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Cellular Biomechanics
Linda Lowe-Krentz
Bioscience in the 21st Century
October 17, 2011
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Outline for today
Tubes
Vessel mechanics and disease Models
Lung mechanics
Technology integration
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Atherosclerosis is Geometrically Focal
Disturbed Flow Region
“Sticky” ECs
“Leaky” Endothelium
Smooth Flow Region
“Non-Sticky” ECs
Intact Endothelium
Flow, along with other factors, contributes to risk.
Inflammed vasculature
Meron Mengistu
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Effects of flow on endothelial cell morphology
• Models are helpful in the study of molecular events in cell culture
12 to 24 hours
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Stress Fiber Alignment
• High Fluid shear stress exposure results in three phases of cell changes: – (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
Phase 1
30’ ↑FSS
Phase 2
60’ – 120’ ↑FSS
Phase 3
Meron Mengistu
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Actin remodeling under shear stress
Low stress for 30 min High stress for 30 min
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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
Low shear flow for 60’
Flow
High shear flow for 60’
Cortical actin No alignment
F-Actin starts to align in the direction of flow
Flow
Actin Remodeling Under Shear Stress
30’
Meron Mengistu
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15 min 30 min 60 min 120 min control
ADINA models
σeff
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
15 min 30 min 60 min 120 min
% E
long
atio
n of
Lon
g-ax
is
Effect of Flow Exposure on Endothelial Cells
4 dyn/cm2 15 dyn/cm2
Jamie Maciaszek, Shannon Alejandro, Josh Slee, Samir Ghadiali
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Finite-Element Models
15 min 30 min 60 min 120 min control
4 dyn/cm2
15 dyn/cm2
σeff
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Add into the model Actin structures we see
No Flow 5’ – 15’ ↑FSS
Phase 1
30’ ↑FSS
Phase 2
60’ – 120’ ↑FSS
Phase 3
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Maximum Effective Stress
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5 min 15 min 30 min 60 min 120 min
Max
imum
Eff
ecti
ve S
tres
s (N
/cm
2)
x 10
000
Effect of Flow Exposure on Basal Surface of Cell
4 dyn/cm2 15 dyn/cm2
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MECHANOSENSING
β/γ α Tyr Tyr Tyr
Tyr Tyr Tyr
Ca2+
Influx
K+ Outflux
Na2+
Influx
Integrin Caveola
PECAM
Src
Raf
MEK1/2
ERK1/2
MEKK1
MKK4/7
JNK
MEKK?
MKK3/6
p38
Ras Rho Rac
MAPK Signaling Cascade
Small GTPases
M E C H A N O T R A N S D U C T I O N
Shc Shp2
Biological Responses
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Burridge, K, & Chrzanowska-Wodnicka , M (1996). FOCAL ADHESIONS, CONTRACTILITY, AND SIGNALING. Annual Review of Cell and Developmental Biology, 12,
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How to continue this work
• Test the model • Do any appropriate signaling proteins turn on? • Are there changes in surface attachment proteins at the
right time? • Use vinculin antibodies • Look for how many patches
of staining are in the cells after flow and where they are.
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0
5
10
15
20
25
30
35
40
45
50
Control 15 min 60 min
Average number FAs/ Cell N
umbe
r of
Foc
al A
dhes
ions
(FA
s)
Time Exposed to high FSS of 15dyn/cm2
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How to continue this work • Block the signaling molecule(s)
• Interfere with the attachment proteins
• Chemically block signaling enzymes
• Refine the model • Alter the attachment?
• Test again
• Identify signal targets
• Test their identities
Infinitely Bounded: Periphery Bounded:
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More tubes • Global Lung Mechanics (the project is from Professor Samir Ghadiali – Ohio State)
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Acute Lung Injury
Ware LB & Matthay MA, New Eng J Med 342(18): 1334-1339 (2000)
• Infections → Necrosis and Detachment of alveolar epithelial cells.
• ↑ permeability of alveolar-capillary barrier → Flooding of small airways/ alveoli
• ↓ 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% mortality rates, 3.6 million hospital days
• Major public health issue, significant burden on health care system
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Quantification of Cellular Morphology
• Laser scanning confocal images of cells with cytoplasmic stain (Calcein AM).
• Cells in 100% monolayer are flatter and thinner.
Subconfluent
1 2 3 4 5 6
Cell Height, z [μm]
Convert confocal cross-sections to
finite element models.
50 μm
ConfluentSubconfluent
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]
Convert confocal cross-sections to
finite element models.
50 μm
Confluent
Ghadiali
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Microbubbles and Cell Injury
• Ventilators reopen fluid-filled lung regions (airways/alveoli) with microbubble flows.
• Microbubbles generate forces that cause cell deformation, death and detachment
• IDEA: Make cell’s more rigid to prevent cell deformation, death and detachment.
Experimental data: Cell death (red cells) Cell detachment (cell loss)
MicrobubbleMicrobubble Fluid-Filled Airway
Type I/II AlveolarEpithelial Cells
(not to scale)
Ghadiali
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Optical Tweezers Methodology
N E
Output: Displacement: D (ω) Phase Shift: δ (ω)
Forced Oscillation
ω: 0.1 – 6,000 Hz
Mengistu and Ou-Yang
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Studying Micro-Mechanical Properties of Endothelial Cells
Microtubules Actin Filaments
Intermediate Filaments
Integrin-bound Bead
PECAM-bound Bead
Endocytosed Bead
Intrinsic Structure
Extracellular Probes Intracellular Probes
Mengistu and Ou-Yang
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Biological/Physiological System are very complex! Biologists provide powerful tools to probe these systems. Engineers provide mathematical tools which can be used to understand how the different components interact.
Integrating Life Science and Engineering
Courtesy HHMI/NIBIB