1. Control of Endothelial Gene Expression via Fluid Induced Shear Stress
Danielle Cook & Adam Siegel
MIT BE.400 Fall 2002
November 14, 2018

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

4. 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

5. 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

6. Model Setup – Overview Stage I cell membrane Stage II G DAG Ca
Shear Stress
cell membrane G
Stage II
DAG
PKCa
PLC
PIP2 Ca
Stage III
IKKa
IP3
NF-kB
IkB
IkB mRNA
nucleus

7. * Model Setup – Stage I Casc Cai Cadc Stage II Model Stage II model
Extracellular Ca
Tau G
kaΦ kb
cell membrane
PLC
Pcai
Casc
PIP2
Cai
Jout
Psd k3 k4
kdag
Stage II Model *
DAG
IP3
P(IP3)
Cadc
Stage II model
cytosol

8. * Model Setup – Stage II Cai Stage I Model Stage III Model
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

9. Model Setup – Stage III IkB Stage II Model cytosol IkB NF-kB NF-kB
IKKa
cytosol
IkB
NF-kB
NF-kB
nucleus
NF-kB
IkBαt
IkBβt
IkBεt
IkB
NF-kB
IkB
IkBα
IkBβ
IkBε

10. 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

11. 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

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

13. Model Results – [Activated NF-kB]

14. Final Output
(short term)
Pulse of activated IKK creates active NFkB in the nucleus which leads to transcription of IkB mRNA

15. Model Results – [IkBα mRNA]
16 hr

16. 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

17. Experimental – Typical Setup
Inject cells with fluorescent NF-kB, IkB plasmids
Grow cells selectively on protein-microstamped surfaces
Enclose live cells in PDMS channels
Induce laminar fluid flow
Measure fluorescence via Fluorescent Resonant Energy Transfer (FRET)

18. FRET Cell Preparation FRET in a nutshell:
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)

19. 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

20. Experimental – Culture/Channel Construction
resist
substrate
μ-stamped
glass slide
PDMS
Create microchannel master using basic photolithography rapid prototyping
Apply PDMS and cure to solidify
Remove PDMS from substrate, align and seal to culture cover slide
Cast PDMS elastomer stamp from master
Coat with adhesion protein (fibronectin, polylysine), contact glass slide
Rinse slide
First Resist Application
Pattern Transfer to Si
Second Resist
Development
Resist Reflow

21. Experimental – Shear Stress Stimulus
Newton’s law of viscosity: τ = μ du/dy
Velocity profile in microchannel
Generate fluid flow in microchannels via automated applied force from syringes
velocity
profile y u
micro/plastic.stm

22. Proposed Experiments FRET GFP expression Cellular mRNA
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 IkBα mRNA as F(t,)
Cellular mRNA
Isolate IkB mRNA on a DNA microarray
Correlate Results with modeling results
Last resort since cells die

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

24. Future Studies
Induced Shear Stress
Controlled velocity
Microfluidic channel endothelial cell tissue
Controlled protein X
produced as a function of
initial applied velocity
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

25. 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

26. Acknowledgements Special thanks to: Dr. Alice Ting Prof. Lauffenburger
Dr. Don Ingber
Willow DiLuzio
Ricardo Brau
Jon Behr
Samantha Sutton
Ty Thompson
Prof. Lauffenburger
Prof. Matsudaira
Ali Khademhosseini
Everyone else in BE400!

27. References

28. Model Results – Cytosolic [Ca]