Robbins Chapter 4: Tissue Repair: Cellular Growth, Fibrosis, and Wound Healing

Robbins Chapter 4:
Tissue Repair: Cellular Growth, Fibrosis, and Wound Healing

Repair of tissues involves regeneration (replacement of damaged cells by cells of the same type) or fibrosis (replacement by connective tissue).
Cell Cycle and Proliferative Potential
G1 – presynthetic
S – DNA synthesis
G2 – premitotic
M – mitotic

Underlying supporting stroma of the parenchymal cells (BM) necessary for organized regeneration.
Nondividing (permanent) cells – left the cell cycle and can’t undergo mitotic division in postnatal life (nerve cells, skeletal and cardiac muscle)
Quiescent (stable) cells – low level of replication (most cells of body)
Continuously dividing cells – have a population of stem cells (epithelium, hematopoietic tissues)

Molecular Events in Cell Growth:
Three signaling methods: autocrine, paracrine and endocrine
Cell Surface Receptors:
· Receptors with intrinsic tyrosine kinase activity: EGF, FGF, PDGF. Dimerization leads to receptor autophosphorylation and interaction with cytolosolic proteins (ras, PI-3 kinase, phospholipase C, src).
· Receptors without intrinsic catalytic activity: cytokine receptor superfamily. Activate cytosolic protein tyrosine kinases.
· G-protein linked receptors: inflammatory chemokines and hormone receptors (epinephrine, glucagon). Have 7 transmembrane loops.

Signal Transduction Systems:
· Mitogen activated protein (MAP) kinase system:
Inactive ras binds GDP/active ras binds GTP: mutant ras binds GTP but can’t hydrolyze it. Permanently “on.”
Ras binds Raf, which phosphorylates MEK (a MAP kinase)
ERK (a MAP kinase) translocates to the nucleus and phosphorylates c-jun and c-fos, activating gene expression
· PI-3 kinase
Generates membrane-associated lipid mediators.
Second messengers recruit and activate intracellular kinases, e.g. Akt.
· Inositol lipid (IP3)
Coupled to tyrosine kinase or G protein linked receptors.
IP3 diffuses into the cytoplasm and associates with Ca+ challens in e.r. membrane
· Cyclic adenosine monophosphate (cAMP)
Coupled through G proteins and generate second messenger cAMP, which activates protein kinase A
· JAK/STAT
Protein kinases in the cytosol (Janus kinases – JAK), phosphorylate downstream proteins (signal transducers and activators of transcription – STATs).

Transcription Factors: c-myc, p53, Rb

Cell Cycle Regulation
Cyclins interact with cyclin-dependent kinases (CDKs)
In G2 cyclin B binds CDK1- necessary to enter M phase. After M over, degraded by ubiquitin-proteasome pathway.
p21 and p27: CDK inhibitors
In G1, cyclin D activates CDKs which hyperphorphorylate Rb, disrupting binding to E2F. Released E2F activates transcription of genes required for S entry.
Checkpoints detect problems in DNA replication, DNA repair and chromosome segregation. Activated checkpoints halt the cell cycle. e.g. p53 activated by DNA damage increases expression of p21, CDK inhibitor.

Growth Inhibition
Contact inhibition
TGF-b binds cell-surface receptors with ser/thr kinase activity; inhib progression to S phase.
Growth Factors
Factor Receptor Characteristics
EGF/TGF-a EGFR (c-erb B1)
PDGF 2 cell- surface receptors a and b 3 isoforms: AA, AB, BB are secreted and biologically active
Stored in platelet a granules.
Causes migration and prolif of fibroblasts, smooth muscle and monocytes
FGF receptor tyrosine kinase Acidic (aFGF/FGF-1) or Basic (bFGF/FGF-2)
Associate with heparan sulfate in angiogenesis, wound repair, development and hematopoiesis
VEGF Promote blood vessel formation
TGF-b Three major isoforms.
Produced by plts, endoth cells, lymphs and macrophages.
Growth inhib to epith cells in vitro and in high concentrations. In vivo, promoted fibroblast chemotaxis and collagen production.
Cytokines Have growth promoting activities to a variety of cells (see Ch.3 or Ch.7)

Extracellular Matrix and Cell-Matrix Interactions
Collagen:
Triple helix a chain gly-x-y repeat. 10% hydroxyproline. Vitamin C needed for hydroxylation of procollagen. Peptidases clip the terminal propeptide chains creating tropocollagen. Failure of aminoprotease to cleave procollagen (because of a collagen mutation) leads to Ehlers-Danlos VII. Lysyl oxidase makes cross-linkages between collagen a chains.
I, II, III – interstitial or fibrillar (most abundant)
IV, V, VI – nonfibrillar or amorphous (in interstitial tissue and BM)
Type Characteristics Distribution
I Fibrillar Skin 80%, Bone 90%
II Fibrillar Cartilage 50%
IV Amorphous Basement Membranes
VIII Amorphous Descemet membrane
IX Cartilage

Elastin: 70 kD protein, present in aorta, uterus, skin & ligaments; 1/3 glycine, with proline and alanine.
Fibrillin: 350 kD; inherited defect responsible for Marfan’s syndrome.
Fibronectin: 450 kD glycoprotein; binds to ECM components (collagen, fibrin, proteoglycans)
Laminin: most abundant glycoprotein in BMs
Integrins: transmembrane glycoproteins, heterodimers; 20 types made from14 a and 8 b chains; cell surface adhesion receptors responsible for leukocyte adhesion. transduce signals to cell interior and form integrin-cytoskeletal complexes with talin, vinculin, a-actinin, tensin and paxillin. “Tensegrity” hypothesis: stress to ECM can be transmitted through integrin receptors to the cell interior.

Matricellular proteins:
· SPARC – tissue remodeling, angiogenesis inhibitor
· thrombospondins – inhibit angiogenesis
· osteopontin – regulates calcification, leukocyte migration
· tenacin – morphogenesis, cell adhesion

Proteoglycans: core protein and glycosaminoglycans
e.g. heparan sulfate, chondroitin sulfate, syndecan (modulators of cell growth)
Hyaluronan: huge disaccharide repeat; binds water forming a viscous gel; provides resilience and lubrication (cartilage)

Fibrosis
Four components to this process:
· Angiogenesis: VEGF (binding to R2 induces endoth cell prolif, R1 stim formation of capillary tubes), angiopoietins (Ang-1 and Ang-2 bind Tie2 receptor),
VEGF expression stim by PDGF, TGF-b, TGF-a and hypoxia.
· Fibroblasts: proliferation triggered by TGF-b, PDGF, EGF, FGF, IL-1, TNF-a
· ECM
· Remodeling: matrix metalloproteases are Zinc containing enzymes that degrade ECM. Inhibited by tissue inhibitors (TIMP).

Wound Healing
By First Intention:
24 hours fibrin, neutrophils, increased basal cell mitoses
24-48 hrs epithelial cell migration, depositing BM
Day 3 macrophages, granulation tissue
Day 5 neovascularization maximal
Week 2 fibroblast proliferation, collagen deposition, blanching
Month 1 scar, regression
Many months maximal strength

By Second Intention: large tissue defect must be filled
Differs from first intention in that:
· inflammatory reaction more intense
· more granulation tissue
· wound contraction (5-10% of original size) from myofibroblasts
Wound strength: 10% at one week, by third month plateau at 70-80% original tensile strength

Factors affecting wound healing:
size, location, type of wound, nutrition, metabolic status, circulatory status, hormones (esp. glucocorticoids), infection, mechanical factors, foreign bodies
Pathologic Aspects: dehiscence or ulceration, hypertrophic scar (keloid), exuberant granulation, aggressive fibromatosis (desmoid), contracture (s/p burn)