T ISSUE R ENEWAL, R EGENERATION, AND R EPAIR Pathology – Chapter 3.

TISSUE RENEWAL, REGENERATION, AND REPAIR

BACKGROUND

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

REGENERATION

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

REGENERATION

Hematopoietic system, skin, GI tract

High proliferative capacity

Renew themselves continuously

Regenerate after injury

REPAIR

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

REPAIR

Chronic inflammation

Accompanies persistent injury

Stimulates scar formation Local production of growth factors and cytokines

Promote fibroblast proliferation and collagen synthesis

FIBROSIS

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)

FIBROSIS

Extracellular matrix (ECM)

Fibroblasts, macrophages, and others Produce growth factors, cytokines, and

chemokines

Critical for regeneration and repair

NORMAL CELL PROLIFERATION

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

NORMAL CELL PROLIFERATION

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

CELL PROLIFERATION

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

CELL PROLIFERATION

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

CELL PROLIFERATION

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

TISSUE PROLIFERATIVE ACTIVITY

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



Vascular endothelial cells

Lymphocytes and other leukocytes

Example Ability of liver to regenerate

Partial hepatectomy

Acute chemical injury



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

STEM CELLS

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

STEM CELLS

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

STEM CELLS

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

STEM CELLS

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

EMBRYONIC STEM 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

EMBRYONIC STEM 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

REPROGRAMMING OF DIFFERENTIATED

CELLS 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


CELLS 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


CELLS 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 STEM CELLS

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

ADULT STEM CELLS

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

ADULT STEM 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 IN TISSUE

HOMEOSTASIS Stem cells

Bone marrow

Skin

Gut

Liver

Brain

Muscle

Cornea

BONE MARROW

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

BONE MARROW

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

BONE MARROW

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

LIVER

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

LIVER

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

BRAIN

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

SKIN

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

SKIN

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

INTESTINAL EPITHELIUM

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

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

CORNEA

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

CELL CYCLE

Replication of cells

Stimulated by growth factors

Stimulated by signaling from ECM components Integrins

CELL CYCLE

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

CELL CYCLE

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

CELL CYCLE

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)

CELL CYCLE

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

CELL CYCLE

Progression through the cell cycle, particularly at the G1/S transition

Tightly regulated by: Proteins called cyclins

Associated enzymes called cyclin-dependent kinases (CDKs)

CELL CYCLE

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

CELL CYCLE

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

CELL CYCLE

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

GROWTH FACTORS

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

EPIDERMAL GROWTH FACTOR (EGF) AND

TRANSFORMING GROWTH FACTOR Α

(TGF-Α) 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

EPIDERMAL GROWTH FACTOR (EGF) AND

TRANSFORMING GROWTH FACTOR Α

(TGF-Α) 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

HEPATOCYTE GROWTH FACTOR

(HGF) 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

HEPATOCYTE GROWTH FACTOR

(HGF) 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

PLATELET-DERIVED GROWTH FACTOR

(PDGF)

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

PLATELET-DERIVED GROWTH FACTOR

(PDGF)

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

VASCULAR ENDOTHELIAL GROWTH

FACTOR (VEGF)

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

FIBROBLAST GROWTH FACTOR

(FGF)

Family of growth factors

Containing more than 20 members

Contribute to:

Wound healing responses Re-epithelialization of skin wounds

FIBROBLAST GROWTH FACTOR

(FGF)

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

TRANSFORMING GROWTH FACTOR Β

(TGF-Β) AND RELATED GROWTH FACTORS

Superfamily of about 30 members

Homodimeric protein

Produced by a variety of different cell types

Platelets, endothelial cells, lymphocytes, and macrophages

TRANSFORMING GROWTH FACTOR Β

(TGF-Β) AND RELATED GROWTH FACTORS

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

CYTOKINES

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

SIGNALING MECHANISMS

Receptor-mediated signal transduction

Activated by binding Ligands, growth factors, and cytokines to specific

receptors

Three general modes of signaling

According to 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

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Paracrine signaling

One cell type produces the ligand Then acts on adjacent target cells that express the

appropriate receptor

Responding cells Close proximity to the ligand-producing cell

Are a different type


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

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Endocrine signaling

Hormones synthesized by cells of endocrine organs Act on target cells distant from their site of synthesis

Usually carried by the blood

Growth factors may also circulate and act at distant sites

HGF

Several cytokines Those associated with systemic aspects of

inflammation

Act as endocrine agents

RECEPTOR TYPES

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

RECEPTORS--INTRINSIC TYROSINE KINASE ACTIVITY

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

RECEPTORS--INTRINSIC TYROSINE KINASE ACTIVITY

Binding of the ligand induces:

Dimerization of the receptor

Tyrosine phosphorylation

Activation of the receptor tyrosine kinase Active kinase then phosphorylates and activates

downstream effector molecules

Molecules that mediate the effects of receptor engagement with a ligand

RECEPTORS--LACKING INTRINSIC

TYROSINE KINASE ACTIVITY

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--LACKING INTRINSIC

TYROSINE KINASE ACTIVITY

Receptors transmit extracellular signals to the nucleus Activating 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

G PROTEIN-COUPLED RECEPTORS

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

G PROTEIN-COUPLED RECEPTORS

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 An enormous number of common pharmaceutical

drugs targets such receptors

STEROID HORMONE RECEPTORS

Receptors are generally 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

STEROID HORMONE RECEPTORS

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

TRANSCRIPTION FACTORS

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

MECHANISMS OF TISSUE AND ORGAN

REGENERATION

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


REGENERATION

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


REGENERATION

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


REGENERATION

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


REGENERATION

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


REGENERATION

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


REGENERATION

Pancreas has a limited capacity to regenerate its exocrine components and islets

Regeneration of pancreatic beta cells Involve beta-cell replication, transdifferentiation

of ductal cells, or differentiation of putative stem cells

LIVER REGENERATION

Human liver

Remarkable capacity to regenerate Demonstrated by its growth after partial hepatectomy

Performed for 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?

LIVER REGENERATION

Human liver

Resection of approximately 60% of the liver in living donors results in the 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 the regrowth of the lobes that were resected at the operation

LIVER REGENERATION

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

LIVER REGENERATION

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

LIVER REGENERATION

Wave of hepatocyte replication

Synchronized

Followed by synchronous replication of nonparenchymal cells Kupffer cells, endothelial cells, and stellate cells

LIVER REGENERATION

Hepatocyte proliferation in the regenerating liver

Triggered by the combined actions of cytokines and polypeptide growth factors

With the exception of the autocrine activity of TGF-α Hepatocyte replication is strictly dependent on

paracrine effects of growth factors and cytokines such as HGF and IL-6 produced by hepatic nonparenchymal cells

LIVER REGENERATION

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

LIVER REGENERATION

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

LIVER REGENERATION

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

LIVER REGENERATION

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

EXTRACELLULAR MATRIX AND CELL-

MATRIX INTERACTIONS

Tissue repair and regeneration

Depend not only on the activity of soluble factors, but also on interactions between cells and the components of the extracellular matrix Regulates the growth, proliferation, movement, and

differentiation of the cells Constantly remodeling, and its synthesis and

degradation accompanies morphogenesis, regeneration, wound healing, chronic fibrotic processes, tumor invasion, and metastasis

Sequesters water Providing turgor to soft tissues, and minerals that give

rigidity to bone


MATRIX INTERACTIONS

The ECMs various functions include:

Mechanical support for cell anchorage and cell migration, and maintenance of cell polarity

Control of cell growth

ECM components can regulate cell proliferation by signaling through cellular receptors of the integrin family

Maintenance of cell differentiation

The type of ECM proteins can affect the degree of differentiation of the cells in the tissue, also acting largely via cell surface integrins


MATRIX INTERACTIONS


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 is critical for the organized regeneration of tissues


MATRIX INTERACTIONS


Establishment of tissue microenvironments Basement membrane acts as a boundary between

epithelium and underlying connective tissue and also forms part of the filtration apparatus in the kidney

Storage and presentation of regulatory molecules Growth factors like FGF and HGF are secreted and

stored in the ECM in some tissues Allows the rapid deployment of growth factors

after local injury, or during regeneration

EXTRACELLULAR MATRIX

Composed of three groups of macromolecules

Fibrous structural proteins, such as collagens and elastins that provide tensile strength and recoil

Adhesive glycoproteins that connect the matrix elements to one another and to cells

Proteoglycans and hyaluronan that provide resilience and lubrication

EXTRACELLULAR MATRIX

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

COLLAGEN

Most common protein in the animal world

Providing the extracellular framework for all multicellular organisms

Without collagen, a human being 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 encoded by 41 genes dispersed on at least 14 chromosomes are known

COLLAGEN

Each collagen is composed of three chains that form a trimer in the shape of a triple helix

Types I, II, III and V, and XI are the fibrillar collagens

Triple-helical domain is uninterrupted for more than 1000 residues

Proteins are found in extracellular fibrillar structures

COLLAGEN

Type IV collagens have long but interrupted triple-helical domains and form sheets instead of fibrils

Main components of the basement membrane, together with laminin

Another collagen with a long interrupted triple-helical domain (type VII) forms the anchoring fibrils between some epithelial and mesenchymal structures, such as epidermis and dermis

COLLAGEN

The messenger RNAs transcribed from fibrillar collagen genes are translated into pre-pro-α chains that assemble in a type-specific manner into trimers.

Hydroxylation of proline and lysine residues and lysine glycosylation occur during translation.

Three chains of a particular collagen type assemble to form the triple helix

Procollagen is secreted from the cell and cleaved by proteases to form the basic unit of the fibrils.

COLLAGEN

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

ELASTIN, FIBRILLIN, AND ELASTIC FIBERS

Tissues such as 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

ELASTIN, FIBRILLIN, AND ELASTIC FIBERS

Peripheral microfibrillar network

Surrounds the core consists largely of 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

CELL ADHESION PROTEINS

Most adhesion proteins, also called CAMs (cell adhesion molecules)

These proteins function as transmembrane receptors but are sometimes stored in the cytoplasm

Can bind to similar or different molecules in other cells, providing for interaction between the same cells (homotypic interaction) or 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

Laminin is the most abundant glycoprotein in the basement membrane Binding domains for both ECM and cell surface

receptors

In the basement membrane, polymers of laminin and collagen type IV form tightly bound networks

Mediate the attachment of cells to connective tissue substrates


Cadherins and integrins

Link the cell surface with the cytoskeleton through binding to actin and intermediate filaments Linkages provide a mechanism for the

transmission of mechanical force and the activation of intracellular signal transduction pathways that respond to these forces

CADHERIN

Name derived from the term "calcium-dependent adherence protein"

Family contains almost 90 members

Participate 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

CADHERIN

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")

Diminished function of E-cadherin contributes to certain forms of breast and gastric cancer

OTHER SECRETED ADHESION

MOLECULES

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

OTHER SECRETED ADHESION

MOLECULES

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

GLYCOSAMINOGLYCANS (GAGS)

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

GLYCOSAMINOGLYCANS (GAGS)

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 and is not linked to a protein backbone

First three of these families are synthesized and assembled in the Golgi apparatus and rough endoplasmic reticulum as proteoglycan

PROTEOGLYCANS

Originally described as ground substances or mucopolysaccharides

Main function was to organize the ECM

Diverse roles in regulating connective tissue structure and permeability

PROTEOGLYCANS

Integral membrane proteins

Act as modulators

Inflammation, immune responses, and cell growth and differentiation

Through their binding to other proteins and the activation of growth factors and chemokines

HYALURONAN

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

HYALURONAN

Provides resilience and lubrication to connective tissue

Notably for the cartilage in joints

Concentration increases in inflammatory diseases

Rheumatoid arthritis, scleroderma, psoriasis, and osteoarthritis

HYALURONAN

Enzymes called hyaluronidases fragment hyaluronan

Lower molecular weight molecules hyaluronan 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

REPAIR BY CONNECTIVE TISSUE

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 BY 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

Tissue repair begins within 24 hours of injury

Stimulate the emigration of fibroblasts

Induction of fibroblasts and endothelial

TISSUE REPAIR

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)

ANGIOGENESIS

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

ANGIOGENESIS

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

ANGIOGENESIS

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

ANGIOGENESIS

Migration of endothelial cells

Toward the angiogenic stimulus

Proliferation of endothelial cells

Just behind the leading front of migrating cells

ANGIOGENESIS

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

ANGIOGENESIS

• Many factors induce angiogenesis

• Most important • bFGF (basic fibroblast growth factor)

• VEGF (vascular endothelial growth factor)

CUTANEOUS WOUND HEALING

Divided into three phases

Inflammation Initial injury causes platelet adhesion and aggregation

Formation of a clot in the surface of the wound

Proliferation Formation of granulation tissue, proliferation and migration

of connective tissue cells, and re-epithelialization of the wound surface

Maturation Involves ECM deposition, tissue remodeling, and wound

contraction

Phases overlap; separation is somewhat arbitrary

WOUND HEALING

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

WOUND HEALING

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

WOUND HEALING

Excisional wounds

Repair process is more complicated

Create large defects on the skin surface Extensive loss of cells and tissue

WOUND HEALING

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

FORMATION OF BLOOD CLOT

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

FORMATION OF BLOOD CLOT

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

FORMATION OF GRANULATION TISSUE

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 is maximal

CELL PROLIFERATION AND COLLAGEN

DEPOSITION

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


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


DEPOSITION

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


DEPOSITION

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


DEPOSITION

Macrophages

Stimulate fibroblasts Produce FGF-7 (keratinocyte growth factor) and

IL-6, which enhance keratinocyte migration and proliferation

Signaling through the chemokine receptor CXCR 3 also promotes skin re-epithelialization


DEPOSITION

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


DEPOSITION

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


DEPOSITION

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


DEPOSITION

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

WOUND CONTRACTION

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 with a scar

Involves changes in the composition of the ECM

RECOVERY OF TENSILE STRENGTH

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 (cross-linking, increased fiber size) after collagen synthesis ceases

FACTORS THAT INFLUENCE WOUND

HEALING 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

FACTORS THAT INFLUENCE WOUND

HEALING

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

FACTORS THAT INFLUENCE HEALING

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

FACTORS THAT INFLUENCE HEALING

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

COMPLICATIONS IN WOUND HEALING

Arise from abnormalities; three categories

Deficient scar formation

Excessive formation of the repair components

Formation of contractures

DEFICIENT SCAR FORMATION

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

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

EXCESSIVE FORMATION

Keloid

Individual predisposition

More common in African Americans

EXCESSIVE FORMATION

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

CONTRACTION

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 after serious burns and can compromise the movement of joints

FIBROSIS

Denote the excessive deposition of collagen and other ECM components in a tissue

Deposition of collagen in chronic diseases

T ISSUE R ENEWAL, R EGENERATION, AND R EPAIR Pathology – Chapter 3.

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Transcript of T ISSUE R ENEWAL, R EGENERATION, AND R EPAIR Pathology – Chapter 3.