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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