Regulation of oxygen consumption by local oxygen concentration in pre-vascular tissue spheroids

1. Regulation of oxygen consumption by local oxygen concentration in pre-vascular tissue spheroids Eric Krauland and Shawdee Eshghi December 12, 2002 BE.400

2. Motivations • Mammalian embryos are served by diffusion until implantantion in uterine wall • Embryos have adapted to survive in low oxygen conditions • Embryoid bodies serve as a good in vitro model of embryogenesis, recreating gastrulation, hematopoiesis, and angiogenesis • Pre-vascular tumors provide another model system to study tissue/cellular response to diffusional transport of oxygen

3. Embryoid Bodies Regulate O2 Consumption • EBs have adapted to O2 diffusion limitations • Average consumption of O2 decreases for larger EBs, suggesting active regulation of consumption Gassmann et al, PNAS 1996

4. Cellular Response to Local Oxygen concentration • Local O2 regulates expression of many genes, including Epo • Monolayers exposed to hypoxia have a lower cellular O2 consumption rate than normoxic cells Wolff et al, Am J Phsiol, 1993

5. HIF-1: A master regulator of the hypoxic response • Binds to erythropoietin DNA in hypoxic conditions • Implicated in upregulation of angiogenic, glycolytic, proliferative, cell adhesion, and stress-response genes • HIF-1 null embryos display vascularization defects Ryan et al, EMBO J, 1998

6. O2 HIF-1 Ub Ub Ub M atp atp atp atp atp atp atp atp atp HIF-1 Regulation of consumption? HIF-1 Mechanism low mitochondria M HIF-1 ARNT HIF-1 ARNT HRE

7. Questions to be addressed: • Do embryoid bodies and tumor spheroids sense O2 concentration and regulate consumption on a cellular/local basis? • Does this control mechanism rely on HIF-1 gene regulation?

8. Monolayer experiment O2 diffusion/ consumption model prediction Spheroid experiment spatial O2, HIF profiles The Forest Local/Cellular3-D tissues fit parameters validation

9. Monolayer experimental design confluent HepG2 and ES cells cultured at normoxic levels 24 hours at experimental PO2 Close system by shutting off pump Measure HIF-1 via quantitative immunohistochemistry Measure PO2 Calculate oxygen consumption rate

10. PO2 pericellular PO2 pump feed Monolayer Experiments: Oxygen Measurement Apparatus PO2(t) O2 O2 O2, CO2, N2 O2 O2 Adapted from: Wolff et al, Am J Physiol, 1993 Yamada et al, Analytical Biochemistry, 1985

11. Empirical Data Fits for Cellular Mechanisms Consumption HIF-1

12. Governing Equations: (1) MO2(PO2) zero BCs: r PO2(r) (2) r = R (3) Empirical Consumption Equation: (4) (1) Under pseudo-steady conditions PO2/t  0: Tissue Diffusion/Consumption Model Geometry: Spherical Tissue Mass PO2(R)=PR K, • PO2 Partial pressure of Oxygen • K  Krogh’s Diffusion coefficient • MO2 Vol. Tissue Consumption of O2 •   Tissue cellular density

13. Model Parameters

14. 2)  Non-dimensionalize Model Defining Dimensionless Parameters: Get non-dimensional governing equation and B.C.: (symmetry) 3 Dimensionless parameters-govern behavior of differential equation: 1) 3)

15. Model Parameter Sensitivity

16. Monolayer experiment O2 diffusion/ consumption model prediction spatial O2, HIF profiles The Forest: Again Local/Cellular3-D tissues fit parameters validation Spheroid experiment

17. Spheroid Experimental Protocol tumor spheroids or EBs expressing HIF-1 -GFP Microelectrode measurement of PO2 every 50 m Confocal imaging of HIF-1 -GFP Spatial map of HIF1 Spatial map of PO2 Compare to model

18. Micromotor-Driven Oxygen Measurement PO2 taken every 50 m embryoid bodies or tumor spheroids O2, CO2, N2 PO2=PR

19. Comparing Model to Experiment • Mmax = 1x10-7 ml O2/cell/min • R = .08 cm • PR = 20 mmHg • = 1x104 cell/ml • K = 6.8x10-8 ml O2/cm/min/mmHg Model parameters • Da = 5.178 • Pm50 = 10 mmHg (Pm50_nd = 0.5) •  = 1 • PH50 = 1 mmHg (PH50_nd = .05) •  = 1 Same except: • Mmax = 5x10-6 ml O2/cell/min • PR = 150 mmHg Model parameters • Da = 31.3 • Pm50 = 10 mmHg (Pm50_nd = 0.07) •  = 1 • PH50 = 1 mmHg (PH50_nd = .007) •  = 1

20. Analysis of Results • Tested local sensing/consumption regulation of cells and correlation between O2 presence and HIF1 persistence in the cell • Correlation between experiment and model validates local sensing and regulation hypothesis • Poor correlation does not disprove local sensing of oxygen but suggest other methods of oxygen regulation

21. Critique: Did our experimental plan address question 2? ->Is O2 consumption modified by HIF-1 gene regulation? No • Experimental design does not directly test role of HIF-1 in regulation of oxygen consumption • Changes in oxygen consumption due to local oxygen sensing could be independent of HIF-1 expression • HIF-1 changes due to O2 could regulate other downstream events (angiogenesis, etc) and not cellular consumption

22. O2 M atp atp atp atp atp atp Regulation of consumption? NO Testing a direct link between regulation of O2 consumption and HIF-1 expression low • RNA interference is post-translational gene silencing via short double-stranded hairpin RNAs • Can introduce into mammalian cells via retroviral vector • Use to knockdown HIF1 and repeat monolayer oxygen consumption experiments M HIF-1 ARNT HIF-1 ARNT HRE

23. Example of Positive Results Mononlayer RNAi induction Comparison to Spheroids Exp. • Suppression of HIF production allows for direct detection ofO2 consumption change in monolayers (spheroid assays provides secondary check) • Quantitative relationships between HIF and O2 consumption require new methods for exact control over post-translation modification of HIF HIF - HIF +

24. O2 diffusion into tissue Signal downregulates outer cell consumption Low O2 M O2  M diffusible signal or cell-cell contact to outer cells Leads to greater oxygen in the tissue Model Critique - continued • Model does not account for possibility of regulation of consumption via cell-cell signaling

25. Possibilities for further experimentation • Assay downstream glycolytic genes for role in regulation of oxygen consumption by repeating monolayer and spheroid experiments • Test (mine) for soluble factors that may control metabolic rates in early embryonic tissues and/or tumors • Explore “community effect” in regulation of oxygen metabolism

26. Project summary • Developed and implemented model for oxygen diffusion in pre-vascular tissue spheroids with consumption rate dependent on local oxygen concentration • Proposed experiments to determine model parameters and validate dependence of oxygen consumption rate on local oxygen concentration • Proposed experiments to determine if regulation of oxygen consumption rate is mediated through HIF1 expression

27. References Bichet S et al, Oxygen tension modulates -glibn switching in embryoid bodies. FASEB J. 1999 Feb;13(2):285-95 Frasch et al, Early Signals in Cardiac Development. Circ Res. 2002 Sep 20;91(6):457-69 Gassmann, M et al. Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells. 1996 PNAS 93:2867-2872 Harris, AL. Hypoxia--A Key Regulatory Factor in Tumour Growth. Nat Rev Cancer 2002 Jan;2(1):38-47 Iyer, NV et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1. Genes Dev. 1998 12:149-162 Kotch LE et al, Defective Vascularization of HIF-1-Null Embryos is Not Associated with VEGF Deficiency but with Mesenchymal Cell Death. Dev Biol. 1999 May 15;209(2):254-67 Krogh, A. The Comparative Physiology of Respiratory Mechanisms. Philadelphia: University of Pennsylvania Press. 1941 Simon et al, Stem Cells. HIF and the Development of Stem Cells of the Cardiovascular System. 2001;19(4):279-86 Ravi R et al, Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1. Genes Dev. 2000 Jan 1;14(1):34-44 Risau et al, Molecular Mechanisms of Vasculogenesis and Embryonic Angiogenesis. J Cell Physiol. 1997 Nov;173(2):206-10 Ryan et al, HIF-1  is required for solid tumor formation and embryonic vascularization. EMBO J. 1998 Jun 1;17(11):3005-15 Wartenberg, M et al, Tumor-induced angiogenesis studied in confrontation cultures of muticellular tumor spheroids and embryoid bodies frown from pluripotent embryonic stem cell. FASEB J 2001 15:995-1005 Wolff, M, J Fandrey and W. Jelkmann. Microelectrode measurements of pericellular PO2 in erythrpoietin-producing human hepatoma cultures. Am J. Physiol 1993 Yamada, T et al, Oxygen Consumption of Mammalian Myocardial Cells in Culture: Measurements in Beating Cells Attached to the Substrate of the Culture Dish, Analytical Biochemistry 1985 145, 302-307 www.visembryo.com