Tissue Repair
“Systems Biology”
8-29-11
Robert A. Anders M.D., Ph.D.
Dept. of Pathology
CRB 2 Rm 346
rander54@jhmi.edu
The Systems
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Tissue homeostasis
Cell Cycle
Soluble mediators
Extracellular matrix
Integration of repair mechanisms
Pathology of repair
Tissue Reactions
• Hypertrophy
– Increase in cell size
• Hyperplasia
– Increase in cell number
• Renewal
– Replacement of cells
• Scar
– Replacement with fibrosis
Intestinal Homeostasis
Epidermal Homeostasis
Liver Homeostasis
Tissue Stem Cells
Cellular Transplant
“let’s repair diseased tissue
with tissue stem cells”
X = tissue 1
Y = tissue 2
1 = Partial phenotype
2 = Unknown progenitor
3 = in vivo not in vitro
in vitro reprogramming
4 = Low frequency events
Ann Rev Cell Dev Biol 17:387403 2001
Cell Cycle
Cell Cycle
• Ordered series of events
• Critical to maintain genome
– Copy number
– Integrity
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Division of cytoplasmic organelles
Quiescent = G0
Ready to divide = G1 to R point
Dividing = R point to S, G2, M
Cell Cycle
Go
Interphase = G1+S+G2
M = mitosis = pro-,meta-,ana-,telophase
Cell Cycle
Cyclins + CDK
Retinoblastoma
Inhibitors
Soluble Mediators
Soluble Mediators
-signaling• Growth factors
– Peptides
– Steroids
– Small molecules
• Extracellular matrix
– Dynamic fluid like environment
– Interstitial space
• Area between cells
– Basement membrane
• Interwoven dense mesh of collagen glycoproteins
Signaling Patterns
Sept 2 2009 NEJM
Signaling In Cancer Cells
Growth Factors
Growth Factors
-classification• Function
– Vascular Endothelial Growth Factor
• Composition
– Protein, steroid
• Receptor
– G-protein coupled, receptor tyrosine kinase
• Structure
– Ion channel, 7 pass transmembrane
• Orphan / Decoy
– Ligand / function unclear
Growth Factors In Regeneration and Wound Healing
-what is in a name?-
Growth Factor Receptors
Steroids
adaptors
P
P
Effectors
Pharm
PKC
Receptor Types
-different signaling pathways-
TGF beta Signaling
-kinases, adaptors, transcription factorsLigand
GS
Receptors
II
GS P
I
Gene Transcription
TGF beta Superfamily
TGF beta Signaling
-epithelial cells-
P
Context Dependent Signaling
Matrix
Extracellular Matrix
• Everything outside the cell
– Interstitial
– Basement membrane
• Dynamic
• Influences cytoplasmic events
• Holds
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Tissues
Water
Minerals
Growth factors
• Three main constituents
– Collagen and elastin
– Adhesive proteins
– Proteoglycans and hyaluronic acid
Preview
Collagen
• Most abundant protein in animals
• Types I, II, III, V form fibrills
• Type IV forms sheets and basement
membrane
• Inherited disorder = Ehlers Danlos
• Acquired defect = Scurvy
Collagen Synthesis
Vit C
Elastic fibers
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Recoil of tissue
Distensible / stretchable
Elastin core protein
Microfibril network of fibrillin
Elastic fibers
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Recoil of tissue
Distensible / stretchable
Elastin core protein
Microfibril network of fibrillin
Elastin
Fibrillin
Mutations = Marfans’ syndrome
Marfan Syndrome
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Autosomal dominant
Mutations in fibrillin 1 FBN1 gene
Abnormal elastin fiber
Normally binds and pools TGF-beta
1896 Anoine Marfan (peds) recognizes
striking features in child
• 1991 Ramierez at Mt. Sinai gene ident.
Marfans Syndrome
Eye lens dislocation
Spontaneous pneumothorax
Spinal cord sac
other matrix proteins….
• Fibronectin
– Large dimeric protein that binds collagen,
proteoglycans and
• Lamin
– Woven into basement membrane
• Proteoglycans and
– Polysaccharide coated protein
– Diverse composition and function!
– Heparin & Chondrotin sulfate
• Hyaluronic acid
– Binds vast amounts of water
Proteoglycans and Hyaluronic acid
Proteoglycans and Growth Factor Reservoirs
Adhesion
Major Classes of Adhesion Molecules
Same cell type
Leukocyte
Cell Adhesion Molecules
• Anchored in the membrane
• Immunoglobin superfamily
– PECAM, VCAM
• Integrins
• Selectins
• Cadherins
Integrins
• Superfamily of ~ 30 homologous members
• Non-covently linked alpha and beta chain
• Globular heads, transmembrane region
and short cytoplasmic tails
• Diverse functions
– Beta1 (VLA) bind collagen, lamins, fibronectin
– Beta 2 (CD11) bind leukocytes
– Beta 3 (GPIIb) involved in clotting
Integrins Stimulate Cytoplasmic Signaling
Cell Adhesion Molecules
• Anchored in the membrane
• Immunoglobin superfamily
– PECAM, VCAM
• Integrins
• Selectins
• Cadherins
Selectin
• Single chain transmembrane
• L-selectin
– Expressed on lymphocytes
– Homing to activated endothelial cells (lymph node >
inflammatory site
– KO mice have small lymph nodes
• E-selectin
– Homing as above < peripheral inflammation
• P-selectin
– Similar to E
– E and P KO mice have poor WBC recruitment
Cell Adhesion Molecules
• Anchored in the membrane
• Immunoglobin superfamily
– PECAM, VCAM
• Integrins
• Selectins
• Cadherins
Cadherins
• 90 members
• Transmembrane proteins
– Extracellular adhesion
– Transmembrane
– Intracellular connection to cytoskeleton
• Identical cell to cell interactions
– Desmosomes linked to intermediate filaments
– Zona adherens / belt desmosome linked to actin
• Roles
– Differentiation, proliferation, and motility
• Contact inhibition in cell culture
General Cadherin Structure
E-Cadherin Function
Summary
Angiogenesis
Fresh and Mature Healing
-vessel density-
Trichrome Stain
Angiogenesis
• Paradigms
– Branch extension
– Stem cell mobilization from bone marrow
Branch Extension
Smooth muscle
Pericytes
Stem Cell Mobilization
Angiogenesis
• Paradigms
– Branch extension
– Stem cell mobilization from bone marrow
• Multiple steps
– Remodeling, migration, proliferation,
maturation
Angiogenesis
Angiogenesis
• Paradigms
– Branch extension
– Stem cell mobilization from bone marrow
• Multiple steps
– Remodeling, migration, proliferation, maturation
• Growth factors
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VEGF (stroma) and VEGFR 2 (endo. cell)
Angiopoietins  stablization, periendothelial cells
PDGF  smooth muscle recruitment
TGF beta  matrix production
Angiogenesis
• Extracellular matrix
– Integrins  cell matrix interaction
– Matricellular proteins  destabilize cell
/matrix
– Proteinases  remodel
• Plasminogen activators
• Matrix metalloproteinases  cleave matrix bound
growth factors
Control of Matrix Metalloproteinase
Tissue Repair
-integration of systemsExamples:
Fresh and Old scar
&
Partial Hepatectomy
Fresh and Mature Healing
-observations-
Trichrome Stain
Healing Phases
Partial Hepatectomy
-mouse liver-
Partial Hepatectomy
-Murine Model-
Intact Liver
B
A
X
C
A
B
C
Partial Hepatectomy
1931
Wild Type
Wild Type
48 hrs post PHX
Decreased DNA Synthesis
-48 hrs Following Partial HepatectomyWild Type
TNFR I -/-
Partial Hepatectomy in Humans
Partial Hepatectomy in Humans
Liver Regeneration
-Cell Cycle-
Priming
G0
Progression
S
Hepatocyte
G1 Division G2
M
Modified from Nelson Fausto
Liver Regeneration
Growth Factors / Cytokines
HGF
TGF
Priming
TNF
Antibody
TNFR I
IL-6
KO
KO
G0
Progression
S
Hepatocyte
G1 Division G2
M
Modified from Nelson Fausto
Model of Liver Regeneration
Integrated Model of Liver Regeneration
Cytokines
LPS
JAK/STAT
IL6
C3a
Stem Cells
Adhesion
TNF
Extracellular Matrix
C5a
Growth Factors
TGF-α
HGF
Metabolic
iNOS
Steroids
Bile acids
G proteins
Sympathetic neural stimulation
Serotonin
FAS
TIMP
Metaloproteinase
Modified from Nelson Fausto Nature Medicine Oct 2005
Integrated Model of Liver Regeneration
Cytokines
LPS
JAK/STAT
IL6
C3a
Stem Cells
Adhesion
TNF
Extracellular Matrix
C5a
Growth Factors
TGF-α
HGF
Metabolic
iNOS
Steroids
Bile acids
G proteins
Sympathetic neural stimulation
Serotonin
FAS
= Science or Nature
TIMP
Metaloproteinase
Modified from Nelson Fausto Nature Medicine Oct 2005
Pathology of Repair
Healing
First Intention
Myocardial Infarction
Myocardial Infarction
Myocardial Infarction
Myocardial Infarction
Myocardial Infarction
Trichrome
Hepatitis and Cirrhosis
Pathways vs Networks
• Nutrition
– Vitamin C
• Systemic illness
– Diabetics angiopathy
• Circulation
– Atherosclerosis
• Hormones
– Glucocorticoids
• Local factors
– Infection, mechanical stress
Summary
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Tissue homeostasis
Cell Cycle
Soluble mediators
Extracellular matrix
Integration of repair mechanisms
Pathology of repair
Thought Experiments
Experiment One
You noticed in todays lecture, TGF-beta
inhibits epithelial cell growth.
You are going to inhibit cancer cell growth
by uncovering TGF-beta dependent cell
cycle inhibitors. How?
Thought Experiment Two
1) What mechanisms can you think of to
disrupt angiogenesis?
2) What might some of the side effects
be?
Experiment Three
• It is difficult to make a lot of hepatocytes
from embryonic stem cells (ESC) using
traditional cell culture techniques.
• Now we use plastic dishes, growth factors
and feeder layers
• Convinced by today's lecture, you are
going to investigate how the extra matrix
could facilitate ESC to liver cell
conversion. How?