The Perspective of Using Flow Cytometry for Unpuzzling Hypoxia-Inducible Factors Signalling

Abstract

Hypoxia-inducible factors (HIFs) are transcription factors that are responsible for adapting to the changes in oxygen levels in the cellular environment. HIF activity determines the expression of cellular proteins that control the development and physiology of the cells and pathophysiology of a disease. Understanding the role of specific HIF (HIF-1-3) in cellular function is essential for development of the HIF-targeted therapies. In this review, we have discussed the use of flow cytometry in analysing HIF function in cells. Proper understanding of HIF-signalling will help to design pharmacological interventions HIF-mediated therapy. We have discussed the role of HIF-signalling in various diseases such as cancer, renal and liver diseases, ulcerative colitis, arthritis, diabetes and diabetic complications, psoriasis, and wound healing. We have also discussed protocols that help to decipher the role of HIFs in these diseases that would eventually help to design promising therapies.

Keywords

flow cytometry - hypoxia inducible factors signalling

Publication History

Received: 11 November 2023

Accepted: 15 January 2024

Article published online:
13 February 2024

• References

• 1 Cossarizza A, Chang HD, Radbruch A. et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol 2017; 47: 1584-1797
• 2 Verschoor CP, Lelic A, Bramson JL. et al. An introduction to automated flow cytometry gating tools and their implementation. Front Immunol 2015; 6: 380
• 3 McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol 2018; 120 5.1
• 4 Shapiro HM. Practical Flow Cytometry, 3rd ed. Wiley; 2003
• 5 Flow Cytometry History. In Internet: Available from: http://www.cyto.purdue.edu/cdroms/cyto5/sponsors/cytomate/flowhist [Last accessed: 01.04.2024].
• 6 Flow Cytometers | Biocompare.com. In Internet: Available from: https://www.biocompare.com/Lab-Equipment/12328-Flow-Cytometer-Flow-Cytometry-Systems-FACS-Machine/ [Last accessed:01.04.2024].
• 7 Cell Sorters / Cell Sorting Instruments | Biocompare. In Internet: Available from: https://www.biocompare.com/8005-Cell-Sorting/24500-Cell-Sorters-Cell-Sorting-Instruments [Last accessed: 01.04.2024].
• 8 Saeys Y, Van Gassen S, Lambrecht BN. Computational flow cytometry: helping to make sense of high-dimensional immunology data. Nat Rev Immunol 2016; 16: 449-462
• 9 Gadalla R, Noamani B, MacLeod BL. et al. Validation of CyTOF against flow cytometry for immunological studies and monitoring of human cancer clinical trials. Front Oncol 2019; 9: 415
• 10 Applications of Flow Cytometry. In Internet: Available from: https://www.seattlechildrens.org/research/resources/flow-cytometry/applications-of-flow-cytometry/ [Last accessed:04.01.2024].
• 11 Young NA, Al-Saleem T. Lymph nodes: Cytomorphology and flow cytometry, Comprehensive Cytopathology 2008; 25: 545–580.
• 12 Barese C, Waring M, Pfeifer R. et al. Flow Cytometry Assay to Monitor Effectiveness of Gene Therapy Correction in Peripheral Blood Mononuclear Cells of Gaucher Disease Type I Patients. Blood 2018; 132: 5791
• 13 Yang SL, Wu C, Xiong ZF. et al. Progress on hypoxia-inducible factor-3: Its structure, gene regulation and biological function (Review). Mol Med Rep 2015; 12: 2411-2416
• 14 Joharapurkar AA, Pandya VB, Patel VJ. et al. Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases. J Med Chem 2018; 61: 6964-6982
• 15 Chowdhury R, Leung IKH, Tian YM. et al Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nature Communications 2016 7:1 2016; 7: 1-10
• 16 Appelhoffl RJ, Tian YM, Raval RR. et al. Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem 2004; 279: 38458-38465
• 17 Smirnova NA, Hushpulian DM, Speer RE. et al. Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases. Biochemistry (Moscow) 2012; 77: 1108-1119
• 18 Cockman ME, Lippl K, Tian YM. et al. Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates. Elife 2019; 8: e46490
• 19 Usategui-Martín R, Rigual R, Ruiz-Mambrilla M. et al. Molecular Mechanisms Involved in Hypoxia-Induced Alterations in Bone Remodeling. Int J Mol Sci 2022; 23: 3233
• 20 Janssens LK, Stove CP. Sensing an Oxygen Sensor: Development and Application of Activity-Based Assays Directly Monitoring HIF Heterodimerization. Anal Chem 2021; 93: 14462-14470
• 21 Joharapurkar AA, Patel VJ, Kshirsagar SG. et al. Prolyl hydroxylase inhibitor desidustat improves anemia in erythropoietin hyporesponsive state. Current Research in Pharmacology and Drug Discovery 2022; 3: 100102
• 22 Jiang X, Gao M, Chen Y. et al. EPO-dependent induction of erythroferrone drives hepcidin suppression and systematic iron absorption under phenylhydrazine-induced hemolytic anemia. Blood Cells Mol Dis 2016; 58: 45-51
• 23 Postovalova EA, Makarova OV, Kosyreva AM. et al. Hypoxia is a key mechanism for regulating inflammation in ulcerative colitis. Russian Open Medical Journal 2020; 9 DOI: 10.15275/rusomj.2020.0101.
• 24 Zhu WJ, Li P, Wang L. et al. Hypoxia-inducible factor-1: A potential pharmacological target to manage psoriasis. Int Immunopharmacol 2020; 86: 106689
• 25 Tashiro N, Segawa R, Tobita R. et al. Hypoxia inhibits TNF-α-induced TSLP expression in keratinocytes. PLoS One 2019; 14: e0224705
• 26 Manresa MC, Smith L, Casals-Diaz L. et al. Pharmacologic inhibition of hypoxia-inducible factor (HIF)-hydroxylases ameliorates allergic contact dermatitis. Allergy 2019; 74: 753-766
• 27 Sharma A, Sinha S, Shrivastava N. Therapeutic Targeting Hypoxia-Inducible Factor (HIF-1) in Cancer: Cutting Gordian Knot of Cancer Cell Metabolism. Front Genet 2022; 13: 673
• 28 Kaelin WG. Von Hippel-Lindau disease: insights into oxygen sensing, protein degradation, and cancer. J Clin Invest 2022; 132: e162480
• 29 Pojani E, Barlocco D. Romidepsin (FK228), A Histone Deacetylase Inhibitor and its Analogues in Cancer Chemotherapy. Curr Med Chem 2021; 28: 1290-1303
• 30 Rodríguez-Jiménez FJ, Moreno-Manzano V. Modulation of hypoxia-inducible factors (HIF) from an integrative pharmacological perspective. Cellular and Molecular Life Sciences 2011; 69: 519-534
• 31 Brugarolas J, Beckermann K, Rini BI. et al. Initial results from the phase 1 study of ARO-HIF2 to silence HIF2-alpha in patients with advanced ccRCC (AROHIF21001). Journal of Clinical Oncology 2022; 40: 339-339
• 32 Arcus Biosciences | Pursuit of Cures for Cancer. In Internet: Available from: https://arcusbio.com/our-science/clinical-candidates/ab521/ [Last accessed: 01.04.2024].
• 33 Kim MH, Lee TH, Lee JS. et al. Hif-1α Inhibitors Could Successfully Inhibit the Progression of Differentiated Thyroid Cancer in Vitro. Pharmaceuticals 2020; 13: 208
• 34 Mylonis I, Simos G, Paraskeva E. Hypoxia-inducible factors and the regulation of lipid metabolism. Cells 2019; 8: 214
• 35 Kerber EL, Padberg C, Koll N. et al. The Importance of Hypoxia-Inducible Factors (HIF-1 and HIF-2) for the Pathophysiology of Inflammatory Bowel Disease. Int J Mol Sci 2020; 21: 1-27
• 36 Marks E, Goggins BJ, Cardona J. et al. Oral delivery of prolyl hydroxylase inhibitor: AKB-4924 promotes localized mucosal healing in a mouse model of colitis. Inflamm Bowel Dis 2015; 21: 267-275
• 37 Thompson AAR, Elks PM, Marriott HM. et al. Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish. Blood 2014; 123: 366-376
• 38 Zhuang H, Lv Q, Zhong C. et al. Tiliroside Ameliorates Ulcerative Colitis by Restoring the M1/M2 Macrophage Balance via the HIF-1α/glycolysis Pathway. Front Immunol 2021; 12: 649463
• 39 Hu Y, Zhang T, Chen J. et al. Downregulation of Hypoxia-Inducible Factor-1α by RNA Interference Alleviates the Development of Collagen-Induced Arthritis in Rats. Mol Ther Nucleic Acids 2020; 19: 1330-1342
• 40 Tambuwala MM, Cummins EP, Lenihan CR. et al. Loss of prolyl hydroxylase-1 protects against colitis through reduced epithelial cell apoptosis and increased barrier function. Gastroenterology 2010; 139: 2093-2101
• 41 Khalil E, Gomaa A, Ismail N. et al. The Expression level of hypoxia inducible factor 1α gene in psoriatic patients in Suez Canal region. Journal of Pakistan Association of Dermatologists 2021; 31: 574-582
• 42 Agamia NF, Sorror OA, Sayed NM. et al. Overexpression of hypoxia-inducible factor-1α in hidradenitis suppurativa: the link between deviated immunity and metabolism. Arch Dermatol Res 2023; 315: 2107-2118
• 43 Feng R, Mayuranathan T, Huang P. et al Activation of γ-globin expression by hypoxia-inducible factor 1α. Nature 2022; 610:7933 2022; 610: 783-790
• 44 Kaul DK, Fabry ME, Suzuka SM. et al. Antisickling fetal hemoglobin reduces hypoxia-inducible factor-1 α expression in normoxic sickle mice: Microvascular implications. Am J Physiol Heart Circ Physiol 2013; 304: 42-50
• 45 Das N, Xie L, Ramakrishnan SK. et al. Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease. J Biol Chem 2015; 290: 23523
• 46 Pedrosa AM, Lemes RPG. Gene expression of HIF-1α and VEGF in response to hypoxia in sickle cell anaemia: Influence of hydroxycarbamide. Br J Haematol 2020; 190: e39-e42
• 47 Meléndez-Rodríguez F, Urrutia AA, Lorendeau D. et al. HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis. Cell Rep 2019; 26: 2257-2265.e4
• 48 Imamura T, Kikuchi H, Herraiz MT. et al. HIF-1α and HIF-2α have divergent roles in colon cancer. Int J Cancer 2009; 124: 763-761
• 49 Ohh M, Taber CC, Ferens FG. et al. Hypoxia-inducible factor underlies von Hippel-Lindau disease stigmata. Elife 2022; 11: e80774
• 50 Iommarini L, Porcelli AM, Gasparre G. et al. Non-canonical mechanisms regulating hypoxia-inducible factor 1 alpha in cancer. Front Oncol 2017; 7: 286
• 51 Zhang Q, Han Z, Zhu Y. et al. Role of hypoxia inducible factor-1 in cancer stem cells (Review). Mol Med Rep 2021; 23: 17
• 52 Semenza GL. Targeting HIF-1 for cancer therapy. Nature Reviews Cancer 2003 3:10 2003; 3: 721-732
• 53 Yu T, Tang B, Sun X. Development of Inhibitors Targeting Hypoxia-Inducible Factor 1 and 2 for Cancer Therapy. Yonsei Med J 2017; 58: 489
• 54 Eckle T, Brodsky K, Bonney M. et al. HIF1A reduces acute lung injury by optimizing carbohydrate metabolism in the alveolar epithelium. PLoS Biol 2013; 11: e1001665
• 55 Brusselmans K, Compernolle V, Tjwa M. et al. Heterozygous deficiency of hypoxia-inducible factor-2alpha protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia. J Clin Invest 2003; 111: 1519-1527
• 56 Burman A, Kropski JA, Calvi CL. et al. Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein. JCI Insight 2018; 3: e99543
• 57 Yu H, Li Q, Kolosov VP. et al. Regulation of cigarette smoke-mediated mucin expression by hypoxia-inducible factor-1α via epidermal growth factor receptor-mediated signaling pathways. J Appl Toxicol 2012; 32: 282-292
• 58 Sun Y, Xia T, Ma J. et al. IL-12 Contributes to the Development of Asthma by Targeting HIF-1α/NLRP3 Pathway through Runx3. Int Arch Allergy Immunol 2022; 183: 1231-1240
• 59 Catrina SB, Zheng X. Hypoxia and hypoxia-inducible factors in diabetes and its complications. Diabetologia 2021; 64: 709-716
• 60 Gunton JE. Hypoxia-inducible factors and diabetes. J Clin Invest 2020; 130: 5063
• 61 Hoppe G, Yoon S, Gopalan B. et al. Comparative systems pharmacology of HIF stabilization in the prevention of retinopathy of prematurity. Proc Natl Acad Sci U S A 2016; 113: E2516-E2525
• 62 Walmsley GG, Maan ZN, Hu MS. et al. Murine Dermal Fibroblast Isolation by FACS. J Vis Exp 2016; 2016: 53430
• 63 Bessho R, Takiyama Y, Takiyama T. et al. Hypoxia-inducible factor-1α is the therapeutic target of the SGLT2 inhibitor for diabetic nephropathy. Sci Rep 2019; 9: 14754
• 64 Takada A, Shibata T, Shiga T. et al. Pharmacokinetic/pharmacodynamic modeling of roxadustat’s effect on LDL cholesterol in patients in Japan with dialysis-dependent chronic kidney disease and anemia. Drug Metab Pharmacokinet 2022; 46: 100461
• 65 Becker KA, Jones JJ. An Emerging Treatment Alternative for Anemia in Chronic Kidney Disease Patients: A Review of Daprodustat. Adv Ther 2018; 35: 5-11
• 66 Pergola PE, Spinowitz BS, Hartman CS. et al. Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in nondialysis-dependent chronic kidney disease. Kidney Int 2016; 90: 1115-1122
• 67 Chen J, Chen J, Huang J. et al. HIF-2α upregulation mediated by hypoxia promotes NAFLD-HCC progression by activating lipid synthesis via the PI3K-AKT-mTOR pathway. Aging 2019; 11: 10839-10860
• 68 Zhang X, Zhang Y, Wang P. et al. Adipocyte Hypoxia-Inducible Factor 2α Suppresses Atherosclerosis by Promoting Adipose Ceramide Catabolism. Cell Metab 2019; 30: 937-951.e5
• 69 Cui Y, Guo C, Xia Z. et al. Exploring the therapeutic potential of a nano micelle containing a carbon monoxide-releasing molecule for metabolic-associated fatty liver disease by modulating hypoxia-inducible factor-1α. Acta Biomater 2023; 169: 500-516
• 70 Dickson AS, Pauzaite T, Arnaiz E. et al. A HIF independent oxygen-sensitive pathway for controlling cholesterol synthesis. Nat Commun 2023; 14: 4816
• 71 Gao F, Yao Q, Zhu J. et al. A novel HIF2A mutation causes dyslipidemia and promotes hepatic lipid accumulation. Pharmacol Res 2023; 194: 106851
• 72 Kobayashi H, Davidoff O, Pujari-Palmer S. et al. EPO synthesis induced by HIF-PHD inhibition is dependent on myofibroblast transdifferentiation and colocalizes with non-injured nephron segments in murine kidney fibrosis. Acta Physiol (Oxf) 2022; 235: e13826
• 73 Jaśkiewicz M, Moszyńska A, Króliczewski J. et al. The transition from HIF-1 to HIF-2 during prolonged hypoxia results from reactivation of PHDs and HIF1A mRNA instability. Cell Mol Biol Lett 2022; 27: 1-19
• 74 Liu Q, Davidoff O, Niss K. et al. Hypoxia-inducible factor regulates hepcidin via erythropoietin-induced erythropoiesis. J Clin Invest 2012; 122: 4635-4644
• 75 Volke M, Gale DP, Maegdefrau U. et al. Evidence for a Lack of a Direct Transcriptional Suppression of the Iron Regulatory Peptide Hepcidin by Hypoxia-Inducible Factors. PLoS One 2009; 4: e7875
• 76 Silva I, Rausch V, Peccerella T. et al. Hypoxia enhances H2O2-mediated upregulation of hepcidin: Evidence for NOX4-mediated iron regulation. Redox Biol 2018; 16: 1-10
• 77 Coffey R, Ganz T. Erythroferrone: An Erythroid Regulator of Hepcidin and Iron Metabolism. Hemasphere 2018; 2: e35
• 78 Schwartz AJ, Das NK, Ramakrishnan SK. et al. Hepatic hepcidin/intestinal HIF-2α axis maintains iron absorption during iron deficiency and overload. J Clin Invest 2019; 129: 336-348
• 79 Hong J, Lai J, Chen X. et al. The effects of hypoxia-inducible factors-1α and -2α and erythroferrone on hepcidin in patients with chronic kidney disease stages 3–5 and renal anemia. Eur J Inflamm 2022; 20 1721727X221103468
• 80 Packer M. Mutual Antagonism of Hypoxia-Inducible Factor Isoforms in Cardiac, Vascular, and Renal Disorders. Basic to Translational Science 2020; 5: 961-968
• 81 Salhiyyah K, Sarathchandra P, Latif N. et al. Hypoxia-mediated regulation of the secretory properties of mitral valve interstitial cells. Am J Physiol Heart Circ Physiol 2017; 313: H14-H23
• 82 Packer M. Mechanistic and Clinical Comparison of the Erythropoietic Effects of SGLT2 Inhibitors and Prolyl Hydroxylase Inhibitors in Patients With Chronic Kidney Disease and Renal Anemia. Am J Nephrol 2023; DOI: 10.1159/000531084.
• 83 Srivani G, Behera SK, Dariya B. et al. Resveratrol binds and inhibits transcription factor HIF-1α in pancreatic cancer. Exp Cell Res 2020; 394: 112126
• 84 McMahon S, Charbonneau M, Grandmont S. et al. Transforming growth factor beta1 induces hypoxia-inducible factor-1 stabilization through selective inhibition of PHD2 expression. J Biol Chem 2006; 281: 24171-24181
• 85 Gogate SS, Fujita N, Skubutyte R. et al. Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: Role of Hsp70 in HIF-1α degradation. Journal of Bone and Mineral Research 2012; 27: 1106-1117
• 86 Richard DE, Berra E, Pouyssegur J. Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 2000; 275: 26765-26771
• 87 Moeller BJ, Cao Y, Li CY. et al. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: Role of reoxygenation, free radicals, and stress granules. Cancer Cell 2004; 5: 429-441
• 88 Dehne N, Brüne B. HIF-1 in the inflammatory microenvironment. Exp Cell Res 2009; 315: 1791-1797
• 89 Qiao H, He X, Zhang Q. et al. Α-synuclein induces microglial cell migration through stimulating HIF-1α accumulation. J Neurosci Res 2017; 95: 1809-1817
• 90 Patel PS, Yang S, Li A. et al. Capsaicin regulates vascular endothelial cell growth factor expression by modulation of hypoxia inducing factor-1alpha in human malignant melanoma cells. J Cancer Res Clin Oncol 2002; 128: 461-468
• 91 Kuschel A, Simon P, Tug S. Functional regulation of HIF-1α under normoxia--is there more than post-translational regulation?. J Cell Physiol 2012; 227: 514-524
• 92 Ogryzko NV, Lewis A, Wilson HL. et al. Hif-1α–Induced Expression of Il-1β Protects against Mycobacterial Infection in Zebrafish. The Journal of Immunology 2019; 202: 494-502
• 93 Scharte M, Jurk K, Kehrel B. et al. IL-4 enhances hypoxia induced HIF-1α protein levels in human transformed intestinal cells. FEBS Lett 2006; 580: 6399-6404
• 94 Jung Y-J, Isaacs JS, Lee S. et al. IL-1beta-mediated up-regulation of HIF-1alpha via an NFkappaB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis. FASEB J 2003; 17: 1-22
• 95 Xing J, Lu J. HIF-1α activation attenuates IL-6 and TNF-α pathways in hippocampus of rats following transient global ischemia. Cellular Physiology and Biochemistry 2016; 39: 511-520
• 96 Huang Z, Zhou M, Wang Q. et al. Mechanical and hypoxia stress can cause chondrocytes apoptosis through over-activation of endoplasmic reticulum stress. Arch Oral Biol 2017; 84: 125-132
• 97 Zhang FJ, Luo W, Lei GH. Role of HIF-1α and HIF-2α in osteoarthritis. Joint Bone Spine 2015; 82: 144-147
• 98 Zhang D, Li J, Costa M. et al. JNK1 mediates degradation HIF-1alpha by a VHL-independent mechanism that involves the chaperones Hsp90/Hsp70. Cancer Res 2010; 70: 813-823
• 99 Zeng CY, Wang XF, Hua FZ. HIF-1α in Osteoarthritis: From Pathogenesis to Therapeutic Implications. Front Pharmacol 2022; 13: 927126
• 100 Venkatesh P. Hypo-angiogenesis: A possible pathological factor in the development of dry age-related macular degeneration and a novel therapeutic target. Medical Hypothesis, Discovery, and Innovation in Ophthalmology 2022; 10: 185-190