1

Reference: ROBBINS BASIC PATHOLOGY, By Kumar, Abbas , Fausto, & Mitchell. 8 th Edition © 2007, SAUNDERS: Tissue repair: Regenerat ion, healing, & Fibrosis, 24 pp Handout. @ 14-9-2008. L ectures prepared by: Dr Mohammad Kamel Alwiswasi, MBChB, PhD, FRCPath

GENERAL PATHOLOGY CHAPTER 3: Tissue repair: Regeneration, h ealing, & Fibrosis •••• The Control of Cell Proliferation: The Cell Cycle, Proliferative Capacities of Tissues, Stem Cells •••• The Nature & Mechanisms of Action of Growth Factor s (GF) Signaling Mechanisms of GF Receptors (GFR) •••• Extracellular Matrix (ECM) & Cell-Matrix Interacti ons Roles of the ECM, Components of the ECM •••• Cell & Tissue Regeneration •••• Repair by Connective Tissue Angiogenesis, Migration of Fibroblasts & ECM Deposition (Scar Formation), ECM & Tissue Remodeling •••• Cutaneous Wound Healing Healing by First Intention, Healing by Second Intention, Wound Strength •••• Pathological Aspects of Repair Overvi ew of Repair Processes Critical to the survival of an organism is the ability to repair the damage caused by toxic insults & inflammation. ☺Repair refers to the restoration of tissue architecture & function after an injury. It occurs by two types of reactions, regeneration & scarring (F3-1): (1) Regeneration : means the ability of injured tissue to replace the damaged components & return to a normal state. (2) Healing by Scarring : means replacement by connective tissue (fibrosis ), resulting in a scar , which occurs (1) if the injured tissues are incapable of complete restitution, or (2) if the supporting structures of the tissues are severely damaged. � Although the fibrous scar is not normal, it provides enough structural stability that the injured tissue is usually able to function. �Commonly, repair involves a combination of both regeneration & scar formation. � Fibrosis term is used to describe the extensive deposition of collagen that occurs in the lungs, liver, kidney, & other organs as a consequence of chronic inflammation, or in the myocardium after extensive MI. � If fibrosis develops in a tissue space occupied by an inflammatory exudate it is called organization (as in organizing pneumonia affecting the lung).

2

� To understand the process of repair, some knowledge of the control of cell proliferation, functions of the ECM & how it is involved in repair, the roles of stem cells in tissue homeostasis, & the roles of GF in the proliferation of different cell types involved in repair. The Control of Cell Proliferation Several cell types proliferate during tissue repair. These include the: (1) Remnants of the injured tissue (which attempt to restore normal structure); the (2) Vascular Endothelial Cells (EC), to create new blood vessels (BV) that provide the nutrients needed for the repair process, & accompanied by the (3) Fibroblasts (the source of the fibrous tissue that forms the scar to fill defects that cannot be corrected by regeneration) (F3-1). ☺The normal size of cell population is determined by a balance of cell (1) proliferation , (2) cell death by apoptosis , & (3) emergence of new differentiated cells from stem cells (F3-2). The Cell Cycle The key process in the proliferation of cells, are DNA replication & mitosis. The sequence of events that control these two processes is known as the cell cycle . � Proliferating cells progress through a series of steps, at which the cell checks for the accuracy of the process & instructs itself to proceed to the next step (F3-3). Cell cycle has multiple controls, both positive & negative. ☺ The cell cycle (F3 -3), consisting of the : (1) Presynthetic growth phase 1 , or (G1); (2) DNA-synthetic phase , or (S); (3) Premitotic growth phase 2, or (G2); & (4) mitotic phase or (M). ☺ Non-dividing cells are either (a) in cell cycle arrest in G1 or (b) they exit the cycle to enter a phase called G0. Any stimulus that initiates cell proliferation, such as exposure to GF, needs to promote the G0 to G1 transition, & the entry of cells into the first, i.e., G1, phase of the cycle. ☺ Checkpoint controls, prevent DNA replication or mi tosis of damaged cells, & either: (1) Transiently stop the cell cycle to allow for DNA repair, or (2) Eliminate irreversibly damaged cells by apoptosis . � Entry & progression of cells through the cell cycle are controlled by changes in The levels of the various cyclins increase at a spe cific stages of the cell cycle, after which they are rapidly degraded as the cell moves on through the cycle. � Cyclins accomplish their regulatory functions by complexing with (& thereby

3

activating) constitutively synthesized proteins called � cyclin-dependent kinases (CDKs). � Different combinations of cyclins & CDKs are associated with each of the important transitions in the cell cycle. CDKs work by promoting DNA replication & various aspects of the mitotic process & are required for the cell cycle progression. CDKs are suppressed during G1 by multiple mechanisms. ☺A major action of GFs is to overcome cell cycle che ckpoint controls, by releasing the suppression of CDK activity. ►Once cells enter the S phase, the DNA is replicated & the cell progresses through G2 and mitosis. �A specific example involves CDK1, which controls the critical transition from G2 to M . As the cell moves into G2, cyclin B is synthesized, & it binds to CDK1. This [inactive ] cyclin B-CDK1 complex, is activated by phosphorylation, & the active kinase [phosphorylated cyclin B-CDK1 complex] then phosphorylates a variety of proteins involved in mitosis, including those involved in DNA replication, depolymerization of nuclear lamina, & mitotic spindle formation. � After cell division, cyclin B degraded by the ubiquitin-proteasomal pathway; leaving behind the inactive CDK1 kinase, which can re-enter the cell cycle at the next G2 stage. Until there is a new growth stimulus & synthesis of new cyclins, the cells do not undergo further mitosis. # In addition to synthesis & degradation of cyclins , the cyclin-CDK complexes are also regulated by the binding of CDK inhibitors . These are particularly important in regulating cell cycle checkpoints (G1 → S & G2 → M), at which the cell takes stock of whether its DNA is sufficiently replicated & all mistakes repaired before progressing. �Failure to adequately monitor the fidelity (accuracy) of DNA replication leads to the accumulation of mutations & possible malignant transformation. # Example, when DNA is damaged (e.g., by ultraviolet irradiation), the tumor suppressor protein TP53 is stabilized. → TP53 induces the transcription of CDKN1A, a CDK inhibitor. This arrests the cells in G1 or G2 until the DNA can be repaired ; at that point, the TP53 levels fall, CDKN1A diminishes, & the cell can proceed through the checkpoint. ▼ If the DNA damage is too extensive , TP53 will initiate a cascade of events to convince the cell to commit suicide (apoptosis ), ♫ Summary, following DNA damage, → TP53 stop progression of cell to mitosis, until DNA damage is repaired, if the damage is not repaired, → TP53 order the cell to commit suicide! REPAIR OR DIE!

4

Proliferative Poten tial of Different Tissues The ability of tissues to repair themselves is critically influenced by their intrinsic proliferative capacity . ☺ Based on this criterion, the tissues of the body are divided into three groups (I) Continuously Dividing Tissues (also called labi le cells . These are continuously dividing (& continuously dying) cells. Replacement is by: (1) maturation from a population of stem cells with relatively unlimited capacity to proliferate, & by (2) Proliferation of mature cells. Labile cells include: (1) hematopoietic cells in the bone marrow, (2) surface epithelia including, the (a) stratified squamous surfaces of the skin, oral cavity, vagina, & cervix; (b) the cuboidal epithelia of the ducts draining exocrine organs (e.g., salivary glands, pancreas, biliary tract); (c) the columnar epithelia of the gastrointestinal tract (GIT), uterus, & fallopian tubes; & (d) the transitional epithelium of the urinary tract. All these tissues can readily regenerate after injury as long as the pool of stem cells is preserved (II) Stable Tissues : Cells of these tissues are considered to be quiescent {in the G0 stage of the cell cycle}, & have only minimal replicative activity in their normal state. However, these cells are capable of proliferating in response to injury or loss of tissue mass. Stable cells constitute the: (1) Parenchyma of most solid glandular tissues , e.g., liver, kidney, & pancreas, (2) Blood vessels EC , & the (3) Fibroblast & smooth muscle connective tissue (mesenchymal) cells, proliferation of which is very important in response to injury & wound healing. N.B. With the exception of liver , all stable tissues have a limited capacity to regenerate after injury. (III) Permanent Tissues : cells of these tissues are considered to be terminally differentiated & nonproliferative (nondividing) cells in postnatal life, those include: (1) The majority of neurons, (2) Cardiac muscle cells. Thus, injury to brain or heart is irreversible & results in only scar since their cells cannot proliferate. ? Limited stem cell replication & differentiation occurs in some areas of the adult brain , & there is some evidence that heart muscle cells may proliferate after myocardial necrosis. Nevertheless, whatever proliferative capacity may exist in these tissues, it is insufficient to produce tissue regeneration after injury.

5

(3) Skeletal muscle is usually classified as a per manent tissue , but satellite cells attached to the endomysial sheath provide some regenerative capacity for this tissue. � In all permanent tissues , repair is typically by scar formation. � With the exception of tissues composed primarily of nondividing permanent cells (e.g., nerve & cardiac muscle), most mature tissues contain variable proportions of three cell types: (1) continuously dividing cells, (2) quiescent cells that can return to the cell cycle, & (3) nondividing cells. Stem Cells ☼ In most continuously dividing tissues, the mature cells are terminally differentiated & short-lived. As mature cells die, the tissue is replenished by the differentiation of cells generated from stem cells. Therefore, there is a homeostatic equilibrium between the (a) replication & differentiation of stem cells, & (b) the death of the mature, fully differentiated cells. ☼ Skin epidermis & the GIT epithelium are good examples. In both, stem cells have been identified near the basal layer of the epithelium. Cells differentiate progressively as they migrate to the upper layers of the epithelium; they ultimately die & are shed from the surface of the tissue. ☼ Stem cells are characterized by two important properties:

(I) Self-renewal capacity , & (II) Asymmetric replication , which means that after each cell division, some progeny cells enter a differentiation pathway, while others remain undifferentiated, retaining their self-renewal capacity.

☼ Stem cells with the capacity to generate multiple cell lineages (pluripotent stem cell) can be isolated from embryos & are called embryonic stem (ES) cells . ☼As mentioned above, stem cells normally present in proliferative tissues & generate cell lineages, specific for the tissue. However, it is now recognized that stem cells with the capacity to generate multiple l ineages are present in the bone marrow & several other tissues of the adult individuals. These cells are called adult stem cells or tissue stem cells . ☼ Bone marrow stem cells have very broad differentiation capabilities, being able to generate fat, muscle, cartilage, bone, & en dothelium. This developmental plasticity was first interpreted as being the consequence of (transdifferentiation), that is, the change in the differentiation program of an already committed cell.

6

Most likely, however, developmental plasticity involves the selection of a specific pathway from the many differentiation pathways available to uncommitted progenitor cells. ☺ The new field of Regenerative Medicine main goal is the regeneration & repopulation of damaged organs using ES or adult stem cells. One exciting prospects in this field is the type of stem cell therapy known as therapeutic cloning. �The main steps involved in therapeutic cloning , using ES cells for cell therapy is illustrated in F3-4. In this procedure: (1) The diploid nucleus of a cell from a patient, is introduced into an enucleated oocyte. (2) The oocyte is activated, & the zygote divides to become a blastocyst containing donor DNA. (3) The blastocyst is dissociated to obtain ES cells; (4) ES cells are capable of differentiating into various tissues (e.g., neurons, muscle cells, or RBC), either in culture or after transplantation into the donor. The goal of the procedure is to reconstitute or repopulate damaged organs from a patient using the cells of the patient, thus avoiding immunologic rejection. ☺ Other potential therapeutic strategies using stem cells involve:

(I) Transplanting stem cells into areas of injury. (II) Mobilization of stem cells from the bone marrow into injured tissues, & the (III) Use of stem cell culture systems to produce large amounts of differentiated cells for transplantation into injured tissue.

THE NATURE AND MECHANISMS OF ACTION OF GR OWTH FACTORS Cell proliferation can be triggered by many chemical mediators, such as hormones, cytokines, & GFs (GF); the first two have many other functions & are discussed separately in other sections of the book. Signals from ECM are also important inducers of cell replication, & they will be discussed later. In this section, we focus on polypeptide GFs, whose major role is to promote cell survival & proliferation & which are important in regeneration & healing. ☼ Expansion of cell population usually involves: (1) an increase in cell size (growth), (2) cell division (mitosis), & (3) protection from apoptotic death (survival). Many GFs have all these three activities. � A GF may act on a specific cell type or on multiple cell types.

7

� Most GFs have pleiotropic effects; (pleio= many, tropic= range or activity), they stimulate: (1) cellular proliferation, (2) migration, (3) differentiation (4) contractility, & (5) enhance the synthesis of specialized proteins (such as collagen in fibroblasts). GF induce cell proliferation by binding to specific receptors & affecting the expression of genes whose products typically have several functions-they: (1) Relieve blocks on cell cycle progression (thus promoting replication), (2) Prevent apoptosis, & (3) Enhance the synthesis of cellular proteins in preparation for mitosis. ☼ A major activity of GFs is to stimulate the function of growth control genes, many of which are called protooncogenes because mutation in them lead to unrestrained cell proliferation characteristic of cancer (Oncogenesis ). ☼ Some GFs stimulate proliferation of some cells & inhibit cycling of other cells. In fact, a growth factor can have opposite effects on the sa me cell depending on its concentration . An example of such GF is TGF-β. → Selected cytokines & GF that are involved in regeneration & wound healing are listed in Table 3-1. ☺ Source: many of the GF that are involved in repair are produced by: (I) leukocytes that are recruited to the site of injury or are activated at this site, as part of the inflammatory process. (II) Other GF are produced by the parenchymal cells or the stromal (connective tissue) cells in response to cell injury or loss. Now, we will discuss how GF work? Signaling Mechanisms of Growth F actor Receptors ☺ The major intracellular signaling pathways, induced by GF receptors, are similar to those of many other cellular receptors that recognize extracellular ligands. The binding of a ligand to its receptor→ triggers a series of events by which extracellular signals are transduced into the cell, leading to the stimulation or repression. Signaling may occur (1) directly in the same cell, (2) between adjacent cells, or (3) over greater distances (F3-5). ♫ Autocrine signaling , in which a soluble mediator acts predominantly (or even exclusively) on the cell that secretes it . This pathway is important in the immune response (e.g., lymphocyte proliferation induced by some cyt okines ) & in compensatory epithelial hyperplasia (e.g., liver regeneration) . ♫♫ Paracrine signaling , in which, a substance affect cells in the immediate vicinity of the cell that released the agent. This pathway is important for (1) recruiting inflammatory cells to the site of infection & for (2) wound healing .

8

♫♫♫ Endocrine signaling, in which a regulatory substance, such as a hormone , is released into the blood stream & acts on target cells at a distance . ☺ Receptor proteins are (I) generally located on the cell surface, but, (II) they may be intracellular; in the latter case, ligands ( i.e., molecules that bind to receptors ) must be sufficiently hydrophobic to enter the cell (e.g., vitamin D, or steroid & thyroid hormones). ☺ For cell surface receptors, the binding of a ligand leads to a cascade of secondary intracellular events that culminate in transcription factor activation or repression, leading to cellular responses. ☺ For some intracellular receptors , ligand binding leads to the formation of receptor-ligand complexes that directly associated with nuclear DNA & subsequently either activate or turn off gene transcription. In some cases, cytoplasmic transcription factors called STATs (discussed later) migrate into the nucleus & bind to DNA directly. → Regardless of their origin, transcription factors bind to gene promoters & enhancers to trigger or inhibit transcription. ☺F3-6 presents an overview of signal transduction originating from three types of receptors: (1) Receptors with intrinsic kinase activity, (2) G-protein-coupled receptors, & (3) Receptors without intrinsic enzymatic activity. (I) Receptors with intrinsic kinase activity �These are usually dimeric transmembrane molecules with an extracellular ligand-binding domain; ligand binding causes stable dimerization with subsequent phosphorylation of the receptor subunits. Once phosphorylated, → the receptors can activate other intracellular proteins (e.g., RAS, P13 kinase pathway, & phospholipase Cγ [PLC-γ]) & stimulate a cascade of events leading to entry into the cell cycle & cell cycle progression, or induction of other transcriptional programs. An especially important pathway stimulated by RAS activation is the mitogen-activated protein (MAP) kinase cascade , which is involved in the intracellular signaling of many GF including: (1) epidermal GF (EGF) , (2) vascular endothelial GF (VEGF), (3) fibroblast GF (FGF), & (4) hepatocyte GF (HGF). (II) G-protein-coup led receptors �These receptors contain seven transmembrane α-helix segments & are also known as {seven transmembrane G-protein-coupled receptors}. After ligand binding, the receptors associate with intracellular guanosine triphosphate (GTP)-binding proteins (G-proteins) that contain guanosine diphosphate (GDP). Binding

9

of the G proteins causes the exchange of GDP with GTP, resulting in activation of the proteins. Among the several transduction pathways activated through G-protein-coupled receptors are those involving cyclic AMP (cAMP ), & the generation of inositol-1,4,5,-triphosphate (IP3), which releases calcium from the endoplasmic reticulum. ☺ Receptors in this category constitute the largest family of plasma membrane receptors (more than 1500 members have been identified) & include those for → epinephrine, vasopressin, serotonin, histamine, glucagon, & the chemokines. (III) Receptors without intri nsic enzymatic activity �These are usually monomeric transmembrane molecules with an extracellular ligand-binding domain; ligand interaction induces an intracellular conformational change that allows association with intracellular protein kinase called Janus kinase (JAKs). Phosphorylation of JAKs activates cytoplasmic transcription factors called STATs (signal transducers & activators of transcription), which shuttle directly into the nucleus. Ligands for these receptors include many cytokines , the interferons, colony-stimulating factors, growth hormone, & erythropoiet in. ▼Not all ligands induce stimulatory signals; in fact, growth-inhibitory signals inducing direct inhibition or inhibition caused by cell-cell contact (contact inhibition) are equally important. ▼ For instance, the TGF-β receptor has intrinsic kinase activity, & when in complex with TGF-β, it phosphorylates specific intracellular proteins, which in turn increase the synthesis of CDK inhibitors (CDKI) & block the activity of transcription factors & cell cycle progression. ♫ To summarize , polypeptide GFs bind to & activate their receptors, many of which possess intrinsic kinase activity . They subsequently phosphorylate a number of substrates involved in signal transduction. The resultant kinase cascade leads to the activation of nuclear transcription factors, initiates DNA synthesis, & ultimately culminates in cell division. The process of cell proliferation is directed by a family of proteins called cyclins that, when complexed with CDKs, control the phosphorylation of proteins involved in cell cycle progression. Extracellular Matrix (ECM) & Cell -Matrix Interactions ☼ ECM is a dynamic, constantly remolding macromolecular complex synthesized locally, arrange into a network that surrounds cells, & constituting a significant

10

proportion of any tissue. ECM sequesters water, providing turgor (firmness) to soft tissue, & minerals, giving rigidity to bone. ☺ By supplying a substratum for cell adhesion & serving as a reservoir for GFs, ECM regulate the proliferation, movement, & differentiation of the cells within it. ECM occurs in two basic forms: interstitial matrix & basement membrane (BM) (F3-7). � Interstitial matrix . This is present in (1) the spaces between cells in connective tissue, & (2) between epithelium & supportive vascular & smooth muscle structures; it is synthesized by mesenchymal cells (e.g., fibroblasts) & tends to form a three-dimensional amorphous gel . ♫ Its major constituents are fibrillar & nonfibrillar collagens, as well as fibronectin, elastin, proteoglycan, hyaluronate & other elements (described later). � Basement membrane (BM). The seemingly random array of interstitial matrix in connective tissues becomes highly organized around (a) epithelial cells, (b) EC, & (c) SMCs, forming the specialized basement membrane . The BM lies (sits) beneath epithelium, & is synthesized by over lying epithelium & underlying mesenchymal cells ; it tends to form a platelike "chicken wire" mesh. Its major constituents are: (a) Amorphous non fibrillar type IV collagen, & (b) Laminin. Roles (functions) of E xtracellular Matrix (1) Mechanical support for cell anchorage (fixation), cell migration, & maintenance of cell polarity (i.e., basolateral (bottom) side versus v.s. apical (top) are important distinctions for most cells in terms of function {e.g., absorption of nutrients from the GIT or release of digestive enzymes in the pancreas}. (2) Control of cell growth . ECM components can regulate cell proliferation by signaling through cellular receptors of the integrin family . (3) Maintenance of cell differentiation . The type of ECM proteins also affects the degree of differentiation of the cells in the tissue, also acting largely via cellular surface receptors of the integrin family . (4) Scaffolding for tissue renewal. The maintenance of normal tissue structure requires BM or Stromal scaffold. The integrity of the BM or the underlying stroma of the parenchymal cells is critical for the organized regeneration of tissues. ▼It is particularly noteworthy that although labile & stable cells are capable of regeneration, injury to these tissues, results in restitution of the normal structure

11

only if the ECM is not damaged. Disruption of these structures (ECM), leads to collagen deposition & scar formation (F3-1, an example of the liver). (5) Establishment of tissue microenvironment . BM acts as a boundary between epithelium & underlying connective tissue & also forms part of the filtration apparatus in the kidney. (6) Storage & presentation of regulatory molecules. For example, GFs like FGF & HGF are excreted & stored in the ECM in some tissues. This allows their rapid deployment after local injury, or during regeneration. Components of the Extracel lular Matrix �There are three basic components of ECM: (1) fibrous structural proteins that confer tensile strength & recoil, (2) water-hydrated gels that permit resilience & lubrication, & (3) adhesive glycoproteins that connect the matrix elements one to another & to cells (F3–7). COLLAGEN �The collagens are fibrous structural proteins that confer tensile strength ; without them human beings would be reduced to a clump of cells connected by neurons! � Collagens are composed of three separate peptide chains braided into a ropelike triple helix. The collagen proteins are rich in hydroxyproline & hydroxylysine. � More than 30 collagen types have been identified, some of which are unique to specific cells and tissues. � Some collagen types (e.g., types I, II, III, & V) form fibrils by virtue of lateral cross-linking of the triple helices. These fibrillar collagens form a major proportion of the connective tissue in healing wounds & particularly in scars. � The tensile strength of the fibrillar collagens derives from their cross-linking , which is the result of covalent bonds catalyzed by the enzyme lysyl-oxidase. This process is dependent on vitamin C ; therefore, children with ascorbate (vitamin C deficiency) have skeletal deformities, bleed easily because of weak vascular wall basement membrane, & heal poorly. � Genetic defects in fibrillar collagens cause diseases such as (1) osteogenesis imperfecta, & (2) Ehlers-Danlos syndrome. � Other collagens are nonfibrillar, & may form: (a) Basement membrane (type IV), or (b) Be component of other structures such as intervertebral discs (type IX), or dermal-epidermal junctions (type VII).

12

ELASTIN After physical stress, the ability of tissue to recoil & return to a baseline structure is conferred by elastic tissue. This is especially important in the walls of large BV (e.g., aorta , which must accommodate recurrent pulsatile blood flow), as well as in the uterus, skin, & ligaments. ☺Morphologically, elastic fibers consist of a central core of elastin protein, surrounded by a mesh like network of fibrillin glycoprotein, which serves as a scaffold for the deposition of elastin & assembly of elastic fibers; defect in the fibrillin synthesis lead to skeletal abnormalities & weakened aortic walls (Marfan syndrome ). PROTEOGLYCANS & HYALURON AN ☺These molecules form highly hydrated compressible gels conferring resilience & lubrication (such as in the cartilage in joints). They consist of long polysaccharides called glycosaminoglycans ( e.g., dermatan sulfate & heparan sulfate ) linked to a protein backbone much like bristles on a test tube brush. � Hyaluronan , a huge molecule, compsed of many disaccaride repeats without a protein core, is also an important constituent of the ECM. Because of its ability to bind water, it forms a viscous, gelatin-like matrix. Besides providing compressibility to a tissue, proteoglycans also serve as reservoirs for GFs secreted into the ECM (e.g., FGF & HGF). �Syndecan, a transmembrane proteoglycan, has attached hyaluronan chains that can bind such matrix GFs as FGF, facilitating the interaction of FGF with cell surface receptors (F3-8). Syndecan also associates with the intracellular actin cytoskeleton & thereby helps to maintain normal epithelial sheet morphology. Adhesive Glycoproteins & Adhesio n Receptors �Both are involved in cell-cell adhesion + the linkage between cells & ECM & + binding between ECM components. The adhesive glycoproteins include fibronectin (major component of the interstitial ECM), & laminin (major constituent of BM). The adhesion receptors, also known as cell adhesion molecules CAMs), are grouped into four families: (I) immunoglobulines, (II) cadherins, (III) selectins, & (IV) integrins. Only the integrins will be discussed here. Fibronectin �Is, a large (450-KD) disulfide heterodimeric, synthesized by firoblasts, monocytes & endothelium. Fibronectin messenger RNA (mRNA) has two splice forms, which generate tissue & plasma fibronectin. Fibronectins have specific domain that bind to a wide spectrum of ECM components (e.g., collagen, fibrin, heparin, & proteoglycans) & can also attach to cell integrins via a tripeptide arginine-glycine-aspartic acid (RGD) motif.

13

� Tissue fibronectin forms fibrillar aggregates at wound healing sites; plasma fibronectin binds to fibrin to form the provisional blood clot of a wound, which serves as substratum for ECM deposition and re-epithelilization. LAMININ �Is the most abundant glycoprotein in BM; it is an 820-kD cross-shaped (†) heterotrimer that connects cells to underlying ECM components such as type IV collagen & heparan sulfate. Besides mediating attachment to BM, laminin also modulates cell survival, proliferation, differentiation, & motility. INTEGRINS � Are a family of transmembrane heterodimeric glycoproteins composed of α & β chains that are the main cellular receptors for ECM components, such as fibronectins & laminins. � Recall ----- that some of the integrins are leukocytes surface molecules that mediate firm adhesion & transmigration across endothelium at sites of inflammation (chapter 2). �Integrins are present in the plasma membrane of most animal cells, with the exception of RBCs. They bind to many ECM components through RGD motifs, initiating signaling cascades that can affect cell locomotion, proliferation, & differentiation. � Integrins intracellular domains link to actin filaments at focal adhesion complexes, through adaptor proteins such as talin & vinculin (F3-9). � Integrin signal transduction utilizes the same intracellular signaling pathways used by GFRs; e.g., integrin-mediated adhesion to fibronectin can trigger elements of the MAP kinase, P13 kinase, & protein kinase C pathways. In this manner, extracellular mechanical forces can be coupled to intracellular synthetic & transcriptional pathways (F3-9). CELL AND TISSUE REGENERATION ☺In labile (continuously dividing) tissues, damage to epithelia or an increased loss of blood cells can be corrected by the proliferation & differentiation of stem cells & , in the bone marrow, by proliferation of more differentiated progenitors. � The renewal of hematopoietic cells is driven by GF called colony-stimulating factors (CSFs), which are produced in response to (a) increased consumption, or (b) loss of, blood cells. �It is not known if GF play a role in the renewal of labile cells.

14

☺In stable cell populations, tissue regeneration can occur in parenchymal organs, but, with the exception of the liver, this regeneration is limited. � Pancreas, adrenal, thyroid, & the lung tissues have some regenerative capacity. � Surgical removal of a kidney elicits in the other, contralateral kidney, a compensatory response (the mechanisms of which is not understood) that consists of both hypertrophy & hyperplasia of the proximal duct cells. � Surgical removal of 40% to 60% of the liver in: (I) Living-donor transplantation , in which a portion of the liver is resected from a normal individual, & is transplanted into a recipient with end-stage liver disease (F3-10), or (II) after partial hepatectomies performed for tumor removal. �In all of these situations, the tissue resection triggers a, dramatic, proliferative response of the remaining hepatocytes (which are normally quiescent), & the subsequent replication of the hepatic non-parenchymal cells. � In experimental systems, hepatocyte replication after partial hepatectomy is initiated by cytokines (e.g., TNF & IL-6 that "prime" the cells for replication by stimulating the transition from G0 to G1 in the cell cycle. Progression through the cell cycle is dependent on the activity of GFs such as HGF & the EGF family of factors, which include TGF-α. ☼ HGF is produced by fibroblasts, endothelial cells, & liver nonparenchymal cells. It induces proliferation of hepatocytes & most epithelial cells, including those in the skin, mammary gland, & lungs. HGF binds to a specific tyrosine kinase receptor (MET), which is frequently overexpressed in human cancers. ☼ EGF & TGF- α, share a common receptor, {epidermal growth factor receptor, EGFR} with intrinsic tyrosine kinase activity. The "EGFR" is actually a family of receptors that respond to EGF, TGF-α, & other ligands of the EGF family. ▲ EGF/TGF-α is (are) mitogenic for hepatocytes & most epithelial cells, including keratinocytes. In cutaneous wound healing EGF is produced by keratinocytes, macrophages, & other inflammatory cells. ☺The main EGFR (referred to as EGFR1 & ER B1) is frequently overexpressed in lung & some brain tumors , & is an important therapeutic target for the treatment of these tumors. ☺ ER B2 (also known as HER-2/NEU ) has received great attention, because of its overexpression in breast cancers , in which it is a target for effective cancer control.

15

☼ It should be emphasized that extensive regeneration , or compensatory hyperplasia can occur only if the residual tissue is structurally & functionally intact , as after partial surgical resection. By contrast, if the tissue is damaged by infection or inflammation, regeneration is incomplete & is accompanied by scarring. Repair by Connective Tissue (a) If tissue injury is severe or chronic, & result in damage to parenchymal & epithelial cells as well as the stromal framework, or, (b) If nondividing cells are injured , ► Then, repair cannot be accomplished by parenchymal regeneration alone. In these conditions, repair occurs by replacement of the nonregenerated parenchymal cells with connective tissue, or by a combination of regeneration & scar formation. � Repair begins within 24 hours of injury by the emigration of fibroblasts & the induction of fibroblast & EC proliferation. � By 3 to 5 days, a specialized type of tissue that is characteristic of healing called granulation tissue is apparent. The term granulation tissue derives from the pink, soft, granular gross appearance, such as that seen beneath the scab of a skin wound. ���� H, granulation tissue is characterized by prolifer ation of fibroblasts & new thin-walled, delicate capillaries, in a loose ECM ( F3-11A). Granulation tissue then progressively accumulates connective tissue matrix, eventually resulting in the formation of scar (F3-11B), which may remolded over time. ☺ Repair by connective tissue deposition consist of four sequential processes (1) Angiogenesis, i.e., formation of new BV (2) Fibroblasts migration & proliferation (3) Deposition of ECM (scar formation) (4) Remolding, i.e., maturation & reorganization of fibrous tissue. Angiogenesis ☺ BVs are assembled by two processes: (a) Vasculogenesis , in which the primitive vascular network is assembled from angioblasts (EC precursors) during embryonic development; &, (b) Angiogenesis , or Neovascularization , by which preexisting BV send out capillary sprouts to produce new BV (F3-12). ☼ Angiogenesis is a critical process in 3 situations, the : (1) Healing at sites of injury, (2) Collateral circulation development at sites of ischemia, & in (3) Allowing tumors to increase in size beyond the constraints (limits) of their original blood supply.

16

� It has recently been found that EC precursor cells may migrate from the bone marrow to areas of injury & participate in angiogenesis at these sites. � Much work has been done in understanding the mechanisms underlying such neovascularization, & therapies to either augment the process (e.g., to improve collateral blood flow to a heart damaged by atherosclerosis) or inhibit it (to interferes with tumor growth) are beginning to come on-line. � Five main steps occur in angiogenesis from preexisting BV (F3–12): (1) Vasodilation in response to nitric oxide (NO) & increased permeability for the preexisting BV induced by VEGF, (2) Migration of EC towards the area of tissue injury, (3) Proliferation of the EC just behind the leading front of migrating cells, (4) Inhibition of EC proliferation & remodeling int o capillary tubes, & (5) Recruitment of periendothelial cells (pericytes for small capillaries, & SMC for larger BV) to form the mature vessel ☺As mentioned bone marrow EC precursor may contribute to angiogenesis. The nature of the homing mechanism by which EC precursor located in the bone marrow migrate into sites of injury is unknown. These cells may participate in the: (1) Replacement of lost EC, (2) Reendothelialization of vascular implants, (3) Neovascularization of cutaneous wounds, ischemic tissues, & in tumor development. ☺ These new BV are leaky because of incompletely formed interendothelial junctions & increased transcytosis. Indeed, this leakiness explains why granulation tissue is often edematous & accounts in part for edema that may persist in healing wounds long after the acute inflammatory response has resolved. � Structural ECM proteins participate in the process of vessel sprouting in angiogenesis, largely through interactions with integrin receptors in ECs. � Nonstructural ECM proteins contribute to angiogenesis by destabilizing cell-ECM interactions to facilitate continued cell migration (e.g., thrombospondin & tenascin C) or degrade the ECM to permit remodeling & ingrowth of vessels (e.g., plasminogen activator & matrix metalloproteinases {MMPs}). ����GFs Involved in angiogenesis : the most important two are: (I) Vascular endothelial growth factor (VEGF) , which is induced mainly by hypoxia , as well as by PDGF, TGF-β, & TGF-α.

17

�VEGF initiate the process of capillary sprouting from preexisting local BV, by stimulating both, proliferation & motility of ECs. �In angiogenesis involving EC precursors from the bone marrow , VEGF acts through receptor-2 (VEGFR-2), which is restricted to ECs, to → mobilize ECs from the bone marrow & to induce proliferation & motility of these ECs at the sites of angiogenesis. �Regardless of the process that leads to capillary formation, new BV need to be stabilized by the recruitment of pericytes & SMC & by the deposition of connective tissue, the (1) PDGF, (2) TGF-β, (3) Angiopoietins 1 & 2, all participate in the stabilization process. (II) Fibroblast growth factors (FGFs), A family with more than 20 members, the best characterized are FGF-1 (acidic FGF) & FGF-2 (basic FGF). These FGFs are produce by many cells. Released FGF can bind to heparan sulfate & be stored in the ECM. FGF-2 participates in angiogenesis by promoting ECs proliferation. It also promotes the migration of macrophages & fibroblasts to the damaged area, & stimulates epithelial cell migration to cover epidermal wounds. Migration of Fibroblasts & ECM Deposition ( Scar Formation) �Fibrosis (scar formation) builds on the granulation tissue framework of new BV & loose ECM that develop early at the repair site. The process of fibrosis occurs in two steps: (1) Emigration & proliferation of fibroblasts into the site of injury, & (2) Deposition of ECM by these cells. ♫The recruitment & stimulation of fibroblasts is driven by many GF described later, including (1) PDGF, (2) bFGF, & (3) TGF-β. ♫Source of these factors are (a) activated EC, (b) macrophages, (c) lymphocytes, & (d) mast cells. �As healing progresses , the number of proliferating fibroblasts & new BV decreases ; however, the fibroblasts progressively assume a more synthetic phenotype, & hence there is increased deposition of ECM. Collagen synthesis, in particular, is critical to t he development of strength in a healing wound site , & its synthesis by the fibroblasts begins early in wound healing (days 3 to 5) & continues for several weeks, depending on the size of the wound. �Many of the same GF that regulate fibroblast proliferation also participate in stimulating ECM synthesis.

18

Collagen synthesis. �Net collagen accumulation, however, depends not only on the increased synthesis but also on diminished collagen degradation (discussed later). Ultimately, the granulation tissue scaffolding evolves into a scar composed of largely inactive, spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue, & other ECM components (F3–11B). As the scar matures , vascular regression eventually transforms the highly vascularized granulation tissue into a pale, largely avascular scar . Growth Factors Involved in ECM Deposition & Scar Formation ♫ Many GF are involved in these processes, including (1) TGF-β, (2) PDGF, & (3) FGF (discussed above). Only the first two are discussed below:

(1) TGF-β has widespread distribution. The active factor binds to two cell surface receptors with serin/threonine kinase activity, triggering the phosphorylation of transcription factors called (smads ). ▲▼ TGF-β has many & often opposite effects , depending on the cell type & the metabolic state of the tissue. In the context of inflammation & repair, TGF-β has two main functions : ▲ (1) TGF-β is a potent fibrogenic agent . It stimulates the production of collage, fibronectin, & proteoglycans, & it inhibits collage degradation by both decreasing proteinase activity & increasing the activity of tissue inhibitors of proteinases known as TIMPs (discussed below). � TGF-β is involved in (a) scar formation after injury, & (b) of fibrosis in lung, liver, & kidneys that follows chronic inflammation. ▼ (2) TGF-β inhibits lymphocyte proliferation & can have a str ong anti-inflammatory effects. Mice lacking TGF-β have widespread inflammation & abundant lymphocyte proliferation. (2) Platelet-derived growth factor (PDGF ) is stored in platelets & released on platelet activation, (hence its name), & produced by activated macrophages, EC & SMC, & many tumor cells. ♫ PDGF causes migration & proliferation of macrophages, fibroblasts, & SMCs. ���� Cytokines , mediators of inflammation, may also function as GF & participate in ECM deposition & scar formation. IL-1 & TNF, for example, induce fibroblast proliferation. They are also chemotactic for fibroblasts & stimulate the synthesis of collagen & collagenase by these cells. The net results of their actions tend to be fibrogenic .

19

Scar Remodeling Scar ECM continues to be modified & remoldeled. The outcome at each stage is a balance between ECM synthesis & degradation. � We have already discussed the cells & factors that regulate ECM synthesis. ▼The degradation of collagen & other ECM components is accomplished by a family of metalloproteinases (so-called because they are dependent on zinc ions for their activity); [these should be distinguished from neutrophil elastase, cathepsin G, plasmin, & other serine proteinases that can also degrade ECM but are not metalloenzymes]. Metalloproteinases include: (I) Interstitial collagenases , which cleave the fibrillar collagen (MMP-1, -2 & -3); (2) Gelatinases (MMP-2 & 9 ), which degrade amorphous collagen & fibronectin; & (3) Stromelysins (MMP-3, -10, & -11), which catabolize a variety of ECM constituents, including proteoglycans, laminin, fibronectin & amorphous collagen. � Metalloproteinases produced by variety of cell types including fibroblasts, synovial cells, macrophages neutrophils & some epithelial cells. Their synthesis & secretion are regulated by GF; cytokines, & other agents (F3–13). Their synthesis is inhibited by TGF-β & may be suppressed pharmacologically with steroids. � The activity of the metalloproteinases is tightly controlled (otherwise, if given the potential, they will wreak havoc & destroy the tissue ). ▼ Thus they are typically elaborated as inactive (zymogen ) precursors that must be first activated ; this is accomplished by certain chemicals or proteases (e.g., plasmin) likely to be present only at sites of injury (F3–13). ▼In addition, activated collagenases can be rapidly inhibited by specific tissue inhibitors of metalloproteinases (TIMPs), pr oduced by most mesenchymal cells . MMPs & their inhibitors are essential in the debridment of injured sites & in the remodeling of the ECM. CUTANEOUS WOUND HEALIN ☼ Here, we specifically describe the healing of skin wounds. This is process involving both (I) epithelial regeneration & the (II) formation of connective tissue scar & is thus illustrative of the general principles that apply to wound healing in all tissues. However, one should be aware that each different tissue in the body has specific cells & features that modify the basic scheme discussed here (e.g., healing of bone fracture).

20

�Wound healing is a complex, but generally orderly process. Sequential waves of specialized cell types first clear the inciting injury & then progressively build the scaffolding to fill in any resulting defect . The events are orchestrated by interplay of soluble GFs & ECM. ☺Cutaneous wound healing has three main phases: (1) Inflammation, (2) Granulation tissue formation, & (3) ECM deposition & remodeling (F3-14). Larger wounds also contract during the healing process. Based on the nature of the wound, the healing of cutaneous wounds can occur by first or second intention. Healing by First Intention One of the simplest examples of wound repair is the healing of a clean, uninfected surgical incision approximated by surgic al sutures (F3–15). This is referred to as primary union or healing by first intention. ☺ The incision causes only focal disruption (loss of continuity) of epithelial basement membrane & death of relatively few epithelial & connective tissue cells. As a result, epithelial regeneration predominates over fibrosis. A small scar is formed, but there is minimal wound contraction. The narrow incisional space first fills with fibrin-clotted blood, which is rapidly invaded by granulation tissue & covered by new epithelium. � Within 24 hours , neutrophils are seen at the incision margin, migrating toward the fibrin clot. Basal cells at the cut edge of the epidermis begi n to exhibit increased mitotic activity. � Within 24 to 48 hours, epithelial cells from both edges have begun to migrate & proliferate along the dermis, depositing basement membrane components as they progress. The cells meet in the midline beneath the surface scab, yielding a thin but continuous epithelial layer. �By day 3, neutrophils have been largely replaced by macrophages , & granulation tissue progressively invades the incisi on space . Collagen fibers are now evident at the incision margins, but these are vertically oriented & do not bridge the incision. Epithelial cell proliferation continues, yielding a thickened epidermal covering layer . �By day 5, angiogenesis (neovascularization) reaches its peak as granulation tissue fills the incisional space. Collagen fibrils become more abundant & begin to

21

bridge the incision . The epidermis recovers its normal thickness as differentiation of surface cells yields a mature epidermal architecture with surface keratinization. �During the second week, there is continued collagen accumulation & fibroblasts proliferation. The WBC infiltrate, edema, & increased vascularity are substantially diminished. The long process of "blanching" begins, accomplished by increasing collagen deposition within the incisional scar & the regression of vascular channels. �By the end of the first month, the scar comprises a cellular connective tissue largely devoid of inflammatory cells & covered by an essentially normal epidermis . � However, the dermal appendages (hair follicles, sweat & sebaceous glands) destroyed in the line of incision are permanently lost . The tensile strength of the wound increases with time, reaching approximately 70% to 80% of the normal strength of unwounded skin by 3 months but usually does not substantially improve beyond that point. Healing by S econdary Intention � When cell or tissue loss is more extensive , as in (1) Infarction, (2) Inflammatory ulceration, (3) Abscess formation, or even in just (4) Large wound or burn, the reparative process is more complex . � In these situations, regeneration of parenchymal cells alone cannot restore the original architecture. As a result, there is an extensive ingrowth of granulation tissue from the wound margins, followed in time by accumulation of ECM & scarring. This form of healing is referred to as secondary union, or healing by second intention (F3–15 & 3-16). � Secondary union Differs from primary union in following aspects: (1) Large tissue defects intrinsically have a greater volume of necrotic debris , exudate, & fibrin that must be removed. Consequently, the inflammatory reaction is more intense, with greater potential for secondary, inflammation- mediated, injury , (e.g., release of lysosomal enzymes into ECM causing cell injury & matrix degradation due to premature degranulation of lysosomes, or e.g., activated leukocytes release of ROS & product of AA metabolism, both of which are capable of causing tissue damage).

22

(2) Much larger amounts of granulation tissue are f ormed. Larger defects require greater volume of granulation tissue to (1) fill the gaps in the stromal architecture & (2) provide the underlying framework for the regrowth of tissue epithelium. A ���� greater volume of granulation tissue generally results ���� in a greater mass of scar tissue = ▓ (3) Wound contraction . Secondary healing exhibits the phenomenon of wound contraction . Within 6 weeks, for example, large skin defects may be reduced to 5% to 10% of their original size !!! Largely by contraction. This process has been ascribed to the presence of myofibroblasts , modified fibroblasts exhibiting many of the ultrastructural & functional features of contractile smooth muscle cells. Wound Str ength � Carefully sutured wounds have approximately 70% of the strength of unwounded skin, largely because of the placement of the sutures . � When sutures are removed , usually at 1 week, wound strength is approximately 10% of that of unwounded skin, but this increases rapidly over the next 4 weeks. � The recovery of tensile strength results from collagen synthesis exceeding degradation during the first 2 months, & from structural modifications of collagen (e.g., cross-linking & increased fiber size) when synthesis declines at later times. ���� Wound strength reaches approximately 70% to 80% of normal by 3 mon ths but usually does not substantially improve beyond t hat point. PATHOLOGIC ASPECTS OF REPAIR In wound healing, normal cell growth & fibrosis may be altered by a variety of factors, frequently reducing the quality or adequacy of the reparative process: ▼Delay in healing may be caused by the following (1) Infection is the single most important cause of delay in healing , by prolonging the inflammation phase of the process & potentially increasing the local tissue injury. (2) Nutrition has profound effects on wound healing, protein deficiency , for example, & particularly vitamin C deficiency , inhibit collagen synthesis & retard healing. (3) Glucocorticoids (steroids) have well-documented anti-inflammatory effects, & their administration may result in poor wound strength owing to diminished fibrosis . In some instances, however, the anti-inflammatory effects of

23

Glucocorticoids are desirable . For example, in corneal infections , glucocorticoids are some times prescribed (along with antibiotics) to reduce the likelihood of opacity that may result from collagen deposition. (4) Mechanical factors such as increased local pressure or torsion may cause wounds to pull apart, or dehisce (e.g. abdominal dehiscence after lapratomy) . (5) Poor perfusion, due either to atherosclerosis (reduced arterial blood supply) or to obstructed venous drainage, also impairs healing. (6) Foreign bodies such as fragments of steel, glass, wood, or even bone impede healing. �The type (& volume) of tissue injured is a critical factor in healing. Complete repair can occur only in tissues composed of labile & sta ble cells; even then, extensive injury will likely result in incomplete tissue regeneration & at least partial loss of function. � Injury to tissue composed of permanent cells must inevitably result in scarring with at most, attempts at functional compensation by the remaining viable elements. Such is the case with healing of a myocardial infarction. �The location of the injury, or the character of the tissue in which the injury occurs, is also important. ☺For example, inflammation arising in tissue spaces (e.g., pleural, pericardial, peritoneal, & synovial cavities ) develops extensive exudates. Subsequent repair may occur by digestion of the exudate, initiated by the proteolytic enzymes of the leukocytes & resorption of the liquefied exudate. This is called resolution , & in the absence of cellular necrosis, the normal tissue architecture is generally restored. ☻However, in the setting of larger accumulations , the exudate undergoes organization , in which granulation tissue grows into the exudate, followed ultimately by fibrous scar = ▓ ���� Aberrations of cell growth and ECM production may occur even in what begins as normal wound healing . For example, ►Keloids (F3-17): the accumulation of exuberant (excessive) amounts of collagen can give rise to prominent, raised scars known as Keloids , more commonly seen in blacks.

24

► Healing wounds may also generate excessive granulation tissue that protrudes above the level of the surrounding skin & in fact hinders reepithelialization , this is called exuberant granulation, or proud flesh, & restoration of epithelial continuity requires cautery or surgical resection o f the granulation tissue. ►The mechanisms underlying the disabling fibrosis associated with chronic inflammatory diseases such as rheumatoid arthritis, pulmonary fibrosis, & liver cirrhosis are essentially identical ton those that are involved in normal wound healing. � However, in these diseases, persistent stimulation of fibrogenesis results from chronic immune/autoimmune reactions that sustain (maintain) the synthesis & secretion of GF, fibrogenic cytokines, & proteases. For example, collagen degradation by collagenases, normally important in wound remodeling, is responsible for much of the joint destruction seen in rheumatoid arthritis. ↓ VASCULAR & CELLULAR RE SPONSE ↓ ACUTE INFLAMMATORY EXUD ATION ↓ ↓ Stimulus promptly destroyed Stimulus not promptly destroyed ↓ ↓ No or minimal necrosis of cells Necrosis of cells ↓ ↓ ↓ ↓ Exudate resolved Exudate organized cells: Labile or Stable Permanent ↓ ↓ ↓ ↓ ↓ ↓ ↓ Framework Fra mework ↓ ↓ ↓ intact destroyed ↓ ↓ ↓ ↓ ↓ ↓ Resolution Scarring Regeneration & Resolution Scarring Mild heat injury Fibrinopurulant Lobar pneumoni a Abscess OR MI Peritonitis ☺ END OF TISSUE REPAIR: CELL REGENERATION, HEALING A ND FIBROSIS, 24 pp Handout. @ 14-9-2008. Lec tures prepared by: Dr Mohammad Kamel Alwiswasi, MBChB, PhD, F RCPath ☺