Lecture 25 Mechanobiology 1 Flashcards
Define mechanobiology
the study of physical forces and changes to cell or tissue mechanics, and how they contribute to development, physiology, and disease
Define mechanotransduction
converting physical forces to biochemical responses (aka mechanosignaling)
What occurs in mechanotransduction
e.g. A physical cue is translated into biochemical signal transduction i.e. activation of kinases that change the chromatin structure and the expression of gene.
Define mechanosensing
a protein or cellular structure’s response to a physical cue, initiating mechanotransduction
What are the 4 key concepts in mechanotransduction
- mechanosensing
- signal transduction
- signal integration at nucleus
- cellular response
Describe each of the 4 key concepts of mechanotransduction
- Mechanosensing:
- Cells test their environment
- Adhesion receptors, membrane proteins probe ECM - Signal transduction:
- Mechanical signal is transduced along a linked network, usually the actin cytoskeleton
- Cytoskeleton is often the force conduit - Signal integration at nucleus:
- Accumulation of signals over time
- Chromatin rearrangement, nuclear pore opening - Cellular response:
- Over a range of timescales from microseconds to minutes
- E.g. Cell shape change, fate, motility, growth
Give 2 examples of mechanotransduction
- Blood pressure autoregulation and coronary artery disease - myogenic tone
- Auditory mechanotransduction and hearing
Define myogenic tone
Myogenic tone is defined as a contraction that originates from the muscle itself rather then ANS or hormone processes
What do arteries use myogenic tone for?
Arteries use a myogenic mechanisms to increase or decrease BP to keep BF in the blood vessel constant
What does an increase in BP cause?
An increase in arterial diameter
What occurs at a certain BP?
At a certain pressure, this increase in blood pressure is sensed and [Ca2+] in smooth muscle increases, which counteracts the increase by reducing arterial diameter.
What do endothelial cells grown in culture detect and respond to? How?
Fluid flow
They change their cytoskeletal organisation
How is the cytoskeleton organised in control (no fluid flow) and with strain (fluid flow)
Control = irregular Strain = organised and ordered
Describe auditory mechanotranduction and hearing
Stereocilia bend in response to sounds, producing an action potential
What is used to investigate mechanotransduction in vitro?
Lung on a chip
Why are lung cells not just grown on a petri dish?
They would lack the physical cues e.g. 3D structure of their environment and the flow of fluid around them
To develop an accurate model of the lungs, the effect of …. must be emulated
Mechanical activity of breathing
What does the lung on the chip emulate?
Stretching force applied to lung cells upon breathing
What is necessary in the lung on chip model?
A flexible porous polydimethylsiloxane (PDMS) membrane to act as a site for cell cultures
What is on top/beneath PDMS membrane?
Top: epithelial layer
Bottom: endothelium
What does the degree of polymerisation of PDMS change?
Flexibility
Describe another vital property of PDMS membranes?
Translucent to visualise the cells
What is put into the upper and lower channels and why?
Air is put in the upper channel and liquid is put in the lower channel, mimicking air in the lungs and blood in the capillaries
What do the side chambers mimic?
The stretching force which would be applied to the cells during breathing is mimicked in the side chambers, by the application of a vacuum, which acts to deform the membrane.
How do we know the epithelial layer has formed properly in lung on chip?
Immunofluorescence analysis
Measuring trans-epithelial resistance
In immunofluorescence analysis what is the black gap in the middle?
The PDMS membrane
Why is immunofluorescence analysis not a perfect model?
In vivo there is an ECM and the epithelial and endothelial layers would be directly adjacent
In immunofluorescence analysis, what would occur if cell layers are stretched?
Monolayers with proper tight junctions
How can the tight junctions in immunofluorescence be visualised?
Stains for occluding and VE cadherin
Where is trans-epithelial resistance measured?
At the air-liquid interface
What sort of evidence is trans-epithelial resistance?
Electrophysical
What will occur in a malformed monolayer in terms of trans-epithelial resistance?
Leakage of electrical current and so a lower resistance
What will occur in a properly formed monolayer in terms of trans-epithelial resistance?
Resistance will be higher as no leakage
What is important when measuring trans-epithelial resistance?
It’s important that the upper channel is filled with air and the lower channel with liquid, as air-liquid interface resistance is higher than liquid-liquid interface resistance.
What can lung on a chip be used to study?
Lung inflammation
How can we study lung inflammation using this technique?
TNF is an inflammatory cytokine
Adding it to epithelium causes inflammatory markers to appear in the endothelium
Neutrophils get stick to the endothelial cells when TNF is present in the epithelium, indicating that something has changed in the endothelium which makes it ‘sticky to neutrophils
What happens when bacteria are added to the epithelium?
There will be an accumulation of neutrophils in the adjacent part of the endothelium. The neutrophil can migrate through the pore to the epithelial layer, contacting, engulfing and destroying the bacteria.
What requires mechanical stretching?
The uptake of nanoparticles
What expression is induced when nanoparticles are present? What is this? Where?
ICAM-1
Inflammatory mediator
In the endothelial layer
What occurs when nanoparticles are added to the epithelium?
Increases ICAM-1 expression independently of strain
What occurs if nanoparticles and the strain are added simultaneously?
ICAM-1 is expressed even further
What also increases when nanoparticles are added alongside the strain?
Generation of reactive oxygen species
Why do we want to identify inhibitors of nanoparticle uptake?
Mechanical activity contributes to the translocation of nanoparticles. We want to avoid this as we do not want to take up nanoparticles and so we can use this model to identify inhibitors of nanoparticle uptake.
Why is the ECM stiffness important?
Cells sense what they are surrounded by - other cells and ECM (inc. collagen, fibronectin, etc.). These main components of the ECM give different degrees of crosslinking changing their physical properties and stiffness.
What are the 3 types of stress?
- Shear stress acts parallel to an area.
- Compression stress is a pushing force (N)
- Tension stress is a pulling force (N)
How is strain defined?
Strain is defined as the change in the length divided by the original length. It is the reaction of the material to the applied stress.
What occurs as stress increases and strain?
As stress and strain increases and so does stiffness.
What is the equation for stress? What is it measured in?
See notes
= force/area
Measured in pascals i.e. N/m^2
What is the equation for strain? What is it measured in?
See notes
= change in length/original length
Unitless
What is the equation for stiffness? Measure in?
See notes
= stress/ strain
Measured in pascals i.e. N/m^2
The stress-strain relationship is … for each material
Specific
Different tissues have … values for …
Different
Stiffness
Stiffness in brain vs bone
Brain, for instance, is a soft tissue, with a lower stiffness; bone is a much stiffer tissue.
Soft vs intermediate vs stiff values for stiffness
Soft = 2kPa Intermediate = 8 kPa Stiff = 80kPa
