Extracellular Matrices Flashcards
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
Why may you purchase collagen? (1) (1/5)
- Fibre-forming molecule (structure to ECM by creating a complex 3D framework of rigid proteins)
- Collagen = most prevalent fibre-forming protein (~77% of fat-free dry weight of human skin)
- Defines the rigidity & elasticity of a T
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
Why may you purchase hyaluronan? (1) (2/5)
- non fibre-forming molecule
- funct to create a charged, dynamic & osmotically active space
- fills majority of interstitial space
- -ive charge & h.philic nature enables proteoglycans & GAGs to funct in: .. within T
- hydration
- buffering
- force dispersion
- key roles in fibrotic & wound-healing
- ↑ XPS’ion of HA & HA-R (CD44) = associated w/ fibrotic diseases in many T
- human oral fibro & fetal scarless wounds = ass w/ ↑ XPS’ion of HA
- HA exerts FXs via CD44 & ‘receptor for HA-mediated motility’ (RHAMM) receptors
- evidence that this large pericellular molecule can trap TGF-b1, close to fibro
- = +ive FB autocrine loop that contributes to fibro → myofibroblast differentiation
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
What may you mix with hyaluronan? (1) (3/5)
- HMW HA (>500 kDa) ass w/
-↓ inflammation
-↑ XPS’ion of collagen-III
-↑activity of anti-fibrotic TGG-b3 - fragmented HA (<400 kDa) ass w/
-↑inflammation
-↑↑ XPS’ion of collagen-III
-↑fibro prolif
-↑myofibro differentiation - controversial bc hexameric fragmented HA (~1 kDa)
- improves fibro migration
- promotes wound closure
- decreases myofibro differentiation
- decreases fibrosis
- hexameric may be of interest in healing chronic wounds
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
Why may you purchase osteonectin? (1) (4/5)
- aka SPARC
- matricellular protein
- group of secreted local proteins that interact in autocrine/paracrine cell-matrix sig
- ≠ contribute to mechanical structure of ECM
- unlike most ECM components that = omnipresent ↷ normal skin & repopulate wounded skin after predicted intervals, matricellular prot can be absent in healthy skin
- XPS’d only after skin wounding
- wound healing
- fibro produce maj of ECM comp, wh/ act simultaneously to mod fibro funct
- interaction of fibro:ECM = thought as autocrine reg that = crucial to wound-healing
- fibro continuously create new ECM-P to heal cutaneous wounds, whilst being reg by prot of its own
- SPARC = found @ ~2d post wound infliction
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
What may you mix with ostenoectin? (1) (5/5)
- more osteonectin in wound healing fibro, rather than healthy
- fibro grown on collagen-I showed ↑XPS’ion of Alpha_vBeta_3 integrin (binds to vitronectin/FN) & ↑XPS’ion of Alpha_2Beta_1 integrin (binds fibros to collagen, req for migration)
- suggest that as an open would contracts & builds tension via tightening of collagen fibrils, myofibro transition away from collagen-mediated binding → non-collagen protein binding in wounds
- collagen may not be necessary
- in this case a non-collagen fibre-forming protein may be best to use, to still ensure mechanical stability i.e., VN/FN
What are the components of the ECM, and what are their functions?
Fibre-forming molecules (2) (1/3)
Collagen
Fibrin
Fibronectin
Vitronectin
Elastin
Fibrillin
- structural element
- tensile strength
- cell adhesion reg
- support chemotaxis & migration
- directs T development
What are the components of the ECM, and what are their functions?
Non fibre-forming molecules (2) (2/3)
Proteoglycans (HA, decorin, versican, dermatopontin)
GAGs
- hydration
- swelling pressure to the T enabling it to withstand compressional forces
- T homeostasis & struct
- modulators of sig pathways, reg cellular processes
What are the components of the ECM, and what are their functions?
Matricellular proteins (2) (3/3)
Glycoproteins
- laminins: basement membrane
- integrins: receptors
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
What other factors may be considered? (1) (6/7) (stiffness)
- mechanical prop of ECM have been shown to promote myofibro diff & lung fibrosis
- matrix stiffness-induced myofibro diff, mediated by intrinsic mechanotransduction
You are working in a lab to study different cells of the skin. You have isolated fibroblasts from healthy skin and wounded skin.
What other factors may be considered? (1) (7/7) (laminin used)
- L-a5 = observed to mod fibro prolif in fibrosis via disruption of the PI3K/Akt/mTOR sig pathway
- downreg of L-a5 = observed to prevent activation of the sig pathway
- PI3K = bridge b/w extra cellular sig & cellular responses
- activated PI3K may promote the transformation of Akt, accelerating phos of downstream mTOR (inhibits cell apoptosis)
Describe the seven different functions of the ECM.
Anchorage, migration barrier/track, signal reservoir (3) (1/2)
Anchorage
- anchorage to the BM = essential for the maintenance of stem cells & establishment of polarity in many T
Migration barrier and track
- context-dependent
- ECM may serve as barrier to or facilitator of cell migration
Signal reservoir
- ECM binding to sig molecules prevents free diffusion
- facilitates establishment of conc gradients
- serves to create reservoir from which they can be liberated during matrix remodelling
Describe the seven different functions of the ECM.
Low affinity co-receptor, signal presenter, biochemical force (3) (2/2)
Low-affinity co-receptor
- ECM comp (e.g., heparan sulfate proteoglycans; HSPG) & ECM receptors (CD44) can bind signalling molecules & serve as co-receptors
Signal presenter
- SAME AS ABOVE
- inv in establishing [growth factor/cytokine] grad → reg their spatial and temporal bioavailability
- fibroblast growth factor family strongly binds to heparan sulfate chains of proteoglycans i.e., perlecan
- HSPG inv in binding, transporting & activation of developmental control factors (Wnt/HH)
- also acts in ligand maturation
- TGF-b = secreted in a latent form & stored in ECM
- activated by MMP-dep proteolysis
Biochemical force
- stiffness exerts profound influence on cells & reg their differentiation
- diff cells/T req diff stiffnesses to grow optimally i.e., bone = one of strongest ECM, neuronal cells grow in elastic modules of 50-209 Pa.
What are the different characteristics in the ECM b/w normal issue & wound healing / fibrotic tissue (4) (1/1)
Normal tissue:
- ECM consists of a basement membrane, interstitial (stromal) ECM
- BM = collagen IV, laminin, fibronectin & several types of proteoglycans
- Role: physical barrier b/w epi cells & connective T (stroma) of the organ, allows gaseous diff & transportation of sig molecules
- normal ECM = highly remodelled after initial set-down = exhibits T-specific composition & organisation
Fibrotic tissue:
- Cancer: rupture of BM permits EMT & migration of epi cells into surrounding stroma → invasion of interstitial ECM
- Epi cells undergoing EMT can activate stromal cells to become pro-tumorigenic, permitting remodelling of the ECM to make a tumour-permissive enviro
- ↑lvls of HA documented in cancer progression & = ass w/ poor prognosis & chemo-resistance
- HA induces EMT by binding CD44 & activating EMT TCF (TWIST-1)
- ↑[HA] has been shown to compromise vascular integrity - imp for metastasis (intravasation & extravasation)
- also shown to be involved in inflam resp that mediate ECM remodelling
- Dermoplasia: linear ECM fibres = observed to provide migratory tracks → cancer cells use to enhance migratory capability
Cell ECM interactions in both norm & fibrotic T are mediated by integrins
Integrins:
- reg CS organisation
- activate intracellular sig path to convey mechanical & chemical signalling
Why are fibroblasts important during wound healing? (5) (1/1)
- pivotal role in reg ECM turnover under norm cond. ∴ in injured T they can be activated = elicit differentiation into myofibroblasts
- they proliferate & chemotax to site of T injury
- Myofibro function during wound healing by ↓size of wound & secreting ECM proteins that elicit appropriate immune resp
- in normal tissue:
- Fibro = stress-shielded by ECM & ≠ develop contractile feat or cell-matrix adhesions
- in injured tissue:
- inflam signals activate fibro to spread into provisional wound matrix → differentiation: TGF-β1 stim proto-myofibroblasts to XPS α-SMA & become differentiated
- myofibros exit cycle when ECM = reconstituted & is able to compensate for the mechanical load (apoptosis when epithelialization completed)
- fibro regulate their collagen & ECM protein syn in resp to mechanical tension:
- ↑mech stress stretches fibros, ↑ing collagen production & ↓ing collagenese production
How is PDGF, IL6, IL13 & eicosanoid leukotrine signalling important in wound healing in fibroblasts? (6) (1/1)
- Cytokines that promote inflammation & development of fibrotic resp.
PDGF
- produced platelets, endothelial cells, smooth muscle cells & macrophages
- acts on mesenchyme & fibroblasts → prolif, differentiation, & ECM production
- imp in wound healing, inflamm, angio, embryonic, fibrosis
IL13
- produced by mast & T-cells
- acts on many cells, inc fibro
- stim TGFβ prod & activation, collagen prod, MMP XPS’ion, fibro proliferation & myofibro differentiation
- TGFβ & other cytokines reg most common molecular mech (EMT) @ the heart of fibrosis
Eicosanoids
- induce fibroblast prolif & ECM production
How is paracrine signalling important in wound healing in fibroblasts? (7) (1/1)
- Paracrine factors antagonise fibrosis i.e., Eicosanoids prostaglandin E2 (PGE_2) & prostacyclin (PGI_2)
- important for reg - ≠ want XS ECM b/c can result in fibrotic diseases i.e., idiopathic pulmonary fibrosis
- involves ↑ matrix stiffness via ↑ deposition of ECM = XS’ive ECM abundance in T microenviro
- PGE_2: inhibits fibro proliferation & collagen prod
- acts on GPC-EP receptors 1-4
- obvs to inhibit fibro differentiation
- acts on GPC-EP receptors 1-4
How and why is the ECM degraded during development & disease? (8) (1/1)
- ECM = ↑ly dynamic struct, constantly subject to remodelling = ECM components are deposited, degraded, modified
- abnormal ECM dynamics = dysregulated cell prolif, failure of cell death, loss of cell diff → congenital defect & pathological processes inc. T fibrosis & cancer
- understanding = essential for therapeutic intervention (T engineering & regenerative medicines)
- ECM high regulation is maintained by redundant mech that modulate XPS’ion & func of ECM modifying enzymes
Regulation by proteinases
- 2 main families of metalloproteases (MPs): MMPs and “disintegrin metalloproteases with thrombospondin motifs” (ADAMTS)
- MMPs
- ~23 memb
- self-inhibitory pro-domain @ N-T
- hemopexin @ C-T (suggested to facilitate binding to variety of prot & are zinc endopeptidases, inv in regulation b/c HX repeats in some matrixins can bind “Tissue Inhibitors of Metalloprotases” (TIMPs))
- catalytic domain & flexible hinge motif
- most have a basic 3-domain structure
- others (MMP2/9) have FN-type-2 repeats in their cata domain, mediating collagen binding
- MMP3/10 targets proteoglycans, FN & laminin
- MMP1 targets collagen-III
- MMP are often pleiotropic (many functions)
Disease examples
- Homeostasis of skin = fundamental for maint of struct, protective & reg functions
- dysregulation of this = disease i.e., atopic dermatitis, psoriasis, epidermolysis bullosa & skin cancer
- impaired skin hydration, thermreg & ↑suscep to infections
- Thermoregulation link:
- Microfibril associated glycoprotein 1 (MAGP1) may reg E metabolism in adipose T
- component of ECM, plays role in thermoregulation by indirectly reg XPS’ion of the thermogenic uncoupling proteins (UCPs); a proton transporter that allow
Atopic dermatitis
- abnormal BM composition
- ↓ BM thickness & ↓ XPS’ion of collagen-IV, integrin-a6, & FN in the BM zone
- Many SNPs in LAMA3 correlate w/ ↑AD incidence
Name the different components of focal adhesions (9) (1/1)
A cell, adherent to FN on the ECM, will have a focal complex and a focal adhesion, the latter consists of:
- actin
- α-actinin
- myosin
- vinculin
- talin
- paxillin
- cell membrane
- integrin dimer
- fibronectin
- cells adhere via their leading edge (integrin) to ligands of the ECM (FN)
- focal complexes form in the lamellipodium, may mature to focal adhesions in the lamella
- FA are typically connected to one another via stress fibres, or connected to the actin network forming dorsal stress fibres
- Talin form actin stress fibres that can contract in the presence of myosin-II motor molecules
- link supported by proteins (vinculin, paxillin, α-actinin)
How do focal adhesions change under different matrix stiffnesses? (10) (1/1)
- sense & respond to physical forces within the ECM (via FA)
- adapt morphology & migrate to other locations
- shape of many mammalian cells = reliant on the matrix stiffness that they grow on
- mediated by FA (dis)assembly, reg biochemically & mechanically
- mechanosensitivity of FA = reg by talin & p130 cas (Δ conformation in resp to mech force)
- disassembly rate = highest on soft ECMs, lower on stiff matrices
- stretching occurs on stiff matrices, stretching of talin reveals vinculin-binding sites, ↑ vinculin binds to FA = ↑ more stable
- integrin (α5-β1) obvs to act as ‘catch-bonds’ & their lifetime ↑ under force
Soft
- forces develop slowly
- FA ≠ grow enough to stick the cell to the SBT
- cell retracts its protrusion
Stiff
- forces build up rapidly
- FA stabilise (more vinculin b/c of talin stretching)
- cell adheres to SBT
- process repeats with other protrusions, allowing for maximal spreading
visualise cell stretching on different matrices with actin staining
- soft (4 kPa): not spread
- stiff (150 kPa): spread
How are HIPPO signalling & the ECM stiffness linked - explain the principle of HIPPO signalling (11) (1/2)
Link?
- HP reg cellular homeostasis
- extracellularly reg by mechanical stimuli & diffusible chemicals
- sig sensed mostly by receptors (i.e., GPCRs) & adherence complexes
- ↑ly specifc resp ensured b/c receptors & junctional complexes become concentrated in distinct membrane structures
- HP also reg by exo- & endocytosis
- stiff matrix sensed by integrins → activation of Rho, leading to formation of stress fibres
- stress fibre formation ass with phos of LIMD1 & LATS1/2
- inhibits kinase that would usually phos YAP/TAZ
- LATS unable to inhibit YAP, ∴ translocates to nucleus = prolif etc
- stiffer matrix means activation of YAP/TAZ & inactivation of HP
How are HIPPO signalling & the ECM stiffness linked - explain the principle of HIPPO signalling (11) (2/2)
Principle of HIPPO.
HP on (YAP inactive):
- LATS1/2 can become phosphorylated by MST1/2-SAV complex
- p-LATS1/2 phos YAP
- p-YAP = degraded & sequestered in cytoplasm
- cell differentiation, apoptosis
HP off (YAP active):
- under stiffer matrix, GPCR signalling via Rho triggers actin stabilisation which inhibits MST1/2-SAV phosphorylation of LATS1/2)
- LATS1/2 cannot phos YAP
- YAP can translocate to nucleus & act with TEAD
- proliferation, anti-apoptosis, EMT
- target genes: NOTCH2, JAG1
What is an important downstream signalling pathway of an ECM receptor? (12) (1/1)
- Different receptors & targets
- HA (different MW) involved in inflammation
- ECM has multitude of macromolecules (growth factors, cytokines, chemokines, matrix-degrading enzymes etc)
- in diff combinations, these are inv in reg of cellular homeostasis
Receptors & their targets
- integrins → laminins, collagens, FN
- discodin domain receptors (DDR) → collagen
- syndecans → FN & intracellular actin-associated protein (α-actinin)
- CD44 → HA, collagens, FN, laminins
Specific example - role of HA fragments in inflammation
- LMW HA
- pro-inflamm & binds CD44 & TLRs
- augments inflam resp via activation of NLRP3/ASC inflammasome
- production of IL-1B
- HMW HA
- many anti-inflam properties
- binding to CD44 → reduces IL8, TNFa & iNOS production
- can block LMW HA binding to TLR2
