First Foundations in Pathology Part 3: Growth and Repair

1. First Foundations in Pathology Part 3: Growth and Repair Paul G. Koles, MD Asst. Prof. Pathology and Surgery Director of Pathology Education Boonshoft School of Medicine at Wright State University

2. Described Menkes “Kinky-hair syndrome” in 1962: Males only, gross abnormalities in hair, deficiency of cupro-enzymes (e.g., lysyl oxidase which crosslinks collagen and elastin), leading to weakened connective tissue. Many untreated patients dies of aortic dissection

4. Overview Part 3 • Control of normal cell growth and regeneration • Extracellular matrix & cell-matrix interactions • Repair by connective tissue (fibrosis) • Wound healing

5. Aaron 17 years later, with Naomi his wife, November, 2008

6. Repair following inflammation: two simultaneous processes • : replacement of injured/necrotic cells by cells of same type, often leaving no evidence of previous injury • : replacement of injured/necrotic cells by connective tissue, leaving a permanent scar (microscopic or macroscopic) Regeneration Fibrosis

7. Control of cellular population Proliferation Differentiation Stem Cells Apoptosis

8. Cell Cycle PHASES of Cell Cycle: G1: presynthetic S: DNA synthesis G2: premitotic M: mitotic G0: quiescent

9. Correlation of Cell Cycle and Tissue Types • Continuously dividing (labile) cells: • Surface epithelium and excretory ducts of glands (skin, gi/gu mucosa, biliary tract, pancreas) • Marrow hematopoietic cells • Stem cells in multiple organs (immature, undifferentiated cells) • Quiescent (stable) cells in G0: • Organ parenchymal cells (liver, kidneys) • Mesenchymal cells (fibroblasts, smooth muscle, endothelium, chondrocytes, osteocytes) • Nondividing permanent cells (can’t re-enter cell cycle) • Neurons, skeletal & cardiac myocytes

10. Control of Passage through Cell Cycle Cyclins • : group of proteins that control cascade of phosphorylation pathways at various points in cell cycle • : surveillance mechanism for ensuring orderly completion of molecular events, sensing problems in DNA replication, DNA repair, and chromosome segregation. If problems identified, progression to next phase of cell cycle can be delayed or stopped. Checkpoints

11. Mechanism: control of cell cycle 2 critical checkpoints to insure normal DNA, regulated by: G1 /S G2 / S Cyclin E / cdk 2 Cyclins A B / cdk 1 2

12. Cell Growth: molecular overview Growth Factors or Cytokines Bind to Cell Surface Receptors Initiate Intracellular Activation of Transcription Factors Promote Changes in Gene Expression (up and down regulation) Resulting in Cell Proliferation or Inhibition

13. Modes of Intercellular Signalling Autocrine Paracrine Endocrine

14. Surface Receptors: 3 classes Receptors without intrinsic tyrosine kinase activity Receptors with intrinsic Tyrosine kinase activity Seven transmembrane (G protein-coupled)

15. Consequences of Receptor Activation • Intrinsic-kinase activity receptors: • Irreversible commitment of cell to enter (proliferative response) • Receptors without intrinsic kinase activity (cytokine superfamily): • Activation cytosolic kinases to mediate functional response (not proliferative) • G-protein coupled (seven spanning) receptors: • Over 1500 receptors identified • Bind various ligands, producing specific intracellular response Cell cycle

16. Signal Transduction by Tyrosine Kinase Receptors Growth factors: coming soon! Clinical application: if mutant ras protein is permanently fixed in active GTP form, what pathologic process may result? Neoplasia

17. Transcription Factors • Definition: intracellular proteins that regulate gene expression, thereby controlling cell growth • Specific domains in transcription factors: • : permits factor to bind specifically to short sequences of DNA • : allows factor to increase transcription of DNA • :allows factor to decrease transcription of DNA • Transcription factors known to be operative in malignant neoplasms: • Growth promoting: c-MYC and c-JUN • Cell cycle inhibiting (tumor suppressor gene): p53 DNA-binding Activation Repression

18. Growth Factors • Definition: proteins that bind to cell surface receptors with generating cascade response that signals cell to enter S-phase (cell division). • These factors can also modulate cell functions: locomotion, contractility, differentiation, etc. Intrinsic tyrosine kinase activity

19. FACTOR EGF = epidermal TGF-a = transforming VEGF=vascular endothelial PDGF= platelet-derived FGF= fibroblast FGF-1=acidic FGF-2=basic EFFECTS Mitogenic for epithelium & fibroblasts Mitogenic for hepatocytes  Mitogenic for endothelial cells Mitogenic for monocytes, fibroblasts, smooth muscle cells; activates neutrophils Angiogenesis, wound repair (mitogenic for both fibroblasts and keratinocytes) Major growth factors / effects

20. Tissue Regeneration Liver from living donor before transplantation, outlining right lobe to be used for grafting into recipient Liver of donor one week post-partial hepatectomy, showing marked growth of left lobe (compensatory hyperplasia) without regrowth of right lobe. Why didn’t right lobe regrow also?

22. Extracellular matrix 1 • Definition: macromolecules outside cells, formed by local secretion and assembled into network surrounding cells • Functions: • Sequester H2O for turgor; minerals for rigidity • Reservoir for growth factors • Scaffolding within which cells adhere, migrate, and proliferate

23. Extracellular matrix (ECM) 2 • Groups of macromolecules in ECM: • Fibrous structural proteins: 2 major families are: • Adhesive glycoproteins • Gel proteins in intercellular junctions and cell surfaces: proteoglycans & hylauronic acid Elastins Collagens

24. Extracellular matrix (ECM) 3 • Macromolecules of ECM assemble into two types of organizational structure: • : fills spaces between cells • : closely associated with cell surfaces Interstitial matrix Basal membranes (basement membranes)

26. Collagen: summary of major types Skin (80%), bone (90%), tendons Genetic deficiency of type IV in: Basal membranes Alport syndrome

27. Collagen synthesis Nutrient required for hydroxylation of alpha chains: Vitamin C Deficiency of this nutrient causes poor wound healing in disease called: Scurvy Inherited disorders of collagen synthesis, leading to defective fibers: Ehlers-Danlos syndromes

28. Elastic Fibers • Definition: fibers capable of stretching and recoiling to original size • Present in tissues requiring elasticity: • Structure: • Central core protein: • Peripheral microfibrillary network: • Inherited defect in synthesis of peripheral microfibrillary network: abnormally weakened elastic fibers. Syndrome? Skin, lung, uterus, ligaments, large blood vessels Elastin Fibrillin Cystic medial degeneration--aortic dissection Associated Vascular disease? Marfan syndrome

29. Adhesion molecules 1 Function: attach cells to ECM matrices; 2 glycoprotein chains held together by disulfide bonds; produced by fibroblasts, endothelial cells, & monocytes Fibronectin

30. Adhesion molecules 2 Most abundant glycoprotein in basement membranes; it spans basal lamina and binds to both cell surfaces and ECM components: Laminin

31. Adhesion molecules 3 Transmembrane glycoproteins with alpha and beta chains that bind to fibronectin, laminin, & collagen. This family of surface receptors mediate attachment of cell membranes to ECM: integrins These also mediate adhesion of which cell type to endothelium? neutrophils

32. Summary: interactions cell-ECM laminin Major EC structural protein: Collagen fibronectin integrins Fig. 3-16, Pathologic Basis of Disease, 7th ed, Elsevier 2005

34. Overview: Repair after injury ACUTE AND CHRONIC INFLAMMATION Damage to parenchymal cells and interstitial framework Regeneration of parenchymal cells whenever possible Replacement of non-regenerated damaged tissue by what? Fibrosis

35. Fibrosis (fibroplasia) • Four components: • : formation new blood vessels • of fibroblasts into damaged tissue • of extracellular matrix • Organization fibrous tissue = angiogenesis Migration and proliferation synthesis remodeling

36. Sequence of events in repair Permanent result: 24 hrs: proliferation of fibroblasts & endothelial cells Within 3-5 days: Mature scar Granulation tissue Little mature collagen Proliferation of young fibroblasts blue-staining collagen (trichrome stain) New capillaries

37. Angiogenesis • Definition: pre-existing vessels send out capillary sprouts to form new vessels • cf. vasculogenesis: the primitive vascular network established during embryogenesis • Clinical importance: • Repair post-inflammation • Formation collateral circulation (post-MI) • Support growth of neoplasms (therapeutic implications)

38. Angiogenesis: 2 mechanisms

39. ECM proteins affecting angiogenesis • Integrins: formation and maintenance new vv. • Matrix proteins which destabilize cell-matrix interactions, promoting angiogenesis: • Thrombospondin • SPARC • Tenascin C • Proteases that remodel matrix • Plasminogen activators • Matrix metalloproteinases • Fragment of collagen that inhibits endothelial proliferation and angiogenesis, with therapeutic application in neoplasia? endostatin

40. Fibrosis (fibroplasia) • Emigration and proliferation of fibroblasts at injury site, triggered by multiple growth factors produced by cells in granulation tissue, most important of which is: • ECM deposition by fibroblasts: fibrillar collagen synthesis enhanced by growth factors and cytokines, thus converting TGF-beta Granulation tissue Into a Fibrous scar

42. Tissue remodeling • Conversion granulation tissue into scar involves changes in composition of ECM. • : enzymes which degrade ECM components for remodeling. These enzymes are dependent on ions for activity. Matrix metalloproteinases zinc

43. Wound Healing Healing by first intention Healing by second intention

44. Summary: phases of wound healing Wound tensile strength: 10% of normal at 7 days; 70-80% of normal at 3 months

46. Factors influencing wound healing Infection • Local Factors • : most important single cause of delay • Mechanical: too early motion can delay • Foreign bodies: may impede or cause abscess • Location: speed of healing proportional to richness of blood supply: face > trunk > extremities • Type of wound: primary intention heals faster than secondary intention

47. Factors influencing wound healing Nutritional deficiency • Systemic factors: • : first of two most important factors. Most important deficiencies (2): • : second of two most important factors. Arterial or venous insufficiency commonly delays healing. • : iatrogenic delay of healing by blunting the normal inflammatory/repair response Protein and vitamin C Inadequate blood supply Steroid therapy

48. Pathologic complications of healing Contracture : exaggeration normal contraction of wound, resulting in deformity (palms, soles, anterior thorax). Common after burns. Deficient granulation tissue/ scar formation: • : rupture of wound, usually due to increased mechanical pressure or inappropriate movement • : usually due to arterial insufficiency caused by atherosclerosis; venous stasis also can contribute. Dehiscence Ulceration/poor healing

49. Pathologic complications 2 Keloid (hypertrophic scar) • Excessive formation of repair components: • Excessive granulation tissue • Desmoid tumor (aggressive fibromatosis) • Best viewed as low grade neoplasm with stubborn tendency for recurrences

50. Fibrosis: Summary