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

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 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 Extracellular Ca Tau G kaΦ 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

8. 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

9. Model Setup – Stage III Stage II Model 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 – [IkBαmRNA] 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: • 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) Modified from: Truong and Ikura, 1999

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. resist substrate PDMS μ-stamped glass slide Experimental – Culture/Channel Construction • Cast PDMS elastomer stamp from master • Coat with adhesion protein (fibronectin, polylysine), contact glass slide • Rinse slide • 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 • 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 www.technet.pnl.gov/dme/ micro/plastic.stm

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

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. 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! Acknowledgements Special thanks to:

27. References

28. Model Results – Cytosolic [Ca]