Lecture 10 - Introduction to fibrosis

Question 1

ECM: what is it, where is it found, what are its most abundant proteins, and what is its role?

Answer 1

The extracellular matrix (ECM) is a three-dimensional network of extracellular macromolecules

Found in multicellular organisms

Collagen - one of the most common proteins in our bodies

The ECM defines the mechanical properties of our tissues (creates tissues along with cells)

Question 2

Young’s modulus

Answer 2

The force/strain required to deform something by a certain amount

Used to show elasticity

Question 3

ECM: how does it communicate with cells and how do cells communicate with it?

Answer 3

Mechanosignalling occurs, conveying mechanical pressure to communicate with cells

Then cells react to this and send out their own signals, instructing the ECM

‘Homeostatic’ systems are actively regulated to maintain a steady state within the ECM

Question 4

How do cells know which cell types they should be/which genes to express?

Answer 4

Their environment tells them - they receive signals that tell them where they are and they express the correct genes based on this

Question 5

Hydrogel: what is it, how is its stiffness controlled, and what is it composed of?

Answer 5

A synthetic polymer gel on which cells can be cultured

It can be made soft or stiff by varying the density of crosslinks

Often made of polyacrylamide (like a gel for SDS-PAGE)

Question 6

Do cells spread more or less, are they harder or weaker, do they grow faster or slower, do they migrate towards or away from, do they promote more or less differentiation, and do they do apoptosis more or less on stiffer substrates?

Answer 6

Stiffer

Harder - more contractile

Faster

Towards stiffer substrates

More differentiation

Less

Question 7

What cell behaviours are affected by mechanical signals?

Answer 7

• Cell morphology (e.g., spreading and shape)
• Contractility (how hard cells pull on their surroundings)
• Propagation rate and apoptosis
• Cell movement
• Differentiation (commitment to lineage)

Question 8

Mechanobiology

Answer 8

An emerging field of science at the interface of biology and engineering [and physics]. It focuses on the way that physical forces and changes in cell or tissue mechanics contribute to development, physiology, and disease.

Question 9

Mechanotransduction

Answer 9

The conversion of a mechanical input into a biochemical signal

Question 10

How do cells receive mechanosignalling?

Answer 10

• Focal adhesions and cytoskeletal remodelling
• Ion channels
• Force-dependent matrix remodelling
• Deposition of a new matrix
• Continuous mechanical linkage to the nucleus
  *Translocation of transcription factors/nuclear remodelling
• Cell-cell contacts

Question 11

What is the process behind cells ‘feeling’ mechanical stress - in regards to focal adhesions and cytoskeleton remodelling specifically?

Answer 11

1 - Force generation (actomyosin contraction)
2 - Force transmission (cytoskeleton)
3 - Mechanosensing (conversion into biochemical signals), this can be done by doing a tug-of-war type of thing

Question 12

What are the key proteins in force generation and transmission?

Answer 12

• Integrins – membrane proteins that form focal adhesion complexes that tether the cytoskeleton to the matrix
• Actin – polymeric filaments; major component of the cytoskeleton; growth of filaments drives cell spreading.
• Myosins – ‘molecular motors’ pull against actin filaments, causing contractility.
• Talin – a protein that deforms when pulled on, activating a signalling cascade (conversion into biochemical signal).

Question 13

How do cellular proteins activate downstream signalling proteins in response to mechanical stress?

Answer 13

Focal adhesion complex: Actin is polymerized at the edge of the cell and pulled by myosin-II (“non-muscle myosin 2A”)

This stretches Talin, causing unfolding which causes it to become more activated and allows Vinculin to bind to its activated domains, activating downstream signalling pathways

Question 14

What signalling proteins are activated in response to stiff ECM and what do they do?

Answer 14

MAPK (“Mitogen-activated protein kinase 1”)

RhoA (“Transforming protein RhoA”)

These cause the cell to make more myosin and actin, so the cell pulls harder - retrograde flow, causes cells to move in like a tank (the wheels)

Question 15

Mechanosensitive ion channels: how do they react to mechanosignalling?

Answer 15

The ion channels are pulled open, allowing an influx/efflux of ions, changing the internal biochemistry of the cell and causing the cell to react

Ie TRPV1 (may-b ggle)

Question 16

Nuclear transmission force: how is it involved with mechanosignalling and what is it mediated by?

Answer 16

Environment attaches to the cytoskeleton which transmits force to the nucleus

The nucleus reacts to the force applied and
changes chromatin formation, resulting in changes in gene expression

This is mediated by the linker of the nuclear skeleton hand cytoskeleton (LINC) complex - composed of Nesprins which bind to SUN proteins which span the nuclear envelope and interact with laminins

Question 17

Translocation of transcription factors: how do they react to mechanosignalling?

Answer 17

YAP1 (transcription factor) can be moved in and out of the nucleus

When moved into the nucleus, in a stiff environment, it drives osteogenic differentiation - bone formation

Mechanical regulation of transcription factors allows control of specific genetic programs

Question 18

Fibrosis: what is it, what does it result in, and what components cause fibrosis formation?

Answer 18

Dysregulation of ECM - Misregulation of feedback and loss of homeostasis causing cells to deposit too much matrix

Stiffer tissues cause mechanical properties to no longer be matched to function

Fibroblasts

Question 19

Fibroblasts: what do they do, what are they primarily used for, and what factors cause fibroblast activation?

Answer 19

Fibroblasts are cells activated at sites of injury which are responsible for synthesising extracellular matrix, such as collagen, after making myofibroblasts which are cells that secrete more ECM and make cells more contractile

They move towards wound sites and are required for wound healing

• Mechanical stimulation
• Transforming growth factor β1 (TGFB1)

Question 20

Name two examples of fibrosis

Answer 20

Atherosclerosis - blocking of the vessels
COPD - chronic obstructive pulmonary disease

Question 21

Scarring: can it be healthy?

Answer 21

Yes, scarring occurs after fibroblast deposits more ECM to cover the wound and prevent infection

When scarring is healthy, the scar will be replaced with healthy cells over time

If too much scar tissue is produced, this can be unhealthy and loss of function may occur

Question 22

IPF: what is its name, what is it, what are its symptoms, what are its risk factors, and what is the timeline of the disease?

Answer 22

Idiopathic pulmonary fibrosis: idiopathic - we don’t know what mechanisms cause the disease

Causes irreversible enlargement of the bronchioles and alveoli, causing a reduction in gas exchange

Common symptoms:
* Shortness of breath
* Chronic dry cough
* Finger clubbing

Occasional symptoms:
* Fatigue
* Weaknesses
* Weight loss

Smoking, environmental exposure, chronic viral infections, abnormal acid reflux, and family history of the disease

Diagnosis after 1-2 years of symptom onset
Death after 2-3 years of suffering occurs in 50% of sufferers