tissue repair. prsntn. updated

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Tissue Repair: Cellular Growth,Tissue Repair: Cellular Growth,

Fibrosis, and Wound HealingFibrosis, and Wound Healing

Facilitator: Dr. Mgaya

Presenters:

Dr. Harrison Chuwa,

Dr. Simon

Dr. Mbonea



IntroductionIntroduction

y Replacement of injured or dead cells is

critical to survival.

y Repair of tissues involves two distinct

processes:

Regeneration: replacement of dead cells by

proliferation of cells of the same type

Replacement by connective tissue orfibroplasia.



CONTROL OF NORMAL CELLCONTROL OF NORMAL CELL

GROWTHGROWTHy The size of a population of cells in adulttissues is determined by the rates of cellproliferation, differentiation, and death by

apoptosis.y Cell proliferation can be stimulated by injury,

mechanical forces acting on tissues, or celldeath.

y The most important factors that regulatecell proliferation are those that recruitquiescent cells into the cell cycle



Cell Proliferation PotentialCell Proliferation Potential

y Cells are divided into 3 groups based on theirproliferation capacity:

i. Continuous dividing (labile) cells e.g.surface epithelia and cells of the bone marrowand hematopoietic cells

ii. Quiescent (stable) cells- normally with slowturnover but capable of rapid division in responseto stimuli e.g. liver, kidney, fibroblasts, smooth

muscle and endothelial cellsiii. Non-dividing (permanent) cells- can·tundergo division in postnatal life e.g. neurons,skeletal muscle, and cardiac muscle



Molecular Events in CellMolecular Events in Cell

ProliferationProliferationy Growth factors induce cell proliferation

by affecting the expression of genes

involved in normal growth control

pathways, the proto-oncogenes.

y Alterations in the structure or expression

of these genes can convert them into

oncogenes, which contribute to theuncontrolled growth characteristic of

cancer.




Proliferation cont«Proliferation cont«y There are 3 schemes of intercellular signalingimportant in the regulation of cell proliferation:

i. Autocrine signaling- cells respond to signalingsubstances that they themselves secrete

ii. Paracrine signaling- a cell produces substancesthat affect only a target cell in close proximity

iii. Endocrine signaling- hormones are synthesizedby cells of endocrine organs and act on target

cells distant from their site of synthesis




Proliferation cont«Proliferation cont«y The chain of molecular events induced by

growth factors include the following

components:

Cell Surface Receptors Interaction

Signal Transduction Systems

Transcription Factors and the regulation of

Gene Expression



Cell Surface Receptors InteractionCell Surface Receptors Interaction

y Cell growth is initiated by binding of a

signaling agent (growth factor) to a

specific receptor frequently located on

the plasma membrane

y The major cell surface receptors involved

in signaling can be grouped into 3 main

categories depending on their activity



Cell Surface ReceptorsCell Surface Receptors

1. Receptors with Intrinsic Kinase

activity- have intrinsic tyrosine kinase

activity, which is activated by ligand

binding.

2. Receptors without Intrinsic Kinase

activity

3. G protein-linked receptors



Signal Transduction SystemsSignal Transduction Systems

y Signal transduction is the process by whichextracellular signals are detected and converted intointracellular signals.

y These systems are typically arranged as networks of sequential protein kinases:

a) Mitogen-activated protein (MAP) kinase pathway

b) Phosphoinositide 3-kinase pathway

c) Inositol-lipid pathway

d) Cyclic adenosine monophosphate (cAMP) pathway

e) JAK/STAT pathwayy These systems transfer information to the nucleus



Transcription Factors and theTranscription Factors and the

Regulation of Gene ExpressionRegulation of Gene Expressiony The signal (information) relayed to the nucleus viathe transduction systems regulates specificchanges that occur in gene expression.

y The regulation is largely achieved at the level of

transcription of genes, a process controlled bytranscription factors.

y Transcription factors are phosphorylated andactivated by specific signaling kinases.

y Among the transcription factors regulating cell

proliferation are a number of proto-oncogenes, inwhich mutation may be associated with tumors,and various types of tumor-suppressor genes (e.g.p53 and Rb)



Cell Cycle and the Regulation of Cell Cycle and the Regulation of

Cell ProliferationCell Proliferationy The cell growth cycle consists of G1

(presynthetic), S (DNA synthesis), G2(premitotic), and M (mitotic) phases.

y Quiescent cells are in a physiologic statecalled G0

y Two types of molecular controls regulate thepassage of cells through specific phases of the cell cycle

A cascade of protein phosphorylation pathwaysinvolving cyclins and CDKs

A set of checkpoints that monitor completion of molecular events



Growth InhibitionGrowth Inhibition

y Regulates cell growth by inhibiting cell

growth

y Inhibitors are also largely polypeptide

factors that use receptors, signal

transduction systems, second messengers,

and transcriptional factors.

y E.g. transforming growth factor- (TGF-);Interferon-



Growth FactorsGrowth Factors

y GFs act by endocrine, paracrine, or

autocrine signaling and in addition to their

growth effects, influence cell movement,

contractility, and differentiation

y The major GFs are as follows:



Major Growth FactorsMajor Growth Factors

Growth

factors

Symbol Source Functions

Epidermal

growth factor

EGF Platelets, macrophages,

saliva, urine, milk,

plasma

Mitogenic for keratinocytes and

fibroblasts; stimulates keratinocyte

migration and

granulation tissue formation

Transforming

growth factoralpha

TGF- Macrophages, T

lymphocytes,keratinocytes,

and many tissues

Similar to EGF; stimulates replication of

hepatocytes and certain epithelial cells

Hepatocytegrowth

factor/scatter

factor

HGF Mesenchymal cells Enhances proliferation of epithelial andendothelial cells, and of hepatocytes;

increases

cell motility



Growth

factors


Vascular

endotheli

al cellgrowth

factor

(isoforms

A, B, C,

D)

VEGF Mesenchymal cells Increases vascular permeability;

mitogenic for endothelial cells

Platelet-

derived

growth

factor

(isoforms

A, B, C, D)

PDGF Platelets, macrophages,

endothelial cells,

keratinocytes, smooth

muscle cells

Chemotactic for PMNs, marcrophages,

fibroblasts, and smooth muscle cells;

activates

PMNs, macrophages, and fibroblasts;

mitogenic for fibroblasts, endothelial cells,

and

smooth muscle cells; stimulates production

of MMPs, fibronectin, and HA; stimulates

angiogenesis and wound contraction;

remodeling; inhibits platelet aggregation;

regulates integrin expression



Growth

factors


Fibroblast

growth

factor-1(acidic), -2

(basic) and

family

FGF Macrophages, mast

cells, T lymphocytes,

endothelial cells,fibroblasts, and many

tissues

Macrophages, mast

cells, T lymphocytes,

endothelial cells,

fibroblasts, and manytissues

Chemotactic for fibroblasts;

mitogenic for fibroblasts and

keratinocytes; stimulateskeratinocyte migration,

angiogenesis, fam wound

contraction and matrix

deposition

Transformin

g growth

factor beta

(isoforms 1,2, 3); other

members of

the family

are BMP

and activin

TGF- Platelets, T lymphocytes,

macrophages,

endothelial cells,

keratinocytes, smoothmuscle cells, fibroblasts

Chemotactic for PMNs, macrophages,

lymphocytes, fibroblasts, and smooth

muscle

cells; stimulates TIMP synthesis,keratinocyte migration, angiogenesis,

and fibroplasia;

inhibits production of MMPs and

keratinocyte proliferation; regulates

integrin

expression and other cytokines;induces TGF- production



row

factors

ym o ource unc ons

Keratinocyte

growth

factor (also

called FGF-7)

K GF Fibroblasts Stimulates keratinocyte migration,

proliferation, and differentiation

Insulin-like

growth factor-

1

IGF-1 Macrophages,

fibroblasts and

other cells

Stimulates synthesis of sulfated proteoglycans,

collagen, keratinocyte migration, and

fibroblast proliferation; endocrine effects

similar to growth hormone

Tumor

necrosis factor

TNF Macrophages,

mast cells, T

lymphocytes

Activates macrophages; regulates other

cytokines; multiple functions

Interleukins IL-1, etc. Macrophages,

mast cells,keratinocytes,

lymphocytes, and

many tissues

Many functions. Some examples: chemotactic

for PMNs (IL-1) and fibroblasts (IL-4),stimulation of MMP-1 synthesis (IL-1),

angiogenesis (IL-8), TIMP synthesis (IL-6);

regulation of other cytokines

Interferons IFN-,

etc.

Lymphocytes and

fibroblasts

Activates macrophages; inhibits fibroblast

proliferation and synthesis of MMPs;



Extracellular Matrix and Cell MatrixExtracellular Matrix and Cell Matrix

InteractionsInteractionsy ECM markedly influences cell growth and

function.

y The ECM consists of fibrous structural

proteins (e.g. collagen) and adhesive glycoproteins embedded in a gel of proteoglycans and hyaluronan.

y These macromolecules assemble into an

interstitial matrix, present in the spacesbetween cells, or into a basementmembrane, located close to the plasmamembrane of some cells.



Collagen and Fiber AssemblyCollagen and Fiber Assembly

y Collagens are divided into 14 types: Type I,II, and II are the fibrillar collagens, andtypes IV,V, andVI are amorphous and

present in interstitial tissue and basementmembranes.

y Collagen synthesis involves first synthesisof chains on ribosomes, followed by a

number of enzymatic hdroxylations, whichare necessary to hold the three chainstogether



Collagen and Fiber Assembly cont«Collagen and Fiber Assembly cont«

y Proteolytic processing of a C-terminalfragment of the procollagen moleculeduring or shortly after secretion from

fibroblasts and smooth muscle cellsresults in the formation of fibrils.

y Extracellular lysyl hydroxylysyl oxidationresults in cross-linkages between chains

of adjacent molecules, which contributeto the tensile strength of collagen(dependent on vitamin C)



Elastin, Fibrillin, and theElastin, Fibrillin, and the

Microfibrillar Network Microfibrillar Network y Elastin provides tissues with elasticity or

the ability to stretch and recoil.

y Elastic fibers consists of a central core of

elastin , and surrounding peripheral

network consisting of fibrillin.

y NB: inherited defects in fibrillin results in

formation of abnormal elastic fibers inMarfan·s syndrome



Adhesive Matrix Glycoproteins andAdhesive Matrix Glycoproteins and

IntegrinsIntegrinsy Adhesive matrix glycoproteins and

Integrins link the ECM with specific

integral cell membrane proteins

y They are several types/forms of the

adhesive matrix glycoproteins . They

include:

Fibronectin Laminin



fibronectinfibronectin

y An adhesion glycoprotein that binds toseveral ECM components ( collagen, heparin,fibrin, proteoglycans) on the one hand and tocell membranes on the other.

y Binding to ECM is mediated by recognitionof a specific amino acid sequence, RGD(arginine, glycine, aspartic acid), present inthe matrix protein.

y

Binding to cells is via integrins or receptorsthat span the cell surface membrane andinteract with the cytoskeleton at points of focal adhesion.



Fibronectin cont«Fibronectin cont«

y Thus, fibronectin is directly involved in

cell attachment, spreading, and

locomotion and interacts with growth

factors to affect growth anddifferentiation.



LamininLaminin

y Is a cross-shaped glycoprotein spanning

basement membranes, also binds to cells

through specific receptors and to collagen

type IV and heparin.

y Thus, is involved in cell attachment,

locomotion, and growth.



IntegrinsIntegrins

y Are the major family of cell surface receptorsthat mediate cellular attachment to the ECM.

y Many integrins are widely expressed, and mostcells have more than one integrins on the cell

surface.y Integrin receptors span the cell membrane and

bind to many components (e.g. fibronectin,laminin, and some collagens) of the ECM byrecognizing the RGD sequence.

y Integrin receptors are important both inorganizing the actin cytoskeleton of cells at pointsof focal adhesion and in transduction of signalsfrom the ECM to the cell interior



Integrins cont«Integrins cont«

y The mechanical linkage between the

Integrin receptors and the cytoskeletal

signaling system may be a mechanism by

which cells convert mechanical force intobiochemical signals



Matricellular ProteinsMatricellular Proteins

y Are secreted proteins that do not function asstructural components of the ECM.

y These proteins interact with matrix components,cell surface receptors, or other molecules (e.g.

growth factors, cytokines, or proteases), whichinteract, in turn, with the cell surface.

y The group shares the ability to disrupt cell-matrixinteractions.

y The family of versatile adapter proteins includesSPARC (secreted protein acidic and r ich incysteine, a.k.a osteonectin), thrombospondins , osteopontin and the tenascin family members.



Proteoglycans and HyaluronanProteoglycans and Hyaluronan

y Proteoglycans are ECM components thatconsists of a core protein linked to one ormore polysaccharides called g lycosamino g lycans.

y Glycosaminoglycans are long repeatingpolymers of modified disaccharides (e.g.heparin sulfate).

y

Proteoglycans can also be integralmembrane proteins, as in the syndecan family,in which the core protein spans the plasmamembrane.



y Hyaluronan is a huge molecule consisting

of many repeats of a disaccharide.

y It serves as a ligand for core proteins and

cell surface receptors.

y Hyaluronan binds large amounts of water,

which helps give connective tissue turgor

pressure and ability to resist compressionforces.



SummarySummary

y Cell growth and differentiation involve the

cellular integration of multiple signals.

y Some of these signals are derived from

growth factors and growth inhibitors.

y Others are derived from components in

the ECM and proceed through integrin-

dependent signaling pathways



REPAIR BY CONNECTIVE TISSUEREPAIR BY CONNECTIVE TISSUE

(FIBROSIS)(FIBROSIS)y Because tissue destruction in wound

healing and chronic inflammation involvesboth parenchymal cells and the stromal

framework, repair cannot beaccomplished solely by regeneration of parenchymal cells.

y Thus, repair involves in large part the

replacement of lost cells and tissues byconnective tissue, which, in time, producesfibrosis and scarring.



REPAIR BY CONNECTIVE TISSUEREPAIR BY CONNECTIVE TISSUE

(FIBROSIS) cont«(FIBROSIS) cont«y Connective tissue repair is the systematic

processes by which unregenerated damage is

replaced by fibrosis and scarring.

y The initial response to a wound consists of the formation of granulation tissue, which

consists of:

Richly vascular connective tissue

New capillaries

Proliferating fibroblasts

Variable numbers of inflammatory cells



Processes involved in repair byProcesses involved in repair by

connective tissueconnective tissuey There are four components to this

orderly process

Formation of new blood vessels

(angiogenesis), spanning the wound Migration and proliferation of fibroblasts filling

and bridging the wound (part of fibroplasia)

Deposition of ECM (part of fibroplasia)

Maturation and re-organization of the fibrous

tissue into a scar (a.k.a remodeling )




connective tissue cont«connective tissue cont«1. Angiogenesis

y Is critical for chronic inflammation,

formation of collateral circulation, and

tumor growth.

y Blood vessels are assembled by two

processes:

x Vasculogenesis- a primitive vascular network isassembled during development

x Neovascularization- pre-existing blood vessels

give rise to capillary buds to produce new vessels



AngiogenesisAngiogenesis

y V asculogenesis- refers to angiogenesis by

mobilization of endothelial precursor cells

(EPCs) from the bone marrow. EPCs are mobilized

from the bone marrow and may migrate to a site of

injury or tumor growth. The homing mechanisms areunknown. At these sites, EPCs differentiate and form

a mature network by linking with existing vessels.

y Neovascularization- i.e. angiogenesis from pre-

existing vessels (capillary growth). Endothelial cellsfrom these vessels become motile and proliferate to

form capillary sprouts



Steps in AngiogenesisSteps in Angiogenesis

y Multiple steps underlie angiogenesis

Proteolytic degradation of the basementmembrane of the parent vessel

Endothelial cell migration and formation of acapillary sprout

Proliferation and maturation of endothelial cells,which includes remodeling into capillary tubes

Recruitment of periendothelial cells, including

pericytes (for small capillaries) and vascularsmooth muscle cells (for larger vessels) tosupport the endothelial tubes



Regulation of AngiogenesisRegulation of Angiogenesis

y The formation, maintenance and remodeling

of blood vessels are controlled by the

following:

Growth factors and receptors: many GFs haveangiogenic activity, but VEGF and the

angiopoietins ( Ang ) are particularly important

in establishing and maintaining new blood vessels.

They interact with the corresponding tyrosinekinase receptors (VEGF-R and Tie) uniquely

expressed by endothelial cells. PDGF and its

receptors are in recruiting periendothelial cells



Regulation of Angiogenesis cont«Regulation of Angiogenesis cont«

ECM proteins, as regulators of angiogenesis: thecell motility and directed migration of endothelialcells that occurs during angiogenesis is regulatedby integrins (e.g. v3), matricellular proteins(e.g. SPARC) and proteases (e.g. plasminogen

activators and matrix metalloproteases) Angiogenesis inhibitors: act to down-regulate

new vessel growth and include certain cytokines(e.g. interf eron-); tissue inhibitors of metalloproteases; certain matricellular proteins

(e.g. thrombospondin); and tumor-derivedfactors, such as angiostatin (a fragment of plasminogen) and endostatin ( a fragment of collagen)




connective tissue cont«connective tissue cont«2. Fibrosis (Fibroplasia)

y Fibrosis occurs within the granulation tissueframework formed at the site of repair andinvolves two processes: Fibroblast migration and proliferation:

increased vascular permeability leads to thedeposition of plasma proteins, such as fibronectinand fibrinogen, which provide a provisionalstroma for ingrowth of fibroblasts. Migration of

fibroblasts and their subsequent proliferation isalso mediated by GFs such as PDGF, EGF, FGFand TGF- and the fibrogenic cytokines IL-1 andTNF-.



Fibroplasia cont«Fibroplasia cont«

ECM deposition: as repair progresses, thenumber of proliferating endothelial cells andfibroblasts decreases. The fibroblasts becomemore synthetic and deposit increased amount of

collagen and other components of ECM. Collagensynthesis is stimulated by GFs (e.g. PDGF, FGF)and by cytokines (IL-1) secreted by fibroblastsand leukocytes in healing wounds. TGF- isthought to play a role in chronic inflammatory

fibrosis. Eventually the granulation tissue scaffolding is converted into a scar composed of

fibroblasts and collagen.




connective tissue cont«connective tissue cont«3. Tissue remodeling

y The replacement of granulation tissue with ascar involves transitions in the composition

of the ECM.y Some of the GFs that stimulate synthesis of

collagen and other connective tissuemolecules modulate the synthesis and

activation of matrix metalloproteinases(MMPs), enzymes that serve to degradethese ECM components.



Tissue remodeling cont«Tissue remodeling cont«

y MMPs consist of:- interstitial collagenases: cleave thefibrillar collagen types I,II, and III

- gelatinases (type IV collagenases):

degrade amorphous collagen as well asfibronectin

- stromelysins: act on a variety of ECMcomponents, including proteoglycans, laminin,

fibronectin, and amorphous collagens- membrane-bound MMPs: cell surface-associated proteases



Tissue remodeling cont«Tissue remodeling cont«

y Secretion of MMPs by fibroblasts and leukocytesis induced by GFs and cytokines and inhibited byTGF-.

y The enzymes are secreted as proenzymes, which

are activated extracellularly.y Activated MMPs can be rapidly inhibited by a

family of specific tissue inhibitors of metalloproteinase (TIMPs).

y The net effect of ECM synthesis versus

degradation results in debridement of injuredsites and remodeling of the connective tissuesframework- important features of both chronicinflammation and wound repair.



WOUND HEALINGWOUND HEALING

y Is a complex but orderly phenomenoninvolving many of the processes: Induction of acute inflammatory process- by

initial injury

Regeneration of parenchymal cells

Migration and proliferation of both parenchymaland connective tissue cells

Synthesis of ECM proteins

Remodeling of connective tissue and parenchymalcomponents

Collagenization and acquisition of woundstrength



Healing by First IntentionHealing by First Intention

y Healing of a clean surgical approximated

incision (1st intention) involves an

orchestrated sequence of events, as follows:

0 hours: the incision is filled with clot

3 to 24 hours: neutrophils from the margins

infiltrate the clot. Mitoses begin to appear in

epithelial basal cells; epithelial closure takes place

by 24 to 48 hours Day 3: neutrophils are replaced by macrophages.

Granulation tissue begins to appear



Healing by First Intention cont«Healing by First Intention cont«

Day 5: the incision space is filled with granulationtissue, neovascularization is maximal, collagenfibrils begin to appear, and epithelial proliferationis now maximal.

Week 2: there is proliferation of fibroblasts andcontinued collagen accumulation to produce ascar. Collagen deposited early in granulationtissue is type III, which is then replaced by adulttype I collagen. Collagen fibers account in largepart for wound strength. Inflammation and newly

formed vessels have largely disappeared. Month 2: scar now consists of connective tissue

devoid of inflammation covered by intactepidermis



Healing by Second IntentionHealing by Second Intention

y Occurs when there is more extensive lossof tissue, such as infarction, ulceration,abscess formation, and large wounds.

y

Abundant granulation tissue grows infrom the margins to fill the defect, but atthe same time the wound contracts; i.e.the defect is markedly reduced from its

original size.y Myofibroblasts contribute to wound

contraction



Summary of Wound HealingSummary of Wound Healing

y Wound healing involves orchestrated

events of inflammation; followed by a

stage of fibroplasia characterized by

granulation tissue; followed by ECMdeposition, tissue remodeling, and scarring



Wound StrengthWound Strength

y Wound strength at the end of first week isapproximately 10% of normal; it is largelydependent on surgical suturing and tissueadhesion.

y The progressive recovery of tensile strengthto 70% to 80% of normal by third month(which may persist for life) is associatedwith: Increased collagen synthesis exceeding collagen

degradation

Cross-linking and increased fiber size of collagenfibers



Factors Influencing Wound HealingFactors Influencing Wound Healing

y There are several factors which can be

categorized as follows:

a. Systemic factors:x Nutritional status of the host (e.g. protein

nutrition and vitamin C intake)

x Metabolic status (e.g. DM delays healing)

x Circulatory status or inadequacy of blood supply

x Hormones, concurrent glucocorticoid therapy,which hinders the inflammatory-reparative

process



Factors Influencing Wound HealingFactors Influencing Wound Healing

cont«cont«

b. Local factors:

- infection, which delays healing

- mechanical factors e.g. motion

directly affecting the wound

- Foreign bodies: impede healing

- size, location, and type of the wound



Pathologic Aspects of WoundPathologic Aspects of Wound

HealingHealingy Complications in wound healing can arise

from abnormalities in any of the basic

repair processes.

y These aberrations can be grouped into 3general categories:

Deficient scar formation

Excessive formation of the repair components Formation of contractures



Deficient Scar FormationDeficient Scar Formation

y This can lead to 2 types of complications:

Wound dehiscence- dehiscence or rupture of awound is most common after abdominal surgery andis due to increased abdominal pressure. This

mechanical stress on the abdominal wound can begenerated by vomiting, coughing, or ileus.

Ulceration- wounds can ulcerate because of inadequate vascularization during healing. e.g. lowerextremity wounds in individuals with atherosclerotic

peripheral vascular disease typically ulcerateNB: Nonhealing wounds also form in areas devoid of

sensation. These neuropathic ulcers are occasionallyseen in patients with diabetic peripheral neuropathy



Excessive Formation of RepairExcessive Formation of Repair

ComponentsComponentsy The formation of excessive amounts of g ranulation tissue,

which protrudes above the level of the surrounding skin

and blocks reepithelialization, has been called

exuberant granulation or proud flesh

y The accumulation of excessive amount of colla g en maygive rise to a raised tumorous scar known as a keloid,

or hypertrophic scar .

y Rarely, incisional scars or traumatic injuries may be

followed by exuberant proliferation of fibroblasts and otherconnective tissue elements that may, in fact, recur after

excision. Called desmoids , or aggressive

fibromatoses , these lie in the interface between benign

proliferations and malignant (though low-grade) tumors.



Formation of ContracturesFormation of Contractures

y Contraction in the size of a wound is animportant part in normal healing process.

y An exaggeration of this process is referred to as acontracture and results in deformities of thewound and the surrounding tissues. e.g. in thehand producing claw deformities and limiting themobility of a joint.

y Contractures are particularly prone to developon the palms, the soles, and the anterior aspect of

the thorax.y Contractures are commonly seen after serious

burns and can compromise the movement of joints.

tissue repair. prsntn. updated

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