Regeneration and Fibrous Repair PDF

Regeneration and Fibrous Repair Mechanism of Disease Ass.Prof. Dr. Mais Almumen Objectives: I. Control of Normal Cell Proliferation A. Regeneration and repair B. Stem cells: biology &...

Regeneration and Fibrous Repair Mechanism of Disease Ass.Prof. Dr. Mais Almumen Objectives: I. Control of Normal Cell Proliferation A. Regeneration and repair B. Stem cells: biology & therapeutic applications C. Cell cycle and regulation of cell replication D. Growth factors and signaling mechanisms II. Mechanisms of Tissue and Organ Regeneration III. Extracellular Matrix and Cell-Matrix Interactions IV. Repair of Tissues After Injury A. Angiogenesis and fibrosis B. Normal wound healing in the skin C. Local and systemic factors influencing wound healing D. Pathologic repair  Definitions: Regeneration vs. Repair REGENERATION: Proliferation of cells and REPAIR: Response to injury involving BOTH tissues to replace lost or damaged cells regeneration and scar formation (fibrosis). and tissues. Normal structure is restored. Normal structure is permanently altered.  What determines regeneration vs. repair with fibrosis? 1. What is capacity of injured cells for regeneration? 2. Is extracellular matrix framework damaged or largely intact? Left: hepatocyte damage with intact matrix: complete regeneration of normal (e.g., after acute hepatitis A) Right: hepatocyte AND matrix damage: some regeneration with reparative scarring (e.g., cirrhosis)  Chronic Injury ➔ Repair (Regeneration with Fibrosis) After years of chronic injury: fibrosis & loss of tissue with inadequate regeneration NORMAL Fibrosis: interstitial and subpleural  Cells proliferate during injury 1-Remannts cell of injured organ- to restore normal structure and function. 2- Vascular endothelial cells – create new blood vessels- give nutrients. 3- Fibroblast –source of fibrosis in scar formation. Mechanisms for regulation of cell populations Proliferation: increase in cell number by replication (hyperplasia) Differentiation: “achievement of adult cell phenotype” Apoptosis: programmed cell death  Stem Cells: Definition Stem cells: normal undifferentiated cells with two features: Self-renewal Can generate differentiated (mature) cells Critical for regeneration of cells in self-renewing tissues Regenerative medicine: therapeutic applications of stem cells to repair damaged tissues Adult stem cell: that do not typically regenerate fibroblast in tissue culture after injury: e.g. heart, brain, skeletal muscle Stem Cells: Origins and Types Embryonic or Multipotent SC: more Adult (somatic) SCs: restricted Pluripotent SC: capable restricted than embryonic SC; capacity to generate certain cell of generating all tissue eventually become “lineage types; thus “lineage-committed” types committed” Stem Cells: therapy Left: “therapeutic cloning” using embryonic stem cells Transfer diploid nucleus from patient’s skin fibroblast into enucleated oocyte ➔ activate oocyte  zygote becomes blastocyst with donor DNA  harvest ESCs, induce differentiation in culture Right: therapy using iPS cells Patient’s skin fibroblast cells cultured transduction with genes encoding transcription factors, cells formed induce differentiation in culture Cell cycle: landmarks and controls Phases of cell cycle G1: presynthetic S: DNA synthesis G2: premitotic M: mitosis 2 ways to enter G1: from G0 or after mitosis Checkpoints (G1/S and G2/M): quality control checks that identify DNA damage and allow DNA repair; if damage too severe, cell is eliminated by apoptosis. Progression through checkpoints is tightly regulated by cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. Defective checkpoints allow cells with mutations to replicate, possibly causing neoplasia Restriction point: rate-limiting step in cell cycle; after this, normal cells are committed to DNA replication. Regulation of cell cycle Cell Type Reprise: Quiescent, Labile, Permanent Cell types: capacity for regeneration Cell type Examples Regenerative capacity Labile Epithelial surfaces Unlimited; characterized (skin, g.i. tract) and by continuous regeneration hematopoietic cells Quiescent Most internal organs Limited, in response to (liver, kidney, stimuli; requires intact endocrine); basement membranes mesenchymal cells (extracellular matrix) for (fibroblast, smooth organized regeneration muscle, vascular) Permanent CNS neurons; Very Little; repaired by skeletal and cardiac replacement with scar muscle cells  Signal Transduction Systems that Require Surface Receptors Chemokines, histamine, serotonin, hormones, many drugs Steroid hormone receptors: in nucleus  Mechanisms Signaling Cell Growth & Replication Polypeptide growth factors Autocrine Endocrine Paracrine Effects on Effects in other Effects on cells nearby same cell organs Growth factors bind to specific receptors on target cells Stimulate transcription Activate genes that regulate of genes that were entry of cells into and through previously silent: protein synthesis the cell cycle: proliferation Signal Transduction Pathways Systems which detect extracellular signals through binding of ligands to specific receptors, initiating an intracellular cascade of events that change gene expression, thus generating a cellular response. Important signal transduction pathways regulating cell growth: (MAP-kinase) (PI3-kinase) (IP3) (cAMP) JAK/STAT Transcription Factors Proteins regulating transcription of genes after signal transduction system transfers information to the nucleus. Contain functional domains DNA-binding domains: recognize short sequences of DNA regulatory domains: for activation or suppression of transcription Transcription factors that regulate cell proliferation: Products of growth-promoting genes: c-MYC, c-JUN Products of cell-cycle-inhibiting genes: p53 Mutations that alter these growth-regulating TFs may result in a dysregulated, uncontrolled proliferation of cells: neoplasia. Liver after Partial Hepatectomy: Compensatory Hyperplasia  In humans, liver remnant doubles in size by one month after a 60% resection After partial hepatectomy, nearly all hepatocytes recruited from G0 into G1; maximal replication and mitosis occurs 24-36 hours after surgery.  Regeneration of Human Liver (living donor) Upper: CT scan before surgery, outlining portion of right lobe to be donated for transplantation Lower: CT scan 1 week after partial hepatectomy; markedly enlarged left lobe due to compensatory hyperplasia (outlined); no regrowth of right lobe 3 phases of response to partial hepatectomy: PRIMING: mediated by cytokines that bind to surface receptors and activate signal transduction pathways PROLIFERATION: primed hepatocytes enter cell cycle, mediated by growth factors and adjuvants GROWTH INHIBITION: poorly understood, but it works at the right time! Most hepatocytes replicate once or twice, then return to G0 Extracellular matrix (ECM) Definition: macromolecules secreted and assembled into an extracelluar supportive network between cells. Two anatomic compartments Interstitial matrix (filling spaces between cells) Basement membrane (closely applied to cell surfaces) 3 Groups of macromolecules in ECM: 1) Fibrous structural proteins: strength and recoil (collagen, elastin) 2) Adhesive glycoproteins: connect cells & matrix (fibronectin, laminin) 3) Gel proteins: resilience and lubrication (proteoglycans, hyaluronan) Functions of ECM Mechanical support: anchors cells; medium for cell migration Control of cell growth: regulate proliferation by ligands binding to surface integrin receptors Modulation of cell differentiation: ECM proteins affect degree of differentiation via integrins Storage of regulatory proteins: growth factors secreted & stored for response to injury and regeneration Scaffolding for regeneration: organized tissue structure requires intact basement membranes  Major Types of Collagen, and its Distribution Collagen Type Tissue Distribution Fibrillar Collagens I Ubiquitous in hard and soft tissues II Cartilage, intervertebral disc, vitreous III Hollow organs, soft tissues V Soft tissues, blood vessels IX Cartilage, vitreous Basement Membrane Collagens IV Basement membranes Adhesion Proteins (“cell adhesion molecules”, CAMs) Function: cell surface receptors that mediate binding with other cells or ECM Types: 1) Adhesive matrix glycoproteins: fibronectin & laminin Fibronectin: binds to both cells and matrix proteins Laminin: glycoprotein in basement membrane; polymerizes with collagen IV 2) Integrins Tissue Repair by Fibrosis The development of a fibrous scar Steps in wound healing: Blood clot forms. Acute inflammation around the edges. Macrophages infiltrate the clot. Capillaries and lymphatics sprout and infiltrate. Myofibroblasts infiltrate and differentiate. Glycoproteins and COLLAGEN are produced Cell population falls, vessels differentiate and are reduced in number. Collagen matures AND CONTRACTS.  Direct observation of fibrous repair 1) Exudate 2) Neutrophils 3) Macrophages and clots. infiltrate lymphocytes are and digest clot recruited  Direct observation of fibrous repair 4) Vessels sprout, 5) Vascular network; 6) Maturity. Cells myofibroblasts collagen much reduced; make synthesised; collagen glycoproteins macrophages matures, contracts, reduced remodels Angiogenesis: 2 Mechanisms Angiogenesis = formation of new blood vessels after infancy (neovascularization) Angioblast: embryonic precursor of endothelial cells, pericytes, vascular smooth muscle cells Mechanism A: sprouting new vessels from pre-existing vessels Mechanism B: angioblast-like endothelial-precursor cells (EPCs) recruited from bone marrow, homing to site of angiogenesis  Tissue Repair: 3 sequential phases Phese1: Inflammation. Phase 2: Deposition of Extracellular Matrix Fibroblasts become less mitotic and more synthetic Collagen synthesis begins 3-7 days post-injury and continues for weeks Growth factors for collagen synthesis similar to fibroblast proliferation. Net collagen deposition depends both on increased synthesis and decreased degradation Phase 3: Maturation and Remodeling Balance between ECM synthesis and degradation ➔ remodeling of tissue Decreasing vascularity and fibroblast proliferation Increasing collagen synthesis and cross-linking: fibrosis gradually acquires tensile strength. Histology of Early & Late Repair: balance of angiogenesis & fibrosis Trichrome histochemical stain (mature collagen stains blue) Early response (3-7 days): Late response (> 4 weeks): fibrosis granulation tissue Mature collagen dominates the proliferating capillaries & fibroblasts picture, with decreased vessel with minimal mature collagen density ECM remodeling by matrix metalloproteinases Matrix Metalloproteinases: family of >20 enzymes that degrade ECM components (interstitial collagenases, gelatinases, stromelysins, membrane-bound MMPs). Fibroblasts synthesize inactive precursors of matrix metalloproteinases. Activation of precursors by plasmin. TIMPs = tissue inhibitors of metalloproteinases (from mesenchymal cells; prevent excessive degradation) 3 Phases of Wound Healing in Skin Wound healing 1- Healing by first intention or primary union 2- Healing by second intention or secondary union Wound Healing by Primary Union (first intention) First intention: Wound damages few keratinocytes and dermal cells, disrupts short segment of basement membrane. Example: surgical incision Result: thin scar  Healing by Secondary Union (Second Intention) Second intention: wounds that create a large defect Healing requires: --more inflammation --larger volume granulation tissue --more collagen deposition --wound contraction Example: deep traumatic abrasion Result: wide scar, often with skin depression or elevation Phases of Wound Healing and Wound Strength Beyond 10 days, accumulation of collagen type I and remodeling control wound healing At 10 days, wound strength is 10% of normal, but increases to 70-80% of normal by 3 months. Therefore, limited activity after surgery is required to avoid separation of wound edges  Systemic Factors Influencing Wound Healing Factor Effect Nutrition Profound effect; deficiencies of protein and vitamin C deficiency inhibit collagen synthesis Metabolic Diabetes mellitus delays healing (insulin status necessary for nucleic acid & protein synthesis) Circulatory Inadequate blood supply slows healing; arterial status atherosclerosis (limiting the inflow of arterial blood) or venous stasis (limiting outflow) Steroid Glucocorticoids inhibit wound healing hormones But can beneficial in certain location ex eye Local Factors Influencing Wound Healing Factor Effect Infection Persistent inflammation; single most important cause of delayed healing Mechanical Early tension applied to wound may separate edges, delaying wound healing Foreign bodies Fragments of metal, glass, wood, bone: prolong the inflammatory response and inhibit healing Anatomic location Sites with rich vascularity (e.g., face) heal faster than sites with reduced vascularity (e.g., foot) Type of wound Sharp incisions (e.g., surgical) heal faster than larger wounds (e.g., traumatic deep abrasion)  Pathologic Wound Repair Deficient scar formation: wound dehiscence: separation of wound edges due to mechanical forces, e.g., vomiting or coughing after abdominal surgical incision wound ulceration: inadequate blood supply (e.g., atherosclerosis) wound necrosis: infection vs. inadequate blood supply. Excessive repair: Hypertrophic scar or keloid Excessive granulation tissue Contracture formation: deformity of tissues due to excessive or exaggerated wound contraction. Deficient Scar Formation: Chronic Ulceration Chronic ulcer associated with venous stasis Chronic ulcer associated with arterial atherosclerosis and compromised inflow  Hypertrophic scar (keloid) Keloid formation has a genetic predisposition; more common in African-Americans Keloid: excess deposition of abnormally thick bundles of collagen in dermis Keloid after ear-piercing Wound Contracture, post-burn Occur in large surface wounds that heal by secondary union Mechanism: network of myofibroblasts at edges of wound, contracting tissues and producing Before After surgical skin excess ECM treatment graft repairs THANK YOU