Molecular Basis of Mechanotransduction in Endothelial Cells

Transcription

1 Molecular Basis of Mechanotransduction in Endothelial Cells Shu Chien, M.D., Ph.D. Departments of Bioengineering and Medicine, and The Whitaker Institute of Biomedical Engineering University of California San Diego, La Jolla, CA May 16, 2006

2 Hemodynamic Forces Acting on The Blood Vessel Blood flow Normal Stress Shear Stress Stretch

3 Effects of Physico-chemical Stimuli on Signal Transduction and Gene Expression Chemical Ligand Mechanical Forces Receptor Signaling pathways Cytoplasm Sensors Trans cis Nucleus Gene Exp. Membrane Protein Exp. Functions Functions: Secretion, Migration, Remodeling, Proliferation, Apoptosis, etc. Mechanotransduction is a fundamental homeostatic process in health and disease.

4 Hemodynamic Factors Blood Cell turnover Monocyte MCP-1* LDL LDL ECs Macrophage ox-ldl Vessel wall Foam cell SMCs * Monocyte Chemotactic Protein-1

5 Atherosclerotic Lesions are Preferentially Located at Regions with Complex Flow Patterns

6 Rectangular Flow Chamber to Study the Effects of Laminar Flows on EC Monolayer Pump Air, 5% CO 2 Microscope Reservoir EC Monolayer

7 MCP-1/GAPDH (Relative Level) Effects of Laminar Shear Stress (12 dyn/cm 2 ) on MCP-1 Gene Expression in HUVECs Time under Shear Stress (hours)

8 Transcription Factor (e.g., AP-1 for MCP-I gene) AP-1 is composed of cjun and cfos Cis element in the promoter region of a gene (e.g., TRE for MCP-I gene)

9 Roles of Ras and MAP Kinases in Shear-Induced Signal Transduction and Gene Expression Shear stress Membrane GDP-Ras GTP- Ras Cytoplasm ERK cfos JNK cjun Nucleus AP-1 MCP-1 TRE Phosphorylation cascade.

10 Relative Degree of Activation Ras JNK MCP Time under Shear Stress (hours)

11 Roles of Adapter Molecules Shc, Grb2 and Sos in Shear-Induced Signal Transduction and Gene Expression Shear stress Flk-1 GTP GDP Tyr- P Shc Grb2 Sos Ras ERK JNK cfos cjun AP-1 TRE MCP-1 Receptor Tyrosine Kinases (RTKs), e.g., the Vascular Endothelial Growth Factor (VEGF) Receptor Flk-1, can mediate the shear-induction of signaling.

12 Mediation of Shear-induced Signal Transduction by Integrins and Focal Adhesion Proteins Shear stress Flk-1 GTP GDP Tyr- P Shc Grb2 Sos Signaling Ras pathways Grb2 Sos Shc FAK c-src Integrins Focal adh. site Trans cis Gene Exp. Protein Exp. e.g., α v β 3 FN, VN Extracellular matrix α v β 3 integrin interacts specifically with fibronectin and vitronectin, but not laminin or collagen. Shear-induced integrin activation leads to its association with FA proteins.

13 Mediation of Shear-induced Signal Transduction by Integrins and Focal Adhesion Proteins Shear stress Flk-1 GTP GDP Tyr- P Shc Grb2 Sos Signaling Ras pathways Grb2 Sos Shc FAK c-src Integrins Focal adh. site Trans cis Gene Exp. Protein Exp. e.g., α v β 3 FN, VN Extracellular matrix Study of temporal and spatial characteristics of Src activation by Fluorescence Resonance Energy Transfer (FRET)

14 Design Strategy for a Novel Reporter for Src Activation by Fluorescence Resonance Energy Transfer (FRET) CFP (1-227) Linker SH2(from c-src) Substrate YFP 433 nm Src Substrate Strong FRET 527 nm QuickTime and a Microsoft Video 1 decompressor are needed to see this picture. CFP YFP SH2 Src Activation

15 Design Strategy for a Novel Reporter for Src Activation by Fluorescence Resonance Energy Transfer (FRET) CFP (1-227) Linker SH2(from c-src) Substrate YFP 433 nm Src Substrate Strong FRET 527 nm 433 nm Weak FRET 490 nm CFP QuickTime and a Microsoft Video 1 decompressor are needed to see this picture. YFP QuickTime and a Microsoft Video 1 decompressor are needed to see this picture. SH2 Src Activation

16 FRET Response of Src Reporter Induced by Pervanadate (A phosphatase inhibitor that increases phosphorylation) Time Src Activation: YFP (2min between images) CFP CFP/YFP Ratio 0.42 (Hi CFP) Pervanadate The results indicate that Src activity is increased by pervanadate (Hi YFP)

17 FRET Response of Src Reporter Is Induced by Shear Stress Time (2min between images) Shear CFP/YFP Ratio 0.42 (Hi CFP) The results indicate that Src activity is increased by shear (Hi YFP)

18 Pulling Fibronectin-coated Beads induced A directional propagation of Src activation (Color changes indicates increases of Src activity after pulling. Time interval between images= 2min) 0.52 Time Force On Ratio (CFP/YFP) 0.25

19 Effects of Physico-chemical Stimuli on Signal Transduction and Gene Expression Chemical Ligand Mechanical Forces Receptor Signaling pathways Cytoplasm Sensors Trans cis Nucleus Gene Exp. Membrane Protein Exp. Functions Functions: Secretion, Migration, Remodeling, Proliferation, Apoptosis, etc.

20 Effects of Laminar and Disturbed Flows on Wound Closure in EC Monolayer Oscillatory Pump Pump Microscope Air, 5% CO 2 Reservoir Step Top View Disturbed Flow Reattachment Zone Direction of Laminar Flow BAEC Monolayer (a) Wound in Disturbed Flow (b) Wound in Laminar Flow

21 Effects of Flow Endothelial Cell Migration (hr) Static Control Laminar Flow Disturbed Flow Laminar flow enhances wound healing, which is much slower under Disturbed flow.

22 Measurement of Cell Traction Force by Using the Beads-in-Membrane Technique Cell Fibronectin Polyacrylamide Gel Embedded with Fluorescent Beads (D= 0.2 µm) 80 µm Glass Cover Slip Side View (not in proportion)

23 EC Traction on Silicon Membrane Containing Fluorescent Beads A. No Flow

24 EC Traction on Silicon Membrane Containing Fluorescent Beads B. 10 min. at 12 dyn/cm 2 Flow

25 Time *: - 4 min 0 - min + 4 min + 10 min + 30 min a c e g i 1.55E-03 cm 1.88E+06 dyn/cm 2 Mag(T EC ) (dyn/cm 2 ) 1.30E E E E E E E E E E E E E E E E+02 b d f h j

26 Force Balance in Mechanically Induced Cell Migration Shear Stress on Membrane Nucleus Intracellular Stress/Strain Junction Proteins (Intercellular Stress) Cytoskeleton Remodeling Cell-Matrix Adhesion Force

27 Multi-factorial Mechanotransduction in Shear-sensing, Signal Transduction and Gene Expression GPCR Channels Junction proteins Shear stress Integrins RTKs (e.g., Flk-1) Membrane (Caveolae) Cbl Akt lkk NFκB Ras JNKK JNK Trans cdc42 cis Rho ROCK Rac Actin Glycocalyx Gene Exp. Protein Exp. Functions Extracellular matrix Functions: Secretion, Migration, Remodeling, Proliferation, Apoptosis, etc.

28 Laminar Flow p53 GADD45 p21 cipl P-Rb Cell cycle arrest in G 0 /G 1

29 Long-term Laminar Shear Stress Causes EC Arrest S Phase G0/G1 Phase % of Total cells (hr) * * * * * (hr) Static Shear Stress

30 Effects of Flow Patterns on Cell Turnover and Permeability Sustained Laminar Flow p53 GADD45 p21 cipl P-Rb Cell cycle arrest in G 0 /G 1 Straight part of the aorta is subjected to sustained laminar shear that leads to cell cycle arrest and hence reduced endothelial permeability. Branch points have unsteady flow pattern, which accelerates cell turnover and increases permeability.

31 Endothelial Cell Mitosis ( ) and Albumin Leaky Spots ( ) In Rabbit Thoracic Aorta Intercostal Orifices Flow

32 Lipid Accumulation and Atherosclerotic Lesions around Intercostal Orifices Lipid accumulation (Rabbit Aorta) D.C. Schwenke & T.E. Carew Arteriosclerosis 9: , 1989 Atherosclerotic lesions (Human Aorta) M. Texon: Hemodynamic Basis of Atherosclerosis

33 MCP-1 Staining and Subintimal WBC Localization around Intercostal Orifices Histochemical Staining of MCP-1 (Rat Aorta) G. Norwich & S. Chien Intimal Distribution of WBC (Rabbit Aorta) Malinauskas et al., Atherosclerosis 115:145, 1995

34 Oscillatory Shears with Different net forward Flows 16 - Pulsatile Shear 16 - Reciprocating Shear (12 ± 4 dyn/cm 2 ) (0.5 ± 4 dyn/cm 2 ) Relative KLF2 mrna Level (% of control) PS RS hour

35 Differential Regulation of KLF2 at Branch Points in Vivo

36 Effects of Local Constriction on KLF2 Expression in Vivo

37 Blood Flow Patterns in Relation to Endothelial Cell Functions and Pathology Straight Part Branch Points Flow Pattern Laminar Disturbed Monocyte Adhesion EC turnover & LDL Permeability Effects on Atherogenesis Anti-Athero. Atherogenic

38 Atherosclerotic Lesions are Preferentially Located at Vessel Bifurcations

39 Hemodynamic Forces Acting on The Blood Vessel Blood flow Normal Stress Shear Stress Stretch

40 Uniaxial and Biaxial Stretch Devices L o +DL D Indenter L o Indenter A B Uniaxial Stretch Biaxial Stretch

41 Uniaxial Stretch Biaxial Stretch 10% linear strain Cell Borders (β-catenin mab / Alexa 488 anti-mouse) F-Actin (Rhodamine Phalloidin)

42 Effects of Uniaxial Stretch on Stress Fiber Orientation Static Control 10% Stretch Cell Images Frequency Distribution of Angle of Orientation Polar Plots

43 Effects of Uniaxial Stretch Magnitude on Stress Fiber Orientation P-value Static % 0.22 Cyclic Uniaxial Stretch 3% 5% 7.5% 0.04 <0.001 < % <0.001

44 Effects of Inhibition of Downstream Effectors of Rho: Rho Kinase (ROCK) and MDia on Stress Fiber Orientation Induced by 10% Stretch Transfection pef (control) pef + Y27632 pef + F1F2 1 % Stretch - 10% - 10% - 10% Cell Images Orientation distribution Inhibition of ROCK (Y27632) or MDia (F1F2 1) changed the 10% stretch-induced stress fiber orientation from perpendicular to parallel.

45 Effects of Active Mutant RhoV14 on Stress Fiber Orientation Transfection GFP (control) % Stretch - 2.5% 10% - 3% 10% Cell Images Orientation distribution

46 Effects of Active Mutant RhoV14 on Stress Fiber Orientation Transfection GFP (control) % Stretch - 2.5% 10% - 2.5% 10% Cell Images Orientation distribution

47 Effects of Active Mutant RhoV14 on Stress Fiber Orientation Transfection GFP (control) % Stretch - 2.5% 10% - 2.5% 10% Cell Images Orientation distribution

48 Effect of Active Rho Mutant (RhoV14) on Stretch-Induced Stress Fiber Orientation Random 0 Orientation Parameter GFP GFP + RhoV14 QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Aligned 1

49 Effect of Active Rho Mutant (RhoV14) on Stretch-Induced Stress Fiber Orientation Random 0 Orientation Parameter GFP +2.9% QuickTime and a RhoV14 has an effect on TIFF (LZW) decompressor are needed to see this picture. stress fiber orientation GFP % equivalent to an RhoV14 additional 2.9% stretch. +2.9% Aligned 1

50 JNK Activation is Transient in Response to Uniaxial Stretch, but Sustained with Biaxial Stretch 10% Uniaxial Stretch 10% Biaxial Stretch GST c-jun Anti-JNK1 Fold JNK Activation (experiment / control) * * Time (hr) * * * * Time (hr)

51 Uniaxial Stretch Biaxial Stretch Cell and Actin Filament Orientation Time Course of JNK Activation Perpendicular to Stretch Transient Random Sustained Hypothesis Remodeling in response to uniaxial stretch leads to the subsidence of JNK activation. Significance Sustained, but not transient, JNK activation causes apoptosis.

52 6 hr

53 6 hr 90 turn 0.5 hr

54 6 hr 90 turn 0.5 hr

55 6 hr 90 turn 0.5 hr 6 hr

56 Regulation of Stress Fiber Orientation and JNK Activation by 10% Uniaxial Stretch: Effects of Change in Stretch Direction Stress Fiber Angle (relative to Current strain direction) * * 0 JNK Activation (Experiment/Control) * * C 0.5 hr 6 hr 6 hr + 90 o 0.5 hr 6 hr + 90 o 6 hr JNK activation subsided following stress fiber realignment

57 Uniaxial Stretch Equibiaxial Stretch Cell and Actin Filament Orientation Time Course of JNK Activation Perpendicular to Stretch Transient Random Sustained Apoptosis Protected Enhanced The Rho-mediated stress fiber orientation perpendicular to the direction of stretch represents a mechanism by which cells adapt to mechanical strain that involves molecular and biomechanical responses.

58 Conclusions: Importance of Directionality in Mechanotransduction Laminar flow with a net forward direction is atheroprotective, whereas disturbed flow with little net forward direction is athergenic. Uniaxial stretch with a definitive direction is antiapoptosis, whereas biaxial stretch without a net direction leads to apoptosis. The directionality of the mechanical stimuli and the consequent directional remodeling of the cytoskeleton play important roles in the modulation of cell functions in response to mechanotransduction.