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What is the molecular environment of a wound?

(healing vs chronic ulcers)
- mitogenic activity (high vs low)
- inflammatory cytokines (low vs high)
- proteases (low vs high
- mitotically competent cells vs senescent cells


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

Clearing of the inflammatory response


What the potential outcomes of an injury?

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)


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

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)


What are examples of renewing tissues?

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


What are examples of stable tissues that undergo compensatory growth?

Liver and kidney


What occurs during cell/tissue homeostasis?

- 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


How do cells know when to proliferate or undergo apoptosis?

- 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


What are some examples of organ normal and abnormal healing?

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

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

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

- 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


What are mitotically active cells?

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


What are quiescent cells?

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


What are long-lived/stable cells?

- '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


What is regeneration?

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

Requires basement membrane.


What is replacement?

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


What do both regeneration and replacement require?

Cell growth, differentiation, and cell-matrix interaction


What are the major cells associated with healing?

- 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


What affects the outcome of healing?

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


What are healing mechanisms controlled by?

- 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


What are the 5 steps of molecular mechanisms of proliferation?

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


What is the role of the ligand-receptor interaction?

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


What are the three classes of receptor types?

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


What are the actions of growth factors?

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


What are some examples of major growth factors?

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


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

- 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


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

- 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)


What is the MAP kinase pathway?

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


What are the ultimate targets of secondary messenger pathways?

- 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


What is an important inhibitor of proliferation?

- 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


Why should we control the cell cycle?

- 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


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

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


How is the cell cycle controlled?

- 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


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

- 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


How is the position of the cell cycle determined?

- 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


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

- 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


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

- 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


What has developmental biology shown us?

- that ECM is critical for correct cell development and proliferation