Tissue repair and renewal

PowerPoint Presentation: 

REGENERATION HEALING (repair)

LEARNING OBJECTIVES: 

LEARNING OBJECTIVES Review the normal physiology and concepts of cell proliferation , cell growth, cell “cycle”, and cell differentiation Understand the basic factors of tissue regeneration Understand the relationships between cells and their ExtraCellular Matrix (ECM) Understand the roles of the major players of healing--- angiogenesis, growth factors (GFs), and fibrosis Differentiate 1 st & 2 nd intention healing

DEFINITIONS:: 

DEFINITIONS: REGENERATION: Growth of cells to replace lost tissues HEALING: A repar ative tissue response to a wound, inflammation or necrosis, often leads to fibrosis GRANULATION TISSUE “ORGANIZING” INFLAMATION

REGENERATION: 

REGENERATION Replacement of lost structures Is dependent on the type of normal turnover the original tissue has Can be differentiated from “compensatory” growth

Tissue Renewal/Regeneration: 

Tissue Renewal/Regeneration

HEALING (repair): 

HEALING (repair) Needs a wound, inflammatory process, or necrosis Many disease appearances anatomically are the result of “healing” such as atherosclerosis Often ends with a scar Fibrosis, as one of the 3 possible outcomes of inflammation, follows “healing” Requires a connective tissue “scaffold” Fibrosis occurs in proportion to the damage of the ECM

Cell Population Fates: 

Cell Population Fates PROLIFERATION Hormonal, especially steroid hormones eg., EPO, CSF DIFFERENTIATION * UNIDIRECTIONAL, GAIN and LOSS APOPTOSIS * One of the most KEY concepts in neoplasia

PowerPoint Presentation: 

Cell proliferation: -May involve physiologic or pathologic stimuli. controlled by either contact-mediated or soluble signals from other cell. -Increased cell proliferation may be accomplished by -Shortening the cell cycle -Recruiting quiescent cells into the cell cycle

Tissues can be divided into three groups depending on their proliferative capability : 

Tissues can be divided into three groups depending on their proliferative capability Continuously dividing (labile): -surface epithelium -hematopoietic tissues -lining mucosa of excretory ducts -columnar epithelium of GIT and uterus -transitional epithelium of urinary tract

PowerPoint Presentation: 

2. Quiescent (stable): -liver -kidney -pancreas -mesenchymal cells e.g.fibroblast,smooth muscle -endothelial cells -resting lymphocytes

PowerPoint Presentation: 

Non-dividing (permanent) -neurons -skeletal muscle - cardiac muscle However there is evidence of neurogenesis from stem cells and differentiation of satellite cells into skeletal muscle

Stem Cells: 

Stem Cells Prolonged self-renewal capacity Asymmetric replication -one daughter differentiates while the other remains a stem cell. Two types -Embyonic stem cells -Adult stem cells

Stem Cells: 

Stem Cells Embryonic Stem Cells Currently embryonic stem cells are used as research tools study of signals and differentiation steps for tissue development production of knockout mice Regenerative medicine

Conceptualization of “Therapeutic Cloning”: 

Conceptualization of “Therapeutic Cloning”

Human Cloning: 

Human Cloning

Human Cloning: 

Human Cloning

Adult Stem Cells: 

Adult Stem Cells -These may be referred to as reservoir cells and in some tissues are sequestered in distinct anatomical niches -These cells possess the ability to differentiate into a limited number of cell types typically those forming the parent tissues ( multipotent ).

Stem Cell Niches: 

Stem Cell Niches Fig 3-5 p 93

Hematopoietic Stem Cells : 

Hematopoietic Stem Cells A notable exception to limited potency are stem cells located in the bone marrow Marrow contains hematopoietic stem cells (HSCs) which are capable of differentiating into all blood elements. Marrow also contains multipotent stromal cells capable of forming bone, fat, cartilage or muscle. HSCs (mesoderm) are capable of transdifferentiating (differentiating) into neurons (ectoderm), hepatocytes (endoderm) and other adult cell lines. Stem cells may perform their own nuclear transfer with host tissue cells which then develop multipotent capabilities- cell fusion

PowerPoint Presentation: 

-True transdifferentiation Vs Cell Fusion -No role in tissue homeostasis under physiological conditions -Their main role appears to be as a local source of growth & reparative factors involved in healing & regeneration. -Multipotent adult progenitor cells (MAPCs) are also found in marrow and at other tissues; isolated in culture these cells show multipotency & limited senescence. - MAPCs as adult counterpart of ES cells

Differentiation pathways for pleuripotent bone marrow stromal cells: 

Differentiation pathways for pleuripotent bone marrow stromal cells Fig 3-6 p 94

The Role of Stem Cells in Tissue Homeostasis: 

The Role of Stem Cells in Tissue Homeostasis Tissue stem cells are responsible for generating mature cells of their parent organ; they have variable potential to differentiate with the capacity to regenerate tissues after a significant injury.

Epithelial Tissue: 

Epithelial Tissue Self renew occurs by 3 nonmutually exclusive strategies 1.Increasing no of actively dividing stem cells 2.Increasing replication of intermediate cells(Amplifying compartment) 3.Decreasing the cell cycle time for cell replication

Liver: 

Liver - Hepatic stem cells give rise to oval cells which are capable of differentiating into hepatic or biliary cells.(Bipotential) - They appear to be active only when hepatocytes are incapable of division (severe hepatic injury).

Brain: 

Brain Neurogenesis first described in birds(Vocal cord neurogenesis) Neural stem cells in human -Olfactory bulb neuronal cells -Dentate gyrus of hippocampus

Striated Muscles: 

Striated Muscles -Skeletal and cardiac myocytes are incapable of division. - Proliferative capability in skeletal muscle is due to satellite ; these cells are located just beneath the basal lamina of myocytes. -Similar cells have not been identified in cardiac muscle .

Growth Factors: 

Growth Factors The key to repair and regeneration is in the regulatory processes involved in initiating stem cell proliferation and differentiation. This is the function of growth factors.

Function of growth factors: 

Function of growth factors Cell proliferation Cell locomotion Contractility Differentiation Angiogenesis

PowerPoint Presentation: 

Epidermal Growth Factor(EGF) -Source- platelets, macrophages, keratinocyte,saliva,urine, milk,plasma,etc. -Receptor-epidermal growth factor receptor(EGFR) -Function-mitogenic for variety of epithelial cells and hepatocytes Transforming Growth Factor(TGF) - Source,receptor and function same as EGF -Receptor-The main EGFR is EGFR1 or ERB B1 -Function-Involved in epithelial cell proliferation in embryos and adults and malignant transformation of cells.

PowerPoint Presentation: 

Hepatocyte Growth Factor(HGF) -Scatter factor,originally isolated from platelets and serum -Source-fibroblast,endothelial cells and liver non parenchymal cells -Receptor-product of c-MET proto-oncogen -Function-mitogenic effect on hepatocytes,biliary epithelium,lung,mammary gland,skin etc. - Morphogen in embryonic development and promotes cell scattering and migration.

PowerPoint Presentation: 

Vascular Endothelial Growth Factor(VEGF) -Various types-VEGF-A,VEGF-B,VEGF-C,VEGF-D and placental growth factor -Receptor-VEGFR-1,VEGF-2(main one) and VEGF-3 -Function-vasculogenesis and angiogenesis(VEGF-2),lymphangiogenesis(VEGF-3) Platelet Derived Growth Factor(PDGF) -Various types each consisting of 2 chains A and B giving rise to 3 isiforms-AA,AB and BB. -2 recently discovered-PDGF-C and PDGF-D -Source-platelet Alfa granule,activated macrophages,endothelial cells,smooth muscle etc.

PowerPoint Presentation: 

-Receptor-PDGFR Alfa and Beta -Function-migration and proliferation of fibroblast,smooth muscle cells,and monocytes -Also activates hepatic stellate cells and cause liver cirrhosis Fibroblast Growth Factor(FGF) -Various types-acidic(aFGF or FGF-1) and (bFGF or FGF-2) are well studied. -When released associate with heparan sulfate in ECM where it is stored -Function -Angiogenesis(FGF-2) -Wound repair-migration of macrophages,fibroblast and endothelial -Development of skeletal muscles and lung maturation -Haematopoesis-differentiation of specific lineages of blood cells

PowerPoint Presentation: 

TGF-beta and Related Growth Factors TGF-beta belongs to a homogenous polypeptides that includes 3 TGF- β isoforms(TGF- β 1,TGF- β 2,TGF- β 3) and other factors e.g. BMPs,activins,inhibins,and mullerin inhibiting substance -Source-platelets,endothelial cells,lymphocytes etc. -Receptor-type 1 and type 2 -Mechanism of action-TGF- β first bind to type 2 receptor͢͢͢͢͢͢͢͢>>the complex binds to type 1 receptor>>phosphorylation of Smad 2 and 3>>forms heterodimer with Smad 4>>enters nucleus and associate with DNA binding proteins to activate or inhibit gene transcription depending upon tissue(pleiotropic)

PowerPoint Presentation: 

-Function -Growth inhibitor for most epithelial cells and for leucocytes(loss will cause tumor) -Growth stimulation of fibroblast and smooth muscle -Strong antiinflammatory function Cytokines -they also function as growth factor for variety of cells

Signaling mechanisms in cell growth: 

Signaling mechanisms in cell growth All growth factors function by binding to specific receptors, which delivers signal to target cell. These signals have 2 general effects 1)Stimulation of transcription of many genes 2)Entry of cell into cell cycle Three types of signaling 1)Autocrine Signaling 2)Paracrine Signaling 3)Endocrine Signaling

PowerPoint Presentation: 

1)Autocrine signaling- liver regeneration,antigen stimulation lymphocytes,some tumors 2) Paracrine signaling -conective tissue repair of wound healing,liver regeneration *Juxtacrine-TNF- ά ,EGF>>promotes cell to cell adhesion 3) Endocrine signaling Hormones

Receptor And Signal Transduction Pathways: 

Receptor And Signal Transduction Pathways A receptor protein has binding specificity for particular ligands Receptor-ligand complex initiate specific cellular response Four types of receptors -Receptors with tyrosine kinase activity-most GFs -Receptors lacking tyrosine kinase activity-cytokines -Seven transmembrane G-protein-coupled receptors vasopressin,epinephrine,norepinephrine,glucagon etc. -Steroid hormone receptors

Signal Transduction Pathways: 

Signal Transduction Pathways

Receptors with intrinsic tyrosine kinase activity: 

Receptors with intrinsic tyrosine kinase activity Most GFs e.g. EGF, TGF- α , HGF, PDGF, VEGF, FGF, c-KIT ligand & insulin 3 parts: extracellular, trnsmembrane & cytoplasmic tail having TK activity Dimerization>tyrosine phosphorylation & activation of TK activity Phosphorylation of downstream effector molecules Phosphorylated residues on the receptor also serve as docking site for adapter molecule

Effector molecules: phospholipase C ƴ & PI3: 

Effector molecules: phospholipase C ƴ & PI3 Membrane inositol phospholipids>>IP3 & DAG IP3>> increase concenteation of Ca>>many effects;K & Cl channel,muscle contration & enzymes activation DAG>>activates protein kinase C(PKC)>>activation of various TFs PI-3K phosphorylate a membrane phospholipid>>product activate PKB/Akt Akt>cell proliferation & inhibition of apoptosis; intermediate in insulin signaling pathway

MAP kinase pathway: 

MAP kinase pathway GRB2>>adaptor protein; Binds a GTP:GDP exchange factor called SOS SOS catalyzes formation of RAS-GTP>>activates mitogen activated protein kinase (MAP kinase) cascade Active MAP kinases>synthesis & phosphorylation of TFs e.g. FOS & JUN>>production of GFs, GFRs & cell cycle regulatory proteins Defects in TK:Cancer, Type 2 DM & atherosclerosis

Receptors lacking TK activity that recruit kinases: 

Receptors lacking TK activity that recruit kinases Ligands: Cytokines e.g. IL-2, IK-3; IF α , β & ƴ; EPO; Granulocyte CSF; GhH & prolactin JAKs phosphorylates cytoplasmic TF called STAT (signal transducer and activation of transcription)>>shuttle in to the nucleus>>activates gene transcription Can also activate MAP kinase pathway

Seven transmembrane G-protein-coupled receptors(GPCRs): 

Seven transmembrane G-protein-coupled receptors(GPCRs) 7 transmembrane α -helices Largest family of plasma membrane receptors (>1500 types identified) G-proteins (GTP binding proteins)>>cAMP pathway Examples: vasopressin, serotonin, histamines, epinephrine & norepinephrine, calcitonin, glucagon, PTH, cortocotrophin, rhodopsin Conformational change>>activation>>interaction between many G-proteins by exchange of GDP>>Ca & adenosine 3, 5 cAMP as 2 nd messenger

Seven transmembrane G-protein-coupled receptors(GPCRs): 

Seven transmembrane G-protein-coupled receptors(GPCRs) Can also produce IP3>>Ca from ER Ca>> cAMP>>PKA & CAMP gated ion channels, important in vision and olfactory sensing Defects>>retinitis pigmentosa, cortocotropin deficiencies & hyperparathyroidism

Steroid harmone receptors: 

Steroid harmone receptors Location of receptors>> nucleus & less frequently cytoplasm Activate transcription of genes Receptor for estrogen>>cytoplasm Other ligands having similar receptors>> thyroid hormone, vitamin D & retinoid

Transcription Factors: 

Transcription Factors Responsible for transfer of information to nucleus and gene expression Genes involved in cell proliferation -Growth promoting-c-MYC and c-JUN -Growth inhibiting- p53 Have DNA binding domains that bind short DNA sequence specific for one or many genes. When rapid response is required the source of TF depend upon post translation modification of TF in cytoplasm>>migration in to nucleus e.g. dimerization of products of cFOS & cJUN>>transcription factor activator protein-1 (AP-1)>>acivated by MAP kinase pathway

The Cell Cycle : 

The Cell Cycle

Cell cycle: 

Cell cycle Role of proto-oncogen & oncogen Replication of cell stimulated by GFs & signalling by ECM components Go to G1: 1 st decision step; gateway to cell cycle>involves the trancriptional activation of large set of genes including protooncogenes and genes required for ribosome synthesis & protein translation G1/S transition: a rate limiting step; after passing, cells are irreversibly committed for replication

Cell cycle: 

Cell cycle Cyclins & CDKs: Many GFs stimulate formation of cyclins CDK inhibitors Cyclins, CDKs & CDK inhibitors orchestrate the cell cycle CDK phosphorylate critical target proteins required for progression of cell cycle to next phase CDKs expressed constitutively but in inactive forms; activated by cyclins Cyclins are produced during specific phase of cell cycle and its level decline rapidly;>15 types discovered

PowerPoint Presentation: 

Control of Cell Cycle

Cyclin D & RB phosphorylation: 

Cyclin D & RB phosphorylation Cyclin D appear in mid G1; 3forms>D1, D2 & D3 Cyclin D like other cyclins are unstable and degraded by ubiquitin-proteasome pathway Cyclin D-CDK4 complex phosphorylates RB protein: molecular on/off switch for cell cycle E2F/DP1/RB complex binds to promoters of E2F responsive genes M phase: phosphate group removed by cellular phosphatase>>regenration of hypophosphorylated form of RB

Cell cycle progression beyond G1/S restriction point: 

Cell cycle progression beyond G1/S restriction point Activated E2F>>cyclin A Cyclin A-CDK2>>regulates events at mitotic prophase Cyclin B-CDK1>>beyond prophase; breakdown of nuclear membrane and initiate mitosis Cyclin B-CDK1 complex activated by phosphatase (Cdc 25)

Cell cycle progression beyond G1/S restriction point: 

Cell cycle progression beyond G1/S restriction point Complexes of CDKs with cyclin A & B regulate critical events at G2/M transition e.g. separation of centrosomes, chromosome condensation etc. Inactivation of cyclin B-CDK1>>cell return to G1 and initiate new cycle or go in to quiescence Proliferating cell: Cyclin E-CDK2 function may be repalced by cyclinA2-CDK2 complex Quiescent cell: absence of both isoforms of E (E1 & E2)

Cell cycle inhibitor: 

Cell cycle inhibitor Two main classes of CDK inhibitors: 1. Cip/Kip family: p21, p27(by TGF β ) & p57 2. INK4/ARF family>>Inhibitor of kinase 4/alternative reading frame (of INK4): p16ARF & p14ARF p16INK4 inactivated by hypermethylation in cancer

Cell cycle checkpoints: 

Cell cycle checkpoints G1/S & G2/M checkpoints S phase>>point of no return G1/S checkpoint: repair of DNA damage; role of p53 gene G2/M checkpoint: completion of replication, checks any damage specially by ionizing radiation; Defects will give rise to chromosomal abnormalities

Cell cycle checkpoints: 

Cell cycle checkpoints Sensors of DNA damage, signal transducer & effectors molecules Sensor & signal transducer similar for G1/S & G2/M checkpoints Sensors: proteins of RAD family & ATM Signal transducers: CHK kinase families G1/S checkpoint: p53 dependent G2/M checkpoint: p53 dependent & independent mechanisms (inactivation of CDC 25 phosphatase) Defects in checkpoints: genetic instability

Mechanism of tissue regeneration: 

Mechanism of tissue regeneration Regeneration in rapidly proliferating cells: Regeneration in quiescent cell: Capacity of whole tissue & organ regeneration is lost in mammals; only compensatory growth process including hypertrophy & hyperplasia do occur

Liver regeneration: 

Liver regeneration Rodent: removal of 70% of liver>>complete regeneration within 10-14 days Restoration of liver mass occurs without regrowth of the lobes that were resected>>compensatory growth Almost all hepatocytes replicate Go to G1 progress takes several hours Wave of hepatocyte replication is synchronous with replication of non paranchymal cells

Liver regeneration: 

Liver regeneration Triggered by combined actions of cytokines and GFs>>by non parenchymal cells>>paracrine TGF α >> autocrine Restriction points: Go/G1 & G1/S Immediate early gene response (Go/G1): transient;>70 genes activated including FOS, JUN & MYC>>TFs>>many genes & activation of other TFs e.g. NFkB, STAT-3 etc. G1/S restriction points: anti apoptotic gene Bcl-X,cylin D>>>

Liver regeneration: 

Liver regeneration Go/G1: TNF & IL-6>>priming G1/S: HGF & TGF α Hepatoytes replicate once or twice and then comes to the quiescent state TGF β >>inhibition of the cycle Intrahepatic stem cell: no role in regeneration after partial hepatectomy No evidence of regeneration from marrow-derived stem cell; some of the endothelial cell may derive from the stem cell

Mechanism Of Tissue Regeneration: 

Mechanism Of Tissue Regeneration

Extracellular Matrix And Cell-Matrix Interaction: 

Extracellular Matrix And Cell-Matrix Interaction Biological Roles of the ECM Mechanical support Determination of cell polarity Control of cell growth Control/maintenance of cell differentiation Scaffolding for tissue renewal Storage and presentation of regulatory proteins

3 groups of macromolecules: 

3 groups of macromolecules 1.Fibrous structural proteins: elastin & collagen 2. Adhesive glycoproteins 3. Proteoglycan & hyaluronic acid

PowerPoint Presentation: 

Major Components of the ECM •Collagen •Elastic fibers •Proteoglycans and hyaluronan •Fibronectin •Laminin •Integrins These assemble into two general organization -Interstitial Matrix -Basement Membrane

Collagen: 

Collagen

Elastin and Fibrillin : 

Elastin and Fibrillin

Cell Adhesion Proteins: 

Cell Adhesion Proteins Classified into 4 main families -Immunoglobulin family CAMs -Cadherins -Intergrins -Selectins Bind to same or different molecules on other cells Homotypic and heterotypic interaction Cadherins - Ca dependent homotypic interaction -Attached to cytoskeleton by catenins -forms zonula adherens and desmosomes

Cell Adhesion Proteins: 

Cell Adhesion Proteins Intergrins: binds fibronectin & laminin>>adhesiveness between cells & ECM as well as to adhesive proteins to other cell>>cell-to-cell contact

Fibronectin and Laminin: 

Fibronectin and Laminin

PowerPoint Presentation: 

Integrins link the ECM to Actin Cytoskeleton through Focal Adhesion Complexes

PowerPoint Presentation: 

Proteoglycans and Hyaluronan -Proteoglycans have a core of protein to which glycosaminoglycans are attached -Glycoproteins are globular proteins with branched monosaccharide chains -Proteoglycans such as syndecan can be transmembrane proteins with ligand binding capacity

PowerPoint Presentation: 

THANK YOU!