Cell BiomechanicsCell Biomechanics

Linda Lowe‐Krentz

Bioscience in the 21st CenturyBioscience in the 21 Century

October 22, 2010

Outline for todayOutline for today

Tubes

Vessel mechanics and diseaseVessel mechanics and disease Models

L h iLung mechanics

Technology integration

Atherosclerosis is  Geometrically Focal

Disturbed Flow Region

“Sticky” ECs

Smooth Flow Region

Sticky  ECs“Non‐Sticky” ECs

“L k ”

Intact Endothelium Inflammed “Leaky” 

Endothelium

Endothelium Inflammed vasculature

Meron Mengistu

Flow, along with other factors, contributes to risk.

Effects of flow on endothelial cell morphology

• Models are helpful in the study of molecular events in cell culture

12 to 24 hours

Stress Fiber AlignmentStress Fiber Alignment

• High Fluid shear stress exposure results inHigh Fluid shear stress exposure results in three phases of cell changes: – (1) increase in stress fiber formation(1) increase in stress fiber formation

– (2) dense cortical band formation of actin cytoskeleton

– (3) stress fiber alignment in the direction of flow

No Flow 5’ – 15’ FSS  30’ FSS 

60’ – 120’ FSS 

Phase 3Phase 1 Phase 2

Meron Mengistu

Actin remodeling under shear stressActin remodeling under shear stress

Low stress for 30 min High stress for 30 minLow stress for 30 min High stress for 30 min

Actin Remodeling Under Shear Stress

Low shear flow for 60’ High shear flow for 60’

Flow

g

F Actin

Flow

Cortical actin No alignment

F‐Actin starts to align in the direction of

30’g direction of 

flow

Cells and their actin filaments begin to align in the direction of flow after 60’ of high shear flow exposure.  No change under low shear stress

Meron Mengistu

60 00%

70.00%

s

Effect of Flow Exposure on Endothelial Cells

40.00%

50.00%

60.00%of Lon

g‐axis

20.00%

30.00%

 Elongation 

0.00%

10.00%

15 min 30 min 60 min 120 min

%

σeff4 dyn/cm2 15 dyn/cm2

ADINA models

15 min 30 min 60 min 120 mincontrol

Jamie Maciaszek, Shannon Alejandro, Josh Slee, Samir Ghadiali

Finite‐Element ModelsFinite Element Models

15 min 30 min 60 min 120 mincontrol

4 dyn/cm2

15 dyn/cm2

σeff

Add into the model ActinAdd into the model Actin structures we see

N Fl 5’ 15’ FSS30’ FSS 

No Flow 5’ – 15’ FSS 60’ – 120’ FSS 

Phase 1 Phase 2Phase 3

Maximum Effective StressMaximum Effective Stress

4

4.5

cm2)

10000

Effect of Flow Exposure on Basal Surface of Cell

2.5

3

3.5

e Stress (N

/c x 1

1

1.5

2

mum

 Effective

0

0.5

5 min 15 min 30 min 60 min 120 min

Maxim

4 dyn/cm2 15 dyn/cm2

MECHANOSENSING K+ OutfluxIntegrin

CaveolaPECAM

/TyrTyrTyr

TyrTyrTyr

Ca2+ 

InfluxNa2+ 

InfluxSrc MInfluxInfluxSrc

Ras  RhoRac SmallGTPases

ECHAN

ShcShp2

Raf

MEK1/2

MEKK1 

MKK4/7

MEKK? 

MKK3/6MAPK 

Si li

GTPases NOTRAMEK1/2 

ERK1/2 

MKK4/7 

JNK

MKK3/6 

p38

Signaling Cascade

NSDUCCTION

Biological Responses

Burridge, K, & Chrzanowska‐Wodnicka , M (1996). FOCAL ADHESIONS, CONTRACTILITY, AND SIGNALING. Annual Review of Cell and Developmental Biology, 12,Developmental Biology, 12, 

How to continue this workHow to continue this work

• Test the modelTest the model• Do any appropriate signaling proteins turn on?• Are there changes in surface attachment proteins at the 

h ?right time?• Use vinculin antibodies • Look for how many patchesLook for how many patches

of staining are in the cells after flow and where  they are.y

Average number FAs/ Cell(FAs)

45

50

sion

s (

30

35

40

 Adh

e

20

25

30

f Focal

5

10

15

berof

0

Control  15 min 60 minNum

Time Exposed to high FSS of 15dyn/cm2

How to continue this workHow to continue this work• Block the signaling molecule(s)

• Interfere with the attachment proteins

• Chemically block signaling enzymes

• Refine the model• Alter the attachment?

I fi it l B d d: P i h B d d:• Test again

• Identify signal targets

T t th i id titi

Infinitely Bounded: Periphery Bounded:

• Test their identities

More tubesMore tubes• Global Lung Mechanics (the project is from Professor Samir Ghadiali – Ohio State)

Acute Lung InjuryAcute Lung Injury

• Infections → Necrosis and Detachment of alveolar epithelial cells.

•↑ permeability of alveolar‐capillary↑ permeability of alveolar capillary barrier → Flooding of small airways/ alveoli

•↓ gas exchange severe hypoxia•↓ gas exchange, severe hypoxia

• Standard of care: Mechanical Ventilation

• Ventilators cause additional cell injury (Ventilator Associated Lung Injury)

• 200 000 patients/yr 30% to 40%• 200,000 patients/yr, 30% to 40% mortality rates, 3.6 million hospital days

Ware LB & Matthay MA,New Eng J Med 342(18): 1334‐1339 (2000)

• Major public health issue, significant burden on health care system

Quantification of Cellular MorphologyQuantification of Cellular Morphology

• Laser scanning confocal images of cells with cytoplasmic stain (Calcein AM).

• Cells in 100% monolayer are flatter and thinner.

Subconfluent ConfluentSubconfluent Confluent

Convert confocal cross-sections to

fi it l t

Convert confocal cross-sections to

fi it l tfinite element models.

finite element models.

1 2 3 4 5 6

Cell Height, z [μm]50 μm

1 2 3 4 5 6

Cell Height, z [μm]

1 2 3 4 5 61 2 3 4 5 6

Cell Height, z [μm]50 μm

Ghadiali

MicrobubblesMicrobubbles and Cell Injuryand Cell Injury

MicrobubbleMicrobubble Fluid-Filled Airway

Type I/II AlveolarEpithelial Cells

(not to scale)

Experimental data:Cell death (red cells)Cell detachment (cell loss)

Ghadiali

• Ventilators reopen fluid‐filled lung regions (airways/alveoli) with microbubble flows.

Ghadiali

• Microbubbles generate forces that cause cell deformation, death and detachment

• IDEA: Make cell’s more rigid to prevent cell deformation, death and detachment.

Optical Tweezers Methodology

Forced 

NEOscillation

: 0.1 – 6,000 Hz

Output:Displacement: D ()Phase Shift: ()

Mengistu and Ou‐Yang

Phase Shift:  ()

Studying Micro-Mechanical Properties of Endothelial CellsEndothelial Cells

Extracellular ProbesIntracellular Probes

MicrotubulesActin FilamentsIntegrin‐bound 

BeadIntrinsic Structure

IntermediatePECAM‐boundEndocytosed Intermediate Filaments

PECAM bound Bead

Endocytosed Bead

Mengistu and Ou‐Yang

Integrating Life Science and EngineeringIntegrating Life Science and Engineering

Biological/Physiological System are very complex!

Biologists provide powerful tools to probe these systems.g p p p y

Engineers provide mathematical tools which can be used to understand how the different components interact.

Courtesy HHMI/NIBIB