Tissue Renewal, Regeneration, and Repair

• Injury to cells and tissues sets in motion a series of events that contain the damage and initiate the healing process. This process can be broadly separated into regeneration and repair.

• Regeneration results in the complete restitution of lost or damaged tissue;

• repair may restore some original structures but can cause structural derangements. In healthy tissues, healing, in the form of regeneration or repair, occurs after any insult that causes tissue destruction, and is essential for the survival of the organism.

• Regeneration refers to the proliferation of cells and tissues to replace lost structures, such as the growth of an amputated limb in amphibians.

• In mammals, whole organs and complex tissues rarely regenerate after injury, and the term is usually applied to processes such as liver growth after partial resection or necrosis, but these processes consist of compensatory growth rather than true regeneration.

• Tissues with high proliferative capacity, such as the hematopoietic system and the epithelia of the skin and gastrointestinal (GI) tract, renew themselves continuously and can regenerate after injury, as long as the stem cells of these tissues are not destroyed.

• Repair most often consists of a combination of regeneration and scar formation by the deposition of collagen. The relative contribution of regeneration and scarring in tissue repair depends on the ability of the tissue to regenerate and the extent of the injury

• For instance, a superficial skin wound heals through the regeneration of the surface epithelium. However, scar formation is the predominant healing process that occurs when the extracellular matrix (ECM) framework is damaged by severe injury.

• Chronic inflammation that accompanies persistent injury also stimulates scar formation because of local production of growth factors and cytokines that promote fibroblast proliferation and collagen synthesis.

• The term fibrosis is used to describe the extensive deposition of collagen that occurs under these situations.

• ECM components are essential for wound healing, because they provide the framework for cell migration, maintain the correct cell polarity for the re-assembly of multilayer structures, and participate in the formation of new blood vessels (angiogenesis).

• Furthermore, cells in the ECM (fibroblasts, macrophages, and other cell types) produce growth factors, cytokines, and chemokines that are critical for regeneration and repair. Although repair is a healing process, it may itself cause tissue dysfunction, as, for instance, in the development of atherosclerosis.

Control of Normal Cell Proliferation and Tissue Growth

• In adult tissues the size of cell populations is determined by the rates of cell proliferation, differentiation, and death by apoptosis and increased cell numbers may result from either increased proliferation or decreased cell death.[5] Apoptosis is a physiologic process required for tissue homeostasis, but it can also be induced by a variety of pathologic stimuli

• Differentiated cells incapable of replication are referred to as terminally differentiated cells.

• The impact of differentiation depends on the tissue under which it occurs: in some tissues differentiated cells are not replaced, while in others they die but are continuously replaced by new cells generated from stem cells.

• Cell proliferation can be stimulated by physiologic and pathologic conditions.

• E.g. The proliferation of endometrial cells under estrogen stimulation during the menstrual cycle and the thyroid-stimulating hormone–mediated replication of cells of the thyroid that enlarges the gland during pregnancy .

• Physiologic stimuli may become excessive, creating pathologic conditions such as BPH resulting from dihydrotestosterone stimulation and the development of nodular goiters in the thyroid as a consequence of increased serum levels of TSH.

• Cell proliferation is largely controlled by signals from the microenvironment that either stimulate or inhibit proliferation. An excess of stimulators or a deficiency of inhibitors leads to net growth and, in the case of cancer, uncontrolled growth.

• The tissues of the body are divided into three groups on the basis of the proliferative activity of their cells: continuously dividing (labile tissues), quiescent (stable tissues), and nondividing (permanent tissues).

• In continuously dividing tissues cells proliferate throughout life, replacing those that are destroyed. These tissues include surface epithelia,the columnar epithelium of the GI tract and uterus; the transitional epithelium of the urinary tract, and cells of the bone marrow and hematopoietic tissues.

• In most of these tissues mature cells are derived from adult stem cells, which have a tremendous capacity to proliferate and whose progeny may differentiate into several kinds of cells.

• Quiescent tissues normally have a low level of replication;

• cells from these tissues can undergo rapid division in response to stimuli and are thus capable of reconstituting the tissue of origin,e.g parenchymal cells of liver, kidneys, and pancreas; mesenchymal cells such as fibroblasts and smooth muscle; vascular endothelial cells; and lymphocytes and other leukocytes.

• Nondividing tissues contain cells that have left the cell cycle and cannot undergo mitotic division in postnatal life,e.g. neurons and skeletal and cardiac muscle cells. If neurons in the central nervous system are destroyed, the tissue is generally replaced by the proliferation of the central nervous system–supportive elements, the glial cells. However, recent results demonstrate that limited neurogenesis from stem cells may occur in adult brains

• . Although mature skeletal muscle cells do not divide, skeletal muscle does have regenerative capacity, through the differentiation of the satellite cells that are attached to the endomysial sheaths.

• Cardiac muscle has very limited, if any, regenerative capacity, and a large injury to the heart muscle, as may occur in myocardial infarction, is followed by scar formation.

STEM CELLS

• Stem cells are characterized by their self-renewal properties and by their capacity to generate differentiated cell lineages so, stem cells need to be maintained during the life of the organism.

• Such maintenance is achieved by two mechanisms: (a) obligatory asymmetric replication, in which with each stem cell division, one of the daughter cells retains its self-renewing capacity while the other enters a differentiation pathway,

• (b) stochastic differentiation, in which a stem cell population is maintained by the balance between stem cell divisions that generate either two self-renewing stem cells or two cells that will differentiate

• In early stages of embryonic development, stem cells, known as embryonic stem cells or ES cells, are pluripotent, that is, they can generate all tissues of the body.

• Pluripotent stem cells give rise to multipotent stem cells, which have more restricted developmental potential, and eventually produce differentiated cells from the three embryonic layers.

Transdifferentiation indicates a change in the lineage commitment of a stem cell.

ADULT STEM CELLS

EMBRYONIC STEM CELLS

• ES cells have been used to study the specific signals and differentiation steps required for the development of many tissues.

• ES cells made possible the production of knockout mice, an essential tool to study the biology of particular genes and to develop models of human disease. ES cells may in the future be used to repopulate damaged organs.

• ES cells may be capable of differentiating into insulin-producing pancreatic cells, nerve cells, myocardial cells, or hepatocytes.

Reprogramming of Differentiated Cells: Induced Pluripotent Stem Cells

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

• may be used for therapeutic cloning in the treatment of human diseases.

• In this technique the nucleus of a skin fibroblast from a patient is introduced into an enucleated human oocyte to generate ES cells, which are kept in culture, and then induced to differentiate into various cell types but is inaccurate.

• One of the main reasons for the inaccuracy is the deficiency in histone methylation in reprogrammed ES cells, which results in improper gene expression.

Cell Cycle and the Regulation of Cell Replication

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

• To achieve DNA replication and division, the cell goes through a tightly controlled sequence of events known as the cell cycle. The cell cycle consists of G1 (presynthetic), S (DNA synthesis), G2 (premitotic), and M (mitotic) phases

• . Quiescent cells that have not entered the cell cycle are in the G0 state.

• the cell cycle has multiple controls and redundancies, particularly during the transition between the G1 and S phases. These controls include activators and inhibitors, as well as sensors that are responsible for checkpoints.

Tissue Repair:Wound Healing

Repair & Regeneration

• Repair begins early in inflammation• Regeneration refers to growth of cells and

tissues to replace lost structure– as liver and kidney growth after partial hepatectomy

and unilateral nephrectomy

• Two processes:– Regeneration– Fibrosis

Repair

• Regeneration of injured cells by cells of same type as with regeneration of skin/oral mucosa (requires basement membrane)

• Replacement by fibrous tissue (fibroplasia, scar formation)

• Both require cell growth, differentiation, and cell-matrix interaction

Tissue Regeneration

• Controlled by biochemical factors released in response to cell injury, cell death, or mechanical trauma– Most important control: inducing resting cells to

enter cell cycle– Balance of stimulatory or inhibitory factors– Shorten cell cycle– Decrease rate of cell loss

Healing

• Healing is usually a tissue response – (1) to a wound (commonly in the skin)– (2) to inflammatory processes in internal

organs– (3) to cell necrosis in organs incapable of

regeneration

• The term fibrosis is used to describe the extensive deposition of collagen that occurs under these situations.

• ECM components are essential for wound healing, because they provide the framework for cell migration, maintain the correct cell polarity for the re-assembly of multilayer structures, and participate in the formation of new blood vessels (angiogenesis).

Repair & Regeneration

• Labile cells: continue to proliferate throughout life : squamous, columnar, transitional epithelia; hematopoitic and lymphoid tissues

• Stable cells: retain the capacity of proliferation but they don’t replicate normally: parenchymal cells of all glandular organs & mesenchymal cells

• Permanent cells: cannot reproduce themselves after birth: neurons, cardiac muscle cells

Cell Cycle and the Regulation of Cell Replication

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

• To achieve DNA replication and division, the cell goes through a tightly controlled sequence of events known as the cell cycle. The cell cycle consists of G1 (presynthetic), S (DNA synthesis), G2 (premitotic), and M (mitotic) phases

Cell Cycle

G1

SDNA SYNTHESIS

G2

M

GAP PHASE CYCLIN B

RESTING PHASE

MITOSIS

G0 CYCLIN D / E RESTRICTION POINT

CYCLIN A / B

CYCLIN

CDK

Intercellular Signaling

• 3 pathways– Autocrine: cells have receptors for their own

secreted factors (liver regeneration)– Paracrine: cells respond to secretion of nearby

cells (healing wounds)– Endocrine: cells respond to factors (hormones)

produced by distant cells

Control of Normal Cell Proliferation and Tissue Growth

In adult tissues the size of cell populations is determined by

• rates of cell proliferation,• differentiation• death by apoptosis • increased cell numbers may result from either

increased proliferation or decreased cell death.[5]

• Apoptosis is a physiologic process required for tissue homeostasis, but it can also be induced by a variety of pathologic stimuli

Cell proliferation can be stimulated by physiologic and pathologic conditions.

• E.g. The 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 during pregnancy .

Physiologic stimuli may become excessive, creating pathologic conditions such as

• BPH resulting from dihydrotestosterone stimulation • development of nodular goiters in the thyroid as a

consequence of increased serum levels of TSH.

STEM CELLS

• Stem cells are characterized by their self-renewal properties and by their capacity to generate differentiated cell lineages so, stem cells need to be maintained during the life of the organism.

• Such maintenance is achieved by two mechanisms: • (a) obligatory asymmetric replication, in which with each stem

cell division, one of the daughter cells retains its self-renewing capacity while the other enters a differentiation pathway

• (b) stochastic differentiation, in which a stem cell population is maintained by the balance between stem cell divisions that generate either two self-renewing stem cells or two cells that will differentiate

Stem Cell Progeny• Capacity for unlimited division• Whose daughters have a choice

DiffeDifferentiate

rentiate 1 2

Stem

Stem

Stem

Stem

StemStem

Stem

3

B lymphocytes

T lymphocytes

Platelets

RBCs

Neutrophils

Macrophages

Pluripotent Stem Cell

Lymphocyte Progenitor

Myeloid Progenitor

• In early stages of embryonic development, stem cells, known as embryonic stem cells or ES cells, are pluripotent, that is, they can generate all tissues of the body.

• Pluripotent stem cells give rise to multipotent stem cells, which have more restricted developmental potential, and eventually produce differentiated cells from the three embryonic layers.

Transdifferentiation indicates a change in the lineage commitment of a stem cell.

Reprogramming of Differentiated Cells: Induced Pluripotent Stem Cells

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

• may be used for therapeutic cloning in the treatment of human diseases.

• In this technique the nucleus of a skin fibroblast from a patient is introduced into an enucleated human oocyte to generate ES cells, which are kept in culture, and then induced to differentiate into various cell types but is inaccurate.

EMBRYONIC STEM CELLS

• ES cells have been used to study the specific signals and differentiation steps required for the development of many tissues.

• ES cells may be capable of differentiating into insulin-producing pancreatic cells, nerve cells, myocardial cells, or hepatocytes.

Imagination is better than knowledge - Einstein

GROWTH FACTORS

Cellular Signalling Pathways

Vital for cell cycle progression, growth, differentiation & death.

Growth Factors – The key stone

A delicate balance between activating and inhibitory signals needs to be maintained normally

Alteration in this balance - Dysregulated cellular proliferation & survival of abnormal cells.

Gene Transcription

G0G1

Priming

S

G2

M

Cell Cycle

Growth Factors

+

Growth Factors & Cell Cycle

Receptors

Growth factors increase synthesis & decrease degradation of

macromolecules

• The proliferation of many cell types is driven by polypeptides known as growth factors.

• can have restricted or multiple cell targets, • promote cell survival• locomotion• contractility• differentiation• angiogenesis.

• All growth factors function as ligands that bind to specific receptors, which deliver signals to the target cells.

• These signals stimulate the transcription of genes that may be silent in resting cells,

• Genes that control cell cycle entry and progression.

Growth Factors and Cytokines Involved in Regeneration and Healing

• EPIDERMAL GROWTH α • platelets, macrophages, saliva, urine,

milk, plasma.• It is mitogenic for keratinocytes and

fibroblasts;• stimulates keratinocyte migration and

granulation tissue formation.

Transforming growth factor α

macrophages T lymphocytes keratinocytes many tissues stimulates replication of hepatocytes and

most epithelial cells

Heparin-binding EGF

• macrophages • mesenchymal cells:• Keratinocyte replication• HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR• mesenchymal cells• Enhances proliferation of hepatocytes, epithelial

cells, and endothelial cells; • increases cell motility,• keratinocyte replication.

Vascular endothelial cell growth factor

• from many types of cells• Increases vascular permeability;• mitogenic for endothelial cells, • angiogenesis

Platelet-derived growth factor• platelets,• macrophages• endothelial cells• keratinocytes• smooth muscle cells• Chemotactic for PMNs, macrophages, fibroblasts, and smooth

muscle cells;• activates PMNs, macrophages, and fibroblasts; • mitogenic for fibroblasts, endothelial cells, and smooth muscle

cells;• stimulates production of MMPs,• fibronectin stimulates angiogenesis and wound contraction

Fibroblast growth factor

macrophages mast cells T lymphocytes endothelial cells fibroblasts Chemotactic for fibroblasts mitogenic for fibroblasts and keratinocytes stimulates keratinocyte migration, angiogenesis, wound contraction matrix deposition.

Transforming growth factor β

• platelets,• T lymphocytes,• macrophages,• endothelial cells,• keratinocytes,• smooth muscle cells• fibroblasts• Chemotactic for PMNs, macrophages, lymphocytes,

fibroblasts, and smooth muscle cells;• stimulates angiogenesis, and fibroplasia;• inhibits production of MMPs and keratinocyte proliferation.

Keratinocyte growth factor

• Fibroblasts• Stimulates keratinocyte migration, proliferation,

and differentiation• TUMOR NECROTIC FACTOR• Macrophages, • mast cells,• T lymphocytes• Activates macrophages;• regulates other cytokines; multiple functions.

SIGNALING MECHANISMS IN CELL GROWTH

• receptor-mediated signal transduction is involved, which is activated by the binding of ligands such as growth factors, and cytokines to specific receptors.

• Different classes of receptor molecules and pathways initiate a cascade of events by which receptor activation leads to expression of specific genes.

Three general modes of signaling

• Autocrine signaling: Cells respond to the signaling molecules that they themselves secrete, thus establishing an autocrine loop

• plays a role in liver regeneration and the proliferation of antigen-stimulated lymphocytes.

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

Paracrine signaling

• : One cell type produces the ligand, which then acts on adjacent target cells that express the appropriate receptor.

• 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).

• It is also necessary for hepatocyte replication

Endocrine signaling

• : Hormones synthesized by cells of endocrine organs act on target cells distant from their site of synthesis, being usually carried by the blood.

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

Receptors and Signal Transduction Pathways

• The binding of a ligand to its receptor triggers a series of events by which extracellular signals are transduced into the cell resulting in changes in gene expression.

• Receptors with intrinsic tyrosine kinase activity. The ligands for receptors with tyrosine kinase activity include

• most growth factors such as EGF, TGF-α, HGF, PDGF, VEGF, FGF, c-KIT ligand, and insulin.

TK Intracellular Domain

Transmembrane Domain

Extracellular Domain

EGFR Structure

TK

EGFR Function in Normal Cell

TKATP ATP

Cell Proliferation Antiapoptosis

Angiogenesis

Gene Transcription

Cell Cycle Progression

+

Normal CellCancerous CellUp Regulation

Mutation

Consequence of proliferation of EGFR receptors

Signaling from tyrosine kinase receptors. Binding of the growth factor (ligand) causes receptor dimerization and autophosphorylation of tyrosine residues. Attachment of

adapter proteins (e.g., GRB2 and SOS) couples the receptor to inactive RAS. Cycling of RAS between its inactive and active forms is regulated by GAP. Activated RAS interacts with and activates RAF (also known as MAP kinase kinase kinase). This

kinase then phosphorylates a component of the MAP kinase signaling pathway, MEK (also known as MAP kinase), Activated MAP kinase phosphorylates other cytoplasmic

proteins and nuclear transcription factors, generating cellular responses.

Receptors lacking intrinsic tyrosine kinase activity that recruit kinases

• . Ligands for these receptors include• many cytokines, such as IL-2, IL-3, and

other interleukins; • interferons α, β, and γ;• erythropoietin;• granulocyte colony-stimulating factor;• growth hormone• prolactin.

G protein–coupled receptors.• These receptors transmit signals into the cell through

trimeric GTP-binding proteins (G proteins).• A large number of ligands signal through this type of

receptor, including• chemokines, • vasopressin,• serotonin, histamine,• epinephrine and norepinephrine,• calcitonin,• glucagon,• parathyroid hormone,• corticotropin, and rhodopsin.

Steroid hormone receptors

• . These receptors are generally located in the nucleus.

• involved in a broad range of responses that include

• adipogenesis ,• inflammation • atherosclerosis.

Transcription Factors

• Many of the signal transduction systems used by growth factors transfer information to the nucleus and modulate gene transcription through the activity of transcription factors.

• Include products of several growth-promoting genes, such as c-MYC and c-JUN, and of cell cycle–inhibiting genes, such as p53.

LIVER REGENERATION

• The human liver has a remarkable capacity to regenerate, as demonstrated by its growth after partial hepatectomy, which may be performed for tumor resection or for living-donor hepatic transplantation.

• resection of approximately 60% of the liver in living donors results in the doubling of the liver remnant in about one month. The portions of the liver that remain after partial hepatectomy constitute an intact “mini-liver” that rapidly expands and reaches the mass of the original liver.

• Restoration of liver mass is achieved without the regrowth of the lobes that were resected at the operation. Instead, growth occurs by enlargement of the lobes that remain after the operation, a process known as compensatory growth or compensatory hyperplasia.

• the end point of liver regeneration after partial hepatectomy is the restitution of functional mass rather than the reconstitution of the original form.

•

• Almost all hepatocytes replicate during liver regeneration after partial hepatectomy. Because hepatocytes are quiescent cells, it takes them several hours to enter the cell cycle, progress through G1, and reach the S phase of DNA replication.

• The wave of hepatocyte replication is synchronized and is followed by synchronous replication of nonparenchymal cells (Kupffer cells, endothelial cells, and stellate cells).

• hepatocyte proliferation in the regenerating liver is triggered by the combined actions of cytokines and polypeptide growth factors.

• Quiescent hepatocytes become competent to enter the cell cycle through a priming phase that is mostly mediated by the cytokines TNF and IL-6, and components of the complement system.

• Priming signals activate several signal transduction pathways to start cell proliferation

• . 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, facilitating the entry of hepatocytes into the cell cycle.

• Individual hepatocytes replicate once or twice during regeneration and then return to quiescence in a strictly regulated sequence of events, but the mechanisms of growth cessation have not been established.

• Growth inhibitors, such as TGF-β and activins, may be involved in terminating hepatocyte replication, but there is no clear understanding of their mode of action,

• Intrahepatic stem or progenitor cells do not play a role in the compensatory growth that occurs after partial hepatectomy.

• Endothelial cells and other nonparenchymal cells in the regenerating liver may originate from bone marrow precursors

Thank you

Extracellular Matrix and Cell-Matrix Interactions

Extracellular Matrix and Cell-Matrix Interactions

• Tissue repair and regeneration depend not only on the activity of soluble factors, but also on interactions between cells and the components of the extracellular matrix (ECM).

• The ECM regulates the growth, proliferation, movement, and differentiation of the cells living within it

• It is constantly remodeling, and its synthesis and degradation accompanies regeneration, wound healing, chronic fibrotic processes, tumor invasion, and metastasis.

• The ECM sequesters water, providing turgor to soft tissues, and minerals that give rigidity to bone, but it does much more than just fill the spaces around cells to maintain tissue structure.

Functions include

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

• Control of cell growth. ECM components can regulate cell proliferation by signaling through cellular receptors of the integrin family.

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

• Scaffolding for tissue renewal. The maintenance of normal tissue structure requires a basement membrane or stromal scaffold. The integrity of the basement membrane or the stroma of the parenchymal cells is critical for the organized regeneration of tissues.

• Although labile and stable cells are capable of regeneration, injury to these tissues results in restitution of the normal structure only if the ECM is not damaged. Disruption of these structures leads to collagen deposition and scar formation

• Establishment of tissue microenvironments. Basement membrane acts as a boundary between epithelium and underlying connective tissue and also forms part of the filtration apparatus in the kidney.

• Storage and presentation of regulatory molecules. For example, growth factors like FGF and HGF are secreted and stored in the ECM in some tissues. This allows the rapid deployment of growth factors after local injury, or during regeneration.

COMPOSITION OF ECM

• fibrous structural proteins, such as collagens and elastins that provide tensile strength and recoil;

• adhesive glycoproteins that connect the matrix elements to one another and to cells;

• proteoglycans and hyaluronan that provide resilience and lubrication.

• These molecules assemble to form two basic forms of ECM: interstitial matrix and basement membranes.

• The interstitial matrix is found in spaces between epithelial, endothelial, and smooth muscle cells, as well as in connective tissue. It consists mostly of fibrillar and nonfibrillar collagen, elastin, fibronectin, proteoglycans, and hyaluronan.

• Basement membranes are closely associated with cell surfaces, and consist of nonfibrillar collagen (mostly type IV), laminin, heparin sulfate, and proteoglycans.

COLLAGEN

• Collagen is the most common protein in the animal world, providing the extracellular framework for all multicellular organisms. Without collagen, a human being would be reduced to a clump of cells interconnected by a few neurons.

• Currently, 27 different types of collagens encoded by 41 genes dispersed on at least 14 chromosomes are known.

• Each collagen is composed of three chains that form a trimer in the shape of a triple helix. The polypeptide is characterized by a repeating sequence in which glycine is in every third position (Gly-X-Y, in which X and Y can be any amino acid other than cysteine or tryptophan).

• Types I, II, III and V, and XI are the fibrillar collagens,these proteins are found in extracellular fibrillar structures.

• Type IV collagens have long triple-helical domains and form sheets instead of fibrils; they are the main components of the basement membrane, together with laminin.

• Type VII collagen forms the anchoring fibrils between some epithelial and mesenchymal structures, such as epidermis and dermis.

• Still other collagens are transmembrane and may also help to anchor epidermal and dermal structures.

FIBRILLAR COLLAGENS

• I in hard and soft tissues• Osteogenesis imperfecta; Ehlers-Danlos

syndrome—arthrochalasias type I• II in Cartilage, intervertebral disk, vitreous• Achondrogenesis type II• III Hollow organs, soft tissues• Vascular Ehlers-Danlos syndrome

• V in Soft tissues, blood vessels• Classical Ehlers-Danlos syndrome• IX in Cartilage, vitreous

BASEMENT MEMBRANE COLLAGENS

• IV,in Basement membranes• Alport syndrome

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

• Vitamin C is required for the hydroxylation of procollagen, a requirement that explains the inadequate wound healing in scurvy.

ELASTIN, FIBRILLIN, AND ELASTIC FIBERS

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

• Proteins of the collagen family provide tensile strength, but the ability of these tissues to expand and recoil depends on the elastic fibers. These 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,a glycoprotein.

• The microfibrils serve as support for deposition of elastin and the assembly of elastic fibers. They 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

• Most adhesion proteins, also called CAMs (cell adhesion molecules), can be classified into four main families:

• immunoglobulin family CAMs,• cadherins• integrins• selectins.

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

• As receptors, CAMs can bind to similar or different molecules in other cells, providing for interaction between the same cells (homotypic interaction) or different cell types (heterotypic interaction).

• other secreted adhesion molecules • (1) SPARC also known as osteonectin,

contributes to tissue remodeling in response to injury and functions as an angiogenesis inhibitor;

• (2) the thrombospondins, a family of large multifunctional proteins, some of which also inhibit angiogenesis;

• .

• (3) osteopontin is a glycoprotein that regulates calcification and is a mediator of leukocyte migration involved in inflammation, vascular remodeling, and fibrosis in various organs

• (4) the tenascin family, which consist of large multimeric proteins involved in cell adhesion

GLYCOSAMINOGLYCANS AND PROTEOGLYCANS

• GAGs make up the third type of component in the ECM, besides the fibrous structural proteins and cell adhesion proteins.

• four structurally distinct families of GAGs: heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate, and hyaluronan.

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

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

• HA is a polysaccharide of the GAG family found in the ECM of many tissues and is abundant in heart valves, skin and skeletal tissues, synovial fluid, the vitreous of the eye, and the umbilical cord.

• It binds a large amount of water, forming a viscous hydrated gel that gives connective tissue the ability to resist compression forces.

• HA helps provide resilience and lubrication to many types of connective tissue, notably for the cartilage in joints.

• Its concentration increases in inflammatory diseases such as rheumatoid arthritis, scleroderma, psoriasis, and osteoarthritis.

THANK YOU

Repair by connective tissuefibrosis

Overview

• Severe or persistent tissue injury with damage both to parenchymal cells and to the stromal framework leads to a situation in which repair cannot be accomplished by parenchymal regeneration alone.

• Repair occurs by replacement of the non regenerated parenchymal cells with connective tissue.

General components of this process are:

Formation of new blood vessels (Angiogenesis) Migration and proliferation of fibroblast Deposition of ECM Maturation and reorganization of the fibrous

tissue (Remodeling)

Angiogenesis from endothelial precursors

Common precursor: hemangioblast that gives rise to angioblast and hematopoietic stem cells.

Angioblasts then proliferate and differentiate into endothelial cells that form arteries,veins and lymphatics.

Epcs (angioblast like cells) in the bone marrow can be recruited into tissues to initiate angiogenesis.

What is VASCULOGENESIS?

Angiogenesis from preexisting vessel• Vasodilation in response to nitric acid and VEGF.

• Increased permeability of pre-existing vessel.

• Proteolytic degradation of the parent vessel BM by metalloproteinases and disruption of cell to cell contact btw endothelial cells of the vessel by plasminogen activator.

• Migration of endothelial cells from the original capillary toward an angiogenic stimulus.

• Proliferation of the endothelial cells behind the leading edge of migrating cells.

• Maturation of endothelial cells with inhibition of growth and organization into capillary tubes.

• Recruitment and proliferation of pericytes (for capillaries) and smooth muscle cells (for larger vessel) to support the endothelial tube.

Growth factors and receptors involved in angiogenesis

VEGF secreted by mesenchymal cells and stromal cells.

Angiopoietins(Ang1 and Ang2)FGF-2

PDGF

TGF-B

Angiopoietins ,PDGF,TGF-B participate in the stabilization process,

PDGF participates in the recruitmenmt of the smooth muscle cells,

TGF-b stabilizes newly formed vessel by

enhancing the production Of ECM production.

ECM proteins as REGULATORS OF ANGIOGENESIS

• Integrins , • Thrombospondin 1 and SPARC, tenascin

C,destabilize cell matrix interactions and promote angiogenesis.

• Proteinases , plasminogen activators and matrix metalloproteinases : important role in tissue remodeling during endothelial invasion.

Scar formation

• Emigration and proliferation of fibroblasts in the site of injury

• Deposition of ECM • Tissue remodeling

Fibroblast Migration and Proliferation

• Exudation and deposition of plasma protein, fibrinogen and plasma fibronectin in the ECM of granulation tissue provides a provisional stroma for fibroblasts and endothelial cell ingrowth.

• Migration of fibroblasts to the site of injury and subsequent proliferation are triggered by multiple growth factors TGF-B, PDGF, EGF, FGF and the cytokines IL-1 and TNF

ECM Deposition and Scar Formation

• TGF-B appears to be the most important because of the multitude of effects that favors fibrous tissue deposition.

• It is produced by most of the cells in granulation tissue and causes fibroblast migration and proliferation, increased synthesis of collagen and fibronectin and decreased degradation of ECM by metalloproteinases.

• Fibrillar collagens form a major portion of the connective tissue in repair sites and development of strength in healing wounds

• As the scar matures, vascular regression continues, eventually transforming the richly vascularized granulation tissue into a pale, avascular scar

Tissue Remodeling

• The balance between ECM synthesis and degradation results in remodeling of the CT framework.

• Degradation of collagen and other ECM proteinS is achieved by a family of matrix metalloproteinases which are dependant on zinc ions for their activity.

• MMPs include interstitial collagenases (MMP1, 2, 3) which cleave the fibrillar collagen type 1, 2 and 3

• Gelatinases (MMP2 and 9) degrade amorphous collagens and fibronectin.

• Stromelysin (MMP-3,10,11) act on proteoglycan , laminin,fibronectin,amorphous collagen.

• MMPS are synthesized as propeptides that require proteolytic cleavage for activation.

• Their secretion is induced by certain stimuli such as growth factors,cytokines,phagocytosis,physical stress and is inhibited by TGF-b and steroids

• Collagenases are synthesized in as a latent precursor (procollagenase) that is activated by chemicals such as free radicals produced in oxidative burst of leukocytes and proteinases

• Once formed activated collagenases are rapidly inhibited by a family of specific tissue inhibitor of metalloproteinases (TIMP).

WOUND HEALING

• Cutaneous wound healing is divided into three phases

• Inflammation (early and late)• Granulation tissue formation and

reepithilialization• Wound contraction ,ECM deposition and

remodeling.

• Wound healing is a fibroproliferative response that is mediated through growth factors and cytokines.

• Skin wounds are classically described to heal by primary or secondary intention.

Healing by first intention (wounds with opposed edges)

• Common example of wound repair is healing of a clean, uninfected surgical wound approximated by sutures. such healing is called as primary union or healing by first intention.

• Incision causes,• Death of limited number of epithelial cells and

connective tissue.• Disruption of epithelial BM.

Process

• Within 24 hours;• Neutrophils appear at the margin of the

incision moving towards the fibrin clot.• In 24 to 48 hours;• Movement of the epithelial cells from wound

edges from dermis cut margins depositing BM components as they move

• Day 3;• Neutrophils are replaced by macrophages.• Invasion of granulaton tissue invading the

incision space.• Collagen fibers are present in the margin of

the incision but does not bridge the incision.• Epithelial cell proliferation thickens the

epidermal layer

• Day5;• Incisional space filled with granulation tissue.• Neovascularization is maximal• Abundant collagen fibers ,bridging the

incision.• Mature epidermal architecture with surface

kertinization.

• Second week;• Continued accumulation of collagen and proliferation

of fibroblast .• Dissapperance of edema ,leukocyte infiltrate, and

increased vascularity.• End of first month;• Scar is made of cellular C.T inflammatory cell

infilterate , covered now by intact epidermis.• Dermal appendages that have been destroyed in the

line of incision are permanently lost.

Healing by second intention (wound with separate edges)

extensive loss of cells and tissue.Large defects.Abundant granulation tissue grows in from the

margin to complete the repair

Difference

• Larger tissue defect• Larger fibrin clot• More necrotic debris and exudate• Inflammatory reaction is more intense.• Abundant granulation tissue is formed

Wound contraction

• Formation of a network of actin containing fibroblasts at the edge of the wound.

• Permanent wound contraction requires the action of myofibroblasts.

• Contraction of these cells at the wound site decreases the gap between the dermal edges of the wound.

Wound strength

• When sutures are removed at the end of the first week ,strength is approximately 10% that of unwounded skin, but strength increases rapidly over the next 4 wks.

• At the end of third month it the rate decreases and the wound strength is about 70 to80% of the unwounded skin.

Factors that influence wound healing

• Systemic factors;• Nutrition (protein def,Zn, vit.C def) inhibit

collagen synthesis• Metabolic status, diabetes mellitis ,delayed

wound healing, ( microangiopathy)• Inadequate B.S (arteriosclerosis, varicose

veins)• Hormones. ( glucocorticoids)

Local factors

• Infection (persistent tissue injury and inflammation)

• Mechanical factors ,(mobility ,compression of B.V)

• Foreign bodies, (sutures ,fragments of bone, steel or glass)

Localization and size of wound (face, foot)

Complications

Inadequate formation of granulation tissue.

Wound dehiscence• Rupture of a wound is most common after

abdominal surgery and is due to increased abdominal pressure

• Vomiting, coughing , ileus

• Exuberant granulation is another deviation in wound healing consisting of the formation of excessive amounts of granulation tissue, which protrudes above the level of the surrounding skin and blocks re-epithelialization called proud flesh.

• Ulceration of the wounds because of inadequate vascularization during healing. (peripheral vascular diseases)

• Non healing wounds are also found in areas of denervation. (diabetic peripheral neuropathy)

Excessive formation of the components of the repair

• Excessive amount of collagen lead to hypertrophic scar.

• Is a raised scar • Keloid ,it is a scar tissue which goes beyond

the boundaries of the original wound and does not regress.

• Incisional scars or traumatic injuries may be followed by exuberant proliferation of fibroblasts and other connective tissue elements that may recur after excision. Called desmoids, or aggressive fibromatoses, these lie in the interface between benign proliferations and malignant (though low-grade) tumors.

• Contracture• Exaggeration in the process of contraction in

the wound is called contracture.• Severe burns• Palms,soles.thorax

• Healing wounds may also generate excessive granulation tissue that protrudes above the level of the surrounding skin and in fact hinders re-epithelialization. This is called exuberant granulation, or proud flesh, and restoration of epithelial continuity requires cautery or surgical resection of the granulation tissue.