Tissue regeneration & Fibrosis

Pooja Goswami

Point to be covered

• Normal Wound healing• Regeneration and repair• Stem cells: biology & therapeutic applications• Cell cycle and regulation of cell replication• Growth factors and signaling mechanisms• Extracellular Matrix and Cell-Matrix Interactions• Summary

Normal wound healing

• Normal healing cascade begins with an orderly process of hemostasis and fibrin deposition i.e.

– Homemostasis

– Inflammation

– Proliferation

– Remodeling

Homeostasis: , After injury, Platelets reaches to wound & release clotting factors and GF.

Inflammation: Within 24 hours neutrophils reaches at wound site and remove foreign materials, (phagocytosis). And macrophages releaes more GF & cytokines

Proliferation: Macrophages releases GF & cytokines. Fibroblasts attach to the cables of the provisional fibrin matrix and begin to produce collagen

Remodeling: Process of ECM, collagen remodeling &, degradation takes place

REPAIR: Response to injury involving both regeneration and scar formation (fibrosis). Normal structure is permanently altered.

REGENERATION: Proliferation of cells and tissues to replace lost or damaged cells and tissues. Normal structure is restored.

Definitions: Regeneration vs. Repair

Persistent insult, there is imbalance between deposition and degradation of collagen termed as fibrosis which is irreversible & leads to tissue dysfunction

Fibrosis

Chronic injury

Acute injuryFibrosis

Cell types: capacity for regeneration

Cell type Examples Regenerative capacity

Labile Physical barrier (skin, GI tract, respiratory tract, urinary tract)

Unlimited; characterized by continuous regeneration

Quiescent Most internal organs (liver, kidney, endocrine); mesenchymal cells (fibroblast, smooth muscle, vascular)

Limited, in response to stimuli; requires intact basement membranes (extracellular matrix) for organized regeneration

Permanent CNS neurons; skeletal and cardiac muscle cells

Very Little; repaired by replacement with scar

Entry of quiescent, labile, permanent in cell cycle

Stem Cells: Origins and TypesTotipotent capable of forming almost 200 different cell types in the adult body”

Pluripotent SC: capable of generating all tissue types

Multipotent SC: more restricted than embryonic SC; eventually become “lineage committed”

Niches: microenvironments in which somatic stem cells reside

Autocrine:Cells have receptors for their own secreted factors (liver regeneration)

Paracrine:cells respond to secretion of nearby cells (healing wounds)

Endocrine:cells respond to factors (hormones) produced by distant cells

How these cells respond to other factors

Connective link

Polypeptide growth factors

Autocrine Paracrine Endocrine

Stimulate transcriptionof genes that were

previously silent: protein synthesis

Activate genes that regulateentry of cells into and through

the cell cycle: proliferation

Growth factors bind to specific receptors

on target cells

Effects on same cell

Effects on cells nearby Effects in other organs

Growth Factors & Effects

Symbol (Factor) EffectsEGF (epidermal growth factor)

Mitogenic for keratinocytes and fibroblasts, stimulates keratinocyte migration and granulation tissue formation

PDGF (platelet-derived growth factor)

Chemotaxis and activation of neutrophils, macrophages, fibroblasts and smooth muscle cells; mitogenic for fibroblasts, endothelial, smooth muscle cells. Stimulates angiogenesis, wound contraction, matrix degradation

FGF (fibroblast growth factor)

Family of >10 factors with many effects: macrophage, fibroblast, and endothelial migration (wound repair), mitogenic for fibroblasts and kertinocytes; stimulates angiogenesis, wound contraction, matrix deposition

VEGF (vascular endothelial growth factor)

Family of factors stimulating vasculogenesis (in embryo), angiogenesis (in repair); increase vessel permeability

TGF-β(transforming growth factor-beta)

Pleiotropic (diverse effects according to tissue and injury): chemotactic for WBCs, fibroblasts, myocytes; normally inhibits epithelial proliferation but potent stimulator of fibroplasia and angiogenesis

HGF (hepatocyte growth factor/scatter factor)

Mitogenic for hepatocytes, epithelial cells, endothelial cells; increases cell motility and promotes cell scattering in embryogenesis

Platelet-derived growth factor (PDGF)

• Secreted by platelet macrophages, & myofibroblast

• Having 4 families, PDGF (A,B,C,D)

• During embryogenesis playing a role in organogenesis i.e. CNS, lung, gonad, gut & kidney

• Pericyte coverage of blood vessel circulation in CNS, skin, lung & heart

• Facilitate migration and proliferation of myofibroblast

• Undetectable in normal condition, only in disease condition they release

• Play major role in lung ,heart and skin fibrosis

T (Transforming) GF-beta

• Made in platelets, endothelial cells, lymphocytes & macrophages

• TGFB is a master switch of fibrosis, it is a profibrotic protein.

• Stimulate fibroblast to myofibroblast by SMA phenotypic expression

• EMT ( Endothelial mesenchymal transition) is also TGF mediated

• Fibrogenic, stimulate fibroblast & enhance production of collagen

• It inhibits ECM degradation by releasing TIMP

• Anti-Inflammatory, enhance immune function

Cross talk between PDGF & TGF is responsible for ECM homeostasis

Fibroblast

Myofibroblast

TGF induced ,SMA expression PDGF induced

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.

• Pathways usually involve sequential activation of protein kinases• Important signal transduction pathways regulating cell growth:– Mitogen Activated Protein-kinase (MAP-kinase)– Phosphatidylinositol 3-kinase (PI3-kinase)– Inositol-triphosphate (IP3)

– Cyclic adenosine monophosphate (cAMP)– JAK/STAT (Janus Kinase/Signal Transducers and Activators of

Transcription)

Signal Transduction Systems that Require Surface Receptors

Chemokines, histamine, serotonin, hormones, many drugs

Steroid hormone receptors: in nucleus

Fibroblast to Myofibroblast• Granulation tissue, which allows the replacement of the injured tissue, is

mainly due to fibroblast proliferation, angiogenesis & ECM deposition. Fibroblasts acquire smooth muscle (SM) features characterizing the myofibroblast which is TGF-β induced

• Myofibroblasts are also responsible for the synthesis of enzymes involved in matrix degradation.

The Extracellular Matrix• ECM is a non-cellular component present in all tissue provide

physical scaffolding and maintain homeostasis at wound healing.

• Excess and uncontrolled remodeling of ECM leads to fibrosis (45% world death)

• ECM is a interlocking mesh of sugar & protein

• Synthesized by: Fibroblasts, myofibroblasts, endothelial cells, adipocytes, chondrocytes, osteocytes

• Major components(1)Fibrous structural proteins: Strength and recoil (collagen & elastin)(2) Adhesive glycoproteins: Connect cells & matrix (fibronectin, laminin, integrin)(3) Gel proteins: Lubrication

(GAGS, proteoglycans, hyaluronan)

Collagen is essential protein for the structural integrity of tissues and organs. Excess collagen deposition leads to fibrosis.

Collagens

The collagens

• They are secreted by connective tissue cells, as well as by a variety of other cell types

• Collagens are extremely rich in proline and glycine

• It is composed mainly of glycine (33%), proline (13%), 4-hydroxyproline (9%)

• Proline stabilizes the helical conformation in each α chain

• Glycine allows the three helical α chains to pack tightly together to form the final collagen superhelix

• Part of the toughness of collagen is accounted by the cross-linking of chains via lysine residues

Collagen cross linking

• Collagen cross-linking maintained in regulated & unregulated way by 2 mechanism

– Enzymatically via LOX (lysyl oxidase) activity is essential in development & wound healing.

– LOX knock out mice died due to fragile diaphragm and CVS

– Non- enzymatically through glycosylation & transglutamination or as a result of increase biglycan and proteoglycan

Elsastin

• The elastin protein is composed largely of two types of short segments

– hydrophobic segments, which are responsible for the elastic properties of the molecule

– alanine- and lysine-rich a-helical segments, which form cross-links between adjacent molecules

• Elastic fibers are abundant in organs requiring stretch & recoil: skin, lung, uterus, aorta, ligaments

• Elastin fibers are damaged by aging and sun exposure

• Resistant to most of the proteolytic enzyme.

Adhesion molecules :Fibronectin• FN fibrils provide a dynamic environment for cells & attach both to the cell

and ECM component

• FN secreted as dimer & activation induced by integrin binding

• Integrin recognition requires Arg-Gly-Asp (RGD) cell-binding sequence at FN

• Produced by fibroblasts, endothelial cells, & monocytes

Laminin: Principal matrix gylcoprotein of basement membrane

• Laminin: glycoprotein in basement membrane; polymerizes with collagen IV

• Cross linked formed by 3 related subfamily α, β, γ as heterotimer

• Laminin molecules self-assemble into two-dimensional sheets that associate with type IV collagen sheets and other basement membrane proteins

• Normal epidermal function and re-epithelialization of wounds.

Entactin

Laminin

GAGS

Integrins : Attaches the cell to ECM • Transmembrane receptors, connecting cells to ECM proteins• Made up of α & β subunit, capable to make 24 combination• RGD (Arg-Gly-Asp ) site serve as attachment site for integrin

mediated cell adhesion • This bidirectional signaling responsible for assembly & disassembly of

other molecules• Integrins can activate several signalling pathways independently but

more frequently they act synergistically with GF receptors

Ligands: fibronectin & laminin

Proteoglycans: Organize the ECM• Fill the extracellular interstitial space within tissue (hydrated

gel) which gives ability to tissue to resist compression forces

• Its conc. ↑es in inflammation.

• Proteoglycans composed of GAGS ( glycosaminoglycans chains) linked to core protein

• GAG are of 2 type, binds to receptors that regulate proliferation & migration – Sulfated ( Heparin, chondrotin & keratin)

– Non sulfated (hyaluronic acid)

Free FGF

ECM degrading enzymes

• Serine & theronine proteases– Heperanase

– Cathepsin

– Hyaluronidase

– Matriptase

• Large super family i.e. Metzincins– ADAMs (A disintegrin & metalloproteases)

– ADAMTs (ADAMs with thrombospondin motifs))

– MMPs & TIMPs ( Matrix metalloproteases & tissue

inhibitor of MMPs)

MMPs: Degradation of ECM• MMPs are Zn endopeptidase , zymogens called pro-MMPs, activation

requires Integrin to affect multiple signaling. MMP family include 25 members

• MMP capable of digesting ECM & control migration, proliferation & apoptosis

• The proteolytic activity of MMPs is regulated by TIMPs

• The balanced b/w MMPs TIMPs is critical for ECM degradation remodeling Fibroblast

ECM Sustains the Repair Process• Three types of ECM contribute to the organization, physical properties, and

function of tissue:

• Basement membrane ( Maintain apicobasal polatity of cell)

• Including collagen IV, laminin, entactin, nidogen, and perlecan, a heparan sulfate proteoglycan.

• Act as filters, cellular anchors, and a surface for migrating epidermal cells after injury & also determine cell shape & morphogenesis

• Repository of growth factor chemotactic peptides

• Provisional matrix

• Include fibrinogen, fibronectin, and vitronectin.

• Serve to stop blood or fluid loss & support of migration of monocyte and fibroblast toward wound site

• Connective tissue (interstitial matrix or stromal matrix)

• Formed from fibrillar collagens type I , elastin, fibrillin, GAGs, proteoglycan and fibronectin

• Provides tensile strength to the tissue

Histologic Structure of ECM

1: BM

2: IM

Mechanisms Used by ECM and Growth Factors to Influence Cells

13

2 4

FibrosisInterstitial Fibrosis

RegenerationNormal lung

FibrosisMycordial scar

Acute injury with intact ECMChronic injury with

Damaged ECM Acute injury

How to maintain ECM integrity

Controlled by specific composition & conc. of matrix and post translational

modification i.e. glycosylation, transglutamination & cross linking

• Composition & conc. of matrix affect cell behavior

• Cross linking of collagen affect mechanical function via 2 ways

1. Regulated Enzymatically via LOX (lysyl oxidase) activity

2. Non- enzymatically through glycosylation & transglutamination

Regulated Enzymatically cross-linking of Collagen via LOX activity

• LOX family member catalyzes the cross-linking of collagen through deamidation of lysine residues

• ↑ LOX activity causes ECM stiffness

• Marker of many tumors

Non- enzymatically collagen cross-linking • Through glycosylation & transglutamination or as a result of ↑

biglycans and proteoglycans

• ↑ GAGS leads to stiffen the ECM , Age related disease, degenerative eye, pulmonary fibrosis, arterial stiffening

• ↑ Tissue stiffness is specific and cue for tissue dysfunction & tumorogenesis

Maintenance of ECM organization & orientation

• Maintained by balance between deposition & degradation of component

• Tightly controlled ECM homeostasis sensitive to altered protease level

• MMP regulate not only ECM turn over but also signalling pathway controlling cell growth, inflammation & angiogenesis. ↑ level of MMPs leads to replacement of normal ECM by tumor derived ECM.

How to maintain cell- ECM interaction • How micro-environmental changes influence the cellular response

• BM provide orientation to cell which affect cell behavior

• Loss of apicobasal polarity of BM causes BM disruption considered to be a driver of tumorogenesis

• ↑ in collagen leads stiffness of ECM promotes focal adhesion

ECM : Local Depot

• For the perfect wound recovery & to avoid fibrosis, ECM should be – Meticulously structured,

– Mechanically functional

– Precise organization & orientation

– Cell –ECM interaction (Cellular response)

– Maintained homeostasis of ECM component

Mechanically signalling :matrix elasticity: Intrinsic elasticity ( Stiffness) regulate cell function and modulate cell response to micro-environmant

Mechanically signalling: ECM sequester Growth factor

Matricelluler signalling:Signals through Adhesive molecule ( collegen, elastin, Integrins & GAGS)

ECM interaction signalling: ECM transmit external forces to the cell (Cell –

Maintained ECM remodeling is important to avoid Fibrosis

Pancreatic stellate cells (PSCs)

• PSCs are 4% cell of total pancreatic cell population

• Vit A containing lipid droplets

• On activation lost vitamin A – Function as maintained matrix turn over

– Protective immune function as phagocytic cell

– Work as progenitor cell (in acute injury secrte insulin after differentiation)

– CCK (Cholecystokinin ) induced pancreatic exocrine function

Pancreatic fibrosis

Lost vitamin A

•Cancer cell induced PSCs •PSCs stimulate cancer cell proliferation increased survival of cancer cell by inhibiting apoptosis•ERK 1/2 is the common signalling pathway regulating cancer cell induced PSC proliferation