1. A review of endothelial cells mechanics (effect of shear stress on EC) Dept. of Experimental Orthopaedics and Biomechanics Bioengineering Reza Abedian (M.Sc.)

2. Flow Mediated Mechanotransduction Fluid flow-induced shear stress (  )  gene transcription (the synthesis of RNA under the direction of DNA) in endothelial cellsRNADNA 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 – there’s no single “mechanosensor” protein Papadaki and Eskin, 1999

3. Flow Mediated Membrane Proteins G-Protein Linked Receptors (transmembrane receptors) –Shear stress activation –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? –Secondary activation by G-proteins Integrins (receptors that mediate attachment between a cell and the tissues surrounding it, which may be other cells or the extracellular matrix (ECM) –Activation via cytoskeletal changes –Activation of MAPK (ERK) pathways (a signal transduction pathway that couples intracellular responses to the binding of growth factors to cell surface receptors. This pathway is very complex and includes many protein components

4. The NF-  B Signaling Pathway NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that acts as a transcription factor 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

5. Cellular deformation leads to a permanent adaptation by changes in the expression of the genetic apparatus (DNA-synthesis) Cell mechanics (I) In the human body various cell types are continuously subjected to passive (stretch, compression) or active deformation (contraction).

6. Section arteriole Endothelial cells Extra cellular matrix Smooth muscle cells

7. Cell mechanics (II) The cytoskeleton plays a key role in the transmission of forces throughout the cell. Understanding of the process of cellular deformation requires detailed measurement of: cellular and cytoskeletal passive characteristics (stiffness)

8. cellular deformation has to be registered development of an in-vitro shear apparatus Effects of shear stress on endothelial cells To unravel shearing effects on EC deformation shear stress has to be controlled visualization cytoskeleton measurement of elasticity changes

9. The cytoskeleton thick filaments microtubuli thin filaments filamentous actin intermediate filaments vimentin is a 3-D intracellular network of filamentous polymers provides a continuous intracellular mechanical coupling

10. Microtubules in normal cardiomyocytes

11. keratin in an epithelial cell

12. nuclear lamin Class V intermediate filaments, are fibrous proteins providing structural function and transcriptional regulation in the cell nucleus

13. actin can form stress fibers focal adhesion pointsstress fibers

14. actin can be identified with stain actin in endothelial cells

15. force indentation Passive cellular stiffness (1)

16. Atomic force microscopy on living cells Multimode SPM cantilever tip x- y-z direction: cell topography z direction: force - indentation curve Passive stiffness of the cytoskeleton

17. focussing objective mirror head tube light source beam splitter Atomic force microscopy on living cells cantilever tip cell sample

18. Ocular inverted confocal laser scanning microscope registration of cytoskeletal deformation In-vitro shearing of endothelial cells tissue culture dish with endothelial cells fluid inlet fluid outlet controllable shear rate

19. Endothelial Cell Alignment

20. t=0 after 12 hrs Development of stress fibers in ECs Shear direction Topographical changes after shearing Cell Mechanics Cytoskeleton - ECM -BME 2001

22. AFM of Endothelial Cells

24. EC shear stress sensation Shear stress is sensed by a cytoskeleton associated flow sensor on the cell surface. Signaling is subsequently transmitted via cytoskeletal elements to various intracellular sites (nucleus, cell-cell adhesion proteins, and focal adhesion sites).

25. Kelvin Body Kelvin bodies have been frequently used to present the mechanical behavior of tissues. EC components are modeled as viscoelastic materials with standard linear solid behavior (Kelvin body).

26. Kelvin bodies in Parallel

27. Kelvin bodies in series

28. Two bodies coupled in Parallel For a given value of F 0, the peak deformation is larger for steady flow than for oscillatory flow.

29. Two bodies coupled in Parallel K 1 of 2 nd body effects F 1 =F 2 F 1 >F 2 F 1 <F 2 Steady force

30. Two bodies coupled in Parallel K 2 of 2 nd body effects F 1 =F 2 F 1 >F 2 F 1 <F 2 Asymptotic value of F 1 for steady flow is independent of k 22 and µ 2. Transient approach does depend on these parameters. Peak deformation under steady flow independent of K 2 and µ 2 Steady force

31. Model EC networks

32. Model Parameters Each structure is modeled as a viscoelastic body with standard linear solid behavior (Kelvin Body) and characterized by its own set of viscoelastic parameters.

33. Typical pattern of an instantaneous deformation followed by gradual creeping µ flow sensor very small  very rapid response F each of actin filament = ½ F flow sensor K 1 actin = ½ K 1 flow sensor  equivalent peak deformation K 1 nucleus = 2 K 1 flow sensor  half peak deformation 4 bodies model results Steady force

34. 6 bodies model results Similar behavior to 4 bodies model. K 1 MT very small  high peak deformation Steady force

35. 6 bodies model results Small   steady flow behavior High   independent of  Intermediate   deformation decrease with  Oscillatory force

36. Conclusions Technology allows time-related detection of cytoskeletal movement in endothelial cells AFM allows measurement of visco-elastic properties of endothelial cells Combination of molecular biological and micro technology can improve our insight into shear-induced adaptations in endothelial cell shape, physical properties and gene transcription

37. References: 1.Evolving concepts from molecular biology for application in the coronary circulation Luc H.E.H. Snoeckx Cardiovascular Research Institute Maastricht, UM Cell Mechanics Unit, Biomedical Technology, TUE June 13, 2002 2.Control of Endothelial Gene Expression via Fluid Induced Shear Stress Danielle Cook & Adam Siegel MIT BE.400 Fall 2002 3.A Model for Shear Stress Sensing and Transmission in Vascular Endothelial Cells (EC), Mazzag et al.