The document summarizes tissue regeneration and fibrosis. It covers the normal wound healing process and definitions of regeneration versus repair. It discusses stem cells, growth factors, signaling pathways, extracellular matrix components and cell-matrix interactions that are important for regeneration. Fibrosis occurs when there is excessive or uncontrolled deposition of extracellular matrix proteins like collagen. Maintaining the proper balance between deposition and degradation of extracellular matrix components is key to avoiding fibrosis and allowing normal tissue regeneration.

1. Tissue regeneration & Fibrosis
Pooja Goswami

2. 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

3. Normal wound healing
• Normal healing cascade begins with an orderly
process of hemostasis and fibrin deposition i.e.
– Homemostasis
– Inflammation
– Proliferation
– Remodeling

4. Inflammation: Within 24 hours
neutrophils reaches at wound site
and remove foreign materials,
(phagocytosis). And macrophages
releaes more GF & cytokines
Homeostasis: , After
injury, Platelets
reaches to wound &
release clotting factors
and GF.
Remodeling: Process of
ECM, collagen
remodeling &, degradation
takes place
Proliferation: Macrophages releases GF
& cytokines. Fibroblasts attach to the
cables of the provisional fibrin matrix and
begin to produce collagen

5. Definitions: Regeneration vs. Repair
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.

6. Fibrosis
Persistent insult, there is imbalance between deposition and degradation of
collagen termed as fibrosis which is irreversible & leads to tissue dysfunction
Chronic injury
Acute injury
Fibrosis

7. 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

8. Entry of quiescent, labile, permanent in cell cycle

9. Stem Cells: Origins and Types
Totipotent 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”

10. Niches: microenvironments in which somatic stem cells reside

11. How these cells respond to other factors
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

12. Connective link
Polypeptide growth factors
Autocrine Paracrine Endocrine
Growth factors bind
to specific receptors
on target cells
Stimulate transcription
of genes that were
previously silent: protein synthesis
Activate genes that regulate
entry of cells into and through
the cell cycle: proliferation
Effects on
same cell
Effects on cells nearby Effects in other
organs

13. Growth Factors & Effects
Symbol (Factor) Effects
EGF
(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-b
(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

14. 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

15. 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

17. Cross talk between PDGF & TGF is responsible for ECM
homeostasis
Fibroblast
Myofibroblast
TGF induced ,SMA
expression PDGF induced
proliferation

18. 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)

19. Signal Transduction Systems that Require Surface Receptors
Chemokines, histamine, serotonin, hormones, many drugs
Steroid hormone
receptors: in nucleus

20. 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.

21. 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)

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

23. 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 a
chain
• Glycine allows the three helical a 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

24. 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

25. 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.

26. 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

27. 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.
Laminin
GAGS
Entactin

28. 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

29. 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

30. 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)

31. 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

33. 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

34. Histologic Structure of ECM
1: BM
2: IM

35. Mechanisms Used by ECM and Growth Factors to Influence Cells
3 1
2
4

36. Fibrosis
Interstitial Fibrosis
Regeneration
Normal lung
Fibrosis
Mycordial scar
Acute injury with intact ECM
Chronic injury with
Damaged ECM Acute injury

37. 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

38. 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

39. 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

40. 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.

41. 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

42. 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

43. Maintained ECM remodeling is important to avoid Fibrosis
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 –

44. 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

45. Pancreatic fibrosis
Lost vitamin A

46. •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