Lecture 7: Tissue Mechanics, Mechanotransduction and Fibrosis

Mechanical properties of Body Tissues

• Mechanical properties of body tissues are diverse:

  • Brain: Soft

  • Bones: Rigid and stiff

  • Tissues (e.g., heart, skin): Must deform to enable movement

Measurement of Mechanical Properties

• Plotted on a scale - Measures on an Elasticity Scale (kPa):

  • Brain and bone marrow: Soft (1 kPa)

  • Non-calcified stiff tissue: 50 kPa

  • Calcified bones with minerals: Stiff, non-deformable (100 kPa)

The Young’s Modulus (E)

• It is a measure of stiffness or elasticity

• It is a measure of how much is required to deform a tissue

  • The large the number, the more force required to deform it

Key Concept of Mechnaincal Properties

• Mechanical properties are matched to the function of tissue

• This gives rise to the diversity in properties e.g. stiff bones to carry weight, robust skin to form a protective barrier

Usefulness of Brain being a Soft Tissue

• The soft nature of brain tissue is beneficial in development

• It allows the remodelling of tissues in the presence of the skull, which offers a source of protection

Tissue Composition

• Composed of cells and extracellular matrix

Extracellular Matrix

• It is a 3D network of extracellular macromolecules found in multicellular organisms

• It is the main composition of humans → occurs for over 50% of dry mass

  • Most of this is collagen (most abundant protein in the body)

• It defines the mechanical properties of our tissue

• Produced by cells and is maintained throughout life

Decellularisation of Tissues

• Organs can have cells washed out using sufactant, leaving behind a collagenous structure and mechanical properties maintained in the tissues, just as if the cells were present

• The cells are removed → it demonstrates the matrix’s responsiblity for the mechanical properties measured

How Do Cells Communicate With the Surroundings

• Cells and ECM exist together in all tissues

• Cells communicate with the surroundings through contact with the ECM, where they take signals (biochemical and through mechanotransduction) and respond

• The signalling processes in response to signals from the surroundings can explain the different functions of cells with similar genetic material

How Can Cells Change Their Environment

• Secreting new proteis

• Remodelling proteins and molecules in the environemnt

ECM-Cell Response Loop In Development

• Extracellular or mechanic signal detected by cell

• This triggers a cellular response

• Cellular response includes: new matrix deposition, matrix degradation and modification

Homeostatic System

• Systems that actively regulated to maintain a steady state

  • In healthy adult bodies, tissues are maintained in a steady state even when challenged by the environment – constantly regenerated and repaired through self correction and regulation via feedback loops

  • Disease processes can disrupt this balance

Cell Morphology

• The shape and appearance of a cell

• It can be determined experimentally by placing cells of a substrate and controlling the mechanical properties of a substrate e.g. hydrogel

Hydrogel

• Synthetic polymer gel, often polyacrylamide on which cells can be cultured

  • Cells can detect the stiffness of the substrate environment and change their shape in response

• It can be made softer of stiffer by varying the density of cross-links

  • softer gels deform more

  • stiffer gels deform less

Cell Morphology Studies: Stiff Hydrogel

• Cells are larger and spread out more

  • Occurs in cartilage (similar stiffness)

Cell Morphology Studies: Soft Hydrogel

• Cells are smaller and balled up

  • Occurs in bone marrow → similar stiffness

Key Concepts of Tissue Stiffness and Cell Morphology

• Cells spread out more of stiffer substrates

  • The cells get bigger and the nuclei is pulled flat

• The shape of the cells changes

Contractility

• How hard the cells are trying to deform their surroundings

  • Cells hold onto their surroundings and pull against them

• Cells detect stiffness by deforming their surroundings → only detect mechanical properties by interacting with the surroundings

Key Concept of Tissue Stiffness Cellular Contractility

• Cells pull harder and are more contractile on stiffer substrates

  • Cell cultured on a stiff and strong spring

• The stiffer the spring the harder the cells pull against this

• This reaches a plateau, where the cell only produces a certain amount of force → reaches a max level due to the amount of machinery present

  • 2 phase behavoiur

Key Concept of Tissue Stiffness and Proliferation

• Cells grow faster on stiffer substrates

• Seen in 3 different types of cells

  • Vascular smooth muscle cells

  • Mammary tissue

  • Fibroblasts

Key Concept of Tissue Stiffness and Apoptosis

• Apoptosis is slower on stiffer substrates

  • In stiffer environments, cells proliferate faster but apoptoses slower → greater rate of cell population increase

Durotaxis

• Movement of cells on different substrates of stiffness

• Investigates using a substrate on a gell with a gradient of stiffness

  • Cell crawls towards the stiff environment

• Cell migration influenced by a gradient of mechanical properties

Key Concept of Tissue Stiffness and Durotaxis

• Cells migrate towards stiffer regions/ environments

Differentiation

• The commitment of a cell to a particular lineage

  • Stem cells can have a n.o of different fates

Key Concept of Tissue Stiffness and Differentiation

• Stiffness can direct cell fate.

  • Choses a fate influenced by the mechanical properties of its surroundings

• E.g. Mesenchymal cells

Mesenchymal Stem Cells In Soft Substrates

• Drives differentiation of MSC to soft tissue types e.g. fat

  • They become adipogenic → forms fat tissue

Mesenchymal Stem Cells In Stiff Substrates

• Drives differentiation of MSC to stiff tissue types e.g. bone

  • They become osteogenic → form bone

• YAP - a TF favours osteogenesis in these cells

Cell Behaviour Influenced by Mechanical Signals

• Cell morphology (e.g., spreading and shape)

• Contractility (how hard cells pull on their surroundings)

• Propagation rate and apoptosis

• Cell movement (durotaxis)

• Differentiation (commitment to lineage)

Mechanotransduction

• The conversion of mechanical input into a biochemical signal

  • Allows the cells to respond to mechanical signal through a series of signalling pathways

How Do Cells Detect Stiffness

• By deforming their surroundings

  • Must apply forces and have a feedback mechanism to detect whether the surroundings have been deformed

Mechanisms Required By Cells

• Force generation (actomyosin cytoskeleton contraction)

• Force transmission (cytoskeleton- structural proteins that make up and form the mechanical backbone of the cell)

• Mechanosensing (conversion into biochemical signals to activate a pathway to modify cell behaviour)

  • This may be done by protein modification or activation of a transcription programme

Adhesion Complex

• A system of proteins that form at the cell membrane and links the cell to its surroundings

  • It acts as a bridge between the ECM where the cells is attached to and the cytoskeleton

• It gives structure to the cell

• It consists of

  • Integrins

  • Actin

  • Myosin

  • Talin

Integrins

• Membrane proteins that form focal adhesion complexes that tether the cytoskeleton to the matrix.

  • Binds to receptors that span the cell membrane

Actin

• Polymeric filaments and a major component of the cytoskeleton;

• They form the growth of filaments that drives cell spreading – push out at the edge of the cell.

Myosins

• Molecular motors proteins that pull against actin filaments, causing contractility.

Talin

• A protein that deforms when pulled on, activating a signalling cascade (conversion into biochemical signal).

  • Provides mechanosensing as it has lots of domains that unfold to reveal cryptic sites in response to ‘pulling’ that can interact with other proteins

Cell Crawling

• Leading edge Actin filaments polymerise and extend, pushing the cell forward.

• Retrograde flow: Myosin pushes filaments back, dissolving them and allowing the cell to pull itself forward and away from its surroundings.

Relationship between focal adhesion, cytoskeletal tension, and cellular mechanics

• Cells pull on their surroundings using actin polymerisation at the leading cell edge and myosin-II (non-muscle myosin 2A).

• Soft surroundings deform under the cell’s pull.

• Stiff surroundings don’t deform, but mechanosensing proteins (like talin) within the cell do, revealing cryptic binding sites.

• This allows vinculin to bind, activating a signalling pathway that involves MAPK and RhoA.

• Signalling leads to more actin and myosin production, increasing tension on the substrate → pulls harder against substrate

Focal Adhesion Complex and Cytoskeletal Tension: Stiff Substrate

• Stiff substrate activates signalling proteins that increase actin and myosin production.

• Actin polymerises, pushing out at the edge of the cell, leading to cell spreading.

• Myosin pulls harder against fibres, increasing cell contractility.

• Key signalling molecules:

  • MAPK (Mitogen-Activated Protein Kinase)

  • RhoA (Transforming Protein RhoA)

• These molecules drive increased actin and myosin production, causing the cell to generate more force and spread further.

Ion Channels

• Protein complexes that perforate the cell membrane to form pores

  • They allow the movement of substances in and out of cells

Transient Receptor Potential Vanilloid 4 (TRPV4) Channel

• A mechanosensitive ion channel

• When the cell membrane is under tension it is transferred to these ion channels, causing them to open

  • It aqllows ions to enter, changing the intracellular concentration, and causes the activation of a signalling pathway – conversion of signal occurs      

• Conversion Process of mechanical input (membrane tension) to Chemical signal (ion concentration)

Mechanotransimission From Outside the Cell To Nuclues

• In the nucleus, chromatin can be remodelled to affect its interaction with transcription factor, transcription and protein expression

• Changes outside in surroundings can be transmitted through the cell and through the cytoskeleton to the nucleus and transferred to chromatin, changing its organisation

  • Changes in chromatin organisation cause dfferent sites to be activated and accessible → can turn on/off different combinations of genes

• Meadted by the transmission system: focal adhesion complex, actin and myosin within the cytoskeleton

LINC Complex

• A complex of proteins that links the cytoskeleton in the cytosol to the nucleus

• It is made up of nespurin → binds to sun proteins

• Sun proteins cross the nuclear envelop and tether to laminin

Laminin

• A network of proteins that forms a cargo net structure on the inside of the nuclear envelope, containing chromatin

• It also provides mechanical robustness to the nucleus

Force Transmission: Cytoskeleton → Nucleus

• Force is applied to the cytoskeleton, where actin then transmits this to the nesprins proteins which bind to SUN proteins and transmitting forces through to the lamina, inside the nucleus and changes the organisation of chromatin, regulating its activity

Disruption of LINC complex

• Due to point mutations in the laminins, nesprins or sun proteins that affect the transmission of force

• It blocks the mechanotransmission to the nuclues

YAP1L:(“yes-associated protein 1”)

• A transcription factor involved in the development, and differentiation of cancer

• Its behaviour is regulated by its localisation inside the nucleus → it is a transcriptional activator and can interact with its target site

  • When localised in the nucleus it drives osteogenic differentiation

• If moved out of the nucleus, it is inactivated → no longer interacts with DNA

  • Regulation of TF by moving it in/out nucleus

Mechanoregulation of Transcription Factors

• It allows control of specific genetic programs and pathways; movement driven by mechanical properties and input → due to control of factors affecting certain genes

  • Translocation can be mechanically regulated by the properties of the environment

• Cell in soft environment: YAP outside the nucleus

• Cell in stiff environment: YAP inside the nucleus - drives oestogeneis

How Do Cells Respond to Physical Properties of Their Environment

• Mechanotransduction signalling pathways allow cells to detect mechanical properties of their environments and respond:

  • Signaling from focal adhesion complexes (MAPK and RhoA)

  • Mechanosensitive ion channels (e.g., TRPV1)

  • Transmission of force to the nucleus (LINC complex)

  • Translocation of transcription factors (e.g., YAP1)

Fibrosis

• Occurs in response to a misregulation of feedback and loss of homeostasis causing cells to deposit too much matrix

  • The mechanical properties are altered

• Tissues become more collagenous and stiffer → changes the environment and biochemical signals with the tissue deviating from mechanical homestatsis

  • Mechanical properties no longer matched to function

Fibroblasts

• Cells responsible for synthesising extracellular matrix, such as collagen.

• They don’t form a continuous layer and can move around when cultured in 2D

• Cells can move towards site of injury (durotaxis → move to scar)

  • Necessary for wound healing

Myofibroblasts

• Activated fibroblasts in response to injury

• These cells are more contractile, pulling harder on their environment and secrete more ECM, contributing to fibrosis (excess matrix)

• They are formed by mechanical stimulation e.g. in response to a stiff environment and by chemical signals

  • e.g. TFGB-1 → cause differentiation of fibroblasts into myofibroblasts

Examples of Fibrotitc Diseases

• Severe atherosclerosis (thickening of blood vessel walls) of the aorta. – constricts blood flow

• COPD (chronic obstructive pulmonary disease) lung tissue – blocks gas exchange

  • Excessive matrix prevents the correct function

Role of collagen and matrix in tissue repair

• Collagen and matrix are produced to fix a wound.

Importance of Scaring

• It can be healthy because it helps wounds heal and prevents further damage by forming a protective layer.

• But it must eventually be resolved by removing excess matrix and collagen to return to the healthy starting point.

  • excess scar tissue can lead to loss of function

Excessive Scarring Fibrosis

• Tissue doesn’t return to healthy tissue function due to the presence of excess matrix and scar tissue

  • The environment becomes too contractile and restricts movement, leading to pathology

Fibrotic Response

• Excessive scarring is not resolved

• Dysregulation of the ECM causes changes in the mechanical environment as the scar tissue is stiff

• An immune response is generated leading to the infiltration of immune cells

• These factors disrupt tissue function, causing further damage; and resulting in a second feedback loop of a fibrotic response

Healthy Lungs

• Air passes through bronchioles into the alveoli which have thin membranes to allow gas exchange

Idiopathic Pulmonary Fibrosis (IPF)

• Fibrosis occurs in the alveoli, resulting in the presence of excess matrix that prevents gas exchange and results in suffocation → low removal and uptake of CO2 and O2

• Not a rare disease → affects 100,000 people aged 14-43

  • Affects men more than women

• Diagnosed by occluded x-rays – matrix build-up or blocking up of the lungs – white trace

• Average diagnosis time: 1-2 years after symptom onset

• Fatal - 50% mortality rate within 2-3 years of diagnosis

Symptoms of Idiopathic Pulmonary Fibrosis

• Common

  • Shortness of breath

  • Chronic dry cough

  • Finger clubbing due to growth factor signalling produced by the scar tissue

• Occasional

  • Fatigue

  • Weakness

  • Weight loss

Cause of IPF

• Idiopathic - unknown but risk factors include

  • Smoking

  • Environmental exposures

  • Chronic viral infection

  • Abnormal acid reflux

  • Family history

  • Ageing – large risk factor for fibrotic disease

Characterisation of IPF

• A group of myofibroblasts – push into healthy tissue, leaving behind scare tissue

  •   Myofibroblasts at the leading edge of the disease region produce too much matrix

Mass Spectrometry Proteomics

• Used to analyse cross-sections of tissue and ECM composition to identify proteins present

• Contains more Collagen I than normal → typical of scarring

  • It builds up in scar tissue, causing stiffness and blocking gas exchange → contributes to disease phenotype

Pathophysiology of Idiopathic Pulmonary Fibrosis (IPF)

• IPF involves tissue insult → causes scarring and dysregulation of the extracellular matrix.

• This leads to collagen-I production, altering tissue mechanics and the formation of a fibrotic front, disrupting alveolar function.

• Immune response causes inflammation and thickening of alveolar walls resulting in impaired gas exchange and disrupted tissue function, leading to organ failure.

• A positive feedback loop ensues, where ongoing injury promotes further fibrosis. → continuing injury that drives the fibrotic response