Tissue Renewal, Regeneration, and Repair

Tissue Renewal, Regeneration, and RepairLisa Stevens, D.O.

Background Injury to cells---series of damaging

events---initiation of healing processRegeneration

Complete restitution of lost or damaged tissueRepair

May restore some original structures Can cause structural derangements

Healthy tissuesHealing (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 structuresGrowth of an amputated limb in

amphibiansMammalian 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 capacityRenew themselves continuously Regenerate after injury

Repair Combination of regeneration and scar

formationDeposition of collagen

Contribution of regeneration and scarringAbility of the tissue to regenerateExtent of the injuryExample

Superficial skin wound • Heals through the regeneration of the surface

epithelium

Repair Chronic inflammation

Accompanies persistent injuryStimulates 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 deathIncreased cell numbers may result

Increased proliferation Decreased cell death

Apoptosis Physiologic process required for tissue

homeostasisInduced 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 conditionsPhysiologic 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 conditionsPathologic 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

microenvironmentStimulate or inhibit proliferationExcess 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)

Tissue Proliferative Activity

Continuously dividing tissues (labile tissues)Cells proliferate throughout life

Replaces destroyed cellsSurface 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 tissuesMature cells are derived from adult stem

cells Tremendous capacity to proliferate


Quiescent tissues (stabile tissues)Low level of replicationCells from these tissues

Undergo rapid division in response to stimuli

Capable of reconstituting the tissue of origin

Parenchymal cells of liver, kidneys, and pancreas

Mesenchymal cells Fibroblasts and smooth muscle


Quiescent tissues (stabile tissues)Vascular endothelial cellsLymphocytes and other leukocytesExample

Ability of liver to regenerate• Partial hepatectomy• Acute chemical injury


Quiescent tissues (stabile tissues)Fibroblasts, endothelial cells, smooth

muscle cells, chondrocytes, and osteocytes Quiescent in adult mammals Proliferate in response to injury Fibroblasts proliferate extensively


Nondividing tissues Contain cells that have left the cell cycleCannot undergo mitotic division in

postnatal lifeNeuronsSkeletal muscle cellsCardiac 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 sheathsCardiac muscle

Very limited regenerative capacity Large injury to the heart muscle• Myocardial infarction

Followed by scar formation

Stem Cells Characterized by:

Self-renewal propertiesCapacity to generate differentiated cell

lineages Need to be maintained during the life

of the organismAchieved 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 organismAchieved 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 tissuesReside 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 developmentContains 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

Reprogramming of Differentiated Cells Great hope that the technique of

nuclear transfer to oocytes may be used for therapeutic cloning in the treatment of human diseasesNucleus 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

Reprogramming of Differentiated Cells In principle, these cells can then be

transplanted into the patient to repopulate damaged organsTherapeutic 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-perpetuationGive 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 plasticityCapacity of a cell to transdifferentiate

into diverse lineages

Stem Cells in Tissue Homeostasis Stem cells

Bone marrowSkinGutLiverBrainMuscleCornea

Bone Marrow Contains hematopoietic stem cells

(HSCs) Contains stromal cells

AKA multipotent stromal cells, mesenchymal stem cells or MSCs

Hematopoietic Stem CellsGenerate 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)

MultipotentPotentially important therapeutic applications

Generate chondrocytes, osteoblasts, adipocytes, myoblasts, and endothelial cell precursors • Depends on the tissue to which they migrate

Migrate to injured tissuesGenerate stromal cells or other cell lineagesDo not participate in normal tissue

homeostasis

Liver Contains stem cells/progenitor cells

in the canals of HeringJunction 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

humansAKA neural precursor cellsCapable of generating neurons,

astrocytes, and oligodendrocytesIdentified 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 epidermisHair 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 epidermisInterfollicular 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

daysVillus

Differentiated compartment Contains cells from multiple crypts

Skeletal Muscle Skeletal muscle myocytes do not

divide, even after injury Growth and regeneration of injured

skeletal muscleOccur 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 factorsStimulated by signaling from ECM

components Integrins

Cell Cycle Cell goes through a tightly controlled

sequence of eventsCell 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 oneCycle stops at a place at which an

essential gene function is deficient Cell cycle has multiple controls and

redundanciesParticularly 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 translationAfter completing mitosis (continuously

replicating cells)

Cell Cycle Cells in G1

Progress through the cycleReach 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 transitionTightly regulated by:

Proteins called cyclins Associated enzymes called cyclin-

dependent kinases (CDKs)

Cell Cycle Activity of cyclin-CDK complexes

Tightly regulated by CDK inhibitorsSome growth factors shut off production of

these inhibitors Embedded in the cell cycle are

surveillance mechanismsGeared primarily at sensing damage to DNA

and chromosomesQuality 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 checkpointChecks DNA after replicationMonitors whether the cell can safely enter

mitosis When cells sense DNA damage…

Checkpoint activation delays the cell cycleTriggers DNA repair mechanisms

Cell Cycle DNA damage--too severe to be

repairedCells are eliminated by apoptosisEnter a nonreplicative state called

senescence Checkpoint defects that allow cells

with DNA strand breaks and chromosome abnormalities to divideProduce 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 receptorsDeliver 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 adultsMalignant transformation of normal cells to

cancerHomology 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 fibroblastsScatter factor

Mitogenic effectsHepatocytes 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 proteinsEach 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 cellsActivated macrophages, endothelial

cells, smooth muscle cells, and many tumor cells

Migration and proliferation of fibroblasts, smooth muscle cells, and monocytesAreas 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

AngiogenesisDevelopment

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

typesPlatelets, endothelial cells, lymphocytes,

and macrophages

Transforming Growth Factor β (TGF-β) and Related Growth Factors

Potent fibrogenic agent Stimulates fibroblast chemotaxisEnhances the production of collagen,

fibronectin, and proteoglycansInhibits collagen degradation

Decreasing matrix proteases Increasing protease inhibitor activities

Development of fibrosis in a variety of chronic inflammatory conditionsLungs, 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

Tissue Renewal, Regeneration, and Repair

Part 2Lisa Stevens, D.O.

Signaling Mechanisms Receptor-mediated signal

transductionActivated by binding

Ligands, growth factors, and cytokines to specific receptors

Three general modes of signalingBased on the source of the ligand and

the location of its receptorsAutocrine, paracrine, and endocrine

Signaling Mechanisms 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 regulationPlays a role in liver regenerationProliferation of antigen-stimulated

lymphocytes

Signaling Mechanisms Paracrine signaling

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

the appropriate receptorResponding cells

Close proximity to the ligand-producing cell

Signaling Mechanisms

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

Signaling Mechanisms Endocrine signaling

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

synthesis• Carried by the blood• Growth factors may also circulate and act at distant

sites HGF

Several cytokines Associated with systemic aspects of

inflammation• Act as endocrine agents

Receptor Types Properties of the major types of

receptorsImportance:

How they deliver signals to the cell interiorPertinent to an understanding of normal

and unregulated (neoplastic) cell growth

Receptors: Intrinsic Tyrosine Kinase Activity

Ligands for receptors with tyrosine kinase activityMost growth factors

EGF, TGF-α, HGF, PDGF, VEGF, FGF, c-KIT ligand, and insulin

Receptors belonging to this familyExtracellular ligand-binding domainTransmembrane regionCytoplasmic tail that has intrinsic tyrosine

kinase activity

Receptors:Intrinsic Tyrosine Kinase Activity

Binding of the ligand induces:Dimerization of the receptorTyrosine phosphorylationActivation of the receptor tyrosine

kinase Active kinase phosphorylates• Activates downstream effector molecules

Molecules that mediate effects of receptor engagement with a ligand

Receptors:Lacking Intrinsic Tyrosine Kinase Activity

Recruit kinases Ligands for these receptors include

many cytokinesIL-2, IL-3, and other interleukinsInterferons α, β, and γErythropoietinGranulocyte colony-stimulating factor

(GCSF)Growth hormoneProlactin

Receptors--Lacking Intrinsic Tyrosine Kinase Activity

Receptors transmit extracellular signals to the nucleus Activates members of the JAK (Janus

kinase) family of proteins JAKs link the receptors and activate

cytoplasmic transcription factors • STATs (signal transducers and activation of

transcription) Directly shuttle into the nucleus and activate

gene transcription

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 receptorsNonodorant 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 receptorChemokines, vasopressin, serotonin,

histamine, epinephrine and norepinephrine, calcitonin, glucagon, parathyroid hormone, corticotropin, and rhodopsin Large number of pharmaceutical drugs

target above receptors

Steroid Hormone Receptors

Receptors located in the nucleus Function as ligand-dependent

transcription factorsLigands diffuse through the cell membraneBind 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 familyThyroid 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 regenerationRapid fibroproliferative response after wounding

Mechanisms of Tissue and Organ Regeneration

Wnt/β-cateninHighly 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


MammalsWnt/β-catenin

Modulates stem cell functions• Intestinal epithelium, bone marrow, and

muscle Participates in liver regeneration after

partial hepatectomy Stimulates oval cell proliferation after liver

injury


Liver illustrates the mechanisms of regenerationEven this process is not one of true

regeneration Resection of tissue does not cause new

growth of liver Triggers a process of compensatory

hyperplasia in the remaining parts of the organ


Other organs capable of compensatory growthKidney, pancreas, adrenal glands,

thyroid, and the lungs of very young animals

Display it in less dramatic form than the liver


New nephrons cannot be generated in the adult kidneyGrowth of the contralateral kidney after

unilateral nephrectomy Involves nephron hypertrophy Replication of proximal tubule cells


PancreasLimited capacity to regenerate exocrine

components and isletsRegeneration of pancreatic beta cells

Beta-cell replication Transdifferentiation of ductal cells Differentiation of putative stem cells

Liver Regeneration Human liver

Remarkable capacity to regenerate Demonstrated by its growth after partial

hepatectomy• Tumor resection or for living-donor hepatic

transplantation Popular image of liver regeneration

Daily regrowth of the liver of PrometheusEaten 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 Doubling of the liver remnant in about one month

Portions of the liver that remain after partial hepatectomy Constitute an intact "mini-liver" Rapidly expands and reaches the mass of the

original liver Restoration of liver mass

Achieved without regrowth of resected lobes

Liver Regeneration Growth occurs by enlargement of the

lobes that remain after the operationCompensatory growth or compensatory

hyperplasia End point of liver regeneration after

partial hepatectomyRestitution 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 cellsSeveral hours to enter the cell cycleProgress through G1Reach the S phase of DNA replication

Liver Regeneration Wave of hepatocyte replication

SynchronizedFollowed by synchronous replication of

nonparenchymal cells Kupffer cells, endothelial cells, and stellate

cells

Liver Regeneration Hepatocyte proliferation in the

regenerating liverTriggered by the combined actions of

cytokines and polypeptide growth factors

Exception: Autocrine activity of TGF-α

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 phasesStarts 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 regenerationReturn 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 Depends on:

Activity of soluble factors Interactions between cells and the

components of the extracellular matrix• Regulates the growth, proliferation,

movement, and differentiation of the cells


The ECMs various functions include: Mechanical support

Cell anchorage and 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 Type of ECM proteins affect the degree of

differentiation of the cells in the tissue


The ECMs various functions include: Scaffolding for tissue renewal

Maintenance of normal tissue structure• Requires a basement membrane or stromal

scaffold Integrity of the basement membrane or

the stroma of the parenchymal cells• Critical for the organized regeneration of

tissues

Extracellular Matrix and Cell-Matrix Interactions The ECMs various functions include:

Establishment of tissue microenvironments Basement membrane• Boundary between epithelium and underlying

connective tissue • Forms part of the filtration apparatus in the

kidneyStorage and presentation of regulatory

molecules Growth factors FGF and HGF are secreted and

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

local injury or during regeneration

Extracellular Matrix Composed of three groups of

macromoleculesFibrous structural proteins

Collagens and elastins Provide tensile strength and recoil

Adhesive glycoproteins Connect the matrix elements to one

another and to cellsProteoglycans and hyaluronan

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 worldProvides extracellular framework for all

multicellular organisms No collagen = human would be reduced

to a clump of cells, like the "Blob" interconnected by a few neurons “Gelatinous horror from outer space" of

1950s movie fame) Currently, 27 different types of collagens

Collagen

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

Types I, II, III and V, and XIFibrillar collagensTriple-helical domain is uninterrupted for

more than 1000 residuesProteins are found in extracellular fibrillar

structures

Collagen

Type IV collagensLong but interrupted triple-helical

domainsForm sheets instead of fibrils Main components of the basement

membrane, together with laminin

Collagen

Collagen fibril formationAssociated 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 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 Fibrillin

350-kD secreted glycoproteinAssociates either with itself or with other

components of the ECMScaffolding 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 AKA CAMs (cell adhesion molecules)

Function as transmembrane receptors Sometimes stored in the cytoplasm

Can bind to similar or different molecules in other cells Interaction between the same cells (homotypic

interaction) Different cell types (heterotypic interaction)

Classified into four main families: Immunoglobulin family CAMs Cadherins Integrins Selectins

Cell Adhesion Proteins 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

Cell Adhesion Proteins ECM Proteins

Fibronectin Fibronectin messenger RNA has two splice

forms• Tissue fibronectin and plasma fibronectin

Plasma form binds to fibrin Stabilize the blood clot that fills the gaps created

by wounds Substratum for ECM deposition and formation of

the provisional matrix during wound healing

Cell Adhesion Proteins ECM Proteins

Laminin Most abundant glycoprotein in the

basement membrane Binding domains for both ECM and cell

surface receptors Mediates the attachment of cells to

connective tissue substrates

Cell Adhesion Proteins Cadherins and integrins

Link the cell surface with the cytoskeleton Binding to actin and intermediate

filaments Linkages • Mechanism for the transmission of

mechanical force• Activation of intracellular signal transduction

pathways

Cadherin Name derived from the term "calcium-

dependent adherence protein" Participates in interactions between cells

of the same typeConnect 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 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 osteonectinContributes to tissue remodeling in

response to injuryFunctions as an angiogenesis inhibitor

ThrombospondinsFamily of large multifunctional proteinsSome of which are similar to SPARCInhibit angiogenesis

Other Secreted Adhesion Molecules

Osteopontin (OPN)Glycoprotein that regulates calcificationMediator of leukocyte migration involved

in inflammation, vascular remodeling, and fibrosis in various organs

Tenascin familyConsist of large multimeric proteinsInvolved 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 GAGsHeparan sulfateChondroitin/dermatan sulfateKeratan sulfateHyaluronan (HA)

Produced at the plasma membrane by enzymes called hyaluronan synthases

Not linked to a protein backbone First three of these families

Synthesized and assembled in the Golgi apparatus and rough endoplasmic reticulum as proteoglycan

Proteoglycans Originally described as ground

substances or mucopolysaccharidesMain 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

Binding to other proteinsActivation 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 tissueNotably for the cartilage in joints

Concentration increases in inflammatory diseasesRheumatoid arthritis, scleroderma,

psoriasis, and osteoarthritis

Hyaluronan Hyaluronidases

Enzymes that fragment hyaluronan Lower molecular weight molecules

Produced by endothelial cellsBinds to the CD44 receptor on leukocytesPromotes recruitment of leukocytes to

sites of inflammationStimulates production of inflammatory

cytokines and chemokines by white cells recruited to the sites of injury

Part 3Lisa Stevens, D.O.

Tissue Renewal, Regeneration, and Repair

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

RepairOccurs by replacement of

nonregenerated parenchymal cells with connective tissue

Repair by Connective Tissue

RepairFour 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 injuryStimulate the emigration of

fibroblastsInduction of fibroblasts and

endothelial

Tissue Repair

By 3-5 days of tissue repair a specialized type of tissue appearsCharacteristic 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

processesVasculogenesis

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 ischemiaStimulate following MI or atherosclerosis

Allows tumors to growInhibit to “starve” tumor growth

Angiogenesis Vasodilation

Response to nitric oxideVEGF-induced increased permeability of

the preexisting vessel Proteolytic degradation of the

basement membrane of the parent vesselMatrix 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

RecruitmentPeriendothelial 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

repairHealing 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 complicatedCreate large defects on the skin surface

Extensive loss of cells and tissue

Wound Healing

Healing of these wounds More intense inflammatory

reactionFormation of abundant

granulation tissueExtensive collagen depositionLeading 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 pathwaysFormation 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 areaRelease of VEGF

Increased vessel permeability and edema

Formation of Blood Clot Dehydration occurs at the external

surface of the clotForms a scab that covers the wound

Within 24 hours, neutrophils appear at the margins of the incisionUse the scaffold provided by the fibrin

clot to infiltrate inRelease proteolytic enzymes that clean

out debris and invading bacteria

Formation of Granulation Tissue

Fibroblasts and vascular endothelial cellsProliferate in the first 24 to 72 hours of

the repair processForm a specialized type of tissue

Granulation tissue• Hallmark of tissue repair


Granulation tissuePink, soft, granular appearance on the

surface of woundsHistologic feature

Presence of new small blood vessels (angiogenesis)

Proliferation of fibroblasts


Granulation tissueNew vessels are leaky

Allow the passage of plasma proteins and fluid into the extravascular space

New granulation tissue is often edematousProgressively invades the incision space


Granulation tissueAmount 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


Migration of fibroblasts to the site of injuryDriven by chemokines, TNF, PDGF, TGF-

β, and FGFProliferation 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

Cell Proliferation and Collagen Deposition Collagen fibers are present at the margins

of the incisionAt first these are vertically oriented

Do not bridge the incision 24 to 48 hours, spurs of epithelial cells

move from the wound edge along the cut margins of the dermis, depositing basement membrane components as they move. Fuse in the midline beneath the surface scab

Producing a thin, continuous epithelial layer that closes the wound


Full epithelialization of the wound surfaceMuch slower in healing by secondary

union Gap to be bridged is much greater Subsequent epithelial cell proliferation

thickens the epidermal layer


MacrophagesStimulate 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


Concurrently with epithelializationCollagen fibrils become more abundantBegin to bridge the incision

Provisional matrix containing fibrin, plasma fibronectin, and type III collagen is formedReplaced by a matrix composed

primarily of type I collagen


TGF-β is the most important fibrogenic agent Produced by most of the cells in

granulation tissueCauses fibroblast migration and

proliferation, increased synthesis of collagen and fibronectin, and decreased degradation of ECM by metalloproteinases


Leukocytic infiltrate, edema, and increased vascularityDisappear during the second weekBlanching begins

Increased accumulation of collagen within the wound area and regression of vascular channels


Original granulation tissue scaffolding is converted into a pale, avascular scar

By the end of the first monthScar 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 areaImportant feature in healing by

secondary union Replacement of granulation

tissue with a scarInvolves 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 sitesEssential 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 strengthSutures 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 strengthHealed wound area may persist for life

Recovery of tensile strengthResults 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 statusModulate 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

InfectionResults 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 bodiesUnnecessary sutures or fragments of steel,

glass, or even bone, constitute impediments to healing

Size, location, and type of woundRichly 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 categoriesDeficient scar formationExcessive formation of the repair

componentsFormation 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 keloidsAccumulation 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 predispositionMore common in African

Americans

Excessive Formation Exuberant granulation

Deviation in wound healingFormation of excessive amounts of

granulation tissueProtrudes above the level of the

surrounding skinBlocks re-epithelializationMust 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

Documents

Tissue Renewal, Regeneration, and Repair