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

Eric Krauland and Shawdee EshghiDecember 12, 2002

BE.400

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

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

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

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

HIF-1 MechanismO2

HIF-1

ARNT

HIF-1 ARNT

HRE

M

UbHIF-1 Ub Ub

HIF-1

atpatp

atp

atpatp

atpatp

atpatp

low

Mmitochondria

Regulation of consumption?

Questions to be addressed:1) Do embryoid bodies and tumor spheroids

sense O2 concentration and regulate consumption on a cellular/local basis?

2) Does this control mechanism rely on HIF-1 gene regulation?

The Forest

fit parameters O2 diffusion/ consumption model

spatial O2, HIF profiles

prediction

Spheroid experiment

validation

Monolayer experiment

Local/Cellular 3-D tissues

Monolayer experimental design confluent HepG2 and ES cells

cultured at normoxic levels

Close system by shutting off pump

24 hours at experimental PO2

Measure PO2

Measure HIF-1 via quantitative

immunohistochemistry

Calculate oxygen consumption rate

Monolayer Experiments: Oxygen Measurement Apparatus

O2, CO2, N2

PO2(t)

Adapted from: Wolff et al, Am J Physiol, 1993

Yamada et al, Analytical Biochemistry, 1985

PO2 pump feed

PO

2 pe

ricel

lula

r

O2

O2

O2

O2

Empirical Data Fits for Cellular Mechanisms

2

2

50max Om

O

PP

PM

M

2

50

50max OH

H

PP

PH

H

Consumption HIF-1

Experimental Data

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140 160

PO2 (mmHg)

Nor

mal

ized

O2

cons

umpt

ion

(M/M

max

)

Experimental Data

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140 160

PO2 (mmHg)

Frac

tiona

l Flu

ores

cenc

e (H

/Hm

ax)

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140 160

PO2 (mmHg)

Nor

mal

ized

O2

cons

umpt

ion

(M/M

max

)

P50=5 beta =1

P50=20 beta=1

P50=20 beta=5

"experimental data"

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120 140 160

PO2 (mmHg)

Frac

tiona

l Flu

ores

cenc

e (H

/Hm

ax)

P50=5 beta =1

P50=20 beta=1

P50=20 beta=5

"experimental data"

Tissue Diffusion/Consumption ModelGeometry: Spherical Tissue Mass

PO2(R)=PR

r = R

r

MO2(PO2)

PO2(r)

(1)

BCs:

Governing Equations:

(1)

(2)

(4)

RRrO PP 2

002

r

O

rP

(3)

PO2

t

Kr 2

r

(r 2PO2

r) M(PO2

)

Empirical Consumption Equation:

2

2

2

50

max)(O

m

OO

PP

PMPM

Under pseudo-steady conditions PO2/t 0:

zero

•PO2 Partial pressure of Oxygen

•K Krogh’s Diffusion coefficient

•MO2 Vol. Tissue Consumption of O2

Tissue cellular density

K,

Model ParametersParameter Description Value/Range Ref./Experiment

R (cm) Outer sphere radius 0.01-0.1 Ref. Gassman/3-D

experiments

PR (mmHg) Bulk O2 partial Pressure at outer sphere surface

10 – 100

Varied experimentally in monolayer experiments

(ml O2/ml tissue /mmHg) O2 solubility coefficient

3.15x10-5 Ref. Wolff

K (ml O2/cm/ min/mmHg) Krogh’s diffusion coefficient ~10-8 Ref Wolff, Secomb

Mmax (ml O2/cell/min) Maximal O2 cellular consumption rate

~10-9-10-5 Fit from monolayer experiments

Pm50 (mmHg) O2 at half-maximal

consumption 0-100 Fit from monolayer

experiments

, (unitless) Cooperitivity cooefficients >1 Fit from monolayer

experiments

Hmax (% Protein/cell) Maximum HIF1- production 0-100 Fit from monolayer

experiments

Ph50(mmHg) O2 at half-maximal

HIF1- production 0-100 Fit from monolayer

experiments

(cells/ml tissue) Cellular density in tissue spheroids ~106-103

Experimentally determined on 3-D cultures

Non-dimensionalize Model

PO2PR;

rR;

˜ t R2K

3 Dimensionless parameters-govern behavior of differential equation:

Defining Dimensionless Parameters:

mPDa

t50

~)(1~

22

Get non-dimensional governing equation and B.C.:

KPRMDa

R

2max

1)1,~( t 0)0,~( dtd (symmetry)

R

mm

PP

P 5050

~ 1) 2) 3)

Model Parameter Sensitivity

The Forest: Again

fit parameters O2 diffusion/ consumption model

spatial O2, HIF profiles

prediction

Spheroid experiment

validation

Monolayer experiment

Local/Cellular 3-D tissues

Spheroid Experimental Protocol tumor spheroids or EBs expressing HIF-1 -GFP

Microelectrode measurement of PO2

every 50 m

Spatial map of PO2

Confocal imaging of HIF-1 -GFP

Spatial map of HIF1

Compare to model

Micromotor-Driven Oxygen Measurement

PO2 taken every 50 m

embryoid bodies or tumor spheroids

PO2=PR

O2, CO2, N2

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 Pm

50 = 10 mmHg (Pm50_nd = 0.5)

= 1 PH

50 = 1 mmHg (PH50_nd = .05)

= 1

Same except: Mmax = 5x10-6 ml O2/cell/min PR = 150 mmHg

Model parameters Da = 31.3 Pm

50 = 10 mmHg (Pm50_nd = 0.07)

= 1 PH

50 = 1 mmHg (PH50_nd = .007)

= 1

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

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

Testing a direct link between regulation of O2 consumption and HIF-1 expression

O2

HIF-1

ARNT

HIF-1 ARNT

HRE

M atpatp

atp

atpatp

atp

low

M

Regulation of consumption?

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

NO

Example of Positive Results

Suppression of HIF production allows for direct detection of O2 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

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120 140 160

PO2 (m m Hg)

Nor

mal

ized

O2

Con

sum

ptio

n

Comsumption Pre RNAi

Consumption Post RNAi

Mononlayer RNAi induction Comparison to Spheroids Exp.

HIF -

HIF +

Model Critique - continued Model does not account for possibility of

regulation of consumption via cell-cell signaling

O2 diffusion into tissue

Low O2diffusible signal or cell-cell contact to outer cells

M

Signal downregulates outer cell consumption

Leads to greater oxygen in the tissue

M O2

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

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

ReferencesBichet 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):279-86Ravi 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

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