3. Contents : session 1
• Introduction
• ECM Origin
• Forms of ECM
• Components of ECM
• Function of ECM
• Collagen
• Elastin
• Fibrillin
• Fibronectin
• laminins
• Integrins
• Proteoglycans & GAGs
• Conclusion
• Bibliography
4. Contents – Sesssion 2
• Degradation of Collagen
• GAGs – types in periodontium (detail)
• MMPs
• TIMPs
• Future applications
• Abnormalities of ECM in diseased state.
• Conclusions
• Reference
5. Collagenases
• It was only 4 years after Gross and Lapiere (1962) discovered the tadpole
collagenase (MMP-1) when the human gingival collagenase was identified by
Fullmer et al., (1966).
• Collagenases have been claimed to occur in animal tissues and fluids in at least
three molecular forms:
a) latent enzvme,
b) free enzyme, and
c) collagen-bound enzyme.
Collagenases and Collagen Degradation STEPHEN M. KRANE THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 79:83s-868, 1982
6. Degradation of collagen
In the course of physiological remodeling and in pathological states, the collagen of
the extracellular matrix is degraded and resorbed.
The possible mechanisms of these resorption processes, which have been extensively
investigated, are not the same for the different collagen types and are subject to
specific tissue regulation.
Considerable evidence has been obtained which indicates, under most circumstances,
that the degradation of the interstitial collagens is mediated by specific collagenases
which act near neutral pH and are capable of cleaving the molecules at specific loci in
the polypeptide chains across the helix
PATHOLOGY OF COLLAGEN DEGRADATION Ruy Pdrez-Tamayo American Journal of Pathology Vol. 92, 511-51 August 197
7. • The action of these collagenases results in solubilization of the molecules at the
surface of the fibrils and eventual dissolution of the fibrils.
• It is possible that proteases other than the collagenases have a synergistic role in
degradation of the fibrils (and fibers) by cleaving peptide bonds in the
telopeptide regions adjacent to sites of intermolecular crosslinks.
• Elastase-type enzymes may not only cleave within telopeptide regions of types I
and III collagens but are also capable of breaking peptide bonds within the type
III collagen helix
8. • A potential role for elastases in degrading type IV and type V collagens has also
been proposed.
• Enzymes with substrate specificity for types IV and V collagen have been
identified in polymorphonuclear leukocytes as well as macrophages.
• Type V collagen has also been shown to be susceptible to cleavage with purified
thrombin, at temperatures >34°C, and it is possible that thrombin could play a
role in degradation of this pericellular collagen during some types of tissue
injury.
9. • The more specific animal collagenases have little action on the type IV and V collagens
• Although there have been examples of processes in which collagen fibrils appear to be
phagocytosed and have been identified within cells, it is probable that this intracellular
digestion is not the critial event in conditions of collagen resorption.
• It is likely that collagen resorption is an important process during growth of most organs in
which the matrix must be removed to allow for increments in size.
• Once adulthood is reached, however, collagen resorption takes place very slowly in most
tissues, with the exception of bone, where remodelling is a continuous process.
10. • Another exception is the involuting uterus
• A considerable proportion of the increase in uterine weight during pregnancy is
accounted for by collagen deposition and the decrease in mass of the uterus
after delivery of the fetus is therefore associated with collagen removal which
correlates with tissue levels of collagenase.
• In bone, however, correlations between mineralized tissue resorption and levels
of collagenase and/or pro collagenase have not been established.
11. DEGRADATION OF COLLAGEN AND
PROTEOGLYCAN BY MACROPHAGES AND
FIBROBLASTS
• Connective tissue remodeling during chronic inflammation is likely to be due
mainly to the action of infiltrating macrophages and fibroblasts.
• In these situations, macrophages are often considered responsible for tissue
breakdown, and fibroblasts for repair by fibrosis and scar formation.
• However both types of cell may secrete enzymes such as collagenase and
proteoglycan-degrading neutral proteases and thus possess degradative
capacities.
DEGRADATION OF COLLAGEN AND PROTEOGLYCAN BY MACROPHAGES AND FIBROBLASTS RUTH LAUB, GHISLAINE
HUYBRECHTS-GODIN, CHANTAL PEETERS-JORIS and GILBERT VAES Biochimica et Biophysica Acta, 721 (1982) 425-433
12. • macrophages produce a factor, a monokine which
stimulates collagenase production and collagen
degradation by fibroblasts, and that the production of
that monokine was itself stimulated by soluble
products (lymphokines) released by lymphocytes upon
stimulation by mitogen.
• It is thus possible to construct a pathway which
indicates the importance of the fibroblasts as an
effector cell in connective tissue destruction resulting
from chronic inflammations, notably those of
immunological origin, as it leads to collagen de-
gradation by fibroblasts via a lymphokine-
macrophage-monokine relay.
13. There are several pathological states in which
collagenolysis is obvious in vivo and can be
demonstrated in cultures of the involved tissue
• Some conditions associated with collagen degradation and collagenase production
• 1. Corneal ulceration (e.g., alkali burns)
• 2. Epidermolysis bullosa
• 3. Cholesteatoma
• 4. Chronic periodontal disease
• 5. Inflammatory joint disease (rheumatoid arthritis, psoriatic arthropathy)
• 6. Other destructive joint disease (pigmented villonodular synovitis, hemophiliac
arthropathy)
14. Diseases of Collagen Degradation :
Excessive
• Corneal ulcers
• Rheumatoid arthritis
• Other arthritis
• Epidermolysis bullosa
• Periodontal disease
• Cholesteatoma
• Tumor invasion
• Paget's disease of bone Pulmonary emphysema
• Wound healing
15. Deficient
• Fibrosis
• Cirrhosis of the liver
• Scleroderma
• Osteopetrosis
• Pulmonary fibrosis
• Diabetes mellitus
Osteopetrosis (Albers-
Schonberg disease, marble
bone disease)
16.
17. Proteoglycans
• The proteoglycans are a large and
diverse group of glycoproteins
that are widely distributed in
mammalian tissues.
• A proteoglycan consists of one or
more sulfated polysaccharides
covalently linked to a protein
core.
18. • The variation of saccharide
composition, length and sulfation as well
as protein core composition affords an
exceptional diversity of structure and
function for proteoglycans.
• The roles ascribed to proteoglycans in
the extracellular matrix have included
everything from physical maintenance of
the matrix to cell-cell interactions.
19. Glycosaminoglycan
• The polysaccharide, called a
glycosaminoglycan, is a linear polymer
consisting of repeating disaccharides
containing an N-acetyl hexosamine and a
hexuronic acid or hexose.
• Glycosaminoglycans are highly anionic
chains that can vary in size from a few
disaccharide units to several thousand
dissacharide units.
20. • The glycosaminoglycans are
represented by several species,
including chondroitin-4-sulfate,
chondroitin-6-sulfate, heparan
sulfate, heparin, hyaluronic acid
and keratin sulfate
21.
22. Hyalarunan• Made up of repeating units of GlcUA
and GlcNAc
• It tends to have enormous carbohydrate
chain
• Not covalently attached to a core protein
• The carbohydrates are not sulfated
• It is a an important constituent of joint
fluid, vitreous body, cartilage
• Important in wound healing
• Hyaluronidase an enzyme secreted by
some bacteria helps with their invasion
of tissue
23. Chondroitin sulfate
• Repeating unit of GlcUA and GalNAc
• Attached to a core protein through xyl-
serine
• Sulfated carbohydrates
• Tends to have shorter polymers
• Provides tensile strength to cartilage,
tendons, ligaments and walls of aorta
24. Dermatan sulfate
• Made up of repeating IdUA and GalNAc.
May also contain GlcUA
• Attached to a core protein through
xylserine
• Widely distributed throughout the body.
Contributes to the pliability of the skin.
25. Keratan Sulfate (KS) I and II
• Repeating units of Gal and GlcNAc
• KS I is attached to core protein through
GlcNAc-Asp
• KS II is attached through GalNAc-Thr .
• Present mainly in cornea, cartilage, bone.
• In CNS they have role in development
and formation of glial scars.
26. Heparin
• Repeating units of GlcN (mostly sulfated but
sometimes acetylated) and either of the
gluconic acids mostly iduronic acid
• Heparin is linked to its core protein (mostly
glycine and serine) through a bond with serine.
27. • Heparin is mostly intracellular unlike rest of
GAGs-in mast cells
• Involved in anticoagulation by binding
factor IX, XI and Plasma antithrombin III
• Binds lipoprotein lipase in endothelial cell
walls and puts them into circulation.
28. Heparan sulfate
• Made up of GlcN and uronic acid
predominantly glucoronic acid
• Attached to its core protein through
xylserine
• Role in developmental processes,
angiogenesis, blood coagulation and
tumor metastasis.
29.
30. Degradation of GAGs and Inborn Errors
of Metabolism
GAGs are degraded by specific
lysosomal enzymes including exo and
endoglycosidases, sulfratases.
31. Inborn error of metabolism
affecting any of these enzymes
results in accumulation of
GAGs in lysosome mucu-
polysaccharidoses Eg. Hurler’s
and Hunter’s syndrome
Hurler’s disease : Abnormal bones in the spine, Claw hand,
Cloudy corneas, Deafness, Halted growth, Heart valve problems,
Joint disease, including stiffness, Intellectual disability that gets worse
over time in severe MPS I.
32.
33.
34. Linkage of GAGs to protein core by
specific trisaccharide linker
35. Synthesis of
Proteoglycans
Starts with core protein synthesis from
ribosomes on the RER
The addition of GAGs takes place in the
Golgi Apparatus
The addtions of the GAGs to their core
protein is of three types
1. O-glycosidic linkage between xylose
and serine
2. O-glycosidic linkage between GalNAc
and serine eg in Keratan sulfate II
3. N-glycosylsamine bond between
GlcNAc and asparagine
36. Elongations
The units in the saccharide chains are elongated in alternating
acidic/amino sugars, donated from UDP (uridine diphosphate) derivatives
through specific glycosyl transferases
Further Modifications
Epimerization of glucoronic acid to iduronic acid catalysed by
epimerases
Sulfation of the amine sugars are catalysed by sulfotransferases.
37. Function of Proteoglycans
organize water molecules
- resistent to compression
- return to original shape
- repel negative molecules
occupy space between cells and collagen
- high viscosity
- lubricating fluid in the joints
38. cell migration and
adhesion
- passageways between cells
anchoring cells to matrix
fibers
specific binding to other
macromolecules
link to collagen fibers
- form network
- in bone combine with
calcium salts (calcium
carbonate, hydroxyapatite)
39.
40. Versican Versican, a principal proteoglycan of loose
connective tissue.
The role of versican in cell adhesion,
migration, and proliferation has been
extensively studied.
Versican is often considered an anti-
adhesion molecule.
41. The protein core contains 14
attachment sites in the central
region for glycosaminoglycans.
This molecule has been shown
to be secreted by fibroblasts.
42. Decorin
• Previously called PG-S2, dermatan sulfate PG-I1 and
PG-40, this small proteoglycan is approximately 120 kDa
and contains only one glycosaminoglycan attachment site
on the core protein.
• Depending on its location in the body, the polysaccharide
is either a dermatan sulfate or a chondroitin sulfate
glycosaminoglycan.
43. • Within the connective tissue, decorin has been identified with collagen fibrils
and fibronectin.
• This protein is a component of connective tissue, binds to type I collagen
fibrils, and plays a role in matrix assembly.
44. Biglycan
• Another relatively small extracellular proteoglycan
similar to decorin is biglycan.
• Although biglycan is strongly bound in the extracellular
matrix, it has no demonstrable collagen- binding activity
• The currently proposed biglycan functions appear to be
dependent on the particular microenvironment and on
the organ in question. It plays a major role in gene
and protein structure, localization,
expression, regulation, and function.
45. Syndecan • Syndecan is a transmembrane proteoglycan that
binds extracellular matrix proteins.
• The structural polymorphism of this cell-
associated proteoglycan depends on whether the
cells are in single layers or are stratified.
• The syndecan-1 protein functions as an integral
membrane protein and participates
in cell proliferation, cell migration and cell-
matrix interactions via its receptor for
extracellular matrix proteins. Syndecan-1 is a
sponge for growth factors, with binding largely
via heparan sulfate chains.
46.
47. In oral tissues, syndecan has been reported to be developmentally regulated in
mesenchyme during tooth organogenesis.
The actions of syndecan in the adult periodontium are currently unknown, but
it may function during wound healing as a requisite or obligate protein for cell
adhesion and growth factor binding.
48. MMP’s (Matrix metalloproteinases )
• Matrix metalloproteinases (MMPs) are members of an enzyme family that
require a zinc ion in their active site for catalytic activity. MMPs are critical for
maintaining tissue allostasis.
• MMPs are active at neutral pH and can therefore catalyze the normal turnover
of extracellular matrix (ECM) macromolecules
• Members of the MMP family include the “classical” MMPs, the membrane-
bound MMPs (MT-MMPs) the ADAMs (a disintegrin and metalloproteinase;
adamlysins) and the ADAMTS ( a disintegrin and metalloproteinase with
thrombospondin motif).
49. • There are more than 20 members in
the MMP and ADAMTS family
including the collagenases,
gelatinases, stromelysins, some
elastases and aggrecanases.
• Adamlysins are membrane-bound
MMPs that also degrade aggrecan,
but more importantly, one ADAM
family member (i.e.ADAM-17) is a
tumor necrosis factor-alpha (TNF-
alpha)-converting enzyme (TACE)
that activates pro-TNF-alpha.
50. • Most of the MMPs are synthesized as inactive latent
enzymes.
• Conversion to the active enzyme is generally mediated
by activator systems that include plasminogen activator
or the pro-hormone convertase, furin.
• MMP activity is regulated by a group of endogenous
proteins, called, tissue inhibitor of metalloproteinases
(TIMPs) that bind to active and alternative sites of the
activated MMP.
51. • MMPs are a family of structurally related
but genetically distinct enzymes that
degrade extracellular matrix (ECM)and
basement membrane(BM) components
• This group of 23 human enzymes is
classified into collagenases, gelatinases,
stromelysins, membrane-type MMPs and
other MMPs, mainly based on the
substrate specificity and molecular
structure.
52. • MMPs are involved in physiological
processes such as tissue development,
remodelling and wound healing (Uittoet al,
2003), and play important roles in the
regulation of cellular communication,
molecular shedding and immune functions
by processing bioactive molecules including
cell surface receptors, cytokines, hormones,
defensins, adhesion molecules and growth
factors.
53. • MMP activity is controlled by changes in the delicate balance between the
expression and synthesis of MMPs and their major endogenous inhibitors,
tissue inhibitors of matrix metalloproteinases (TIMPs).
• The catalytic competence of MMPs is controlled through the activation of
proenzymes, and the inhibition of the activation or activity by TIMPs (Uitto et
al, 2003)
54. Regulation Of MMP’s Synthesis
• MMP gene expression is regulated principally by transcription.
• Post-transcriptional regulation exemplified by alterations in MMP mRNA
stability can also modulate MMP synthesis .
• These cellular processes are responsible for up-regulation (positive regulation)
or down-regulation (negative regulation) of MMP synthesis.
• receptor-activated intracellular protein kinase signaling pathways that control
MMP promoter activity.
55. MMP activation and inhibition
• MMPs are mostly produced in latent, non-active form, and activation through a
so-called cysteine switch is required for the enzyme function.
• In most cases, activation involves removal of the prodomain, resulting into
lower molecular weight active forms (reviewed by Nagase, 1997), although the
most recent studies indicate that in vivo, the proforms of at least certain MMPs
may also be active while in full-size or in complex with certain proteins
(Bannikov et al, 2002; Fedarko et al, 2004)
56. • Non-proteolytic activation of MMP proforms can be accomplished in vitro, e.g.
by SH-reactive agents, such as mercurial compounds, detergents, gold(I)-
compounds, or oxidation.
• Proteolytic activation can be attained by several proteolytic enzymes, including
serine proteinases together with other MMPs
• MMP activation and activity can be controlled by inhibition in several
ways: proteolytic degradation and inactivation, non-specific endogenous
inhibitors such as a2-macroglobulin, and especially by specific tissue
inhibitors of MMPs, TIMPs (reviewed by Brew et al, 2000). Currently, four
TIMPs (TIMP 1–4) are known to be expressed in vertebrates.
57. • TIMPs inhibit MMPs by forming 1:1 stoichiometric enzyme-inhibitor
complexes. TIMP-1, -2 and -4 are secreted, while TIMP-3 is sequestred to the
ECM. The substrate specificity of TIMPs varies (Brew et al, 2000)
58. MMPs in the pathogenesis of
periodontitis and peri-implantitis
• Since putative periodontopathogenic bacteria are always present in periodontitis, it was
logical to assume that collagenases in periodontal disease would originate from microbial
sources (Sorsa et al, 1987)
• Sorsa et al (1988) demonstrated that the major collagenase in periodontitis was human
collagenase-2, MMP-8, accompanied by MMP-9 (Sorsa et al, 1995).
• Previously it was thought that the expression and release of MMP-8 was limited to
neutrophils (Uitto et al, 2003), but at present it is clear that many non PMN-lineage cell
types present in the normal and diseased human periodontium (gingival sulcular epithelial
cells, fibroblasts and endothelial cells, monocyte/ macrophages and plasma cells) can be
induced to express distinct MMPs including MMP-8 (Hanemaaijer et al, 1997; Tervahartiala et
al, 2000; Wahlgren et al, 2001; Kiili et al, 2002; Prikk et al, 2002).
59. • It also early became evident that TIMPs are not sufficient to down-regulate the
pathologically elevated MMPs (Ingman et al, 1996). Therefore, the possibility of
selective MMP inhibition by synthetic inhibitors as a method to avoid or limit
the periodontal tissue destruction was advanced.
• MMP-8, -9, -13 and -14, all considered as (at least potentially) important in
periodontitis, are far more sensitive to inhibition by doxycycline and chemically
modified non-antimicrobial tetracycline (CMT)-derivatives than MMP-1 and -2
(Golub et al, 1998)
60. • Further work demonstrated that the inhibition can be obtained by
therapeutically attainable serum concentrations of these drugs (Golub et al,
1998).
• While MMP inhibition is a promising approach in periodontal disease
treatment, further work including other approaches need to be evaluated
(reviewed by Reddy et al, 2003).
• for a chair-side test for diagnosis and monitoring of periodontal diseases MMP-
8 is a potential candidate (Sorsa et al, 1988, 1999; Kiili et al, 2002).
61. • A chair-side dipstick test for MMP-8 that allowed the development of a novel
sensitive, specific, rapid and practical immunological chair-side dip-stick test for
MMP-8 in GCF and peri-implant sulcular fluid (PISF) (Kivela¨ -Rajama¨ ki et al,
2003 ; Ma¨ ntyla¨ et al, 2003).
• The test, bearing resemblance to pregnancy home test kits , can be performed
by a dentist without specific equipment, and measures the GCF MMP-8 level in
5 min (Ma¨ ntyla¨ et al, 2003).
62. • It differentiates healthy and gingivitis sites from periodontitis sites (Ma¨ ntyla¨ et al, 2003),
and reduction of GCF MMP-8 levels can be observed after successful periodontal
treatment (Ma¨ ntyla¨ et al, 2003).
• GCF MMP-8 level testing is a very useful tool to monitor the beneficial effects of
adjunctive sub-antimicrobial doxycycline-medication for periodontitis patients (Emingil et
al, 2004).
• This rapid point of-care test developed for periodontitis is obviously a useful tool also for
monitoring of peri-implantitis (Sorsa et al, 1999; Kivela¨ -Rajama¨ ki et al, 2003).
63. TIMP’s
• Tissue inhibitors of metalloproteinases (TIMPs)
are endogenous inhibitors of these
metalloproteinases and are consequently
important regulators of ECM turnover, tissue
remodeling and cellular behavior.
• Designated as “tissue inhibitor of
metalloproteinases” or “TIMP” as it inhibited not
only collagenases, but also gelatinases and
proteoglycanase (now called matrix
metalloproteinase 3/MMP-3)
64. • All four TIMPs inhibit MMPs, but with affinities that vary for different inhibitor-protease
pairs. Among the four TIMPs, TIMP-3 has the broadest inhibition spectrum as it inhibits
several members of the ADAM and ADAMTS families. It also differs from the others in
being tightly bound to the extracellular matrix.
• In addition to their metalloproteinase inhibitory activity, TIMPs have various biological
activities such as promoting cell proliferation, anti-angiogenic, pro- and anti-apoptotic and
synaptic plasticity activities, many of which are independent of metalloprotease inhibition
65. Functional specialization for
metalloproteinase inhibition
• The four human TIMPs are, in general terms, broad-spectrum inhibitors of the 23
MMPs found in humans, but there are some differences in specificity among them.
• TIMP-1 is more restricted in its inhibitory range than the other three TIMPs, having a
relatively low affinity for the membrane-type MMPs, MMP-14, MMP-16, and MMP-24
as well as for MMP-19.
• Also, there are some relatively subtle differences between the affinities of different
TIMPs for other MMPs. For example, TIMPs-2 and -3 are weaker inhibitors than
TIMP-1 for MMP-3 and MMP-7, contrasting with their affinities for other MMPs
66. • TIMP-3 is unique among the mammalian TIMPs in inhibiting a broader array of
metalloproteinases including several members of the ADAM and ADAMTS families .
• Other TIMPs have limited inhibitory activities for ADAMs: TIMP-1 and TIMP-2
inhibit ADAM10 and ADAM12, respectively.
• TIMP-3 and N-TIMP-4, but not full-length TIMP-4, inhibit ADAM17.
• TIMP-4 was also reported to inhibit ADAM28.
67. • It has now been established that TIMP-3 is an effective inhibitor of ADAM10, 12, 17, 28
and 33, and ADAMTS-1, -2, -4 and -5. ADAMTS-4 and -5 are also called aggrecanases 1
and 2, respectively. The inhibition of ADAM17 and aggrecanases by TIMP-3 appears to
be important in regulating inflammatory processes, affecting the progression of disease
processes such as cancer, rheumatoid arthritis and osteoarthritis
70. ECM – future prospects
• genomics, proteomics, glycomics, metabolomics, and signalomics are all
legitimate “omics” under intense study; basically, such systems analyses illustrate
how molecular components interact to create dynamic functional activities.
• Potentially, a systemic biology strategy can help decipher the pathogenesis of
complex diseases and provide insight into tailoring medications that interfere
with critical pathways, thereby stopping disease progression.
• Ideally, early interception of disease states before significant irreversible damage
occurs may become possible by developing tools that identify individuals at risk
for specific diseases.
71. • Initially, genomic analyses can be implemeted; later, as other tools such as
proteomics and glycomics come on stream, they will provide important ancillary
information. In this way, individual profiles can be made at one’s request, similar
to contemporary genetic testing to detect hereditary diseases.
• Progressive developments in technology will make this idea into reality in the
near future, opening a new dimension in disease prevention.
72. • Time and spatially resolved matrisome : analysis of the component of ECM
can reveal novel or unsuspected component of ECM characteristics of a
diseased state or casual of a diseased state.
• Development of high throughput approaches to map post-translational
modifications of ECM proteins : ECM can undergo proteolytic cleavage and
release fragments as a part of their psychological (or pathological) turnover.
Increased ECM degradation is the hallmark of pathologies such as
osteoarthiritis, fibrosis and cancers.
73. Translational applications
• ECM proteins as biomarkers : the composition of ECM can lead to identification
of novel biomarkers for other diseases in addition to cancer.
• ECM proteins are favorable biomarkers for immunohistochemically based assays
since they are readily accessible, abundant and laid down in characteristic patterns.
• Once identified disease based ECM proteins, or protein isoforms could serve as
anchors for imaging molecules (e.g. fluorescent molecules , radiotracers) or
therapeutic (drugs, cytokines and radioisotopes) that could be coupled to an anti-
ECM antibody, on models of system developed in Nori Lab.
• Proteolytic fragments of ECM proteins can be released in body fluids and could be
used as read-outs for disease progression or treatment efficacy.
74. • ECM proteins used as therapeutic targets : there are successful precendents
for inhibiting integrins to treat thrombosis, auto immune disease and
inflammatory diseases.
• However , there are also unsuccessful examples, like the use of MMP’s in
clinical trails for cancer patients.
• ECM and regenerative medicine : it is a fundamental component of stem
cell niches. In fact, many stem calls markers are ECM receptors. (e.g. CD49a-f
are integrins α 1-6, CD29 is the integrin β1 & Lgr5 is an R-spondin receptor )
and laminins have been demonstrated to affect pluripotency and stem cell
differentiation.
The extracellular matrix : Tools and insights for the “omics” era Naba A. et al. 2015
75. Conclusion
• The great success in understanding the building blocks of cells and the ECM
emerged when a reductionist strategy was used to identify, isolate, and
characterize molecular components.
• with our understanding of periodontal homeostasis, the etiology and
progression of periodontal diseases and the regeneration of the periodontium
we can say that the ECM provides the microenvironment the information feed-
back loop for cellular functions.
76. Bibliography…
• Structure and function of MMP and TIMPs Nagase et. Al cardiovascular research 69
(2006) 562-73
• Pathology of Collagen Degradation : A Review Ruy Pdrez-Tamayo
• Degradation of collagen and proteoglycan by macrophages and fibroblasts Laub et.al
BiochimicaetBiophysicaActa, 721 (1982) 425-433
• Extracellular Matrix Degradation and Remodeling in Development and Diseases Lu
et.al September 14, 2011 2011; doi: 10.1101
• Matrix metalloproteinases (MMPs) in health and disease: an overview Charles J.
Malemud Frontiers in Bioscience 11, 1696-1701, May 1, 2006
77. • The tissue inhibitors of metalloproteinases (TIMPs): An ancient family with structural
and functional diversity Keith Brewa and Hideaki Nagaseb Biochim Biophys Acta.
2010 January ; 1803(1): 55–71. doi:10.1016
• Diabetes-Induced Alterations in the Extracellular Matrix and Their Impact on
Myocardial Function Brittany Law†, Vennece Fowlkes†, Jack G. Goldsmith, Wayne
Carver, and Edie C. Goldsmith Microsc Microanal. 2012 February ; 18(1): 22–34.
doi:10.1017
• Collagenases and Collagen Degradation STEPHEN M. KRANE the journal of
investigative dermatology, 79:83s-868, 1982
• Degradation Products of Extracellular Matrix Affect Cell Migration and Proliferation
Reing et.al. TISSUE ENGINEERING: Part A Volume 15, Number 3, 2009
80. Role of Vitamin C in collagen formation
• Vitamin C (Vit.C) plays a critical role in the maintenance of a normal mature
collagen network in humans (antiscurvy properties) by preventing the auto-
inactivation of lysyl and prolyl hyroxylase, two key enzymes in collagen
biosynthesis.
• Biochemical studies demonstrated that vitamin C is a co-factor for lysyl and
prolyl hydroxylase, two essential enzymes in the collagen biosynthesis pathway.
Effect of vitamin C and its derivatives on collagen synthesis and cross-linking by normal human fibroblasts N. BOYERA*, I. GALEY and B.A. BERNARD
International Journal of Cosmetic Science 20, 151–158 (1998)
81. • The process of hydroxylation is achieved by two iron
enzymes, lysyl and prolyl hydroxylase. Vitamin C, as a
co-factor, prevents iron oxidation, and thus protects
these enzymes against auto-inactivation.
• Thus, vitamin C promotes the synthesis of a normal
mature collagen network by maintaining optimal lysyl
and prolyl hydroxylase activity
Effect of vitamin C and its derivatives on collagen synthesis and cross-linking by normal human fibroblasts N. BOYERA*, I. GALEY and B.A. BERNARD
International Journal of Cosmetic Science 20, 151–158 (1998)
82. Gingival ablation
• Friction from soft tissue (gingival ablation).
• Laser ablation cut and coagulate soft tissues tissues leading to haemostasis.
83. Collagen markers for disease
• Collagen I propeptide (PICP),
• Collagen III propeptide (PIIINP) and
• the cross-linked telopeptide of type I collagen (ICTP)
• CII synthesis markers include the procollagen type II C-propeptide (PIICP) and the
procollagen type IIA N-propeptide (PIIANP).
• CII degradation markers include CII C-telopeptide (CII-X), CII neoepitope (TIINE),
helix II, C2C, CNBr 9.7, Coll 2-1, and Coll 2-1 NO2.
84. Factors inhibiting collagen
synthesis• Age – decrease with age
• Liver Injury – cirrhosis of liver
The collagen content at the typical stage of liver cirrhosis was more than fivefold
higher than that of the normal state.
• Radiation exposure - Collagen synthesis also increases as a result of radiation
therapy or accidental radiation. This results in a condition called fibrosis
characterized by increased accumulation of extra cellular matrix, especially
collagen. The irradiation mediated collagen synthesis is mediated by a fibrogenic
cytokine called transforming growth factor (TGFb).
Factors Influencing Collagen Biosynthesis O. Kavitha* and Raghava Varman Thampan Journal of Cellular Biochemistry 104:1150–1160 (2008)
85. • ASCORBIC ACID AS A REGULATOR OF COLLAGEN SYNTHESIS - Study
conducted by Schwarz and Bissell [1977] reported the role of ascorbate in regulating
collagen synthesis. Ascorbate is essential for the collagen molecule to maintain its triple
helical structure.
• PROTEOGLYCANS AS REGULATORS OF COLLAGEN SYNTHESIS - A mixture
of chondroitin sulfate and glucosamine has been extensively tested for the clinical efficacy
of symptomatic relief in patients with osteoarthritis. This is because of the
chondroprotective effect of this mixture. Since collagen is the major component of the
connective tissues, the mixture may also have some collagen regenerative property
86. • GLUCOSE AS A REGULATOR OF COLLAGEN SYNTHESIS - Aging and age related
diseases alter the extra cellular matrix components considerably. The effect of high glucose
concentration (characteristic of diabetes) on collagen synthesis was checked by Benazzoug
et al. [1998].
• It was observed that in control conditions (5 mM glucose) collagen III production increased
along with in vitro cell aging. High glucose concentrations (10 and 15 mM) increased
specifically collagen III synthesis both at the mRNA and protein levels, without altering
collagen I production.Fibronectinsynthesiswasalsoincreased both during in vitro cell aging
and in high glucose exposures of fibroblasts.
87. • CELLULAR GROWTH FACTORS AND THEIR INFLUENCE ON
COLLAGEN SYNTHESIS : Recombinant human epidermal growth factor
specifically inhibited the production and accumulation of type I collagen in
cultured human fibroblasts while it stimulated the production of non-
collagenous proteins [Kurata and Hata, 1991]. In this experiment it is clear that
human EGF reduced steady state levels of mRNAs for both proa1(I) and
proa2(I) chains and also influenced the levels of transcription of these genes.
However, the actual mechanism involved in the regulation mediated by human
EGF is not clear.
88. • THE MINERAL MICROENVIRONMENT THAT REGULATES
COLLAGEN SYNTHESIS : In the study carried out by Deshmukh and Kline
[1976] it was observed that monolayer culture of chondrocytes produced type I
collagen and in suspension cultures the choice depended on the presence or
absence of calcium. In the absence of calcium, the cells produced their specific
type II collagen and in the presence of calcium type I collagen was synthesized.
The increased influx of calcium (induced) in monolayer culture stimulated these
cells to produce type I collagen in suspension culture even in the absence of
calcium in the extracellular matrix [Deshmukh et al., 1976]
89. • PHYTOESTROGENS THAT INFLUENCE COLLAGEN SYNTHESIS :
Studies at the cellular level have provided insight into the ability of soybeans to
prevent osteoporosis [Akiyama et al., 1987; Bames, 1998]. Recent studies have
reported the effect of genistein on the induction of increased collagen synthesis
[Varani et al., 2004; Morris et al., 2006].
• ESTROGEN AS A REGULATOR OF COLLAGEN SYNTHESIS : Estrogen
replacement is one of the most common and effective strategies used in
preventing osteoporosis in postmenopausal women.
90. • WOUND HEALING BY HERBS : Cell proliferation and collagen synthesis at the
wound site is also promoted by the plant extract along with the increase in total
protein content. Hexuronic acid and hexosamine, which are the components of
glycosaminoglycans, are the other factors which are increased by Ocimum extract.
• MISCELLANEOUS FACTORS THAT INFLUENCE COLLAGEN
BIOSYNTHESIS : Histone deacetylase (HDAC) has the ability to modulate gene
expressions. HDAC promotes type II collagen expression. This is by inactivation of
the factor Wnt-5, which inhibits type II collagen transcription. HDAC deacetylates
the promoter of Wnt-5 thus blocking its transcription [Huh et al., 2007].
92. Gorlin sign
• Gorlin sign is the ability to touch
the tip of the nose with the
tongue.
• Approximately ten percent of the
general population can perform
this act, whereas fifty percent of
people with the inherited
connective tissue disorder, Ehlers-
Danlos syndrome, can.
93. Enzyme responsible for Ehlers
Danlos syndrome
• D4ST1-deficient EDS is caused
by mutations in CHST14, which
encodes an enzyme responsible
for post-translational
modification of GAG.
94. Stains used for ECM
• H & E
• Safranin O stain
• Alcian blue stain
