Chap3 Tissuerep f

CHAPTER 3TISSUE RENEWAL, REGENERATION, and REPAIR

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Injury to cells and tissues sets in motion a series of events that contain the damage and initiate the healing process

Regeneration- Refers to the proliferation of cells and tissues to replace

lost structures. Repair

- most often consists of a combination of regeneration and scar formation by the deposition of collagen

CONTROL OF NORMAL CELL PROLIFERATION AND TISSUE GROWTH

In adult tissues, the size of populations is determined by the:1. rates of cell proliferation2. differentiation and3. death by apoptosis

Increased cell number result from either:a. increased proliferationb. decreased cell death

Terminally differentiated cells- Refers to differentiated cells incapable of replication.

cell proliferation - can be stimulated by physiologic - Largely controlled by signals from the

microenvironment that either stimulate or inhibit proliferation

TISSUE PROLIFERATIVE ACTIVITY

tissues of the body are divided into 3 group on the basis of the proliferative activity of their cells:

1. continuously dividing tissues- labile tissues- cells proliferate throughout life, replacing those that are

destroyed - include:

o surface epithelia such stratified squamous epithelia of the sin, oral cavity, vagina and cervix

o lining mucosa of all the excretory ducts of the glands of the body

o the columnar epithelium of the GI tract and uterus

o transitional epithelium of the urinary tract, and cells of the bone marrow and hematopoietic capacity

2. Quiescent - stable- low level of replication- cells from these tissues can undergo rapid division in

response to stimuli- capable of reconstituting the tissue of origin- this includes:

o parenchymal cells of liver, kidneys and pancreas

o mesenchymal cells such as fibroblasts and smooth muscle

o vascular endothelial cellso lymphocytes and other leukocytes

3. Nondividing - permanent tissues - contain cells that have left the cell cycle an cannot

undergo mitotic division in the postnatal life. - this belongs:

o neuronso skeletal and cardiac muscle cells

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

cardiac muscle has a very limited, regenerative capacity

STEM CELLS Stem cells

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

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

1. obligatory asymmetric replication- with each stem cell division, one of the

daughter cells retain its self- renewing capacity while the other enters a differentiation pathway

2. stochastic differentiation - stem cell population is cell maintained by a balance

between stem cell divisions that generate either two self-renewing stem cells or two cells that will differentiate

In the early stages of developmento stem cells are known as embryonic stem cells or

ES cells and are pluripotent. Pluripotent

o can generate all tissues of the bodyo give rise to multipotent stem cells

have more restricted development potential

produce differentiated cells from the 3 embryonic layers

Transdifferentiationo a change in the lineage commitment of a stem cell

In adultso stem cells are often referred to as adult stem cells

or somatic stem cellso have more restricted capacity to generate

different cell typeso some somatic cell reside in microenvironments

called nicheso niche cells are:

composed of mesenchymal, endothelial and other types of cells

generate or transmit stimuli that regulate stem cell self-renewal and the generation of the progeny cells

Induced pluripotent stem cells (iPS cells)- differentiated cells that are reprogrammed into

pluripotent cells, similar to ES cells- induced by transduction of genes encoding ES cell

transcription factors

EMBRYONIC STEM CELLS

Embryonic Stem Cells- pluripotent stem cells- found in the inner cell mass of blastocyst in early

embryonic development

REPROGRAMMING OF DIFFERENTIATED CELLS: INDUCED PLURIPOTENT STEM CELLS (iPS)

Differentiated cells of adult tissues can be reprogrammed to become pluripotent by transferring their nucleus to an enucleated oocyte.

Human fibroblasts from adults and newborns have been reprogrammed into pluripotent cells by the transduction of four genes encoding transcription factors:1. Oct ¾2. Sox 23. c-myc4. Klf4

homebox Nanog acts to prevent differentiation Reprogrammed cells

- also known as iPS cells- able to generate cells from endodermal, mesodermal

and ectodermal origin- becomes a source of cells from patient-specific stem

cell therapy without the involvement of nuclear transfer into oocytes.

ADULT (SOMATIC) STEM CELLS

In adult, stem cells are present in tissues that continuously divide such as:- bone marrow- skin


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- lining of the GI tract Transdifferentiation - change in differentiation of a cell from

one type to another. Developmental capacity - capacity of a cell to

transdifferentiate into diverse lineage

STEM CELLS IN TISSUE HOMEOSTASIS

Bone marrow- The bone marrow contains HSCs and stromal cells (also

known as multipotent stromal cells, mesenchymal stem cells or MSCs).

o Hematopoietic Stem Cells. HSCs generate all of the blood cell

lineages can reconstitute the bone marrow

after depletion caused by disease or irradiation

widely used for the treatment of hematologic diseases

o Marrow Stromal Cells. MSCs are multipotent. potentially important therapeutic

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

migrate to injured tissues generate stromal cells or other cell

lineages do not seem to participate in

normal tissue homeostasis Liver.

- The liver contains stem cells/progenitor cells in the canals of Hering.

- Canals of Heringo the junction between the biliary ductular

system and parenchymal hepatocytes o Cells located in this niche can give rise to a

population of precursor cells known as oval cells

- Oval cellso bipotential progenitorso capable of differentiating into hepatocytes

and biliary cells. - In contrast to stem cells in proliferating tissues, liver

stem cells function as a secondary or reserve compartment activated only when hepatocyte proliferation is blocked.

- Oval cell proliferation and differentiation are prominent in the livers of patients recovering from fulminant hepatic failure, in liver tumorigenesis, and in some cases of chronic hepatitis and advanced liver cirrhosis.

Brain. - Neurogenesis from neural stem cells (NSCs) occurs in

the brain of adult rodents and humans. - the long-established dogma that no new neurons are

generated in the brain of normal adult mammals is now known to be incorrect.

- NSCs (also known as neural precursor cells), capable of generating neurons, astrocytes, and oligodendrocytes, have been identified in two areas of adult brains1. the subventricular zone (SVZ) and the 2. dentate gyrus of the hippocampus.

Skin- Stem cells are located in three different areas of the

epidermis: 1. the hair follicle bulge

The bulge area of the hair follicle constitutes a niche for stem cells that produce all of the cell lineages of the hair follicle

2. interfollicular areas of the surface epidermis, and

Interfollicular stem cells are scattered individually in the epidermis and are not contained in niches.

They divide infrequently but generate transit amplifying cells that generate the differentiated epidermis

The human epidermis has a high turnover rate of about 4 weeks.

3. sebaceous glands Intestinal epithelium.

- In the small intestineo crypts are monoclonal structures derived

from single stem cells: o the villus is a differentiated compartment

that contains cells from multiple crypts. - Stem cells in small intestine crypts regenerate the crypt

in 3 to 5 days. - As with skin stem cells, the Wnt and BMP pathways are

important in the regulation of proliferation and differentiation of intestinal stem cells.

- Stem cells may be located immediately above Paneth cells in the small intestine, or at the base of the crypt, as is the case in the colon.

Skeletal and cardiac muscle. - Skeletal muscle myocytes do not divide, even after

injury- growth and regeneration of injured skeletal muscle

occur by replication of satellite cells. - Satellite cells

located beneath the myocyte basal lamina constitute a reserve pool of stem cells that

can generate differentiated myocytes after injury.

Active Notch signaling, triggered by up-regulation of delta-like (Dll) ligands, stimulates the proliferation of satellite cells

Cornea. - The transparency of the cornea depends on the

integrity of the outermost corneal epithelium, which is maintained by limbal stem cells (LSCs).

- Limbal stem cells are located at the junction between the

epithelium of the cornea and the conjunctiva Hereditary or acquired conditions that result

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

CELL CYCLE AND REGULATION OF CELL REPLICATION

A. The replication of cells is stimulated by growth factors or by signaling from ECM components through integrins

B. To achieve DNA replication and division, the cell goes through a tightly regulated controlled sequence of events known as cell cycle

C. The cell cycle consist of 1. G1 (presynthetic)2. S (DNA Synthesis)3. G2 (premiotic)4. M (mitotic)

D. Quiescent cells that have not entered the cell cycle are in the G0 state- Each phase is dependent on the proper activation and

completion of the previous one- the cycle stops at a place at which an essential gene

function is deficientE. The cell cycle has multiple controls and redundancies,

particularly during the transition between the G1 and S phase- Cell cycle’s central role is maintaining tissue

homeostasis and regulating physiologic growth processes such as regeneration and repair.

- Cells can enter G1 either from G0 or after completing meiosis (continuously

replicating cells)- Quiescent cells first must go through the transition

from G0 to G1 o first decision step o functions as gateway to the cell cycle


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o involves the transcriptional activation of pro oncogenes and genes required for ribosome synthesis and protein translation

- Restriction pointo Cells in G1 progress through the cycle and

reach a critical stage at the G1/S transition known as restriction point.

o rate limiting step for replicationo regulated by cyclins and associated enzymes

called cyclin-dependent kinase- RB Protein - - Checkpoints - ensure that cells with damaged DNA or

chromosomes do not complete replication- G1/S checkpoint - monitors the integrity of DNA before

replication- G2/M checkpoint checks DNA after replication; monitors

whether the cell can safely enter mitosis. - Senescence

o non replicative stageo p53 dependent mechanism

GROWTH FACTORS

Growth Factors- polypeptides- restricted or multiple targets- promote survival, locomotion, contractility,

differentiation and angiogenesis all GF function as ligands that binds to specific receptors

which deliver signals to the target cells- signals stimulate the transcription of genes that may be

silent in resting cells

1. Epidermal Growth Factor (EGF) and Transforming Growth Factor α (TGF-α)

EGF family common receptor EGFR EGF

- mitogenic for EC, hepatocytes and fibroblasts- widely distributed in tissue secretions and fluids- healing wounds of the skin produced by

keratinocytes, macrophages and other inflammatory cells that migrate into the area

TGF-a - extracted from the sarcoma virus-transformed cells- involved in EC proliferation in embryos and adults- also in malignant transformation of normal cells to

cancer

Best characterized EGFR (EGF receptor) is EGFR1, ERB B1 or simply EGFR.- responds to EGF, TGF-a and other ligands such as HB -

EGF and amphiregulin- EGFR 1

o causes cancer of the lungs, head and neck, and breast

o glioblastomao other cancers

- ERB B2 receptoro HER-2/HER2/Neuo overexpressed in subset of breast cancer

2. Hepatocyte Growth Factor (HGF) originally isolated from platelets and serum Scatter factor - HGF is identical to; factor isolated from

fibroblasts HGF/SF mitogenic functions:

a. acts as morphogen in embryonic developmentb. promotes cell scattering and migrationc. enhances the survival of hepatocytes

produced by a. fibroblasts b. most mesenchymal cellsc. endothelial cellsd. liver nonparenchymal cells

produced as inactive single-chain form activated by serine protease

c-Meto receptor for HGFo highly expressed or mutated in human tumors

(renal and thyroid papillary carcinomas)

3. Platelet Derived Growth Factor (PDGF) closely related proteins consisting of 2 chains isoforms of PDGF: AA, AB and BB - secreted as biologically

active molecules exert effects by binding 2 cell surface receptors: PDGFRα

and β stored in platelet granules released on platelet activation produced by

a. activated macrophagesb. ECc. SMCd. tumor cells

causes migration and proliferation of fibroblasts, smooth muscle cells and monocytes to areas of inflammation and healing skin wounds.

PDGF-B and C participate in the activation of hepatic stellate cells in the initial steps of liver fibrosis; stimulate wound contraction

4. Vascular Endothelial Growth Factor (VEGF) homodimeric proteins VEGF A to D, PIGF (placental growth factor) VEGF A/VEGF

- potent inducer of blood vessel formation in early development (vasculogenesis)

- has a central role in growth of new blood vessels (angiogenesis) in adults

- promotes angiogenesis in chronic inflammation, healing of wounds and in tumors.

VEGF family signal through 3 tyrosine kinase receptors: VEGFR1 to 3.- VEGFR-2

o located in ECo main receptor for vasculogenic and

angiogenic effects of VEGF- VEGFR-1

o facilitate the mobilization of endothelial stem cells

o has a role in inflammation- VEGFR-3

o VEGF-C and VEGF-D bind to this receptoro act on lymphatic endothelial cells to induce

the production of lymphatic vessels (lymphangiogenesis)

5. Fibroblasts Growth Factor acidic FGF (aFGF, FGF-1) basic FGF (bFGF, FGF-2) transduce signal through four tyrosine kinase receptors

(FGF1-4) FGF-1 - binds to all receptors FGF-7 - referred to as keratinocyte growth factors (KGF) FGFs associated with heparaj sulfate in the ECM which can

serve as reservoir for the storage of inactive factors. - contribute to :a. wound repair

o FGF-2 and KGF contribute to reepithelialization of skin wounds

b. angiogenesiso FGF-2 - has the ability to induce new blood

vessel formationc. hematopoiesis

o implicated in blood lineages as well as development of blood marrow stroma

d. developmento skeletal and cardiac muscle development,

lung maturation and specification of liver.

6. Transforming Growth Factor -β- includes 3 isoform (TGF-β1, TGFβ2 and TGFβ3)- BMPs. activins, inhibins, mullerian inhibiting substance- TBGFβ


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o most widespread distributiono homodimeric protein produced by a variety of

different cell types e.g.:a. plateletsb. ECc. lymphocytesd. macrophages

- Native TGF-β is synthesized as precursor protein secreted proteolytically cleaved to yield:a. active TGF-β

o binds to 2 cell surface receptors with serine threonine kinase activity triggers the phosphorylated of cytoplasmic transcription factor called Smads

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

b. second latent component- TGF-β has multiple opposing factors - pleiotropica. TGF-β is a growth inhibitor for most epithelial cells.

o It blocks the cell cycle by increasing the expression of cell cycle inhibitors of the Cip/Kip and INK4/ARF families.

o The effects of TGF-β on mesenchymal cells depend on the tissue environment, but it can promote invasion and metastasis during tumor growth.

o Loss of TGF-β receptors frequently occurs in human tumors, providing a proliferative advantage to tumor cells.

o At the same time TGF-β expression may increase in the tumor microenvironment, creating stromal-epithelial interactions that enhance tumor growth and invasion.

b. TGF-β is a potent fibrogenic agento stimulates fibroblast chemotaxis o enhances the production of

collagen fibronectin, and proteoglycans.

o inhibits collagen degradation by decreasing matrix proteases increasing protease inhibitor

activities. o involved in the development of fibrosis in a

variety of chronic inflammatory conditions particularly in the lungs, kidney, and liver.

o High TGF-β expression also occurs in hypertrophic scars, systemic sclerosis , and the Marfan syndrome.

c. TGF-β has a strong anti-inflammatory effect but may enhance some immune functions

Cytokines

TNF and IL-1 : participate in wound healing reactions TNF and IL-6: participate in initiation of liver regeneration

SIGNALING MECHANISMS IN CELL GROWTH

According to the source of the ligand and the location of its receptors (i.e., in the same, adjacent, or distant cells), three general modes of signaling, named autocrine, paracrine, and endocrine, can be distinguished:

a. Autocrine signaling: o Cells respond to the signaling molecules that they

themselves secrete, thus establishing an autocrine loop.

o Autocrine growth regulation plays a role in liver regeneration and the proliferation of antigen-stimulated lymphocytes.

o Tumors frequently overproduce growth factors and their receptors, thus stimulating their own proliferation through an autocrine loop.

b. Paracrine signaling: o One cell type produces the ligand, which then acts

on adjacent target cells that express the appropriate receptor.

o The responding cells are in close proximity to the ligand-producing cell and are generally of a different type.

o Paracrine stimulation is common in connective tissue repair of healing wounds, in which a factor produced by one cell type (e.g., a macrophage) has a growth effect on adjacent cells (e.g., a fibroblast).

o It is also necessary for hepatocyte replication during liver regeneration (discussed later), and for Notch effects in embryonic development, wound healing, and renewing tissues.

c. Endocrine signaling: o Hormones synthesized by cells of endocrine

organs act on target cells distant from their site of synthesis, being usually carried by the blood.

o Growth factors may also circulate and act at distant sites, as is the case for HGF.

Receptor and Signal Transduction Pathways

a. Receptors with intrinsic tyrosine kinase activityb. Receptors lacking intrinsic tyrosine kinase activity that

recruit kinasesc. G protein–coupled receptorsd. Steroid hormone receptors.

EXTRACELLULAR MATRIX AND CELL-MATRIX INTERACTIONS

ECMo regulates growth, proliferation, movement and

differentiation of the cells living withino constantly remodelingo synthesis and degradation accompanies

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

o sequesters watero functions:

a. mechanical supportb. control of cell growthc. maintenance of cell differentiationd. scaffolding for tissue renewale. establishment of tissue microenvironmentsf. storage and presentation of regulatory molecules

ECM is composed of 3 groups of macromolecules:1. FIBROUS STRUCTURAL PROTEINS

a. collagensb. elastins

- provide tensile strength and recoil2. ADHESIVE GLYCOPROTEINS- connect the matrix elements to one another and to

cells3. PROTEOGLYCANS AND HYALURONAN- provides resilience and lubrication

TWO Basic Forms of ECMI. Interstitial MatrixII. Basement Membrane

Interstitial Matrixo found in spaces between epithelial, endothelial

and smooth muscle cells; connective tissueo consists mostly of

a. fibrillar and non fibrillar collagenb. elastinc. fibronectind. proteoglycans e. hyaluronan

Basement Membraneo associated with cell surfaceso consist of

a. non fibrillar collagen (type IV)b. lamininc. heparin sulfated. proteoglycan

COLLAGEN


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most common protein provides the extracellular framework Each collagen is composed of three chains that form a

trimmer at a shape of a helix.- polypeptide is characterized by repeating sequence- glycine is in every 3rd position- contains specialized amino acids 4-hydroxyproline and

hydroxylysine

Collagen Type Tissue Distribution Genetic Disorders

FIBRILLAR COLLAGENS

I Ubiquitous in hard and soft tissues

Osteogenesis imperfecta; Ehlers-Danlos syndrome—arthrochalasias type I

II Cartilage, intervertebral disk, vitreous

Achondrogenesis type II, spondyloepiphysea dysplasia syndrome

III Hollow organs, soft tissues

Vascular Ehlers-Danlos syndrome

V Soft tissues, blood vessels

Classical Ehlers-Danlos syndrome

IX Cartilage, vitreous Stickler syndrome

BASEMENT MEMBRANE COLLAGENS

IV Basement membranes Alport syndrome

OTHER COLLAGENS

VI Ubiquitous in microfibrils

Bethlem myopathy

VII Anchoring fibrils at dermal-epidermal junctions

Dystrophic epidermolysis bullosa

IX Cartilage, intervertebral disks

Multiple epiphyseal dysplasias

XVII Transmembrane collagen in epidermal cells

Benign atrophic generalized epidermolysis bullosa

XV and XVIII

Endostatin-forming collagens, endothelial cells

Knobloch syndrome (type XVIII collagen)

Collagen and fibril formation is associatied with the oxidation of lysine and hydroxylysine residues by lysyl oxidase

Vitamin C is required for hydroxylation of procollagen

ELASTIN, FIBRILLIN AND ELASTIC FIBERS

Tissues such as blood vessels, skin, uterus, and lung require elasticity for their function.

elastic fibers.- can stretch and then return to their original size after

release of the tension. - Morphologically, elastic fibers consist of a central core

made of elastin, surrounded by a peripheral network of microfibrils.

Substantial amounts of elastin are found in the walls of large blood vessels, such as the aorta, and in the uterus, skin, and ligaments.

The peripheral microfibrillar network that surrounds the core consists largely of fibrillin

The microfibrils a. serve, in part, as scaffolding for deposition of elastin

and the assembly of elastic fibers. b. also influence the availability of active TGFβ in the ECM.

inherited defects in fibrillin result in formation of abnormal elastic fibers in Marfan syndrome, manifested by changes in the cardiovascular system (aortic dissection) and the skeleton

CELL ADHESION PROTEINS

CAMS- cell adhesion molecules- four main families

i. immunoglobulin family CAMSii. cadherinsiii. integrinsiv. selectins

these proteins function as transmembrane receptors

sometimes stored in cytoplasm

- provide interaction between the same cells or different cell types (homotypic and heterotypic respectively)

Integrins o bind to ECM proteins such as

fibronectin laminin, and osteopontin

o providing a connection between cells and ECM, and also to adhesive proteins in other cells, establishing cell-to-cell contact.

Fibronectin o is a large protein that binds to many molecules,

such as collagen, fibrin, proteoglycans, and cell surface receptors.

o It consists of two glycoprotein chains, held together by disulfide bonds.

o Fibronectin messenger RNA has two splice forms, giving rise to tissue fibronectin and plasma fibronectin.

o The plasma form binds to fibrin, helping to stabilize the blood clot that fills the gaps created by wounds, and serves as a substratum for ECM deposition and formation of the provisional matrix during wound healing

Laminin o is the most abundant glycoprotein in the

basement membrane and has binding domains for both ECM and cell surface receptors.

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

o Laminin can also mediate the attachment of cells to connective tissue substrates.

cadherin o calcium-dependent adherence protein.” o participate in interactions between cells of the

same type. o These interactions connect the plasma membrane

of adjacent cells, forming two types of cell junctions called a. zonula adherens

small spotlike junctions located near the apical surface of

epithelial cells, and b. desmosomes

stronger and more extensive junctions

present in epithelial and muscle cells.

o Migration of keratinocytes in the re-epithelialization of skin wounds is dependent on the formation of dermosomal junctions.

o Linkage of cadherins with the cytoskeleton occurs through two classes of catenins.

o β-catenin links cadherins with α-catenin, which, in

turn, connects to actin, thus completing the connection with the cytoskeleton.

o Cell-to-cell interactions mediated by cadherins and catenins play a major role in regulating cell motility, proliferation, and differentiation and account for the inhibition of cell proliferation that occurs when cultured normal cells contact each other (“contact inhibition”).

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


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o free β-catenin acts independently of cadherins in the Wnt signaling pathway, which participates in stem cell homeostasis and regeneration.

o Mutation and altered expression of the Wnt/β-catenin pathway is implicated in cancer development, particularly in gastrointestinal and liver cancers

SPARCo secreted protein acidic and rich in cysteineo aka osteonectino contributes to tissue remodeling in response to

injuryo functions as an angionesis inhibitor

Thrombospondinso inhibit angiogenesis

Osteopontin o glycoprotein that regulates calcificationo mediator of leukocyte migration involved in

inflammation, vascular remodeling and fibrosis Tenascin

o morphogenesiso cell adhesion

GLYCOSAMINOGLYCANS

GAGs o consist of long repeating polymers of specific

disaccharides. o With the exception of hyaluronan GAGs are linked

to a core protein, forming molecules called proteoglycans.

Proteoglycans o originally described as ground substances or

mucopolysaccharides, o main function was to organize the ECM, o now recognized that these molecules have diverse

roles in regulating connective tissue structure and permeability

o can be integral membrane proteins o through their binding to other proteins and the

activation of growth factors and chemokines act as modulators of inflammation, immune responses, and cell growth and differentiation.

There are four structurally distinct families of GAGs: a. heparan sulfateb. chondroitin/dermatan sulfatec. keratan sulfate, and d. hyaluronan (HA).

The first three of these families are synthesized and assembled in the Golgi apparatus and rough endoplasmic reticulum as proteoglycans.

HA is produced at the plasma membrane by enzymes called hyaluronan synthases and is not linked to a protein backbone.

HEALING BY REPAIR, SCAR FORMATION AND FIBROSIS

If tissue injury is severe or chronic results in damage of both parenchymal cells and the stromal framework of the tissue, healing can not be accomplished by regeneration main healing process is repair by deposition of collagen and other ECM components, causing the formation of a scar.

In contrast to regeneration which involves the restitution of tissue components, repair is a fibroproliferative response that “patches” rather than restores the tissue.

scar o wound healing in the skin o the replacement of parenchymal cells in any tissue

by collagen, as in the heart after myocardial infarction.

Repair by connective tissue deposition includes the following basic features:a. inflammationb. angiogenesisc. migration and proliferation of fibroblastsd. scar formatione. connective etissue remodeling

The relative contributions of repair and regeneration are influenced by- the proliferative capacity of the cells of the tissue- the integrity of the extracellular matrix; and - the resolution or chronicity of the injury and

inflammation.

MECHANISMS OF ANGIOGENESIS

blood vessels are assembled during embryonic development by vasculogenesis

o in which a primitive vascular network is established from endothelial cell precursors (angioblasts), or from dual hemopoietic/endothelial cell precursors called hemangioblasts.

Blood vessel formation in adults, known as angiogenesis or neovascularization

oo involves the branching and extension of adjacent

pre-existing vesselso can also occur by recruitment of endothelial

progenitor cells (EPCs) from the bone marrow

Angiogenesis from Preexisting Vessels

- In this type of angiogenesis there is a. vasodilationb. increased permeability of the existing vesselc. degradation of ECMd. migration of endothelial cells.

- The major steps are: a. Vasodilation in response to nitric oxide, and VEGF-

induced increased permeability of the preexisting vessel

b. Proteolytic degradation of the basement membrane of the parent vessel by matrix metalloproteinases (MMPs) and disruption of cell-to-cell contact between endothelial cells by plasminogen activator

c. Migration of endothelial cells toward the angiogenic stimulus

d. Proliferation of endothelial cells, just behind the leading front of migrating cells

e. Maturation of endothelial cells, which includes inhibition of growth and remodeling into capillary tubes

f. Recruitment of periendothelial cells (pericytes and vascular smooth muscle cells) to form the mature vessel

Angiogenesis from Endothelial Precursor Cells (EPCs) EPCs can be recruited from the bone marrow into tissues to

initiate angiogenesis The nature of the homing mechanism is uncertain. These cells express some markers of hematopoietic stem

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

EPCs may contribute to the re-endothelization of vascular implants and the neovascularization of ischemic organs, cutaneous wounds, and tumors.

The number of circulating EPCs increases greatly in patients with ischemic conditions, suggesting that EPCs may influence vascular function and determine the risk of CVD.

GROWTH FACTORS and RECEPTORS INVOLVED IN ANGIOGENESIS

VEGF o the most important growth factor in adlt tissues

undergoing physiologic angiogenesis as well as angiogenesis occurring in chronic inflammation, wound healing, tumors and diabetic retinopathy.

o secreted by mesenchymal and stromal cells VEGFR-2

- most important in angiogenesis- tyrosine kinase receptor- expressed by EC - aka KDR

VEGF functions:


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a. induces migration of EPCs in the none marrowb. enhances proliferation and differentiation of cells at

sites of angiogenesis In angiogenesis from preexisting local vessels, VEGF:

a. stumlates the survival of endothelial cells, proliferation, motility

b. initiating the sprouting of new capillaries Notch Pathway

- mechanism for modulation of vasculogenesis- promtes proper branching of new vessels - prevents excessive angiogenesis by decreasing the

responsiveness to VEGF

ECM PROTEINS AS REGULATORS OF ANGIOGENESIS

integrins- especially αvβ- critical for the formation and maintenance of newly

formed blood vessels, (2) matricellular proteins,

- including thrombospondin 1, SPARC, and tenascin C- which destabilize cell-matrix interactions - promote angiogenesis

proteinases- plasminogen activators and MMPs- important in tissue remodeling during endothelial

invasion. Additionally, these proteinases cleave extracellular proteins,

releasing matrix-bound growth factors such as VEGF and FGF-2 that stimulate angiogenesis.

Proteinases can also release inhibitors such as endostatin, a small fragment of collagen that inhibits endothelial proliferation and angiogenesis

CUTANEOUS WOUND HEALING

divided into 3 phases:1. INFLAMMATION2. PROLIFERATION3. MATURATION

these phases overlap.

1. Inflammationo initial injury causes platelet adhesion and

aggregationo formation of clot in the surface of the wound

inflammation2. Proliferation

o formation of fgranulation tissueo proliferation and migration of connective

tissue cello re epithelialization of wound surface.

3. Maturationo ECM depositiono tissue remodelingo wound contraction

Cutaneous wound repairo is of two types:

1. Healing by primary union or by first intention

2. Healing by secondary union or by second intention

Healing by primary union or first intentiono little loss of tissueo small amount of granulation tissue o formation of a thin scar with minimal

contraction

o causes death of a limited munmer of epithelial and connective tissue cells

o disruption of epithelial basement membrane continuity

o re epithelialization to close the wound occurs with formation of a relatively thin scar

o healing of a clean, uninfected surgical incision by surgical sutures : simplest type of cutaneous wound repair

Healing by secondary union or by secondary intentiono excisional woundso create large defects on the skin surfaceo extensive loss of cells and tissueso healing of wounds involves an intense

inflammatory reactiono formation of abundant granulation tissueo extensive collagen depositiono formation of substantial scar that generaly

contracts Basic mechanisms are still the same.

SEQUENCE OF EVENTS IN WOUND HEALING

1. Formation of Blood clot wounding rapid activation of coagulation pathways

formation of a blood clot in the wound surface clot contains:

a. entrapped red cellsb. fibrinc. fibronectind. complement components

Clot serves to:a. stop bleedingb. scaffold for migrating cells migrating cells are

attracted by GF, cytokines, and chemokines released in the area.

Release of VEGF increased vessel permeability edema Dehydration occurs at the external surface of the clot

scab that covers the wound Within 24 hours: neutrophils appear at the margins of the

incision neutrophils use the scaffold provided by the fibrin clot to

march in neutrophils release proteolytic enzymes thtt clean out debris

and invading bacteria

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

hallmark of tissue repair term derives from its pink, soft, granular appearance on the surface of the woundscharacteristic histologic feature: a. presence of new small blood vessels

(angiogenesis)b. proliferation of fibroblast

new vessels are leaky allowing the passage of plasma proteins and fluids into the extravascular space

new granulation tissue is often edematous. progressively invades the incision spacedepends on the size of the tissue deficit created by the wound and the intensity of inflammationmore prominent in healing by 2° unionby 5 to 7 days, granulation tissue fills the wound area and neovascularization is maximal.

3. Cell Proliferation and Collagen Deposit by 48 to 96 hours, macrophage replaced by neutrophils Macrophage

o are key cellular constituents of tissue repairo clearing extracellular debris, fibrin, and other

foreign material at the site of repair, and o promoting angiogenesis and ECM deposition

Migration of fibroblasts to the site of injury is driven by chemokines


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a. TNFb. PDGFc. TGF-bd. FGF

Macrophages are main source of these factors

Collagen fibers are now present at the margins of the incision (vertically oriented and do not bridge the incision)

24 to 48 hourso spurs of epithelial cells move from the wound edge

along the cut margins of the dermis deposit basement membrane components as they move fuse in the midline beneath the surface scab producing a thin, continuous epithelial layer that closes the wound

o full epithelialization of the wound surface is much slower in healing by secondary union because the gap to be bridged is much greater

Macrophages stimulate fibroblast to produce:a. FGF-7 (Keratinocyte growth factor) b. IL 6 - enhance keratocyte migration and proliferation

HGF and HB-EGF CXCR-3 - promotes skin reepithelialization TGF β

- most important fibrogenic agent- produced by most cells in granulation tissue- causes

a. fibroblast migration proliferationb. increased synthesis of collagen and fibronectionc. decreased degradation of ECM by

metaloproteinases

Growth Factors and Cytokines Affecting Various Steps in Wound HealingMonocyte Chemotaxis Chemokines, TNF, PDGF, FGF,

TGF-βFibroblast Migration/ Replication

PDGF, EGF, FGF, TGF-β, TNF, IL-1

Keratinocyte Replication HB-EGF, FGF-7, HGFAngiogenesis VEGF, angiopoietins, FGFCollagen synthesis TGF-β, PDGFCollagenase secretion PDGF, FGF, TNF; TGF-β, inhibits

4. Scar Formation 2nd week - leukocytic infiltrate, edema and increased

vascularity disappear. Blanching begins

- blanching is accomplished by:a. increased accumulation of collagen within the

wound areab. regression of vascular channles

Original granulation tissue scaffolding is converted into a pale, avascular scar, composed of:a. spindle shaped fibroblastsb. dense collagenc. fragments of elastic tissued. other ECM components

Dermal appendages have been destroyed in the line of the incision are permanently lost

By the end of first month, the scar is made up of acellular connective tissue devoid of inflammatory infiltrate, covered by intact epidermis

5. Wound Contraction Wound contraction generally occurs in large surface wounds Contraction helps to close the wound by:

a. decreasing the gap between its dermal edgesb. reducing the wound surface area

contraction is an important feature in healing by secondary union

Initial steps of wound contraction involves the formation of myofibroblasts at the edge of the wound.

Myofibroblasts- expresse smooth mucle α-actin and vimentin- Ultrastructural characteristics of a smooth muscle cell- contract in the wound tissue- produce large amounts of ECM components such as a. type I collagenb. tenascin-Cc. SPARCd. extra-domain fibronection

- formed from tissue fibroblasts through the effects of PDG, TGF β and FGF-2

released by macrophages at the wound site

can also irigioante from bone marrow precursors (fibrocytes)

can also be from epithelial cells (epithelial-to-mesenchymal transition)

6. Connective Tissue Remodeling replacement of granulation tissue with a scar involves

changes in the composition of ECNM Remodeling of the connective tissue framework balance between ECM synthesis and degradation

- important feature of tissue repair Degradation of collagen and other ECM proteins is achieved

by matrix metalloproteinase Metalloproteinase

- common 180-reside zinc protease domain- include:

a. interstitial collagenases (MMP-1, 2 and 3) - cleave fibrillar collagen types I, II and III

b. gelatinase (MMP-2 and 9) - degrade amorphous collagen as well as fibronectin

c. stromelysins (MMP-3,10 and 11) - act on a variety of ECM components including proteoglycans, laminin, fibronectin and amorphous collagens

d. membrane bound MMPs (ADAM)- produced by fibroblasts, macrophages, neutrophils,

synovial cells and some epithelial cells- secretion is induced by

GF (PDGF, FGF) cytokines (IL-1 and TNF) phagocytosis in macrophages

- secretion is inhibited by TGF-β steroids

Collagenaseo cleave collagen under physiologic conditions. o synthesized as a latent precursorso activated by chemicals

ADAMo disintegrin and metalloproteinase-domain

familyo anchored by a single transmembrane domain

to cell surfaceo ADAM-17

(aka TACE, TNF-converting enzyme)

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

releasing the active molecules ADAM -17 deficiency

7. Recovery of Tensile Strength Type I collagen form a major portion of the connective tissue

in repair sites and are essential for the development of strength in healing wounds

Net collagen accummuationdepends on:a. increased collagen synthesisb. decreased collagen degradation

Sutures are removed from an incisional surgical wound at the end of 1st week - wound strength: 10% that of unwounded skin.

Wound strength a. increases rapidly over the next 4 weeksb. slows down at approximately the 3rd month after the

original incisionc. reaches a plateau at about 70% to 80% of the tensile

strength of the unwounded skin Recovery of tensile strength

- results from the excess of collagen synthesis over collagen degradation during the first 2 months of healing

- strucutal modifications of collagen fibers (cross linking) after collagen synthesis ceases


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LOCAL AND SYSTEMIC FACTORS THAT INFLUENCE WOUND HEALING

A. Systemic Factors:

Nutrition- protein deficiency- vitamin C deficiency -inhibit collagen synthesis and

retard healing Metabolic Status

- change wound healing- DM - associated with delated wound healing

(consequence of microangiopathy) Circulatory Status

- modulate wound healing- inadequate blood supply (caused by arterioscelorosis)

or venous abnormalities that retard venous drainaige, also impairs healing

Hormones- glucocorticoids

o anti inflammatory effectso influence components of inflammationo inhibits collagen synthesis

B. Local Factors

Infection- most important cause of delay in healing- it results in persistent tissue injury and inflammation

Mechanical Factors- e.g. early motion of wounds- delay healing by compressing blood vessels and

separating edges of the wounds Foreign bodies

- unnecessary sutures or fragments of steel, glass or bone

Size, location and type of wound4. wounds in richly vascularized areas (e.g. face) - heal faster

than poorly vascularized area (foot)

PATHOLOGIC ASPECTS OF REPAIR

3 general categories of aberrations in wound healing:a. deficient scar formationb. excessive formation of repair componentsc. Formation of contractures

Deficient Scar Formation inadequate formation of granulation tissue or assembly of a

scar leads to 2 types of complications:1. Dehiscence

o rupture of a woundo most common after an abdominal surgery due

to increased abdominal pressureo vomiting, coughing, or ileus can generate

mechanical stress on the abdominal wound2. Ulceration

o inadequate vascularization during healingo lower extremity wounds in px with atherosclerotic

peripheral vascular disease o nonhealing wounds also form in areas devoid of

sensationo diabetic peripheral neuropathy

Excessive Formation of Components of Repair Give rise to:

a. hypertrophic scarsb. keloids

Hypertrophic Scarso accumulation of excessive amounts of collageno raised scaro generally develop after thermal or traumatic injury

that involves the deep layer of the dermis Keloid

o scar tissue grows beyond the boundaries of the original wound

o does not regresso individual predispositiono common in African American

Exuberant granulationo formation of excessive amounts of granulation

tissueo protrudes above the level of the surrounding skino blocks reepithelializationo proud flesho must be removed by cautery or surgical excision

to permit restoration of the continuity of the epithelium

o Desmoids exuberant proliferation of fibroblasts

and other connective tissue elements that recur after excision

occurs after incisional scars or traumatic injuries

aka aggressive fibromatoses lie in the interface between benign

hyperplasias characteristic of repair and neoplasia

Contractions Contractures

exaggeration in the contraction of the size of the wound results in deformities of the wound and the surrounding

tissues prone to develop in palms, solesand the anterior aspect of

the thorax commonly seen after injurious burns; can compromise the

movement of jointsFIBROSIS

deposition of collagen is part of normal wound healing Fibrosis

- excessive deposition of collagen in chronic diseases. - the injurious stimulus caused by infections,

autoimmune reactions, trauma and other types of tissue injury persists in chronic diseases organ dysfunction and organ failure

Note that:a. the initial wave of the host response to external

invaders and tissue injury generates classically activated macrophages

- effective in ingesting and destroying microbes and dead tissues

b. alternatively activated macrophages- suppress the microbicidal activities- remodel tissues- promote angionesis and scar formation

TGF-b is always involved as an important fibrogenic agent Osteopontin

- impt role in healing and fibrosis- strongly expressed in the fibrosis of heart, lung, liver,

kidney and some other tissues Fibrotic disorders include:

a. liver cirrhosisb. systemic sclerosisc. fibrosing disease of the lungs

o idiopathic pulmonary fibrosis


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o pneumoconiosiso drug,radiation induced pulmonary fibrosis

d. chronic pancreatitise. glomerulonephritisf. constrictive pericarditis

SUMMARY

Chap3 Tissuerep f

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