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Flashcards in lecture 8 Deck (37):
1

What is the extracellular matrix?

- macromolecules outside cells, formed by local secretion and assembled into network surrounding cells

2

What are the functions of the ECM?

- 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

What are the three stages of after wounding?

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

What is the importance of the matrix in regeneration and repair?

- 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

What are the two types of cellular-macromolecular organisation?

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

What are the three groups of molecules that form the matrix?

1. fibrous structural proteins e.g. collagen, elastins
2. adhesive glycoproteins e.g. fibronectin, laminin
3. gelatinous-like molecules e.g. proteoglycans, hyaluronan

7

What is important to note about the ECM?

- not a layering
- there is an interaction between all aspects
- e.g. between epithelium and basement membrane via integrins

8

What is collagen?

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

How is collagen synthesised?

- 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

What is elastin/fibrillin?

- 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

What is marfan syndrome?

- 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

What are the 4 major families of adhesive glycoproteins/integrins?

- 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

What is fibronectin?

- 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

What is laminin?

- 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

What are integrins?

- 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

What are cadherins?

- 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

What is SPARC?

- secreted protein acidic and rich in cysteine
- aka osteonectin
- important in tissue remodelling: angiogenesis inhibitor

18

What are thrombospondins?

- angiogenesis inhibitors

19

What is osteopontin?

- leukocyte migration, regulates calcification

20

What is tenascin?

- morphogenesis
- cell adhesion

21

What are proteoglycans?

- 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

What are essential processes in repair?

- 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

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)

24

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

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