the subsequent angiogenic stage of progressive tumor growth. Unlike other cancer cells, multiple myeloma (MM) cells have to survive and to grow in a microenvironment which is already hypoxic by nature. This hypoxic bone marrow (BM) microenvironment is essential for normal hematopoiesis. However, the role of BM hypoxia in myeloma tumor progression is not known. Herein we addressed this topic in the 5T2MM mouse model. Flow cytometric analysis of control mice and 5T2MM diseased mice injected with pimonidazole hypoxyprobe indicated that both normal BM and myeloma infiltrated BM are hypoxic. However, in myelomatous BM the hypoxia was significantly decreased. Analysis of HIF-1a expression, a surrogate marker of hypoxia, by flow cytometry also demonstrated significantly lower levels of hypoxia in myeloma infiltrated BM. HIF-1a expression was found in 5T2MM-cells and was significantly higher compared to the non-tumor cell fraction. In vitro culturing of 5T2MM cells under hypoxic conditions, indicated increased activation of apoptosis inducing caspase-3 in the CD45− MM-fraction, but not in the CD45+ 5T2MM-cells, suggesting that native BM hypoxia selects the tumor population for tumor initiating CD45+ 5T2MM-cells. Although angiogeneic switch and angiogeneic heterogeneity has been reported in MM, the role of myeloma associated angiogensis is remains unclear. The decreased hypoxia in myeloma infiltrated BM adds strength to the hypothesis that myeloma associated neovascularization is functional by increasing BM oxygenation. The data also suggest that the angiogenesis allows expansion of CD45− 5T2MM-cells by decreasing BM hypoxia. All together, these findings suggest an important role of BM hypoxia in myeloma tumor progression.
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Watt, Suzanne M., Sarah Hale, Dilair Baban, Maria Roubelakis, Meis Moukayed, Jaeseung Lim, Dacey J. Ryan, Kay Davies, Adrian L. Harris, and Enca Martin-Rendon. "The Centromeric Protein, CEN(P)-F, a Marker of Cell Proliferation Is Regulated by Hypoxia in Human Mesenchymal Stem Cells and Their Bone Marrow Stromal Progeny." Blood 106, no. 11 (November 16, 2005): 1385. http://dx.doi.org/10.1182/blood.v106.11.1385.1385.
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Abstract Human bone marrow mesenchymal stem cells (MSC) are multipotent progenitors that generate osteoblasts, chondrocytes, adipocytes, myoblasts and the bone marrow stromal cells that support hematopoiesis. Although the bone marrow microenvironment is hypoxic, little is known about the maintenance and response of MSC and their bone marrow stromal progeny to hypoxia. Using cDNA microarray hybridization technologies, we show, for the first time, that a total of 231 mRNAs in cultured MSCs are regulated by short exposures (4–48hrs) to hypoxia. These include known hypoxia-responsive genes, such as BHLHB2, PGK1, GLUT-1 and VEGF. Interestingly, we demonstrate that a significant proportion of genes involved in cell growth, proliferation or survival are also regulated by hypoxia in these cells. Amongst these, we have identified the centromeric protein CENP-F as a novel gene up-regulated in cultured MSCs by hypoxia. This up-regulation results in an increased level of CENP-F protein. The hypoxic stimulus also enhances cell division in the bone marrow MSC-derived stromal cells, resulting in a doubling in cell number within 24hrs. This contrasts with the effects of hypoxia on mature endothelial cells (EC), where recruitment into cell cycle is unchanged. Our demonstration that hypoxia promotes cell division and cell proliferation in the bone marrow stromal progeny of human MSC may have wider implications for the regulated growth and survival of other stem cells in hypoxic microenvironmental niches.
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Fuhrmann, Dominik C., Michaela Tausendschön, Ilka Wittig, Mirco Steger, Martina G. Ding, Tobias Schmid, Nathalie Dehne, and Bernhard Brüne. "Inactivation of Tristetraprolin in Chronic Hypoxia Provokes the Expression of Cathepsin B." Molecular and Cellular Biology 35, no. 3 (December 1, 2014): 619–30. http://dx.doi.org/10.1128/mcb.01034-14.
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Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.
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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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Jiang, Yide, Michael J. Vasconcelles, Sharon Wretzel, Anne Light, Charles E. Martin, and Mark A. Goldberg. "MGA2 Is Involved in the Low-Oxygen Response Element-Dependent Hypoxic Induction of Genes inSaccharomyces cerevisiae." Molecular and Cellular Biology 21, no. 18 (September 15, 2001): 6161–69. http://dx.doi.org/10.1128/mcb.21.18.6161-6169.2001.
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ABSTRACT Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example,OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-inducedOLE1 expression, we examined OLE1expression under normoxia, hypoxia, and cobalt treatment conditions in Δmga2 or Δspt23 deletion strains. The results of OLE1promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Δmga2 strain but not in a Δspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Δmga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Δmga2 strain. These results indicate thatMGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.
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Brown, Rachel C., Karen S. Mark, Richard D. Egleton, and Thomas P. Davis. "Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium." American Journal of Physiology-Cell Physiology 286, no. 5 (May 2004): C1045—C1052. http://dx.doi.org/10.1152/ajpcell.00360.2003.
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Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity.
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Bell, Eric L., Tatyana A. Klimova, James Eisenbart, Paul T. Schumacker, and Navdeep S. Chandel. "Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Inducible Factor-Dependent Extension of the Replicative Life Span during Hypoxia." Molecular and Cellular Biology 27, no. 16 (June 11, 2007): 5737–45. http://dx.doi.org/10.1128/mcb.02265-06.
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ABSTRACT Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span.
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Zeng, Chih-Wei, Jin-Chuan Sheu, and Huai-Jen Tsai. "Hypoxia-Responsive Subtype Cells Differentiate Into Neurons in the Brain of Zebrafish Embryos Exposed to Hypoxic Stress." Cell Transplantation 31 (January 2022): 096368972210779. http://dx.doi.org/10.1177/09636897221077930.
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Severe hypoxia results in complete loss of central nervous system (CNS) function in mammals, while several other vertebrates, such as zebrafish, can regenerate after hypoxia-induced injury of CNS. Since the cellular mechanism involved in this remarkable feature of other vertebrates is still unclear, we studied the cellular regeneration of zebrafish brain, employing zebrafish embryos from transgenic line huORFZ exposed to hypoxia and then oxygen recovery. GFP-expressing cells, identified in some cells of the CNS, including some brain cells, were termed as hypoxia-responsive recovering cells (HrRCs). After hypoxia, HrRCs did not undergo apoptosis, while most non-GFP-expressing cells, including neurons, did. Major cell types of HrRCs found in the brain of zebrafish embryos induced by hypoxic stress were neural stem/progenitor cells (NSPCs) and radial glia cells (RGs), that is, subtypes of NSPCs (NSPCs-HrRCs) and RGs (RGs-HrRCs) that were induced by and sensitively responded to hypoxic stress. Interestingly, among HrRCs, subtypes of NSPCs- or RGs-HrRCs could proliferate and differentiate into early neurons during oxygen recovery, suggesting that these subtype cells might play a critical role in brain regeneration of zebrafish embryos after hypoxic stress.