lecture 8 Flashcards
1
Q
What is the extracellular matrix?
A
- macromolecules outside cells, formed by local secretion and assembled into network surrounding cells
2
Q
What are the functions of the ECM?
A
- reservoir for growth factors
- scaffolding within which cells adhere, migrate, and proliferate
- sequester H2O for turgor (physical pressure that ECM exerts on cells within the matrix); minerals for rigidity
3
Q
What are the three stages of after wounding?
A
Inflammation (2-6days)
- clot formation
- chemotaxis
Proliferation (6-12 days)
- re-epithelialization
- angiogenesis and granulation tissue
- provisional matrix
Maturation (12-16 days)
- collagen deposition
- collagen matrix
- wound contraction
4
Q
What is the importance of the matrix in regeneration and repair?
A
- ECM regulates proliferation, movement and differentiation of the cells living in it
- If the ECM is destroyed you can’t regenerate –> a scar forms instead
5
Q
What are the two types of cellular-macromolecular organisation?
A
Interstitial matrix/a.k.a stroma
- spaces between epithelial, endothelial and smooth muscle cells and in connective tissue
Basement membrane
- associated with cell surfaces (epithelial and mesenchymal)
6
Q
What are the three groups of molecules that form the matrix?
A
- fibrous structural proteins e.g. collagen, elastins
- adhesive glycoproteins e.g. fibronectin, laminin
- gelatinous-like molecules e.g. proteoglycans, hyaluronan
7
Q
What is important to note about the ECM?
A
- not a layering
- there is an interaction between all aspects
- e.g. between epithelium and basement membrane via integrins
8
Q
What is collagen?
A
most common protein in the animal world
- each molecule
- 3 right handed alpha (triple) helices
- gly-X-Y repeating sub-units
- mutations: collagens lose stability and vulnerable to proteinase digestion: bone structure can be affected, flexibility/elasticity, skin/hair/eye integrity
- 27 types: 41 genes, 14 chromosomes
- I, II, III, V, XI: fibrillar = extracellular structures
- IV non-fibrillar:amorphous - forms sheets - Basement membrane
- other collagens:
- meshworks/anchors at epidermis/epidermis junction, cartilage, blood-platelet activation; vessel wall
9
Q
How is collagen synthesised?
A
- procollagen 3 strands put together
- enzymes cleave off terminal ends - forms tropocollagen
- lots of glycine residues
- cross-linking with other triple helices - collagen fibre formed
10
Q
What is elastin/fibrillin?
A
- blood vessels, skin, uterus, lung - need elasticity
- elastic fibres: stretch to several times length - return after release of push/pull force
- elastic fibres = central core (elastin), surrounded by fibrillin
- 70 kD protein
- rich in amino acids: glycine, proline, alanine
- cross-links enable and regulate elasticity
associated with:
- fibrillin = microfibrillar network - surrounds core
- 350kD glycoprotein
- self-associating
- scaffolding for elastin/elastic fibres
11
Q
What is marfan syndrome?
A
- connective tissue genetic disorder affecting the FBN1 gene that encodes Fibrillin-1
- tall stature and long limbs resulting in defects of the aorta and heart valves, lungs, eyes, skeleton
- affected individuals exhibit increased chronic inflammatory disease (e.g. severe RhA) since Fibrillin-1 regulates TGF-beta dependent inflammation
12
Q
What are the 4 major families of adhesive glycoproteins/integrins?
A
- in membranes = receptors - homotypic or heterotypic binding OR
- in cytoplasm (stored
immunoglobulin cell adhesion molecules
- ICAMs
- hetero/homotypic
Cadherins
= Ca++ dependent adherence protein, homotypic
- connect plasma membranes of adjacent cells - regulate motility, proliferation and differentiation
Integrins
- cell adhesion
- bind to fibronectin and laminin
- bind cells/ECM and cell/cell
Selectins
- e.g. neutrophil margination and rolling (E and P selectin)
13
Q
What is fibronectin?
A
- adhesive glycoprotein - stromal
- multifunctional - attaches cells to matrix
- 450 kD glycoprotein - 2 chains - disulphide bonds
- from: fibroblasts, monocytes, endothelial cells
- binds to: collagen, fibrin, proteoglycans - domains
- binds to: cells via receptors/membrane e.g. bone marrow stroma
- cell attachment and spreading, cell migration
- enhances sensitivity to growth factors
14
Q
What is laminin?
A
- most abundant glycoprotein in basement membrane
- = family - 820kD, heterotrimeric, cross shaped
- bind with receptors on cell surface
- binds with matrix e.g. collagen type IV, heparan sulphate
- mediates attachment: cells to connective tissue substrate
- in vitro: growth, survival, morphology, motility cells e.g. alignment of endothelial cells - capillaries
- binds to integrins
15
Q
What are integrins?
A
- major family of cell surface receptors
- transmembrane, 2 chains: alpha (14 types) Beta (8 types)
- at least 30 different heterodimers
attachment
- cell-ECM, cell-cell e.g. leukocytes
- via recognition of RGD sequence by extracellular domain
crucial in
- development
- leukocyte extravasation, platelet aggregation, wound healing
- lack of attachment via integrins leads to apoptosis
- transduction of signals: ECM -> cell interior
- organise actin cytoskeleton into focal adhesion complexes
- complexes - activate signal transduction pathways
- possible similar to growth factor pathways e.g. MAP kinase, PI3 kinase.
16
Q
What are cadherins?
A
- CA++ dependent adherence protein - 90 members
- interactions between cells of same type via specialised junctions e.g. desmosomes
- integrins - bind to actin/intermediate filaments and link cell surface with cytoskeleton
17
Q
What is SPARC?
A
- secreted protein acidic and rich in cysteine
- aka osteonectin
- important in tissue remodelling: angiogenesis inhibitor
18
Q
What are thrombospondins?
A
- angiogenesis inhibitors
19
Q
What is osteopontin?
A
- leukocyte migration, regulates calcification
20
Q
What is tenascin?
A
- morphogenesis
- cell adhesion
21
Q
What are proteoglycans?
A
- core protein and polysaccharides (=glycosaminoglycans/GAGs)
- polysaccharides have -ve charge, occupy large volume, hydrophilic
- in all ECM, on cell surfaces, in biological fluids
- diverse
- named for polysaccharide e.g. heparan sulphate, chondroitin sulphate, dermatan sulphate
- regulate connective tissue structure and permeability
- modulate cell growth and differentiation
- in ECM of many cells – huge molecules
- associates with receptors
- proliferation/migration
- binds large amount of water –> viscous gel
- gives connective tissue its ‘turgor pressure’
- resists compression - joints
- inhibits cell-cell adhesion
- facilitates migration
hyaluronan = MW 8x10^6
- act as a reservoir for growth factors
- regulates timing - damage will cause release of growth facts
22
Q
What are essential processes in repair?
A
- angiogenesis = formation of new blood vessels
- proliferation, migration, differentiation
- fibroplasia = formation of new connective tissue
- proliferation + migration + synthetic activity = ECM deposition
- remodelling - maturation and organisation off the new fibrous tissue
23
Q
What is vasculogenesis?
A
Occurs during development: formation of first blood vessels
1. Hemangioblasts (haemopoietic stem cells)
2. angioblasts + HSCs (form R/WBCs) + Endothelial Precursor Cells (stored in bone marrow, used in adults when undergoing angiogenesis)
angioblasts proliferate, migrate, differentiate
3. endothelial cells
+ pericytes
+ smooth muscle cells
4. vessels (arterioles, venules, capillaries)
24
Q
What are the two basic ways angiogenesis can happen in adults?
A
- endothelial precursor cells triggered by chemotactic signal –> follow this (site of injury)
migrate and form new capillary network - endothelial cells at site of injury can form new cells - limited response, less efficient than EPCs
25
How is angiogenesis controlled?
- VEGF is most important GF
- VEGFR-2 is most important receptor
1. proteolysis of ECM
2. migration and chemotaxis
3. proliferation (VEGF-2 + VEGFR-2)
4. lumen formation, maturation, and inhibition of growth
5. increased permeability through gaps and transcytosis
26
What is vasculogenesis?
Occurs during development: formation of first blood vessels
1. Hemangioblasts (haemopoietic stem cells)
2. angioblasts + HSCs (form R/WBCs) + Endothelial Precursor Cells (stored in bone marrow, used in adults when undergoing angiogenesis)
angioblasts proliferate, migrate, differentiate
3. endothelial cells
+ pericytes
+ smooth muscle cells
4. vessels (arterioles, venules, capillaries)
27
What are the two basic ways angiogenesis can happen in adults?
1. endothelial precursor cells triggered by chemotactic signal --> follow this (site of injury)
migrate and form new capillary network
2. endothelial cells at site of injury can form new cells - limited response, less efficient than EPCs
28
What is fibroplasia?
- organisation/healing/repair
- - scar production
- - scar production replacing necrotic tissue
- fibrosis: any abnormal deposition of connective tissue – occurs in chronic disease
- - e.g. cirrhosis
- - rheumatoid arthritis
- - chronic obstructive lung disease
- - chronic glomerulonephritis
- in granulation tissue - new blood vessels + ECM
- migration and proliferation of fibroblasts at injury site
- deposition of ECM
- after a few months huge amounts of fibrosis that can never be recovered
deposition of ECM
- with time - fibroblasts produce ECM
- fibrillar collagens - strength
- begins and 3-5 days for weeks
- synthesis stimulated by growth factors e.g. TGF-beta, PDGF, TNF and cytokines e.g. IL-1, IL-4
- amount of collagen is a balance between synthesis and degradation
- as scar matures: vascular regression occurs
29
What is VEGF?
- family (A-D)
- low levels in many adult tissues produced by mesenchymal and stromal cells (connective tissue and blood vessel cells)
- induced by hypoxia, TGF-beta, PDGF, TGF-alpha
- receptors e.g. VEGFR-2 (endothelial cells), VEGFR-3 - lymphatic cells
- functions
- - angiogenesis
- - vascular permeability
- - endothelial migration and proliferation
- - endothelial differentiation and sprouting of new capillaries
- - endothelial expression of collagenase etc.
30
What is the role of ECM in angiogenesis?
- motility and directed migration of endothelial cells
- integrins - (alpha-v, beta-3) - formation and maintenance of newly formed vessels - regulates VEGFR-2
- matricellular proteins e.g. thrombospondin - destabilise cell-matrix interactions (i.e. destroy newly formed vessels that are not required)
- proteinases e.g. plasminogen activator and matrix metalloproteinases - tissue remodelling - release growth factors and inhibitors from storage
31
What is fibroplasia?
- organisation/healing/repair
- - scar production
- - scar production replacing necrotic tissue
- fibrosis: any abnormal deposition of connective tissue – occurs in chronic disease
- - e.g. cirrhosis
- - rheumatoid arthritis
- - chronic obstructive lung disease
- - chronic glomerulonephritis
32
How is fibroplasia regulated?
TGF-beta
- fibroblast migration, proliferation, increased synthesis
- decreased degradation of ECM by metalloproteinases
- increased expression in chronic fibrotic disease
Macrophages
- clear extracellular debris, fibrin, foreign material
- secrete growth/survival factors
33
What occurs during remodelling?
- vascular granulation tissue changes into an avascular scar --> changes in composition of ECM
- balance between proliferation of fibroblasts and synthesis of ECM vs activation of matrix metalloproteinases which degrade ECM
34
Why is it good to understand ECM and the signalling going on?
- attempts to mimic
- synthetic ECM for cardiomyocytes to grow back, liver, even artificial bone
- a few years ago they replaced patient's oesophagus ECM from donor --> fully formed
35
How does a thin wound heal?
- occurs in small wounds that close easily
- epithelial regeneration predominates over fibrosis
- healing is fast, with minimal inflammation, scarring/infection
- e.g. small cuts, minor surgical procedures
36
How does healing with scar formation and contraction occur?
- occurs in larger wounds that have gaps between wound margins
- fibrosis predominates over epithelial regeneration
- healing is slower, with more inflammation and granulation tissue formation, and more scarring e.g. infarction, large burns and ulcers
37
Why is good to understand ECM and the signalling going on?
- attempts to mimic
| - synthetic ECM
