Transcript Tissue Renewal, Regeneration, and Repair

Lisa Stevens, D.O.
 Injury to cells---series of damaging events---
initiation of healing process

Regeneration


Complete restitution of lost or damaged tissue
Repair
May restore some original structures
 Can cause structural derangements

 Healthy tissues
 Healing (regeneration/repair)
Occurs after any insult that causes tissue destruction
 Essential for the survival of the organism

 Proliferation of cells and tissues to replace
lost structures
Growth of an amputated limb in amphibians
 Mammalian whole organs and complex tissues

 Rarely
regenerate after injury
 Applied to liver growth after partial resection or
necrosis
• Compensatory growth rather than true regeneration
 Hematopoietic system, skin, GI tract
High proliferative capacity
 Renew themselves continuously
 Regenerate after injury

 Combination of regeneration and scar
formation

Deposition of collagen
 Contribution of regeneration and scarring
Ability of the tissue to regenerate
 Extent of the injury
 Example

 Superficial
skin wound
• Heals through the regeneration of the surface epithelium
 Chronic inflammation
Accompanies persistent injury
 Stimulates scar formation

 Local production of growth
factors and cytokines
• Promote fibroblast proliferation and collagen synthesis
 Extensive deposition of collagen
 Extracellular matrix (ECM)

Components are essential for wound healing
 Provide
the framework for cell migration
 Maintain the correct cell polarity for the re-assembly
of multilayer structures
 Participate in angiogenesis (formation of new blood
vessels)
 Extracellular matrix (ECM)

Fibroblasts, macrophages, and others
 Produce growth
factors, cytokines, and chemokines
• Critical for regeneration and repair
 Adult tissues

Size of cell populations
 Determined
by rate of cell proliferation, differentiation,
and death

Increased cell numbers may result
 Increased
proliferation
 Decreased cell death
 Apoptosis
Physiologic process required for tissue homeostasis
 Induced by a variety of pathologic stimuli

 Terminally differentiated cells
Differentiated cells incapable of replication
 Impact of differentiation

 Depends
on the tissue under which it occurs
• Differentiated cells are not replaced
• Differentiated cells die but are continuously replaced
by new cells generated from stem cells
 Stimulated by physiologic and pathologic
conditions

Physiologic proliferation
 Proliferation
of endometrial cells under estrogen
stimulation during the menstrual cycle
 Thyroid-stimulating hormone-mediated replication
of cells of the thyroid that enlarges the gland
 Stimuli may become excessive, creating pathologic
conditions
 Stimulated by physiologic and pathologic
conditions

Pathologic proliferation
 Nodular prostatic
hyperplasia
• Dihydrotestosterone stimulation
 Nodular
goiters in the thyroid
• Increased serum levels of thyroid-stimulating hormone
 Controlled by signals from the microenvironment
Stimulate or inhibit proliferation
 Excess of stimulators or a deficiency of inhibitors

 Leads
to net growth and, in the case of cancer,
uncontrolled growth
 Tissues of the body

Divided into three groups
 Basis
of the proliferative activity of their cells
• Continuously dividing (labile tissues)
• Quiescent (stable tissues)
• Nondividing (permanent tissues)
 Continuously dividing tissues (labile tissues)

Cells proliferate throughout life
 Replaces

destroyed cells
Surface epithelia
 Stratified
squamous epithelia of the skin, oral cavity,
vagina, and cervix
 Lining mucosa of all the excretory ducts of the
glands of the body
• Salivary glands, pancreas, biliary tract
 Continuously dividing tissues (labile tissues)

Surface epithelia, cont’d
 Columnar epithelium
of the GI tract and uterus
 Transitional epithelium of the urinary tract
 Cells of the bone marrow and hematopoietic tissues

Mature cells are derived from adult stem cells
 Tremendous
capacity to proliferate
 Quiescent tissues (stabile tissues)
Low level of replication
 Cells from these tissues

 Undergo
rapid division in response to stimuli
 Capable of reconstituting the tissue of origin
Parenchymal cells of liver, kidneys, and pancreas
 Mesenchymal cells

 Fibroblasts
and smooth muscle
 Quiescent tissues (stabile tissues)
Vascular endothelial cells
 Lymphocytes and other leukocytes
 Example

 Ability
of liver to regenerate
• Partial hepatectomy
• Acute chemical injury
 Quiescent tissues (stabile tissues)

Fibroblasts, endothelial cells, smooth muscle cells,
chondrocytes, and osteocytes
 Quiescent
in adult mammals
 Proliferate in response to injury
 Fibroblasts proliferate extensively
 Nondividing tissues
Contain cells that have left the cell cycle
 Cannot undergo mitotic division in postnatal life
 Neurons
 Skeletal muscle cells
 Cardiac muscle cells

 Nondividing tissues

Neurons in the central nervous system (CNS)
 Destruction
of cells
• Replaced by the proliferation of the CNS-supportive
elements

Glial cells
 Nondividing tissues
 Mature skeletal muscle
 Cells
do not divide
 Regenerative capacity
• Through the differentiation of the satellite cells


Attached to the endomysial sheaths
Cardiac muscle
 Very
limited regenerative capacity
 Large injury to the heart muscle
• Myocardial infarction

Followed by scar formation
 Characterized by:
Self-renewal properties
 Capacity to generate differentiated cell lineages

 Need to be maintained during the life of the
organism

Achieved by two mechanisms
 Obligatory
asymmetric replication
• With each stem cell division, one of the daughter cells
retains its self-renewing capacity while the other
enters a differentiation pathway
 Need to be maintained during the life of the
organism

Achieved by two mechanisms
 Stochastic
differentiation
• Stem cell population

Maintained by the balance between stem cell divisions
that generate either two self-renewing stem cells or two
cells that will differentiate
 Embryonic stem cells (ES cells)

Pluripotent
 Generate
all tissues of the body
 Give rise to multipotent stem cells
• More restricted developmental potential
• Eventually produce differentiated cells

Three embryonic layers
 Adult stem cells (somatic stem cells)
Restricted capacity to generate different cell types
 Identified in many tissues
 Reside in special microenvironments

 Niches
• Composed of mesenchymal, endothelial, and other
cell types
• Niche cells generate or transmit stimuli that regulate
stem cell self-renewal and the generation of progeny
cells
 Inner cell mass of blastocysts in early
embryonic development
Contains pluripotent stem cells (ES cells)
 Cells isolated from blastocysts

 Maintained
in culture as undifferentiated cell lines
 Induced to differentiate into specific lineages
• Heart and liver cells
 ES cells may in the future be used to
repopulate damaged organs
 Effectiveness of these procedures in animals

Under intense study
 Much debate about the ethical issues
associated with the derivation of ES cells
from human blastocytes
 Induced Pluripotent Stem Cells

Differentiated cells of adult tissues can be
reprogrammed to become pluripotent
 Transferring
their nucleus to an enucleated oocyte
 Oocytes implanted into a surrogate mother can
generate cloned embryos that develop into
complete animals
• Reproductive cloning

Successfully demonstrated in 1997 by the cloning of Dolly
the sheep
 Great hope that the technique of nuclear
transfer to oocytes may be used for
therapeutic cloning in the treatment of
human diseases

Nucleus of a skin fibroblast from a patient
 Introduced
into an enucleated human oocyte
• Generate ES cells, which are kept in culture, and then
induced to differentiate into various cell types
 In principle, these cells can then be
transplanted into the patient to repopulate
damaged organs

Therapeutic as well as reproductive cloning are
inefficient and often inaccurate
 Deficiency
in histone methylation in reprogrammed
ES cells
• Results in improper gene expression
 Adult organism

Stem cells are present in tissues
 Continuously
divide
• Bone marrow, skin, and the lining of the GI tract
 Stem
cells may also be present in organs
• Liver, pancreas, and adipose tissue

Do not actively produce differentiated cell lineages
 Transit amplifying cells
Rapidly dividing cells generated by somatic stem
cells
 Lose the capacity of self-perpetuation
 Give rise to cells with restricted developmental
potential

 Progenitor
cells
 Transdifferentiation

Change in the differentiation of a cell from one
type to another
 Developmental plasticity

Capacity of a cell to transdifferentiate into diverse
lineages
 Stem cells
Bone marrow
 Skin
 Gut
 Liver
 Brain
 Muscle
 Cornea

 Contains hematopoietic stem cells (HSCs)
 Contains stromal cells

AKA multipotent stromal cells, mesenchymal
stem cells or MSCs
 Hematopoietic Stem Cells
Generate all of the blood cell lineages
 Reconstitute the bone marrow after depletion

 Caused
by disease or irradiation
 Hematopoietic Stem Cells
Widely used for the treatment of hematologic
diseases
 Collected directly from:

 Bone
marrow
 Umbilical cord blood
 Peripheral blood of individuals receiving cytokines
• Granulocyte-macrophage colony-stimulating factor,
which mobilize HSCs
 Marrow Stromal Cells (MSCs)
Multipotent
 Potentially important therapeutic applications

 Generate
chondrocytes, osteoblasts, adipocytes,
myoblasts, and endothelial cell precursors
• Depends on the tissue to which they migrate
Migrate to injured tissues
 Generate stromal cells or other cell lineages
 Do not participate in normal tissue homeostasis

 Contains stem cells/progenitor cells in the
canals of Hering
Junction between the biliary ductular system and
parenchymal hepatocytes
 Give rise to a population of precursor cells

 Oval
cells
• Bipotential progenitors
• Capable of differentiating into hepatocytes and biliary
cells
 Oval cells
Function as a secondary or reserve compartment
 Activated only when hepatocyte proliferation is
blocked
 Proliferation and differentiation

 Fulminant
hepatic failure
 Liver tumorigenesis
 Chronic hepatitis and advanced liver cirrhosis
 Neurogenesis from neural stem cells (NSCs)
Occurs in the brain of adult rodents and humans
 AKA neural precursor cells
 Capable of generating neurons, astrocytes, and
oligodendrocytes
 Identified in two areas of adult brains

 Subventricular
zone (SVZ)
 Dentate gyrus of the hippocampus
 Human epidermis has a high turnover rate

About 4 weeks
 Stem cells are located in three different areas
of the epidermis

Hair follicle bulge
 Constitutes
a niche for stem cells that produce all of
the cell lineages of the hair follicle
 Stem cells are located in three different areas
of the epidermis

Interfollicular areas of the surface epidermis
 Stem
cells are scattered individually in the epidermis
and are not contained in niches
 Divide infrequently
 Generate transit amplifying cells
• Generate the differentiated epidermis

Sebaceous glands
 Small intestine

Crypts
 Monoclonal structures
 Derived
from single stem cells
 Stem cells regenerate the crypt in 3 to 5 days

Villus
 Differentiated
compartment
 Contains cells from multiple crypts
 Skeletal muscle myocytes do not divide, even
after injury
 Growth and regeneration of injured skeletal
muscle

Occur by replication of satellite cells
 Located beneath
the myocyte basal lamina
 Constitute a reserve pool of stem cells
 Generate differentiated myocytes after injury
 Transparency of the cornea

Integrity of the outermost corneal epithelium
 Maintained
by limbal stem cells (LSCs)
• Located at the junction between the epithelium of the
cornea and the conjunctiva
 Replication of cells
Stimulated by growth factors
 Stimulated by signaling from ECM components

 Integrins
 Cell goes through a tightly controlled
sequence of events

Cell cycle
 G1 (presynthetic)
S (DNA synthesis)
 G2 (premitotic)
 M (mitotic) phases
 Quiescent cells that have not entered the cell cycle
are in the G0 state

 Each cell cycle phase
Dependent on the proper activation
 Dependent on completion of the previous one
 Cycle stops at a place at which an essential gene
function is deficient

 Cell cycle has multiple controls and
redundancies

Particularly during the transition between the G1
and S phases
 Cells can enter G1

From G0 (quiescent cells)
 Cells
first must go through the transition from G0 to
G1
• Involves the transcriptional activation of a large set of
genes



Including various proto-oncogenes
Genes required for ribosome synthesis and protein
translation
After completing mitosis (continuously replicating
cells)
 Cells in G1
Progress through the cycle
 Reach a critical stage at the G1/S transition

 Restriction
point
• Rate-limiting step for replication

Upon passing this restriction point
 Normal
cells become irreversibly committed to DNA
replication
 Progression through the cell cycle,
particularly at the G1/S transition

Tightly regulated by:
 Proteins
called cyclins
 Associated enzymes called cyclin-dependent
kinases (CDKs)
 Activity of cyclin-CDK complexes
 Tightly regulated by CDK inhibitors
 Some growth factors shut off production of these
inhibitors
 Embedded in the cell cycle are surveillance
mechanisms
Geared primarily at sensing damage to DNA and
chromosomes
 Quality control checks are called checkpoints

 Ensure
that cells with damaged DNA or
chromosomes do not complete replication
 G1/S checkpoint

Monitors the integrity of DNA before replication
 G2/M checkpoint
Checks DNA after replication
 Monitors whether the cell can safely enter mitosis

 When cells sense DNA damage…
Checkpoint activation delays the cell cycle
 Triggers DNA repair mechanisms

 DNA damage--too severe to be repaired
Cells are eliminated by apoptosis
 Enter a nonreplicative state called senescence

 Checkpoint defects that allow cells with DNA
strand breaks and chromosome
abnormalities to divide

Produce mutations in daughter cells that may lead
to neoplasia
 Proliferation of many cell types driven by
polypeptides
 Restricted or multiple cell targets
 Promote cell survival, locomotion, contractility,
differentiation, and angiogenesis
 Function as ligands that bind to specific
receptors

Deliver signals to the target cells

Stimulate the transcription of genes that may be silent in
resting cells
 Belong to the EGF family
 Share a common receptor (EGFR)
 EGF
Mitogenic for a variety of epithelial cells,
hepatocytes, and fibroblasts
 Widely distributed in tissue secretions and fluids

 TGF-α
 Originally extracted from sarcoma virus-transformed
cells
 Involved in epithelial cell proliferation in embryos and
adults
 Malignant transformation of normal cells to cancer
 Homology with EGF, binds to EGFR, and shares
biologic activities of EGF
 EGFR1 mutations and amplification
 Detected in cancers of the lung, head and neck, and
breast, glioblastomas, and other cancers
 Originally isolated from platelets and serum
 Identical to a previously identified growth
factor isolated from fibroblasts

Scatter factor
 Mitogenic effects

Hepatocytes and most epithelial cells
 Biliary
epithelium, and epithelial cells of the lungs,
kidney, mammary gland, and skin
 Morphogen in embryonic development
 Promotes cell scattering and migration
 Enhances survival of hepatocytes
 Produced by fibroblasts and most
mesenchymal cells, endothelial cells, and
liver nonparenchymal cells
 Family of several closely related proteins

Each consisting of two chains
 Three isoforms of PDGF (AA, AB, and BB) are
secreted as biologically active molecules
 Produced by a variety of cells

Activated macrophages, endothelial cells, smooth
muscle cells, and many tumor cells
 Migration and proliferation of fibroblasts,
smooth muscle cells, and monocytes

Areas of inflammation and healing skin wounds
 Family of homodimeric proteins
 Potent inducer of blood vessel formation in
early development (vasculogenesis)
 Central role in the growth of new blood
vessels (angiogenesis) in adults
 Promotes angiogenesis in chronic
inflammation, healing of wounds, and in
tumors
 Family of growth factors
 Containing more than 20 members
 Contribute to:

Wound healing responses
 Re-epithelialization
of skin wounds
 Contribute to:

Hematopoiesis
 Differentiation
of specific lineages of blood cells and
development of bone marrow stroma
Angiogenesis
 Development

 Skeletal
and cardiac muscle development
 Lung maturation
 Specification of the liver from endodermal cells
 Superfamily of about 30 members
 Homodimeric protein
 Produced by a variety of different cell types

Platelets, endothelial cells, lymphocytes, and
macrophages
 Potent fibrogenic agent
 Stimulates fibroblast chemotaxis
 Enhances the production of collagen, fibronectin,
and proteoglycans
 Inhibits collagen degradation
 Decreasing
matrix proteases
 Increasing protease inhibitor activities
 Development of fibrosis in a variety of
chronic inflammatory conditions

Lungs, kidney, and liver
 Important functions as mediators of
inflammation and immune responses
 Tumor necrosis factor (TNF) and IL-1

Participate in wound healing reactions
 TNF and IL-6

Involved in the initiation of liver regeneration
Part 2
Lisa Stevens, D.O.
 Receptor-mediated signal transduction

Activated by binding
 Ligands,
growth factors, and cytokines to specific
receptors
 Three general modes of signaling
Based on the source of the ligand and the location
of its receptors
 Autocrine, paracrine, and endocrine

 Autocrine signaling

Cells respond to the signaling molecules that they
themselves secrete
 Establishes
an autocrine loop
• Tumors overproduce growth factors and their
receptors

Stimulating their own proliferation
 Autocrine growth regulation
Plays a role in liver regeneration
 Proliferation of antigen-stimulated lymphocytes

 Paracrine signaling

One cell type produces the ligand
 Acts
on adjacent target cells that express the
appropriate receptor

Responding cells
 Close
proximity to the ligand-producing cell
 Paracrine signaling

Paracrine stimulation
 Common in
connective tissue repair of healing
wounds
• Factor produced by one cell type (macrophage)
has a growth effect on adjacent cells (fibroblast)
 Necessary
for:
• Hepatocyte replication during liver regeneration
• Notch effects in embryonic development, wound
healing, and renewing tissues
 Endocrine signaling

Hormones synthesized by cells of endocrine
organs
 Act
on target cells distant from their site of
synthesis
• Carried by the blood
• Growth factors may also circulate and act at distant
sites


HGF
Several cytokines
 Associated
with systemic aspects of inflammation
• Act as endocrine agents
 Properties of the major types of receptors

Importance:
 How

they deliver signals to the cell interior
Pertinent to an understanding of normal and
unregulated (neoplastic) cell growth
 Ligands for receptors with tyrosine kinase
activity

Most growth factors

EGF, TGF-α, HGF, PDGF, VEGF, FGF, c-KIT ligand,
and insulin
 Receptors belonging to this family
 Extracellular ligand-binding domain
 Transmembrane region
 Cytoplasmic tail that has intrinsic tyrosine kinase
activity
 Binding of the ligand induces:
Dimerization of the receptor
 Tyrosine phosphorylation
 Activation of the receptor tyrosine kinase

 Active
kinase phosphorylates
• Activates downstream effector molecules

Molecules that mediate effects of receptor
engagement with a ligand
 Recruit kinases
 Ligands for these receptors include many
cytokines
IL-2, IL-3, and other interleukins
 Interferons α, β, and γ
 Erythropoietin
 Granulocyte colony-stimulating factor (GCSF)
 Growth hormone
 Prolactin


Receptors transmit extracellular signals to the
nucleus
 Activates
members of the JAK (Janus kinase) family
of proteins
 JAKs link the receptors and activate cytoplasmic
transcription factors
• STATs (signal transducers and activation of
transcription)

Directly shuttle into the nucleus and activate gene
transcription
 Receptors transmit signals into the cell
through trimeric GTP-binding proteins (G
proteins)
 Contain seven transmembrane α-helices
 Constitute the largest family of plasma
membrane receptors

Nonodorant G protein-coupled receptors
accounting for about 1% of the human genome
 A large number of ligands signal through this
type of receptor

Chemokines, vasopressin, serotonin, histamine,
epinephrine and norepinephrine, calcitonin,
glucagon, parathyroid hormone, corticotropin,
and rhodopsin
 Large
number of pharmaceutical drugs target above
receptors
 Receptors located in the nucleus
 Function as ligand-dependent transcription
factors
Ligands diffuse through the cell membrane
 Bind the inactive receptors

 Causes
their activation
• Activated receptor then binds to specific DNA
sequences


Hormone response elements within target genes
Bind to other transcription factors
 Other ligands that bind to members of this
receptor family

Thyroid hormone, vitamin D, and retinoids
 Group of receptors belonging to this family

Peroxisome proliferator-activated receptors
 Nuclear
receptors
 Involved in a broad range of responses
• Adipogenesis, inflammation, and atherosclerosis
 Transfer of information to the nucleus
 Modulate gene transcription

Through action of these factors
 Transcription factors that regulate cell proliferation
 Products of several growth-promoting genes


c-MYC and c-JUN
Products of cell cycle-inhibiting genes

P53
 Modular design
 Contain domains for DNA binding and for
transcriptional regulation
 Urodele amphibians
 Newt can regenerate their tails, limbs, lens, retina,
jaws, and even a large portion of the heart
 Capacity for regeneration of whole tissues
and organs has been lost in mammals
 Inadequacy of true regeneration in mammals

Absence of blastema formation
 Source of cells

for regeneration
Rapid fibroproliferative response after wounding
 Wnt/β-catenin
Highly conserved pathway
 Participates in the regeneration of:

 Planaria
flatworms
 Fin and heart regeneration in zebra fish
 Blastema and patterning formation in limb
regeneration in newts
 Mammals

Wnt/β-catenin
 Modulates
stem cell functions
• Intestinal epithelium, bone marrow, and muscle
 Participates
in liver regeneration after partial
hepatectomy
 Stimulates oval cell proliferation after liver injury
 Liver illustrates the mechanisms of
regeneration

Even this process is not one of true regeneration
 Resection
of tissue does not cause new growth of
liver
 Triggers a process of compensatory hyperplasia in
the remaining parts of the organ
 Other organs capable of compensatory
growth
Kidney, pancreas, adrenal glands, thyroid, and the
lungs of very young animals
 Display it in less dramatic form than the liver

 New nephrons cannot be generated in the
adult kidney

Growth of the contralateral kidney after unilateral
nephrectomy
 Involves
nephron hypertrophy
 Replication of proximal tubule cells
 Pancreas
Limited capacity to regenerate exocrine
components and islets
 Regeneration of pancreatic beta cells

 Beta-cell
replication
 Transdifferentiation of ductal cells
 Differentiation of putative stem cells
 Human liver
 Remarkable capacity to regenerate

Demonstrated by its growth after partial hepatectomy
• Tumor resection or for living-donor hepatic transplantation
 Popular image of liver regeneration
 Daily regrowth of the liver of Prometheus
 Eaten every day by an eagle sent by Zeus

Zeus was angry at Prometheus for stealing the secret of
fire
• Did he know that Prometheus's liver would regenerate?
 Human liver
 Resection of approximately 60% of the liver in living
donors

Doubling of the liver remnant in about one month
 Portions of the liver that remain after partial
hepatectomy


Constitute an intact "mini-liver"
Rapidly expands and reaches the mass of the original
liver
 Restoration of liver mass
 Achieved without regrowth of resected lobes
 Growth occurs by enlargement of the lobes
that remain after the operation

Compensatory growth or compensatory hyperplasia
 End point of liver regeneration after partial
hepatectomy

Restitution of functional mass rather than the
reconstitution of the original
 Almost all hepatocytes replicate during liver
regeneration after partial hepatectomy
 Hepatocytes are quiescent cells
Several hours to enter the cell cycle
 Progress through G1
 Reach the S phase of DNA replication

 Wave of hepatocyte replication
Synchronized
 Followed by synchronous replication of
nonparenchymal cells

 Kupffer cells,
endothelial cells, and stellate cells
 Hepatocyte proliferation in the regenerating
liver
Triggered by the combined actions of cytokines
and polypeptide growth factors
 Exception: Autocrine activity of TGF-α

 Two major restriction points for hepatocyte
replication
G0/G1 transition that bring quiescent hepatocytes into
the cell cycle
 G1/S transition needed for passage through the late G1
restriction point

 Gene expression in the regenerating liver
proceeds in phases

Starts with the immediate early gene response

Transient response that corresponds to the G0/G1
transition
 Quiescent hepatocytes

Become competent to enter the cell cycle through
a priming phase
 Mediated
by the cytokines TNF and IL-6, and
components of the complement system

Priming signals activate several signal
transduction pathways as a necessary prelude to
cell proliferation
 Quiescent hepatocytes
Under the stimulation of HGF, TGFα, and HB-EGF,
primed hepatocytes enter the cell cycle and
undergo DNA replication
 Norepinephrine, serotonin, insulin, thyroid and
growth hormone

 Act
as adjuvants for liver regeneration
• Facilitates the entry of hepatocytes into the cell cycle
 Individual hepatocytes
 Replicate once or twice during regeneration
 Return to quiescence in a strictly regulated
sequence of events
 Intrahepatic stem or progenitor cells
 Do not play a role in the compensatory growth
that occurs after partial hepatectomy
 No evidence for hepatocyte generation from
bone marrow-derived cells during this
process
 Tissue repair and regeneration

Depends on:
 Activity
of soluble factors
 Interactions between cells and the components of
the extracellular matrix
• Regulates the growth, proliferation, movement, and
differentiation of the cells
 The ECMs various functions include:
 Mechanical support


Control of cell growth


Cell anchorage and migration, and maintenance of cell
polarity
ECM components can regulate cell proliferation by
signaling through cellular receptors of the integrin family
Maintenance of cell differentiation

Type of ECM proteins affect the degree of differentiation
of the cells in the tissue
 The ECMs various functions include:

Scaffolding for tissue renewal
 Maintenance
of normal tissue structure
• Requires a basement membrane or stromal scaffold
 Integrity
of the basement membrane or the stroma
of the parenchymal cells
• Critical for the organized regeneration of tissues
 The ECMs various functions include:
 Establishment of tissue microenvironments

Basement membrane
• Boundary between epithelium and underlying connective
tissue
• Forms part of the filtration apparatus in the kidney

Storage and presentation of regulatory molecules

Growth factors FGF and HGF are secreted and stored in
the ECM in some tissues
• Allows rapid deployment of growth factors after local injury
or during regeneration
 Composed of three groups of
macromolecules

Fibrous structural proteins
 Collagens
and elastins
 Provide tensile strength and recoil

Adhesive glycoproteins
 Connect the
matrix elements to one another and to
cells

Proteoglycans and hyaluronan
 Provide
resilience and lubrication
 Molecules assemble to form two basic forms of
ECM:

Interstitial matrix
Found in spaces between epithelial, endothelial, and
smooth muscle cells, as well as in connective tissue
 Consists mostly of fibrillar and nonfibrillar collagen,
elastin, fibronectin, proteoglycans, and hyaluronan


Basement membranes
Closely associated with cell surfaces
 Consist of nonfibrillar collagen (mostly type IV), laminin,
heparin sulfate, and proteoglycans

 Most common protein in the animal world

Provides extracellular framework for all multicellular
organisms
 No collagen = human would be reduced to a
clump of cells, like the "Blob" interconnected by
a few neurons

“Gelatinous horror from outer space" of 1950s movie
fame)
 Currently, 27 different types of collagens
 Each collagen is composed of three chains

Form a trimer in the shape of a triple helix
 Types I, II, III and V, and XI
Fibrillar collagens
 Triple-helical domain is uninterrupted for more than
1000 residues
 Proteins are found in extracellular fibrillar structures

 Type IV collagens
 Long but interrupted triple-helical domains
 Form sheets instead of fibrils
 Main components of the basement membrane,
together with laminin
 Collagen fibril formation
 Associated with the oxidation of lysine and hydroxylysine
residues by the extracellular enzyme lysyl oxidase
 Cross-linking between the chains of adjacent molecules

Major contributor to the tensile strength of collagen
 Vitamin C
 Required for the hydroxylation of procollagen

Requirement that explains the inadequate wound healing in
scurvy
 Genetic defects in collagen production
 Inherited syndromes

Ehlers-Danlos syndrome and osteogenesis imperfecta
 Blood vessels, skin, uterus, and lung

Require elasticity for their function
 Morphologically

Elastic fibers consist of a central core made of
elastin
 Surrounded by a
peripheral network of microfibrils
 Substantial amounts of elastin

Found in the walls of large blood vessels
 Aorta,
and in the uterus, skin, and ligaments
 Fibrillin
 350-kD secreted glycoprotein
 Associates either with itself or with other components
of the ECM
 Scaffolding for deposition of elastin and the assembly
of elastic fibers
 Influence the availability of active TGFβ in the ECM
 Inherited defects in fibrillin

Formation of abnormal elastic fibers in Marfan syndrome
• Changes in the cardiovascular system (aortic dissection) and
the skeleton
 Most adhesion proteins
 AKA CAMs (cell adhesion molecules)
 Function as transmembrane receptors
 Sometimes stored in the cytoplasm
 Can bind to similar or different molecules in other cells
 Interaction between the same cells (homotypic interaction)
 Different cell types (heterotypic interaction)
 Classified into four main families:
 Immunoglobulin family CAMs
 Cadherins
 Integrins
 Selectins
 Integrins
 Bind to ECM proteins such as fibronectin, laminin, and
osteopontin
 Provides a connection between cells and ECM and adhesive
proteins in other cells

Establishing cell-to-cell contact
 ECM Proteins
 Fibronectin



Large protein
Binds to many molecules (collagen, fibrin, proteoglycans, and
cell surface receptors)
Consists of two glycoprotein chains, held together by disulfide
bonds
 ECM Proteins

Fibronectin
 Fibronectin
messenger RNA has two splice forms
• Tissue fibronectin and plasma fibronectin

Plasma form binds to fibrin
 Stabilize the blood clot that fills the gaps created by wounds
 Substratum for ECM deposition and formation of the
provisional matrix during wound healing
 ECM Proteins

Laminin
 Most
abundant glycoprotein in the basement
membrane
 Binding domains for both ECM and cell surface
receptors
 Mediates the attachment of cells to connective
tissue substrates
 Cadherins and integrins

Link the cell surface with the cytoskeleton
 Binding
to actin and intermediate filaments
 Linkages
• Mechanism for the transmission of mechanical force
• Activation of intracellular signal transduction pathways
 Name derived from the term "calcium-
dependent adherence protein"
 Participates in interactions between cells of the
same type

Connect the plasma membrane of adjacent cells
forming two types of cell junction

Zonula adherens
• Small, spotlike junctions located near the apical surface of
epithelial cells

Desmosomes
• Stronger and more extensive junctions, present in epithelial
and muscle cells
 Diminished function of E-cadherin

Contributes to certain forms of breast and gastric
cancer
 SPARC (secreted protein acidic and rich in
cysteine)
AKA osteonectin
 Contributes to tissue remodeling in response to
injury
 Functions as an angiogenesis inhibitor

 Thrombospondins
 Family of large multifunctional proteins
 Some of which are similar to SPARC
 Inhibit angiogenesis
 Osteopontin (OPN)
Glycoprotein that regulates calcification
 Mediator of leukocyte migration involved in
inflammation, vascular remodeling, and fibrosis in
various organs

 Tenascin family
Consist of large multimeric proteins
 Involved in morphogenesis and cell adhesion

 Make up the third type of component in the
ECM
 Consist of long repeating polymers of specific
disaccharides
 Linked to a core protein, forming molecules
called proteoglycans
 Four structurally distinct families of GAGs
 Heparan sulfate
 Chondroitin/dermatan sulfate
 Keratan sulfate
 Hyaluronan (HA)
Produced at the plasma membrane by enzymes called
hyaluronan synthases
 Not linked to a protein backbone


First three of these families

Synthesized and assembled in the Golgi apparatus and rough
endoplasmic reticulum as proteoglycan
 Originally described as ground substances or
mucopolysaccharides

Main function was to organize the ECM
 Diverse roles in regulating connective tissue
structure and permeability
 Integral membrane proteins
 Act as modulators
Inflammation, immune responses, and cell growth
and differentiation
 Binding to other proteins
 Activation of growth factors and chemokines

 Polysaccharide of the GAG family
 Found in the ECM of many tissues
 Abundance in:
 Heart valves, skin and skeletal tissues
 Synovial fluid, vitreous of the eye, and umbilical cord
 Huge molecule
 Many repeats of a simple disaccharide stretched end-to-end
 Binds a large amount of water
 About 1000-fold its own weight
 Forms a viscous hydrated gel

Gives connective tissue the ability to resist compression forces
 Provides resilience and lubrication to
connective tissue

Notably for the cartilage in joints
 Concentration increases in inflammatory
diseases

Rheumatoid arthritis, scleroderma, psoriasis, and
osteoarthritis
 Hyaluronidases
 Enzymes that fragment hyaluronan
 Lower molecular weight molecules
 Produced by endothelial cells
 Binds to the CD44 receptor on leukocytes
 Promotes recruitment of leukocytes to sites of
inflammation
 Stimulates production of inflammatory cytokines and
chemokines by white cells recruited to the sites of
injury
Part 3
Lisa Stevens, D.O.
 Severe or persistent tissue injury
 Damage to parenchymal and stromal
cells
 Leads
to a situation in which repair
cannot be accomplished by
parenchymal regeneration alone
 Repair
 Occurs by replacement of
nonregenerated parenchymal cells
with connective tissue
 Repair

Four components of this process
 Angiogenesis
 Migration
and proliferation of
fibroblasts
 Deposition of ECM
 Remodeling (maturation and
reorganization of the fibrous tissue)
 Tissue repair begins within 24 hours
of injury
Stimulate the emigration of fibroblasts
 Induction of fibroblasts and endothelial

 By 3-5 days of tissue repair a
specialized type of tissue appears

Characteristic of healing  “granulation
tissue”
Name from pink soft appearance of tissue
(seen beneath scab, for example)
 Characterized by fibroblast proliferation and
new, thin walled delicate capillaries
 Outcome is formation of dense fibrosis
(scarring)

 Blood vessels are assembled by two
processes

Vasculogenesis
 Assembly
of primitive vascular network - from
angioblast

Angiogenesis or neovascularization
 Pre-existing
blood vessels send out capillary sprouts
 Critical process in the healing at sites of injury
 Development of collateral circulations at sites
of ischemia

Stimulate following MI or atherosclerosis
 Allows tumors to grow

Inhibit to “starve” tumor growth
 Vasodilation
 Response to nitric oxide
 VEGF-induced increased permeability of the
preexisting vessel
 Proteolytic degradation of the basement
membrane of the parent vessel


Matrix metalloproteinases (MMPs)
Disruption of cell-to-cell contact between endothelial
cells by plasminogen activator
 Migration of endothelial cells

Toward the angiogenic stimulus
 Proliferation of endothelial cells

Just behind the leading front of migrating cells
 Maturation of endothelial cells

Includes inhibition of growth and remodeling into
capillary tubes
 Recruitment

Periendothelial cells, pericytes and vascular
smooth muscle cells to form the mature vessel
• Many factors induce angiogenesis
•
Most important
•
•
bFGF (basic fibroblast growth factor)
VEGF (vascular endothelial growth factor)
 Divided into three phases
 Inflammation



Proliferation


Formation of granulation tissue, proliferation
and migration of connective tissue cells, and reepithelialization of the wound surface
Maturation


Initial injury causes platelet adhesion and
aggregation
Formation of a clot in the surface of the wound
Involves ECM deposition, tissue remodeling, and
wound contraction
Phases overlap; separation is somewhat
arbitrary
 Simplest type of cutaneous wound repair
Healing of a clean, uninfected surgical incision
 Approximated by surgical sutures
 Referred to as healing by primary union or by first
intention

 Incision
Death of a limited number of epithelial and
connective tissue cells
 Disruption of epithelial basement membrane
continuity
 Re-epithelialization to close the wound

 Occurs with
formation of a relatively thin scar
 Excisional wounds
Repair process is more complicated
 Create large defects on the skin surface

 Extensive
loss of cells and tissue
 Healing of these wounds
More intense inflammatory reaction
 Formation of abundant granulation tissue
 Extensive collagen deposition
 Leading to the formation of a substantial
scar

 Generally
contracts
 Healing by secondary union or by second
intention
 Wounding causes the rapid activation
of coagulation pathways

Formation of a blood clot on the wound
surface
 Entrapped
red cells, fibrin, fibronectin,
and complement components
 Clot serves to stop bleeding and as a
scaffold for migrating cells
• Attracted by growth factors, cytokines and
chemokines released into the area

Release of VEGF
 Increased vessel
permeability and edema
 Dehydration occurs at the external surface of
the clot

Forms a scab that covers the wound
 Within 24 hours, neutrophils appear at the
margins of the incision
Use the scaffold provided by the fibrin clot to
infiltrate in
 Release proteolytic enzymes that clean out debris
and invading bacteria

 Fibroblasts and vascular endothelial cells
Proliferate in the first 24 to 72 hours of the repair
process
 Form a specialized type of tissue

 Granulation
tissue
• Hallmark of tissue repair
 Granulation tissue
Pink, soft, granular appearance on the surface of
wounds
 Histologic feature

 Presence
of new small blood vessels (angiogenesis)
 Proliferation of fibroblasts
 Granulation tissue

New vessels are leaky
 Allow
the passage of plasma proteins and fluid into
the extravascular space
 New granulation tissue is often edematous

Progressively invades the incision space
 Granulation tissue

Amount of granulation tissue that is
formed depends on:
 Size
of the tissue deficit created by the
wound
 Intensity of inflammation
Much more prominent in healing by
secondary union
 By 5 to 7 days, granulation tissue fills
the wound area and neovascularization

 Neutrophils

Largely replaced by macrophages by 48 to 96
hours
 Macrophages
are key cellular constituents of tissue
repair
• Clearing extracellular debris, fibrin, and other foreign
material at the site of repair
• Promoting angiogenesis and ECM deposition
 Migration of fibroblasts to the site of injury
Driven by chemokines, TNF, PDGF, TGF-β, and
FGF
 Proliferation is triggered by multiple growth
factors

 PDGF,
EGF, TGF-β, FGF, and the cytokines IL-1 and
TNF
• Macrophages are the main source for these factors
 Collagen fibers are present at the margins of the
incision

At first these are vertically oriented

Do not bridge the incision
 24 to 48 hours, spurs of epithelial cells move
from the wound edge along the cut margins of
the dermis, depositing basement membrane
components as they move.

Fuse in the midline beneath the surface scab

Producing a thin, continuous epithelial layer that closes
the wound
 Full epithelialization of the wound surface

Much slower in healing by secondary union
 Gap
to be bridged is much greater
 Subsequent epithelial cell proliferation thickens the
epidermal layer
 Macrophages

Stimulate fibroblasts
 Produce FGF-7 (keratinocyte
growth factor) and IL6, which enhance keratinocyte migration and
proliferation
 Signaling through the chemokine receptor
CXCR 3 also promotes skin reepithelialization
 Concurrently with epithelialization
Collagen fibrils become more abundant
 Begin to bridge the incision

 Provisional matrix containing fibrin, plasma
fibronectin, and type III collagen is formed

Replaced by a matrix composed primarily of type I
collagen
 TGF-β is the most important fibrogenic agent
Produced by most of the cells in granulation tissue
 Causes fibroblast migration and proliferation,
increased synthesis of collagen and fibronectin,
and decreased degradation of ECM by
metalloproteinases

 Leukocytic infiltrate, edema, and increased
vascularity
Disappear during the second week
 Blanching begins

 Increased
accumulation of collagen within the
wound area and regression of vascular channels
 Original granulation tissue
scaffolding is converted into a pale,
avascular scar
 By the end of the first month

Scar is made up of acellular connective
tissue devoid of inflammatory infiltrate,
covered by intact epidermis
 Generally occurs in large surface
wounds
 Contraction helps to close the
wound by decreasing the gap
between its dermal edges and by
reducing the wound surface area

Important feature in healing by
secondary union
 Replacement of granulation tissue
 Fibrillar collagens (mostly type I collagen)
Form a major portion of the connective tissue in
repair sites
 Essential for the development of strength in
healing wounds

 Net collagen accumulation

Depends not only on increased collagen synthesis
but also on decreased degradation
 Length of time for a skin wound to achieve its
maximal strength

Sutures are removed from an incisional surgical wound
End of the first week, wound strength is approximately
10% that of unwounded skin
 Wound strength increases rapidly over the next 4 weeks
 Slows down at approximately the third month after the
original incision
 Reaches a plateau at about 70% to 80% of the tensile
strength of unwounded skin

 Lower tensile strength

Healed wound area may persist for life
 Recovery of tensile strength
Results from the excess of collagen synthesis over
collagen degradation during the first 2 months of
healing
 Structural modifications of collagen fibers (crosslinking, increased fiber size) after collagen
synthesis ceases

 Adequacy of wound repair may be
impaired by systemic and local host
factors
 Systemic factors include:

Nutrition
 Protein
deficiency: Esp vitamin C
deficiency, inhibit collagen synthesis
and retard healing

Metabolic status
 Diabetes
mellitus is associated with
delayed healing
• Consequence of the microangiopathy
 Circulatory status
 Modulate wound healing
 Inadequate blood supply, usually caused
by arteriosclerosis or venous
abnormalities (e.g., varicose veins) that
retard venous drainage, also impairs
healing
 Hormones
 Glucocorticoids
Well-documented anti-inflammatory
effects
 Influence various components of
inflammation
 Agents also inhibit collagen synthesis

 Infection

Results in persistent tissue injury and
inflammation
 Mechanical factors
Early motion of wounds, can delay healing
 Compressing blood vessels and separating the
edges of the wound

 Foreign bodies
 Unnecessary sutures or fragments of steel, glass, or
even bone, constitute impediments to healing
 Size, location, and type of wound
 Richly vascularized areas, such as the face, heal faster
than those in poorly vascularized ones, such as the
foot
 Small incisional injuries heal faster and with less scar
formation than large excisional wounds or wounds
caused by blunt trauma
 Arise from abnormalities; three categories
Deficient scar formation
 Excessive formation of the repair components
 Formation of contractures

 Lead to two types of complications
 Wound dehiscence
 Rupture
of a wound is most common after
abdominal surgery
 Due to increased abdominal pressure
• Vomiting, coughing, or ileus

Ulceration
 Inadequate
vascularization during healing
 Areas devoid of sensation
 Excessive formation of the
components of the repair process
can give rise to hypertrophic scars
and keloids

Accumulation of excessive amounts of
collagen may give rise to a raised scar
 Hypertrophic scar
• Develop after thermal or traumatic injury

Involves the deep layers of the dermis
 Keloid
Individual predisposition
 More common in African Americans

 Exuberant granulation
 Deviation in wound healing
 Formation of excessive amounts of granulation
tissue
 Protrudes above the level of the surrounding skin
 Blocks re-epithelialization
 Must be removed by cautery or surgical excision
 Permit
restoration of the continuity of the
epithelium
 Important part of the normal healing
process
 Exaggeration of this process

Gives rise to contractures

Results in deformities of the wound and the
surrounding tissues
 Contractures are particularly prone to
develop on the palms, the soles, and
the anterior aspect of the thorax
 Contractures are commonly seen
 Denote the excessive deposition of collagen
and other ECM components in a tissue
 Deposition of collagen in chronic diseases