CHAPTER 3

TISSUE RENEWAL, REGENERATION, and REPAIR

1.

Injury to cells and tissues sets in motion a series of events

that contain the damage and initiate the healing process
Regeneration
Refers to the proliferation of cells and tissues to replace
lost structures.
Repair
most often consists of a combination of regeneration
and scar formation by the deposition of collagen

CONTROL
GROWTH

OF

NORMAL

CELL

PROLIFERATION

AND

TISSUE

In adult tissues, the size of populations is determined by the:

1.
rates of cell proliferation
2.
differentiation and
3.
death by apoptosis
Increased cell number result from either:
a.
increased proliferation
b.
decreased cell death
Terminally differentiated cells
Refers to differentiated cells incapable of replication.
cell proliferation
can be stimulated by physiologic
Largely
controlled
by
signals
from
the
microenvironment that either stimulate or inhibit
proliferation

2.
-

TISSUE PROLIFERATIVE ACTIVITY

tissues of the body are divided into 3 group on the basis of
the proliferative activity of their cells:
1. continuously dividing tissues
labile tissues
cells proliferate throughout life, replacing those that are
destroyed
include:
o
surface epithelia such stratified squamous
epithelia of the sin, oral cavity, vagina and
cervix
o
lining mucosa of all the excretory ducts of the
glands of the body
o
the columnar epithelium of the GI tract and
uterus
o
transitional epithelium of the urinary tract,
and cells of the bone marrow and
hematopoietic capacity
2. Quiescent
stable
low level of replication
cells from these tissues can undergo rapid division in
response to stimuli
capable of reconstituting the tissue of origin
this includes:
o
parenchymal cells of liver, kidneys and
pancreas
o
mesenchymal cells such as fibroblasts and
smooth muscle
o
vascular endothelial cells
o
lymphocytes and other leukocytes
3. Nondividing
permanent tissues
contain cells that have left the cell cycle an cannot
undergo mitotic division in the postnatal life.
this belongs:
o
neurons
o
skeletal and cardiac muscle cells

skeletal
muscle
have
a
regenerative capacity through the
differentiation of the satellite cells
that
are
attached
to
the
endomysial sheath

cardiac muscle has a very limited,

regenerative capacity
STEM CELLS

Stem cells
self- renewal properties
capacity to generate differential cell lineage

to give rise to cell lineage, stem cells need to be achieved by

two mechanism:

obligatory asymmetric replication

with each stem cell division, one of the
daughter cells retain its self- renewing
capacity
while
the
other
enters
a
differentiation pathway

stochastic differentiation
stem cell population is cell maintained by a balance
between stem cell divisions that generate either two
self-renewing stem cells or two cells that will
differentiate
In the early stages of development
o
stem cells are known as embryonic stem cells or
ES cells and are pluripotent.
Pluripotent
o
can generate all tissues of the body
o
give rise to multipotent stem cells

have more restricted development

potential

produce differentiated cells from the 3

embryonic layers
Transdifferentiation
o
a change in the lineage commitment of a stem cell
In adults
o
stem cells are often referred to as adult stem cells
or somatic stem cells
o
have more restricted capacity to generate
different cell types
o
some somatic cell reside in microenvironments
called niches
o
niche cells are:

composed of mesenchymal, endothelial

and other types of cells

generate or transmit stimuli that

regulate stem cell self-renewal and the
generation of the progeny cells
Induced pluripotent stem cells (iPS cells)
differentiated cells that are reprogrammed into
pluripotent cells, similar to ES cells
induced by transduction of genes encoding ES cell
transcription factors

EMBRYONIC STEM CELLS

Embryonic Stem Cells

pluripotent stem cells
found in the inner cell mass of blastocyst in early
embryonic development

REPROGRAMMING OF DIFFERENTIATED CELLS: INDUCED PLURIPOTENT

STEM CELLS (iPS)

Differentiated cells of adult tissues can be reprogrammed to

become pluripotent by transferring their nucleus to an
enucleated oocyte.
Human fibroblasts from adults and newborns have been
reprogrammed into pluripotent cells by the transduction of
four genes encoding transcription factors:
1.
Oct
2.
Sox 2
3.
c-myc
4.
Klf4
homebox Nanog acts to prevent differentiation
Reprogrammed cells
also known as iPS cells
able to generate cells from endodermal, mesodermal
and ectodermal origin
becomes a source of cells from patient-specific stem
cell therapy without the involvement of nuclear transfer
into oocytes.

ADULT (SOMATIC) STEM CELLS

In adult, stem cells are present in tissues that continuously

divide such as:
bone marrow
skin
lining of the GI tract

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR

Transdifferentiation - change in differentiation of a cell from

one type to another.
Developmental capacity - capacity of a cell to
transdifferentiate into diverse lineage

STEM CELLS IN TISSUE HOMEOSTASIS

Bone marrow
The bone marrow contains HSCs and stromal cells (also
known as multipotent stromal cells, mesenchymal stem
cells or MSCs).
o
Hematopoietic Stem Cells.

HSCs generate all of the blood cell

lineages

can reconstitute the bone marrow

after depletion caused by disease
or irradiation

widely used for the treatment of

hematologic diseases
o
Marrow Stromal Cells.

MSCs are multipotent.

potentially important therapeutic

applications, because they can
generate
chondrocytes,
osteoblasts, adipocytes, myoblasts,
and endothelial cell precursors
depending on the tissue to which
they migrate.

migrate to injured tissues

generate stromal cells or other cell

lineages

do not seem to participate in

normal tissue homeostasis
Liver.
The liver contains stem cells/progenitor cells in the
canals of Hering.
Canals of Hering
o
the junction between the biliary ductular
system and parenchymal hepatocytes
o
Cells located in this niche can give rise to a
population of precursor cells known as oval
cells
Oval cells
o
bipotential progenitors
o
capable of differentiating into hepatocytes
and biliary cells.
In contrast to stem cells in proliferating tissues, liver
stem cells function as a secondary or reserve
compartment
activated
only
when
hepatocyte
proliferation is blocked.
Oval cell proliferation and differentiation are prominent
in the livers of patients recovering from fulminant
hepatic failure, in liver tumorigenesis, and in some
cases of chronic hepatitis and advanced liver cirrhosis.
Brain.
Neurogenesis from neural stem cells (NSCs) occurs in
the brain of adult rodents and humans.
the long-established dogma that no new neurons are
generated in the brain of normal adult mammals is now
known to be incorrect.
NSCs (also known as neural precursor cells), capable of
generating neurons, astrocytes, and oligodendrocytes,
have been identified in two areas of adult brains
1.
the subventricular zone (SVZ) and the
2.
dentate gyrus of the hippocampus.
Skin
Stem cells are located in three different areas of the
epidermis:
1.
the hair follicle bulge

The bulge area of the hair follicle

constitutes a niche for stem cells that
produce all of the cell lineages of the
hair follicle
2.
interfollicular areas of the surface epidermis, and

Interfollicular stem cells are scattered

individually in the epidermis and are not
contained in niches.

They divide infrequently but generate

transit amplifying cells that generate the
differentiated epidermis

The human epidermis has a high

turnover rate of about 4 weeks.
3.
sebaceous glands
Intestinal epithelium.
In the small intestine
o
crypts are monoclonal structures derived
from single stem cells:
o
the villus is a differentiated compartment
that contains cells from multiple crypts.
Stem cells in small intestine crypts regenerate the crypt
in 3 to 5 days.
As with skin stem cells, the Wnt and BMP pathways are
important in the regulation of proliferation and
differentiation of intestinal stem cells.
Stem cells may be located immediately above Paneth
cells in the small intestine, or at the base of the crypt,
as is the case in the colon.
Skeletal and cardiac muscle.
Skeletal muscle myocytes do not divide, even after
injury
growth and regeneration of injured skeletal muscle
occur by replication of satellite cells.
Satellite cells

located beneath the myocyte basal lamina

constitute a reserve pool of stem cells that

can generate differentiated myocytes after
injury.

Active Notch signaling, triggered by upregulation

of
delta-like
(Dll)
ligands,
stimulates the proliferation of satellite cells
Cornea.
The transparency of the cornea depends on the
integrity of the outermost corneal epithelium, which is
maintained by limbal stem cells (LSCs).
Limbal stem cells

are located at the junction between the

epithelium of the cornea and the conjunctiva

Hereditary or acquired conditions that result

in LSC deficiency and corneal opacification
can be treated by limbal transplantation or
LSC grafting.

CELL CYCLE AND REGULATION OF CELL REPLICATION

A.
B.
C.

D.

E.

The replication of cells is stimulated by growth factors or by

signaling from ECM components through integrins
To achieve DNA replication and division, the cell goes
through a tightly regulated controlled sequence of events
known as cell cycle
The cell cycle consist of
1.
G1 (presynthetic)
2.
S (DNA Synthesis)
3.
G2 (premiotic)
4.
M (mitotic)
Quiescent cells that have not entered the cell cycle are in
the G0 state
Each phase is dependent on the proper activation and
completion of the previous one
the cycle stops at a place at which an essential gene
function is deficient
The cell cycle has multiple controls and redundancies,
particularly during the transition between the G1 and S
phase
Cell cycles central role is maintaining tissue
homeostasis and regulating physiologic growth
processes such as regeneration and repair.
Cells can enter G1 either from

G0 or

after
completing
meiosis
(continuously
replicating cells)
Quiescent cells first must go through the transition
from G0 to G1
o
first decision step
o
functions as gateway to the cell cycle
o
involves the transcriptional activation of pro
oncogenes and genes required for ribosome
synthesis and protein translation
Restriction point

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR
Cells in G1 progress through the cycle and
reach a critical stage at the G1/S transition
known as restriction point.
o
rate limiting step for replication
o
regulated by cyclins and associated enzymes
called cyclin-dependent kinase
RB Protein 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.
Senescence
o
non replicative stage
o
p53 dependent mechanism
o

o
3.

GROWTH FACTORS

1.

2.

Growth Factors
polypeptides
restricted or multiple targets
promote
survival,
locomotion,
contractility,
differentiation and angiogenesis
all GF function as ligands that binds to specific receptors
which deliver signals to the target cells
signals stimulate the transcription of genes that may be
silent in resting cells
Epidermal Growth Factor (EGF) and Transforming Growth
Factor (TGF-)
EGF family
common receptor EGFR
EGF
mitogenic for EC, hepatocytes and fibroblasts
widely distributed in tissue secretions and fluids
healing wounds of the skin produced by
keratinocytes, macrophages and other inflammatory
cells that migrate into the area
TGF-a
extracted from the sarcoma virus-transformed cells
involved in EC proliferation in embryos and adults
also in malignant transformation of normal cells to
cancer
Best characterized EGFR (EGF receptor) is EGFR1, ERB B1 or
simply EGFR.
responds to EGF, TGF-a and other ligands such as HB
-EGF and amphiregulin
EGFR 1
o
causes cancer of the lungs, head and neck,
and breast
o
glioblastoma
o
other cancers
ERB B2 receptor
o
HER-2/HER2/Neu
o
overexpressed in subset of breast cancer
Hepatocyte Growth Factor (HGF)
originally isolated from platelets and serum
Scatter factor - HGF is identical to; factor isolated from
fibroblasts
HGF/SF
mitogenic
functions:
a.
acts as morphogen in embryonic development
b.
promotes cell scattering and migration
c.
enhances the survival of hepatocytes
produced by
a.
fibroblasts
b.
most mesenchymal cells
c.
endothelial cells
d.
liver nonparenchymal cells
produced as inactive single-chain form activated by serine
protease
c-Met
o
receptor for HGF

4.

5.

6.

highly expressed or mutated in human tumors

(renal and thyroid papillary carcinomas)

Platelet Derived Growth Factor (PDGF)

closely related proteins consisting of 2 chains
isoforms of PDGF: AA, AB and BB - secreted as biologically
active molecules
exert effects by binding 2 cell surface receptors: PDGFR
and
stored in platelet granules
released on platelet activation
produced by
a.
activated macrophages
b.
EC
c.
SMC
d.
tumor cells
causes migration and proliferation of fibroblasts, smooth
muscle cells and monocytes to areas of inflammation and
healing skin wounds.
PDGF-B and C participate in the activation of hepatic stellate
cells in the initial steps of liver fibrosis; stimulate wound
contraction

Vascular Endothelial Growth Factor (VEGF)

homodimeric proteins
VEGF A to D, PIGF (placental growth factor)
VEGF A/VEGF
potent inducer of blood vessel formation in early
development (vasculogenesis)
has a central role in growth of new blood vessels
(angiogenesis) in adults
promotes angiogenesis in chronic inflammation, healing
of wounds and in tumors.
VEGF family signal through 3 tyrosine kinase receptors:
VEGFR1 to 3.
VEGFR-2
o
located in EC
o
main
receptor
for
vasculogenic
and
angiogenic effects of VEGF
VEGFR-1
o
facilitate the mobilization of endothelial stem
cells
o
has a role in inflammation
VEGFR-3
o
VEGF-C and VEGF-D bind to this receptor
o
act on lymphatic endothelial cells to induce
the
production
of
lymphatic
vessels
(lymphangiogenesis)
Fibroblasts Growth Factor
acidic FGF (aFGF, FGF-1)
basic FGF (bFGF, FGF-2)
transduce signal through four tyrosine kinase receptors
(FGF1-4)
FGF-1 - binds to all receptors
FGF-7 - referred to as keratinocyte growth factors (KGF)
FGFs associated with heparaj sulfate in the ECM which can
serve as reservoir for the storage of inactive factors.
contribute to :
a.
wound repair
o
FGF-2
and
KGF
contribute
to
reepithelialization of skin wounds
b.
angiogenesis
o
FGF-2 - has the ability to induce new blood
vessel formation
c.
hematopoiesis
o
implicated in blood lineages as well as
development of blood marrow stroma
d.
development
o
skeletal and cardiac muscle development,
lung maturation and specification of liver.
Transforming Growth Factor -
includes 3 isoform (TGF-1, TGF2 and TGF3)
BMPs. activins, inhibins, mullerian inhibiting substance
TBGF
o
most widespread distribution
o
homodimeric protein produced by a variety of
different cell types e.g.:

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR

a.

b.

c.

a.
platelets
b.
EC
c.
lymphocytes
d.
macrophages
Native TGF- is synthesized as precursor protein
secreted proteolytically cleaved to yield:
a.
active TGF-
o
binds to 2 cell surface receptors with
serine threonine kinase activity

triggers
the
phosphorylated
of
cytoplasmic transcription factor called
Smads
o
Smads

(Smad 1,2,3,5 and 8)

b.
second latent component
TGF- has multiple opposing factors - pleiotropic
TGF- is a growth inhibitor for most epithelial cells.
o
It blocks the cell cycle by increasing the
expression of cell cycle inhibitors of the
Cip/Kip and INK4/ARF families.
o
The effects of TGF- on mesenchymal cells
depend on the tissue environment, but it can
promote invasion and metastasis during
tumor growth.
o
Loss of TGF- receptors frequently occurs in
human tumors, providing a proliferative
advantage to tumor cells.
o
At the same time TGF- expression may
increase in the tumor microenvironment,
creating stromal-epithelial interactions that
enhance tumor growth and invasion.
TGF- is a potent fibrogenic agent
o
stimulates fibroblast chemotaxis
o
enhances the production of

collagen

fibronectin, and

proteoglycans.
o
inhibits collagen degradation by

decreasing matrix proteases

increasing
protease
inhibitor
activities.
o
involved in the development of fibrosis in a
variety of chronic inflammatory conditions
particularly in the lungs, kidney, and liver.
o
High TGF- expression also occurs in
hypertrophic scars, systemic sclerosis , and
the Marfan syndrome.
TGF- has a strong anti-inflammatory effect but may
enhance some immune functions

Cytokines

a.

b.

a.
b.
c.
d.

ECM

a.
b.
c.
d.
e.
f.

regulates growth, proliferation, movement and

differentiation of the cells living within
o
constantly remodeling
o
synthesis
and
degradation
accompanies
morphogenesis, regeneration, wound healing,
chronic fibrotic processes, tumor invasion and
metastasis
o
sequesters water
o
functions:
mechanical support
control of cell growth
maintenance of cell differentiation
scaffolding for tissue renewal
establishment of tissue microenvironments
storage and presentation of regulatory molecules
o

According to the source of the ligand and the location of its

receptors (i.e., in the same, adjacent, or distant cells), three
general modes of signaling, named autocrine, paracrine, and
endocrine, can be distinguished:
Autocrine signaling:
o
Cells respond to the signaling molecules that they
themselves secrete, thus establishing an autocrine
loop.
o
Autocrine growth regulation plays a role in liver
regeneration and the proliferation of antigenstimulated lymphocytes.
o
Tumors frequently overproduce growth factors and
their receptors, thus stimulating their own
proliferation through an autocrine loop.
Paracrine signaling:
o
One cell type produces the ligand, which then acts
on adjacent target cells that express the
appropriate receptor.
o
The responding cells are in close proximity to the
ligand-producing cell and are generally of a
different type.
o
Paracrine stimulation is common in connective
tissue repair of healing wounds, in which a factor
produced by one cell type (e.g., a macrophage)

Receptors with intrinsic tyrosine kinase activity

Receptors lacking intrinsic tyrosine kinase activity that
recruit kinases
G proteincoupled receptors
Steroid hormone receptors.

EXTRACELLULAR MATRIX AND CELL-MATRIX INTERACTIONS

TNF and IL-1 : participate in wound healing reactions

TNF and IL-6: participate in initiation of liver regeneration
SIGNALING MECHANISMS IN CELL GROWTH

has a growth effect on adjacent cells (e.g., a

fibroblast).
o
It is also necessary for hepatocyte replication
during liver regeneration (discussed later), and for
Notch effects in embryonic development, wound
healing, and renewing tissues.
c.
Endocrine signaling:
o
Hormones synthesized by cells of endocrine
organs act on target cells distant from their site of
synthesis, being usually carried by the blood.
o
Growth factors may also circulate and act at
distant sites, as is the case for HGF.
Receptor and Signal Transduction Pathways

ECM is composed of 3 groups of macromolecules:

1.
FIBROUS STRUCTURAL PROTEINS
a.
collagens
b.
elastins
provide tensile strength and recoil
2.
ADHESIVE GLYCOPROTEINS
connect the matrix elements to one another and to
cells
3.
PROTEOGLYCANS AND HYALURONAN
provides resilience and lubrication
TWO Basic Forms of ECM
I.
Interstitial Matrix
II.
Basement Membrane
Interstitial Matrix
o
found in spaces between epithelial, endothelial
and smooth muscle cells; connective tissue
o
consists mostly of
a.
fibrillar and non fibrillar collagen
b.
elastin
c.
fibronectin
d.
proteoglycans
e.
hyaluronan
Basement Membrane
o
associated with cell surfaces
o
consist of
a.
non fibrillar collagen (type IV)
b.
laminin
c.
heparin sulfate
d.
proteoglycan

COLLAGEN

most common protein

provides the extracellular framework
Each collagen is composed of three chains that form a
trimmer at a shape of a helix.
polypeptide is characterized by repeating sequence
glycine is in every 3rd position

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR
-

contains specialized amino acids 4-hydroxyproline and

hydroxylysine

Collagen
Type
Tissue Distribution

i.
ii.
iii.
iv.

Genetic Disorders

FIBRILLAR COLLAGENS
I

Ubiquitous in hard and

soft tissues

Osteogenesis imperfecta; EhlersDanlos syndrome

arthrochalasias type I

II

Cartilage,
intervertebral disk,
vitreous

Achondrogenesis type II,

spondyloepiphysea dysplasia
syndrome

III

Hollow organs, soft

tissues

Vascular Ehlers-Danlos syndrome

Soft tissues, blood

vessels

Classical Ehlers-Danlos syndrome

IX

Cartilage, vitreous

Stickler syndrome

BASEMENT MEMBRANE COLLAGENS

IV

Basement membranes

Alport syndrome

OTHER COLLAGENS
VI

Ubiquitous in
microfibrils

Bethlem myopathy

VII

Anchoring fibrils at
dermal-epidermal
junctions

Dystrophic epidermolysis bullosa

IX

Cartilage,
intervertebral disks

Multiple epiphyseal dysplasias

XVII

Transmembrane
collagen in epidermal
cells

Benign atrophic generalized

epidermolysis bullosa

XV and
XVIII

Endostatin-forming
collagens, endothelial
cells

Knobloch syndrome (type XVIII

collagen)

Collagen and fibril formation is associatied with the oxidation

of lysine and hydroxylysine residues by lysyl oxidase
Vitamin C is required for hydroxylation of procollagen

ELASTIN, FIBRILLIN AND ELASTIC FIBERS

Tissues such as blood vessels, skin, uterus, and lung require

elasticity for their function.
elastic fibers.
can stretch and then return to their original size after
release of the tension.
Morphologically, elastic fibers consist of a central core
made of elastin, surrounded by a peripheral network of
microfibrils.
Substantial amounts of elastin are found in the walls of large
blood vessels, such as the aorta, and in the uterus, skin, and
ligaments.
The peripheral microfibrillar network that surrounds the core
consists largely of fibrillin
The microfibrils
a.
serve, in part, as scaffolding for deposition of elastin
and the assembly of elastic fibers.
b.
also influence the availability of active TGF in the ECM.
inherited defects in fibrillin result in formation of abnormal
elastic fibers in Marfan syndrome, manifested by changes in
the cardiovascular system (aortic dissection) and the
skeleton

CELL ADHESION PROTEINS

CAMS
cell adhesion molecules
four main families

immunoglobulin family CAMS

cadherins
integrins
selectins

these proteins function as

transmembrane receptors

sometimes
stored
in
cytoplasm
provide interaction between the same cells or different
cell types (homotypic and heterotypic respectively)

Integrins
o
bind to ECM proteins such as

fibronectin

laminin, and

osteopontin
o
providing a connection between cells and ECM,
and also to adhesive proteins in other cells,
establishing cell-to-cell contact.
Fibronectin
o
is a large protein that binds to many molecules,
such as collagen, fibrin, proteoglycans, and cell
surface receptors.
o
It consists of two glycoprotein chains, held
together by disulfide bonds.
o
Fibronectin messenger RNA has two splice forms,
giving rise to tissue fibronectin and plasma
fibronectin.
o
The plasma form binds to fibrin, helping to
stabilize the blood clot that fills the gaps created
by wounds, and serves as a substratum for ECM
deposition and formation of the provisional matrix
during wound healing
Laminin
o
is the most abundant glycoprotein in the
basement membrane and has binding domains for
both ECM and cell surface receptors.
o
In the basement membrane, polymers of laminin
and collagen type IV form tightly bound networks.
o
Laminin can also mediate the attachment of cells
to connective tissue substrates.
cadherin
o
calcium-dependent adherence protein.
o
participate in interactions between cells of the
same type.
o
These interactions connect the plasma membrane
of adjacent cells, forming two types of cell
junctions called
a.
zonula adherens

small

spotlike junctions

located near the apical surface of

epithelial cells, and
b.
desmosomes

stronger
and more extensive
junctions

present in epithelial and muscle

cells.
o
Migration
of
keratinocytes
in
the
reepithelialization of skin wounds is dependent on
the formation of dermosomal junctions.
o
Linkage of cadherins with the cytoskeleton occurs
through two classes of catenins.
o
-catenin

links cadherins with -catenin, which, in

turn, connects to actin, thus completing
the connection with the cytoskeleton.
o
Cell-to-cell interactions mediated by cadherins and
catenins play a major role in regulating cell
motility, proliferation, and differentiation and
account for the inhibition of cell proliferation that
occurs when cultured normal cells contact each
other (contact inhibition).
o
Diminished function of E-cadherin contributes to
certain forms of breast and gastric cancer.
o
free -catenin acts independently of cadherins in
the Wnt signaling pathway, which participates in
stem cell homeostasis and regeneration.
o
Mutation and altered expression of the Wnt/catenin pathway is implicated in cancer
development, particularly in gastrointestinal and
liver cancers

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR

MECHANISMS OF ANGIOGENESIS

SPARC
o
o
o

secreted protein acidic and rich in cysteine

aka osteonectin
contributes to tissue remodeling in response to
injury
o
functions as an angionesis inhibitor
Thrombospondins
o
inhibit angiogenesis
Osteopontin
o
glycoprotein that regulates calcification
o
mediator of leukocyte migration involved in
inflammation, vascular remodeling and fibrosis
Tenascin
o
morphogenesis
o
cell adhesion

GLYCOSAMINOGLYCANS

GAGs
o

consist of long repeating polymers of specific

disaccharides.
o
With the exception of hyaluronan GAGs are linked
to a core protein, forming molecules called
proteoglycans.
Proteoglycans
o
originally described as ground substances or
mucopolysaccharides,
o
main function was to organize the ECM,
o
now recognized that these molecules have diverse
roles in regulating connective tissue structure and
permeability
o
can be integral membrane proteins
o
through their binding to other proteins and the
activation of growth factors and chemokines act
as
modulators
of
inflammation,
immune
responses, and cell growth and differentiation.
There are four structurally distinct families of GAGs:
a.
heparan sulfate
b.
chondroitin/dermatan sulfate
c.
keratan sulfate, and
d.
hyaluronan (HA).

The first three of these families are

synthesized and assembled in the Golgi
apparatus and rough endoplasmic
reticulum as proteoglycans.

HA is produced at the plasma

membrane
by
enzymes
called
hyaluronan synthases and is not linked
to a protein backbone.

HEALING BY REPAIR, SCAR FORMATION AND FIBROSIS

If tissue injury is severe or chronic results in damage of

both parenchymal cells and the stromal framework of the
tissue, healing can not be accomplished by regeneration
main healing process is repair by deposition of collagen and
other ECM components, causing the formation of a scar.
In contrast to regeneration which involves the restitution of
tissue components, repair is a fibroproliferative response
that patches rather than restores the tissue.
scar
o
wound healing in the skin
o
the replacement of parenchymal cells in any tissue
by collagen, as in the heart after myocardial
infarction.
Repair by connective tissue deposition includes the following
basic features:
a.
inflammation
b.
angiogenesis
c.
migration and proliferation of fibroblasts
d.
scar formation
e.
connective etissue remodeling
The relative contributions of repair and regeneration are
influenced by
the proliferative capacity of the cells of the tissue
the integrity of the extracellular matrix; and
the resolution or chronicity of the injury and
inflammation.

blood vessels are assembled during embryonic development

by vasculogenesis
o
in which a primitive vascular network is
established from endothelial cell precursors
(angioblasts),
or
from
dual
hemopoietic/endothelial cell precursors called
hemangioblasts.
Blood vessel formation in adults, known as angiogenesis or
neovascularization
o
o
involves the branching and extension of adjacent
pre-existing vessels
o
can also occur by recruitment of endothelial
progenitor cells (EPCs) from the bone marrow

Angiogenesis from Preexisting Vessels

-

In this type of angiogenesis there is

a.
vasodilation
b.
increased permeability of the existing vessel
c.
degradation of ECM
d.
migration of endothelial cells.
The major steps are:
a.
Vasodilation in response to nitric oxide, and VEGFinduced increased permeability of the preexisting
vessel
b.
Proteolytic
degradation
of
the
basement
membrane of the parent vessel by matrix
metalloproteinases (MMPs) and disruption of cellto-cell contact between endothelial cells by
plasminogen activator
c.
Migration of endothelial cells toward the
angiogenic stimulus
d.
Proliferation of endothelial cells, just behind the
leading front of migrating cells
e.
Maturation of endothelial cells, which includes
inhibition of growth and remodeling into capillary
tubes
f.
Recruitment of periendothelial cells (pericytes and
vascular smooth muscle cells) to form the mature
vessel
Angiogenesis from Endothelial Precursor Cells (EPCs)

EPCs can be recruited from the bone marrow into tissues to

initiate angiogenesis

The nature of the homing mechanism is uncertain.

These cells express some markers of hematopoietic stem

cells as well as VEGFR-2, and vascular endothelialcadherin
(VE-cadherin).

EPCs may contribute to the re-endothelization of vascular

implants and the neovascularization of ischemic organs,
cutaneous wounds, and tumors.

The number of circulating EPCs increases greatly in patients

with ischemic conditions, suggesting that EPCs may
influence vascular function and determine the risk of CVD.

GROWTH FACTORS and RECEPTORS INVOLVED IN ANGIOGENESIS

VEGF
o

o
VEGFR-2

the most important growth factor in adlt tissues

undergoing physiologic angiogenesis as well as
angiogenesis occurring in chronic inflammation,
wound healing, tumors and diabetic retinopathy.
secreted by mesenchymal and stromal cells

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR

most important in angiogenesis

tyrosine kinase receptor
expressed by EC
aka KDR

VEGF functions:
a.
induces migration of EPCs in the none marrow
b.
enhances proliferation and differentiation of cells at
sites of angiogenesis
In angiogenesis from preexisting local vessels, VEGF:
a.
stumlates the survival of endothelial cells, proliferation,
motility
b.
initiating the sprouting of new capillaries
Notch Pathway
mechanism for modulation of vasculogenesis
promtes proper branching of new vessels
prevents excessive angiogenesis by decreasing the
responsiveness to VEGF

o
o
o
o
o
o
o

little loss of tissue

small amount of granulation tissue
formation of a thin scar with minimal
contraction
causes death of a limited munmer 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
healing of a clean, uninfected surgical
incision by surgical sutures : simplest type of
cutaneous wound repair

Healing by secondary
o
o
o
o

union or by secondary intention

excisional wounds
create large defects on the skin surface
extensive loss of cells and tissues
healing of wounds involves an intense
inflammatory reaction
o
formation of abundant granulation tissue
o
extensive collagen deposition
o
formation of substantial scar that generaly
contracts
Basic mechanisms are still the same.
SEQUENCE OF EVENTS IN WOUND HEALING

ECM PROTEINS AS REGULATORS OF ANGIOGENESIS

integrins
especially v
critical for the formation and maintenance of newly
formed blood vessels, (2)
matricellular proteins,
including thrombospondin 1, SPARC, and tenascin C
which destabilize cell-matrix interactions
promote angiogenesis
proteinases
plasminogen activators and MMPs
important in tissue remodeling during endothelial
invasion.
Additionally, these proteinases cleave extracellular proteins,
releasing matrix-bound growth factors such as VEGF and
FGF-2 that stimulate angiogenesis.
Proteinases can also release inhibitors such as endostatin, a
small fragment of collagen that inhibits endothelial
proliferation and angiogenesis

1.

CUTANEOUS WOUND HEALING

1.

divided into 3 phases:

1.
INFLAMMATION
2.
PROLIFERATION
3.
MATURATION
these phases overlap.

Inflammation
o
o

2.

Proliferation
o
o

2.

Formation of Granulation Tissue

Fibroblast and vascular endothelial cells
proliferate in the first 24 to 72 hours
form a specialized type of tissue called granulation tissue
Granulation tissue

hallmark of tissue repair

term derives from its pink, soft, granular

appearance on the surface of the wounds

characteristic histologic feature:

a.
presence of new small blood vessels
(angiogenesis)
b.
proliferation of fibroblast

new vessels are leaky

allowing
the
passage
of
plasma proteins and fluids
into the extravascular space

new granulation tissue is often edematous.

progressively invades the incision space

depends on the size of the tissue deficit created by

the wound and the intensity of inflammation

more prominent in healing by 2 union

by 5 to 7 days, granulation tissue fills the wound

area and neovascularization is maximal.

3.

Cell Proliferation and Collagen Deposit

by 48 to 96 hours, macrophage replaced by neutrophils
Macrophage
o
are key cellular constituents of tissue repair

initial injury causes platelet adhesion and

aggregation
formation of clot in the surface of the wound
inflammation
formation of fgranulation tissue
proliferation and migration of connective
tissue cell
re epithelialization of wound surface.

o
Maturation
o
ECM deposition
o
tissue remodeling
o
wound contraction

Cutaneous wound repair

o
is of two types:
1.
Healing by primary union or by first
intention
2.
Healing by secondary union or
by
second intention
Healing by primary union or first intention

Formation of Blood clot

wounding rapid activation of coagulation pathways
formation of a blood clot in the wound surface
clot contains:
a.
entrapped red cells
b.
fibrin
c.
fibronectin
d.
complement components
Clot serves to:
a.
stop bleeding
b.
scaffold for migrating cells migrating cells are
attracted by GF, cytokines, and chemokines released in
the area.
Release of VEGF increased vessel permeability edema
Dehydration occurs at the external surface of the clot scab
that covers the wound
Within 24 hours: neutrophils appear at the margins of the
incision
neutrophils use the scaffold provided by the fibrin clot to
march in
neutrophils release proteolytic enzymes thtt clean out debris
and invading bacteria

3.

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR
clearing extracellular debris, fibrin, and other
foreign material at the site of repair, and
o
promoting angiogenesis and ECM deposition
Migration of fibroblasts to the site of injury is driven by
chemokines
a.
TNF
b.
PDGF
c.
TGF-b
d.
FGF
Macrophages are main source of these
factors
Collagen fibers are now present at the margins of the
incision (vertically oriented and do not bridge the incision)
24 to 48 hours
o
spurs of epithelial cells move from the wound edge
along the cut margins of the dermis deposit
basement membrane components as they move
fuse in the midline beneath the surface scab
producing a thin, continuous epithelial layer that
closes the wound
o
full epithelialization of the wound surface is much
slower in healing by secondary union because the
gap to be bridged is much greater
Macrophages stimulate fibroblast to produce:
a.
FGF-7 (Keratinocyte growth factor)
b.
IL 6 - enhance keratocyte migration and proliferation
HGF and HB-EGF
CXCR-3 - promotes skin reepithelialization
TGF
most important fibrogenic agent
produced by most cells in granulation tissue
causes
a.
fibroblast migration proliferation
b.
increased synthesis of collagen and fibronection
c.
decreased
degradation
of
ECM
by
metaloproteinases

a.
b.
c.
d.
-

Growth Factors and Cytokines Affecting Various Steps in Wound Healing

Monocyte Chemotaxis
Chemokines, TNF, PDGF, FGF,
TGF-
Fibroblast
Migration/
PDGF, EGF, FGF, TGF-, TNF, IL-1
Replication
Keratinocyte Replication
HB-EGF, FGF-7, HGF
Angiogenesis
VEGF, angiopoietins, FGF
Collagen synthesis
TGF-, PDGF
Collagenase secretion
PDGF, FGF, TNF; TGF-, inhibits
4.

5.

6.

Scar Formation
2nd week - leukocytic infiltrate, edema and increased
vascularity disappear.
Blanching begins
blanching is accomplished by:
a.
increased accumulation of collagen within the
wound area
b.
regression of vascular channles
Original granulation tissue scaffolding is converted into a
pale, avascular scar, composed of:
a.
spindle shaped fibroblasts
b.
dense collagen
c.
fragments of elastic tissue
d.
other ECM components
Dermal appendages have been destroyed in the line of the
incision are permanently lost
By the end of first month, the scar is made up of acellular
connective tissue devoid of inflammatory infiltrate, covered
by intact epidermis

Wound Contraction
Wound contraction generally occurs in large surface wounds
Contraction helps to close the wound by:
a.
decreasing the gap between its dermal edges
b.
reducing the wound surface area
contraction is an important feature in healing by secondary
union
Initial steps of wound contraction involves the formation of
myofibroblasts at the edge of the wound.
Myofibroblasts
expresse smooth mucle -actin and vimentin
Ultrastructural characteristics of a smooth muscle cell
contract in the wound tissue

7.

produce large amounts of ECM components such as

type I collagen
tenascin-C
SPARC
extra-domain fibronection
formed from tissue fibroblasts through the effects of
PDG, TGF and FGF-2

released by macrophages at
the wound site

can also irigioante from bone

marrow
precursors
(fibrocytes)

can also be from epithelial

cells
(epithelial-tomesenchymal transition)

Connective Tissue Remodeling

replacement of granulation tissue with a scar involves
changes in the composition of ECNM
Remodeling of the connective tissue framework
balance between ECM synthesis and degradation
important feature of tissue repair
Degradation of collagen and other ECM proteins is achieved
by matrix metalloproteinase
Metalloproteinase
common 180-reside zinc protease domain
include:
a.
interstitial collagenases (MMP-1, 2 and 3) - cleave
fibrillar collagen types I, II and III
b.
gelatinase (MMP-2 and 9) - degrade amorphous
collagen as well as fibronectin
c.
stromelysins (MMP-3,10 and 11) - act on a variety
of ECM components including proteoglycans,
laminin, fibronectin and amorphous collagens
d.
membrane bound MMPs (ADAM)
produced by fibroblasts, macrophages, neutrophils,
synovial cells and some epithelial cells
secretion is induced by

GF (PDGF, FGF)

cytokines (IL-1 and TNF)

phagocytosis in macrophages
secretion is inhibited by

TGF-

steroids
Collagenase
o
cleave collagen under physiologic conditions.
o
synthesized as a latent precursors
o
activated by chemicals
ADAM
o
disintegrin and metalloproteinase-domain
family
o
anchored by a single transmembrane domain
to cell surface
o
ADAM-17

(aka TACE, TNF-converting enzyme)

cleaves
the
membrane-bound
precursor forms of TNF and TGF-

releasing the active molecules

ADAM -17 deficiency

Recovery of Tensile Strength
Type I collagen form a major portion of the connective tissue
in repair sites and are essential for the development of
strength in healing wounds
Net collagen accummuationdepends on:
a.
increased collagen synthesis
b. decreased collagen degradation
Sutures are removed from an incisional surgical wound at the
end of 1st week - wound strength: 10% that of unwounded
skin.
Wound strength
a.
increases rapidly over the next 4 weeks
b. slows down at approximately the 3rd month after the
original incision
c.
reaches a plateau at about 70% to 80% of the tensile
strength of the unwounded skin
Recovery of tensile strength
results from the excess of collagen synthesis over
collagen degradation during the first 2 months of
healing

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR
-

strucutal modifications of collagen fibers (cross linking)

after collagen synthesis ceases

LOCAL AND SYSTEMIC FACTORS THAT INFLUENCE WOUND HEALING

A.

Systemic Factors:

Nutrition
protein deficiency
vitamin C deficiency -inhibit collagen synthesis and
retard healing
Metabolic Status
change wound healing
DM - associated with delated wound healing
(consequence of microangiopathy)
Circulatory Status
modulate wound healing
inadequate blood supply (caused by arterioscelorosis)
or venous abnormalities that retard venous drainaige,
also impairs healing
Hormones
glucocorticoids
o
anti inflammatory effects
o
influence components of inflammation
o
inhibits collagen synthesis

B.

Local Factors

Infection
most important cause of delay in healing
it results in persistent tissue injury and inflammation
Mechanical Factors
e.g. early motion of wounds
delay healing by compressing blood vessels and
separating edges of the wounds
Foreign bodies
unnecessary sutures or fragments of steel, glass or
bone
Size, location and type of wound
wounds in richly vascularized areas (e.g. face) - heal faster
than poorly vascularized area (foot)

4.

PATHOLOGIC ASPECTS OF REPAIR

3 general categories of aberrations in wound healing:

a.
deficient scar formation
b.
excessive formation of repair components
c.
Formation of contractures

Deficient Scar Formation

inadequate formation of granulation tissue or assembly of a

scar leads to 2 types of complications:
1.
Dehiscence
o
rupture of a wound
o
most common after an abdominal surgery due
to increased abdominal pressure
o
vomiting, coughing, or ileus can generate
mechanical stress on the abdominal wound
2.
Ulceration
o
inadequate vascularization during healing
o
lower extremity wounds in px with atherosclerotic
peripheral vascular disease
o
nonhealing wounds also form in areas devoid of
sensation
o
diabetic peripheral neuropathy
Excessive Formation of Components of Repair

Give rise to:

a.
hypertrophic scars
b. keloids

Hypertrophic Scars
o
accumulation of excessive amounts of collagen
o
raised scar
o
generally develop after thermal or traumatic injury
that involves the deep layer of the dermis

Keloid

scar tissue grows beyond the boundaries of the

original wound
o
does not regress
o
individual predisposition
o
common in African American
Exuberant granulation
o
formation of excessive amounts of granulation
tissue
o
protrudes above the level of the surrounding skin
o
blocks reepithelialization
o
proud flesh
o
must be removed by cautery or surgical excision
to permit restoration of the continuity of the
epithelium
o

Desmoids

exuberant proliferation of fibroblasts and

other connective tissue elements that
recur after excision

occurs after incisional scars or traumatic

injuries

aka aggressive fibromatoses

lie in the interface between benign

hyperplasias characteristic of repair and
neoplasia

Contractions

Contractures
exaggeration in the contraction of the size of the wound

results in deformities of the wound and the surrounding

tissues

prone to develop in palms, solesand the anterior aspect of

the thorax

commonly seen after injurious burns; can compromise the

movement of joints
FIBROSIS

deposition of collagen is part of normal wound healing

Fibrosis
excessive deposition of collagen in chronic diseases.
the
injurious
stimulus
caused
by
infections,
autoimmune reactions, trauma and other types of
tissue injury persists in chronic diseases organ
dysfunction and organ failure

Note that:
a.
the initial wave of the host response to external
invaders and tissue injury generates classically
activated macrophages
effective in ingesting and destroying microbes and dead
tissues
b.
alternatively activated macrophages
suppress the microbicidal activities
remodel tissues
promote angionesis and scar formation
TGF-b is always involved as an important fibrogenic agent
Osteopontin
impt role in healing and fibrosis
strongly expressed in the fibrosis of heart, lung, liver,
kidney and some other tissues

CHAPTER 3
TISSUE RENEWAL, REGENERATION, and REPAIR

Fibrotic disorders include:

a.
liver cirrhosis
b.
systemic sclerosis
c.
fibrosing disease of the lungs
o
idiopathic pulmonary fibrosis
o
pneumoconiosis
o
drug,radiation induced pulmonary fibrosis
d.
chronic pancreatitis

e.
f.
SUMMARY

glomerulonephritis
constrictive pericarditis

10