lecture 7

Question 1

What is the molecular environment of a wound?

Answer 1

(healing vs chronic ulcers)

• mitogenic activity (high vs low)
• inflammatory cytokines (low vs high)
• proteases (low vs high
• mitotically competent cells vs senescent cells

Question 2

What is one the most important things that needs to happen in order to heal?

Answer 2

Clearing of the inflammatory response

Question 3

What the potential outcomes of an injury?

Answer 3

Stimulus removed (acute injury)
1. Parenchymal cell death (intact tissue framework), superficial wounds, some inflammatory processes:
Regeneration: restitution of normal structure
E.g. liver regeneration after partial hepatectomy
superficial skin wounds
resorption of exudate in lobar pneumonia
2. parenchymal cell death (damaged tissue framework), deep wounds
Repair: scar formation
e.g. deep excisional wounds
myocardium infarction

Persistent tissue damage
Fibrosis: tissue scar
e.g. chronic inflammatory diseases (cirrhosis, chronic pancreatitis, pulmonary fibrosis)

Question 4

What is an aspect of normal homeostasis that relates to wound healing?

Answer 4

In normal tissues there is an equilibrium of proliferation vs apoptosis

• low level of proliferation in many tissues but not all
• brain: nerve cells are essentially life long (almost no proliferation)

Question 5

What are examples of renewing tissues?

Answer 5

• Epidermis
• GI tract epithelium
• Haematopoietic system
• constant homeostatic replacement

Question 6

What are examples of stable tissues that undergo compensatory growth?

Answer 6

Liver and kidney

Question 7

What occurs during cell/tissue homeostasis?

Answer 7

• baseline cell population that replaces itself (proliferation vs apoptosis)
• certain level of plasticity - may have a function of their own but can divide/differentiate into cells of more specified functions
• differentiated cells no longer able to divide
• stem cells: small number of cells that are able to replace baseline population

Question 8

How do cells know when to proliferate or undergo apoptosis?

Answer 8

• autocrine (same cell) e.g. liver regeneration
• paracrine (next door neighbour) (many Growth Factors) e.g. wound healing
• endocrine (far away, delivered by blood), e.g. hormones

Question 9

What are some examples of organ normal and abnormal healing?

Answer 9

Skin

• normal healing: wounds, burns
• abnormal healing: chronic ulcers – pressure, venous stasis, diabetes

Fractures

• normal healing (slower)
• abnormal healing - chronic non-healing fractures

GIT

• normal healing - mechanical or toxic lesions
• abnormal healing - chronic peptic ulcer, inflammatory bowel disease

Lung

• normal healing: simple acute injuries
• abnormal healing: ARDS, chronic obstructive pulmonary disease

Myocardial infarction: very difficult to heal, often the site of injury will retain fibrotic tissue

Question 10

What are mitotically active cells?

Answer 10

• always dividing (?)
• replace dying cells
• epithelia: skin, oral cavity, exocrine ducts, GI tract, hematopoietic

Question 11

What are quiescent cells?

Answer 11

• stable
• usually G0 and low rate of division
• driven into G1 and rapid proliferation
• liver, kidney, pancreas, endothelium, fibroblasts

Question 12

What are long-lived/stable cells?

Answer 12

• ‘permanently’ removed from cell cycle
• irreversible injury leads only to scar (replacement with non-specialised/non-functional cells/tissue)
• nerve cells, myocardium
• excluding ‘tissue-specific’ stem cells

Question 13

What is regeneration?

Answer 13

Regeneration of injured cells by cells of same type as with regeneration of skin/oral mucosa.

Requires basement membrane.

Question 14

What is replacement?

Answer 14

Injured tissue replaced by fibrous tissue (fibroplasia, scar formation) – ‘non-functional’

Question 15

What do both regeneration and replacement require?

Answer 15

Cell growth, differentiation, and cell-matrix interaction

Question 16

What are the major cells associated with healing?

Answer 16

• fibroblasts: produce growth factors important in the healing process
• endothelial cells: every cell requires a blood supply
• epithelial cells: esp GIT
• osteoblasts: cell types of bone involved in regenerating bone tissue

Question 17

What affects the outcome of healing?

Answer 17

• organ affected
• cells: labile/stable/permanent
• ? loss of matrix ~ severity of injury

Question 18

What are healing mechanisms controlled by?

Answer 18

• controlled by biochemical factors released in response to cell injury, cell death, or trauma
• most important control: inducing resting cells to enter cell cycle, i.e. to ‘awaken’ them
• balance of stimulatory or inhibitory factors
• shorten cell cycle i.e. faster cell division
• decrease rate of cell loss i.e. inhibit apoptosis

Question 19

What are the 5 steps of molecular mechanisms of proliferation?

Answer 19

1. ligand (growth factors, matrix proteins, hormones, cytokines, chemokines etc) and receptor (at cell membrane)
2. 2nd messenger cascade (cytoplasm)
3. transcription factor activation (nucleus)
4. gene expression (nucleus)
5. Action

Question 20

What is the role of the ligand-receptor interaction?

Answer 20

designed to amplify the signal cascade: the binding of just a few ligands can trigger a rapid and sustained response

Question 21

What are the three classes of receptor types?

Answer 21

growth factor receptors

• intrinsic tyrosine kinase activity
• receptors exist as monomers (ligand binding, transmembrane, cytoplasmic)
• growth factors bind
• binding –> dimerisation –> autophosphorylation at tyrosine
• activation of tyrosine kinase activity
• phosphorylates/activates 4 types cytoplasmic proteins (adaptor molecules)
• leads to a variety of cytoplasmic pathways (effector molecules)
• leads to proliferation

seven transmembrane G-protein coupled receptors

• inflammatory chemokines and hormones bind
• e.g. vasopressin, serotonin, histamine, many drugs

cytokine receptors

• w/o intrinsic tyrosine kinase activity
• don’t have any capacity on their own to trigger a response
• rather they respond to a secondary messenger which then has enzyme activity
• bind to cytokines
• e.g. IL-2, IL-3, IFN-alpha, beta, gamma, GM-CSF, growth hormone etc
• activate cytosolic protein tyrosine kinases
• phosphorylation of receptor leads to functional activities e.g. synthesis, secretion, migration etc OR inhibition of these things
• all involve ligand interaction

Question 22

What are the actions of growth factors?

Answer 22

• stimulate cell division and proliferation
• cell migration
• promote cell survival

Question 23

What are some examples of major growth factors?

Answer 23

EGF/TGF-alpha (epidermal growth factor/transforming growth factor-alpha)

• from macrophages, platelets, epithelia and found in tissue fluids and secretions (e.g. sweat, saliva, urine)
• mitogenic for epithelial cells and fibroblasts

PDGF (platelet derived…)

• family - 3 isoforms, from platelets, macrophages, endothelial cells, smooth muscle cells and tumour cells
• proliferation and migration of fibroblasts, smooth muscle cells, monocytes, pro-inflammatory

FGFs (fibroblast…)

• family - > 10 types, acidic and basic most studied
• from macrophages, T cells, endothelial cells and fibroblasts
• angiogenesis and [mac, fibroblast, epithelial] migration during wound repair
• development - skeletal muscle and lung
• haematopoiesis

HGF (hepatocyte…)

• from fibroblasts, endothelial cells, liver non-parenchymal cells
• mitogenic for epithelial cells - liver, bile, duct, lung, breast, skin
• also causes cell migration
• required for survival during embryogenesis

VEGF (vascular endothelial…)

• family – from fibroblasts and endothelial cells
• induces blood vessel formation
• binds to receptor on endothelial cells

Question 24

What is a case of cell growth that is independent of growth factor signals?

Answer 24

• cells can also respond to loss of cell-cell contact signals
• ‘loss of their neighbourhood’
• e.g. cells anchor to dish surface and divide
• when cells have formed a complete single layer, they stop dividing (density-dependent inhibition)
• if some cells are scraped away, the remaining cells divide to fill the gap and then stop
• this is as opposed to cells which require growth factors to grow in a lab

Question 25

What are examples of second messenger cascades in the context of proliferation?

Answer 25

- MAP (mitogen associated) kinase pathway - Inositol-lipid (IP3) - cyclic AMP (adenosine monophosphate) - major pathway emanating from G-protein couple receptor - phosphoinositide-3-kinase (PI3) - JAK/STAT (janus kinase/signal transduction and activators of transcription)

Question 26

What is the MAP kinase pathway?

Answer 26

- GF binds receptor tyrosine kinase | - chain of adaptor proteins/phosphorylation events that ultimately lead to the nucleus and stimulate growth

Question 27

What are the ultimate targets of secondary messenger pathways?

Answer 27

- signal transduction pathways aim to transmit signals to the nucleus and cause changes in regulation of gene expression - thus their targets are transcription factors - e.g. protooncogenes: c-fos, c-jun, c-myc = growth promoting - e.g. tumour suppressor genes: p53, Rb (retinoblastoma), PTEN = inhibit growth promoting pathways

Question 28

What is an important inhibitor of proliferation?

Answer 28

TGF-beta - inhibits progress through S phase - transforming growth factor - beta - pleiotropic = multiple actions depending on tissue/cell and type of injury - decreases epithelial cell proliferation, increases collagen production, anti-inflammatory

Question 29

Why should we control the cell cycle?

Answer 29

- cells duplicate their genetic material before they divide, ensuring that each daughter cell receives an exact copy of the genetic material - a dividing cell duplicates its DNA, allocates the two copies to opposite ends of the cell, and only then splits into daughter cells - it is important that these events flow in a precise order - timing is crucial - regulation of the cell cycle itself is cell autonomous - cell cycle is complex - therefore control is complex

Question 30

What is a potential outcome of the loss of cell cycle control?

Answer 30

- cancer | - tumour cell has multiple nuclei - cell cycle totally screwed up

Question 31

How is the cell cycle controlled?

Answer 31

- cycle is initiated by signals from growth factors or matrix - two critical control point - G1 - S: check for DNA damage - G2 - M: check for damaged or unduplicated DNA

Question 32

What are the two types of control in regards to the cell cycle?

Answer 32

- cascade of protein phosphorylation pathways involving cyclins and cyclin dependent kinases (CDKs). Key cyclins are present at different phases of the cell cycle. - a set of checkpoints - completion of molecular events monitored - damage induces delay to allow for DNA repair or activation of apoptosis

Question 33

How is the position of the cell cycle determined?

Answer 33

- the cyclin present 1. Cdk1/cyclin B initiates mitosis 2. mitotic activators stimulate the synthesis of cyclin D 3. cyclins D and E and Cdk4 and Cdk2 phosphorylate Rb, inactivating it 4. Once R (restriction point) is passed, the cell cycle proceeds 5. Cdk2/cyclin A stimulate DNA replication

Question 34

(briefly) how does regeneration of the liver occur in rats?

Answer 34

- organ has the capacity to regenerate after partial hepatectomy - can experimentally show that hepatocytes near the region that has been excised will be moved from a G0 state to G1, - rapid and efficient response - ultimately get a relatively normal sized liver

Question 35

What is the importance of the matrix in terms of healing?

Answer 35

- if you have injury to the cells but the ECM that underlies these is relatively normal you can get complete recovery - if there is a destruction of underlying ECM, the cells don't know where to go - fibrosis occurs more than in normal regeneration

Question 36

What has developmental biology shown us?

Answer 36

- that ECM is critical for correct cell development and proliferation