Mechanotrasduction and its role in atherosclerosis

Mechanotransduction

Arun.V.MSc.BMB14368005

And its Role in Atherosclerosis

© Arun Viswanathan

What is Mechanotransduction?

“The processes through which cells sense and respond to mechanical stimuli by converting them to biochemical signals that elicit specific cellular responses.”

Defines this subject area as

© Arun Viswanathan

How MT happens?

transduction of physical •forces occur through changes in protein conformation

Protein folding favors the •conformation that yields the lowest free energy

physical forces that •modify the energy landscape will directly alter protein folding.

©Mechanisms of MechanotransductionA. Wayne Orr, Brian P. Helmke, Brett R. Blackman, Martin A. Schwartz

Change in confirmation

Change in folding

© Arun Viswanathan

“Force-induced effects on conformation of protein is a general mechanism by which

enzymatic activity or protein interactions can be modified to mediate signaling in MT.”

A unifying principle

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Stretch-sensitive channels Example

• Increasing tension within the lipid bilayer from 10–12 to 20 dyn/cm increases channel opening probability.

• open state occupies a greater area in the bilayer, membrane tension will result directly in lower free energy.

Mechanisms of Mechanotransduction; A. Wayne Orr, Brian P.

Helmke, Brett R. Blackman, Martin A. Schwartz; Developmental Cell,

Volume 10, Issue 3, March 2006, Page 407 © Arun Viswanathan

Mechanosensistive Channel-L Example

(A) Structure of the MscL channel in the open state viewed from the extracellular side. It opens and close like an iris of camera

(B) MscL is a homopentamer whose subunits have two α-helical TM domains, TM1 and TM2, cytoplasmic N- and C-terminal domains and a central periplasmicdomain. Side views of the transmembrane domains TM1 and TM2 of the open channel. The inset shows the structure of the channel monomer.

Open channel structure of MscL and the gating mechanism of mechanosensitivechannels, Eduardo Perozo, D. Marien Cortes, Pornthep Sompornpisut, Anna Kloda & Boris Martinac; Nature 418, 942-948 (29 August 2002) | doi:10.1038/nature00992; Received 4 April 2002 © Arun Viswanathan

Mechanosensistive Channel-L Example

Changes in the spectral line shape from position Ile24 obtained by the EPR spectroscopy were used to monitor the influence of different bilayer tension gradients produced by different lipid environments on the conformation of MscL: • Symmetric phosphatidylcholine

(PC18; closed), • Asymmetric

lysophosphatidylcholine (LPC; open),

• Detergent solution (closed)• Symmetric LPC (closed)

Note, narrowing of the spectral line after addition of LPC to one monolayer

is characteristic of the open channel.

Underlying the transduction of bilayer deformation forces during mechanosensitive channel gating ;Eduardo Perozo, Anna Kloda2 D. MarienCortes & Boris Martinac; Nature Structural Biology 9, 696 - 703 (2002) Published online: 12 August 2002; | doi:10.1038/nsb827 Physical principles © Arun Viswanathan

(1) phosphorylation & activation of MAP kinase signaling pathways (denoted by MEKK and JNK) that lead to ERK1/2 phosphorylation,

(2) activation of PLC leading to IP3

generation and gating of intracellular Ca2+ stores

(3) alterations in the actin cytoskeleton network. PhosphorylatedERK1/2 causes the activation of the AP-1 family of TF

Signaling : mechanical to chemical signals

© Arun Viswanathan

Signaling : mechanical to chemical signals

There is a common mechanism for MT in cells, regardless of the cell type. Integrins, interacting with their matrix/environment, mediate increases in intracellular Ca2+ levels and activate MAP kinase cascades to cause ERK1/2 phosphorylation. Phosphorylated ERK1/2 causes the activation of AP-1 family of TFs that are necessary for the pro-growth response. © Arun Viswanathan

(1) Glycocalix : mediate MT signalling in response to fluid shear stress ECs

(2) Cell-cell junctional receptors(3) cell-matrix focal adhesions allow the

cells to probe its environment. (4) Force-induced unfolding of

extracellular matrix proteins (fibronectin) : initiate MT signallingoutside the cell.

(5) Intracellular strain can induce conformational changes in cytoskeletal elements by changing binding affinities to specific molecules & activating signallingpathways.

(6) nucleus has been proposed to act as a mechanosensor. Intracellular deformations could alter chromatin conformation and modulate access to TFs & TMs. (direct evidence for this mechanism is still lacking)

(7) Compression of the intercellular space can alter the effective concentration of autocrine and paracrine signalling molecules.

(8) changes in G-protein coupled receptors, lipid fluidity, and even mitochondrial activity have been proposed as mechanosensors

Myriad ways of MT

short term effects :Increases (or decreases) in intracellular tension, adhesion, spreading or migration

long term effects : Protein synthesis/secretion, structural reorganization, proliferation, viability mediated through multiple, overlapping & crosstalking signalling pathways.

© Arun Viswanathan

Diseases related to MT

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Atherosclerosis

Area of bifurcation of blood vessels are

prone to atherosclerosis

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Forces acting on blood vessels

shear stress parallel (tangential to the endothelial cell surface) by blood flow and the generations of normal stress (perpendicular to the endothelial cell surface) and

circumferential stretch due to the action of pressure.

Circumferential Stress(Cyclic Stress or stretch)

Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell; Shu Chien; American Journal of Physiology - Heart and Circulatory Physiology Published 1 March 2007 Vol. 292 no. 3, H1209-H1224 DOI: 10.1152/ajpheart.01047.2006 © Arun Viswanathan

Disturbed flow at bifurcation

Contours of the wall shear stress (N/m2) magnitude at the main and D1-S1 bifurcations of coronary segments flow divider (FD) and lateral walls (LW).

Wall shear stress oscillation and its gradient in the normal left coronary artery tree bifurcations; Soulis JV, Fytanidis DK, Seralidou KV, Giannoglou GD; Hippokratia 2014, 18, 1: 12-16

© Arun Viswanathan

Disturbed flow at bifurcation

Monocyte Adhesion(+)LDL permeability(+)

Monocyte Adhesion(-)LDL permeability(-)

Mechanotransduction in vascular physiology and atherogenesis; Cornelia Hahn & Martin A. SchwartzNature Reviews Molecular Cell Biology 10, 53-62 (January 2009); doi:10.1038/nrm2596 © Arun Viswanathan

MT in response to shear stress


MT in response to shear stress

• shear stress can activate a number of mechanosensors . These include membrane proteins such as – RTK(e.g., VEGFR Flk-1)

– the integrins (αvβ3, α2β1, α5β1, and α6β1)

– G proteins and G protein-coupled receptors

– Ca2+ channel

– intercellular junction proteins

– Membrane lipids and membrane glycocalyx may also play a role.

© Arun Viswanathan

Effects of laminar and disturbed flow on EC proliferation

Laminar Flow p53

GADD45

p21P-Rb

Cell CycleArrestin G0 and G1

Disturbed Flow p53

GADD45

p21

P-RbCell Cycleprogression

© Arun Viswanathan

MT in response to stretch

Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell; Shu Chien; American Journal of Physiology - Heart and Circulatory Physiology Published 1 March 2007 Vol. 292 no. 3, H1209-H1224 DOI: 10.1152/ajpheart.01047.2006

© Arun Viswanathan

Cell borders : Beta-catenin mAbF-Actin : Rhodamine Phalloidin

Uniaxial stretch causes an orientation of the stress fibers in a direction perpendicular to that of stretch, whereas biaxial stretch does not result in any specific orientation of the stress fibers

MT in response to stretch

Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell; ShuChien; American Journal of Physiology -Heart and Circulatory Physiology Published 1 March 2007 Vol. 292 no. 3, H1209-H1224 DOI: 10.1152/ajpheart.01047.2006 © Arun Viswanathan

Stretch and signaling


JNK1 activation

ECs to uniaxial stretch for 6 h, which has led to a

perpendicular stress fiber alignment and subsidence of

JNK activation and then change the direction of stretch by 90°. Thus the uniaxial stretch is now applied in a direction parallel to the oriented stress fibers. This change in direction results in a

transient reactivation of JNK when the stress fibers are not

yet realigned. After 6 h of uniaxial stretch in this new

direction, however, the stress fibers become again oriented

perpendicular to the new direction of stretch, and the

JNK activity then subsides once again


JNK1 activation is associated with apoptosis in EC’s

• Lack of a clear stretch direction in branch points would not induce the feedback minimization of intracellular stress • Result: sustained activation of JNK• The blockade of JNK activity by using the negative interfering mutant JNK(K-R) markedly decreased the apoptosis induced by colchicine.•Activation of JNK is associated with apoptosis*

*Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress; Ying-Li Hu, Song Li, John Y.-J. Shyy, Shu ChienAmerican Journal of Physiology - Heart and Circulatory Physiology Published 1 October 1999 Vol. 277 no. 4, H1593-H1599 DOI:

© Arun Viswanathan

Monocyte Chemotactic Protein-1

• expression of MCP-1 gene is modulated by the Ras-mitogen-activated protein kinases (MAPKs) pathway.

• The activation of MAPKs entails the phosphorylation of a series of serine-threonine protein kinases with Ras serving as an upstream molecule

• ERK, JNK, and p38 as three key downstream molecules• application of a steady shear stress (12 dyn/cm2) to ECs causes Ras

to become bound with GTP instead of GDP, • followed by the activation of MAPKs and the MCP-1 expression. • sustained laminar shear stress causes the downregulation of Ras (a

few seconds), MAPKs (on the order of an hour), and MCP-1 gene expression, which decreases to below the preshear level by 5 h

• laminar shear stress has a short-term effect of upregulation of MCP-1, it has a long-term effect of downregulation when its application is sustained

© Arun Viswanathan

MCP-1

ECs in static culture respond to the applied shear flow by a transient MCP-1 activation, which then vanishes when the cells adapt to the long-term shear stress. The induction of MCP-1 expression by oxidative stress has been shown to be attenuated by pulsatile shear stress and augmented by reciprocating shear stress

© Arun Viswanathan

KLK-2 gene Expression

• Krüppel-like factor (Klf)-2 belongs to the zinc finger family of DNA-binding transcription factors.

• Klf2 was shown to be a potent inhibitor of cytokine-mediated induction of VCAM-1 and E-selectin expression in endothelial cells

• Anti-proliferative and Anti-inflamatory

© Arun Viswanathan

three pictures showing enlarged views of regions 1, 2, and 3 in B. The expression of KLF2 was high and continuous on ECs of the abdominal aorta (labeled 3) and the medial aspect (labeled 2) of the celiac branch, but virtually absent on the lateral aspect (labeled 1) of the branch. These results are representative of three independent experiments. In C, the short filled arrows (in 2 and 3) indicate the positive staining for KLF2 protein, and the open arrow (in 1) shows the absence of KLF2 protein in the endothelium.



Lipid Metabolism in EC

•The application of laminar shear stress (12 dyn/cm2) causes a transient activation of SREBP1 •Disturbed flow causes a sustained activation of SREBP1 •Cause translocation of TF domain into the nucleus •Blockade of β1-integrin with AIIB2 blocking-type MAbor disruption of actin cytoskeleton with cytochalasinD inhibits the shear-activation of SREBP1 •indicating that integrins and the actin cytoskeleton play significant roles in the modulation of EC lipid metabolism in response to shear stress

© Arun Viswanathan

After being sheared for 1 or 12 h, the cells were

fixed and immunostained for SREBP1. Whereas

disturbed flow induced a sustained activation

SREBP1, as indicated by its translocation into the nuclei (B, lower left), laminar flow

activated SREBP1 in a transient manner, as

evidenced by the lack of nuclear staining of SREBP1 at 12 h (B,

lower right).

© Arun Viswanathan

Leaky endotheliumMechanical

Factors

Cell TurnoverLDL

LDL

Ox-LDL

Blood

Vessel Wall

MCP-1

Low KLK-2

Macrophage

Foam Cell

Endothelial Cells

SMCs

© Arun Viswanathan

Complete signaling

Straight Part Branching Point

Flow Pattern Laminar Disturbed

EC turnover Low High

LDL Permeability Low High

JNK1 Transient/optimum High/stable

MCP-1 Expression Low High

Klk-2 Expression Optimum Low

SREBP1 Not activated Activated

Effects on atherosclerosis Anti-Atherosclerosis Atherogenic

Summary

© Arun Viswanathan

• Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell; ShuChien; American Journal of Physiology - Heart and Circulatory Physiology Published 1 March 2007 Vol. 292 no. 3, H1209-H1224 DOI: 10.1152/ajpheart.01047.2006

• Mechanotransduction in vascular physiology and atherogenesis; Cornelia Hahn & Martin A. Schwartz Nature Reviews Molecular Cell Biology 10, 53-62 (January 2009); doi:10.1038/nrm2596

• Mechanisms of Mechanotransduction A. Wayne Orr, Brian P. Helmke, Brett R. Blackman, Martin A. Schwartz

• Wall shear stress oscillation and its gradient in the normal left coronary artery tree bifurcations; Soulis JV, Fytanidis DK, Seralidou KV, Giannoglou GD; Hippokratia 2014, 18, 1: 12-16

• Diana E. Jaalouk and Jan Lammerding; Mechanotransduction gone awry; Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2009 Jul 1. Published in final edited form as: Nat Rev Mol Cell Biol. 2009 Jan; 10(1): 63–73. doi: 10.1038/nrm2597

• Shear Stress Activation of SREBP1 in Endothelial Cells Is Mediated by Integrins; Yi Liu, Benjamin P.-C. Chen, Min Lu, Yi Zhu, Michael B. Stemerman, Shu Chien, John Y.-J. Shyy

References

© Arun Viswanathan

Mechanotrasduction and its role in atherosclerosis

Science

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