Control of Endothelial Gene Expression via Fluid Induced Shear Stress

1
Control of Endothelial Gene Expression via Fluid
Induced Shear Stress

• Danielle Cook Adam Siegel
• MIT BE.400 Fall 2002

2
Overview

• Fluid flow-induced shear stress (?)
• ? gene transcription in endothelial cells
• Our Project Aims
• MODEL a pathway from shear stress to gene
  expression
• Design EXPERIMENTS employing microfluidics with
• fluorescence detection techniques to quantify the
  relationship
• UTILIZE modeling and experimentation results of
  system to engineer a new tool for flow-controlled
  gene expression
• Future applications flow-mediated gene
  therapies, gradient tissue engineering,
  cell-based flow sensor

3
Flow Mediated Mechanotransduction

• Endothelial cells
• Form monolayer between blood and arterial wall
• Hemodynamic forces regulate cell via flow
  mediated signal transduction
• Several applicable forces
• Fluid Shear Stress
• Compressive Stress
• Circumferential Stress
• Mechanisms by which cells identify and respond to
  shear stress forces are still unclear no single
  mechanosensor protein

Papadaki and Eskin, 1999
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Flow Mediated Membrane Proteins

• G-Protein Linked Receptors
• Shear stress activation alters concentrations of
  2nd messengers
• Exact mechanism unclear - plasma membrane itself
  may activate G-proteins from changes in lipid
  bilayer fluidity
• Ion Channels
• Ca and K are the primary channels
• Stretch induced? Asymmetries in trans-bilayer
  pressure profile
• Secondary activation by G-proteins
• Integrins
• Activation via cytoskeletal changes
• Activation of MAPK (ERK) pathways

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The NF-?B Signaling Pathway

• NEEDED
• Simple biochemical pathway linking ? to gene
  expression
• Model-able with known parameters
• Experimental detection of mechanoresponsive
  behavior possible
• FOUND
• G-proteins activated by ? lead
• to activation of the NF-?B
• transcription factor
• NF-?B binds to the Shear-Stress
• Response Elements (SSREs) in
• some gene promoters

pps98.cryst.bbk.ac.uk/assignment/
projects/ruiz/PROJ/nfkb.gi
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Model Setup Overview
Stage I
Shear Stress
cell membrane
G
Stage II
DAG
PKCa
PLC
PIP2
Ca
Stage III
IKKa
IP3
NF-kB
IkB
IkB mRNA
nucleus
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Model Setup Stage I
Extracellular Ca
Tau
G
kaF
cell membrane
kb
PLC
Pcai
Casc
PIP2
Cai
Jout
Psd
k3
k4
kdag
Stage II Model

DAG
IP3
P(IP3)
Cadc
Stage II model
cytosol
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Model Setup Stage II
cell membrane
PKC-DAG memb
DAG
PKC-Ca memb

PKC-Ca DAG
PKC-basal
Cai
Stage I Model
PKC-Ca
IKKi-PKCa
PKC-cyto
IKKi
IKKa
Stage III Model
cytosol
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Model Setup Stage III
IKKa
Stage II Model
cytosol
NF-kB
IkB
NF-kB
nucleus
NF-kB
IkBat IkBßt IkBet
IkB
NF-kB
IkB
IkBa
IkBe
IkBß
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Model Formulation

• Typical reactions
• A B AB E S ES P E

Equations also describe translocation and
mechanical deformation of molecules

• 39 first-order ODEs
• 83 Parameters, all from literature
• Equations solved in Matlab v.6.5 using ode23s
• Function for no stress conditions to retrieve
  initial conditions
• Function for applied stress conditions

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Model Modifications from Lit.

• P(Cai) redefined to balance Jout
• maintains resting calcium level when ? 0
  dyne/cm2
• does not produce a calcium transient like the
  original model
• makes the dc compartmental calcium the only
  source for intracellular calcium
• AA-dependent components eliminated from Stage II
• Initial concentrations estimated
• from steady state runs under no applied shear
  stress
• to match concentrations of comparable molecules

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Model Assumptions

1. Active PKC is the sole enzyme activating IKK
2. NF-?B-induced promotion of I?B? is not an
   atypical example of NF-?B action
3. Active NF-?B binds to I?B? promoter and nowhere
   else on DNA
4. No other pathways modulate any of our pathway
   molecules as a function of shear stress
5. Cells do not change shape or move during fluid
   flow
6. Cells that do not grow, divide, or do anything
   unusual over 16 hour simulation period

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Model Results Activated NF-kB
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Final Output (short term) Pulse of activated IKK
creates active NFkB in the nucleus which leads to
transcription of IkB mRNA
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Model Results IkBa mRNA
16 hr
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Model Conclusions

• Pathway is sensitive to magnitude of ? until
  activation of IKK
• Inactive IKK is quickly consumed by enzymatic
  reaction with active PKC, rendering downstream
  reaction independent of ? level
• NF-?B is activated in pulses by active IKK
• I?B? mRNA and protein levels produced in distinct
  periods at decreasing levels

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Experimental Typical Setup

1. Inject cells with fluorescent NF-kB, IkB
   plasmids
2. Grow cells selectively on protein-microstamped
   surfaces
3. Enclose live cells in PDMS channels
4. Induce laminar fluid flow
5. Measure fluorescence via Fluorescent Resonant
   Energy Transfer (FRET)

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FRET Cell Preparation
Modified from Truong and Ikura, 1999

• FRET in a nutshell
• fluorophores apart ? see both colors
• fluorophores together ? see one color
• Plasmids Buy from Clonetech CFP with NF-kB, YFP
  with IKB
• Cells Human UVEC (Umbilical Cord Endothelial
  Cells) or BAECs (Bovine Arterial Endothelial
  Cells)

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Experimental FRET Fluorescence Detection

• CFP intensity over population of cells
  proportional to the average activated NF-?B in a
  single cell
• Monitor YFP and CFP intensity difference over time

more bound
less bound
Modified from Truong and Ikura, 1999
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Experimental Culture/Channel Construction

1. Cast PDMS elastomer stamp from master
2. Coat with adhesion protein (fibronectin,
   polylysine), contact glass slide
3. Rinse slide

1. Create microchannel master using basic
   photolithography rapid prototyping
2. Apply PDMS and cure to solidify
3. Remove PDMS from substrate, align and seal to
   culture cover slide

1. First Resist Application
2. Pattern Transfer to Si
3. Second Resist
4. Development
5. Resist Reflow

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Experimental Shear Stress Stimulus

• Newtons law of viscosity t µ du/dy
• Velocity profile in microchannel
• Generate fluid flow in microchannels via
  automated applied force from syringes

velocity profile
y u
www.technet.pnl.gov/dme/ micro/plastic.stm
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Proposed Experiments

• FRET
• Intermolecular For unbound cytosolic NF-kB
• Intramolecular NF-kB may change conformation
  with IkB dissociation or DNA association
• Plot NF-kB(t,?)
• GFP expression
• Transfect cells with GFP, expressed under NF-kB
  regulation
• Plot steady-state concentrations of GFP
• Use to determine IkBa mRNA as F(t,?)
• Cellular mRNA
• Isolate IkB mRNA on a DNA microarray
• Correlate Results with modeling results
• Last resort since cells die

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FRET Measurements Checklist

1. Fluorophore-fused NF-kB and IkB
2. function like native proteins
3. are expressed at similar levels to native
   proteins
4. are expressed at higher levels than untagged
   proteins, but not so high that cell pathway
   reactions are changed
5. Examine conformational change upon un/binding of
6. NF-kB from IkB for intermolecular FRET
7. NF-kB with DNA for intramolecular FRET
8. Measure background signal from unattached
   proteins, i.e. find signal-to-noise ratio

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Future Studies

• Power our device is in developing novel
  technology producing biological response with
  mechanical stimuli
• Technology I Flow sensor
• Cell lights up upon mechanical stimulus
• Potential for cells that sense multiple
  directions
• Technology II Flow mediated Gene Expression
• Cell expresses a gene to a level based upon shear
  stress
• Possibilities in gene therapy
• Technology III Spatially variable expression in
  single tissues via multiple laminar flow streams
  over tissue
• Uses laminar flow to stream flows upon a tissue
  at different stresses
• Flow induces on/off gene expression in each of
  the cells of the tissue

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In Conclusion

• How endothelial cells sense shear stress
• Use of the NF-kB Signaling Pathway
• Model Formulation
• Model Results Conclusions
• Experiments to Verify Model
• Future Studies

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Acknowledgements
Special thanks to

• Dr. Alice Ting
• Dr. Don Ingber
• Willow DiLuzio
• Ricardo Brau
• Jon Behr
• Samantha Sutton
• Ty Thompson

• Prof. Lauffenburger
• Prof. Matsudaira
• Ali Khademhosseini
• Everyone else in BE400!

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References
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Model Results Cytosolic Ca