MATRIX METALLOPROTEINASES IN HEALTH AND DISEASE

OVERVIEW▪ Introduction

▪ Definition

▪ Anatomy

▪ Types

▪ Role in maintaining health

▪ Role in diseases and malignancy

INTRODUCTION▪ Living tissues are not just accumulations of tightly packed cells.

▪ Much of a tissue's volume is made up of extracellular space .This void is filled with a complex meshwork called the extracellular matrix.

▪ If cells were seeds then the extracellular matrix (ECM) would be the soil.

▪ In addition to providing structural support for the cells embedded within a tissue, the extracellular matrix guides their division, growth, and development.

▪ Hence ,the extracellular matrix largely determines how a tissue looks and functions.

▪ CONTENTS OF ECM

Collagen Elastin Fibronectin Laminin Growth factors

and Metalloproteinases

CONTENTS OF ECM

▪ Collagen-Main structural protein in the extracellular space.

Type I: skin, tendon, bone Type II: cartilage

Type III: main component of reticular fibers, commonly found alongside type I.

Type IV: basement membrane Type V: cell surfaces, hair and placenta

▪ Elastin-Highly elastic protein in connective tissue and allows many tissues in the body to resume their shape after stretching or contracting.

CONTENTS OF ECM

▪ Fibronectin-located within the basement membrane and key role in cell adhesion and wound healing response to injury.

▪ Laminins-are among the first proteins of the ECM to appear in embryos ,role in cell differentiation and migration

▪ Growth Factors-The ECM has been referred to as a ‘reservoir’ for GFs. VEGF, FGF and TGF are tied to the ECM through heparan sulphate.

▪ Matrix Metalloproteinases (MMPs)

TALE(TAIL) OF MATRIX METALLOPROTEINASES▪ MMPs were discovered, in 1962, by Jerome Gross while studying the

degradation of triple-helical collagen during the metamorphosis of a tadpole tail.

Tad pole tail

Area of clearing

Collagen gel

METALLOPROTEINASES▪ The timely breakdown of extracellular matrix (ECM) is essential for embryonic

development, morphogenesis, reproduction, and tissue resorption and remodeling.

▪ Metalloproteinases /Matrixins are a large group of zinc-dependent proteases responsible for cleaving and rebuilding connective tissue.

N-terminal propeptide domain-MMPs are initially synthesized as inactive zymogens with a pro-peptide domain that must be removed before the enzyme is active -Cysteine switch

C-Terminal Domain-Structurally similar to proteins of the hemopexin family. The domain has a relatively large surface area for protein-protein interactions.

Catalytic Domain-Contains two zinc ions and two or three calcium ions. The first Zn ion present in the active site directly participates in catalytic processes. Calcium ions are necessary to stabilize the domain structure

TYPES

▪ Collagenases

▪ Gelatinases

▪ Stromelysins and

▪ Membrane-type metalloproteinases.

COLLAGENASES

▪ Collagenases are enzymes that break the peptide bonds in collagen.

▪ MMP-1, MMP-8, and MMP-13 are the enzymes with the capacity to cleave the triple helix of fibrillar collagen.

▪ Interstitial collagenase(MMP-1) has preferential activity against type III collagen.

▪ MMP-1 is produced and secreted predominantly by cells prevalent in the post acute stages of wound healing (fibroblasts, endothelial cells)

COLLAGENASES

▪ Polymorphonuclear collagenase (MMP-8) has an affinity for type I collagen.

▪ MMP-8 is stored in granules within the neutrophils and once activated can be released in seconds.

▪ MMP-13 appears to have the unique ability to cleave type I, type II, and type III collagen.

GELATINASES▪ Gelatinase is a proteolytic enzyme that allows a living organism to hydrolyse gelatin

into its sub-compounds .

▪ The gelatinases (MMP-2 and MMP-9) cleave collagen types IV, V, VII, and X, elastin, basement membranes and denatured collagen.

▪ The gelatinases may also act synergistically with the collagenase family by further degrading types I, II, and III.

▪ MMP-2 is secreted by fibroblasts, MMP-9 is produced predominantly by leukocytes and keratinocytes.

STROMELYSINS▪ Due to their broad base of substrate specificity, stromelysins (MMP-3, MMP-7,

MMP-10, MMP-11, and MMP-12) play a varied role in degradation of the extracellular matrix.

▪ Degrades collagen types II, III, IV, IX, an X, proteoglycans, fibronectin, laminin, and elastin.

MEMBRANE-TYPE MATRIX METALLOPROTEINASES

▪ Members of this class of matrix metalloproteinases (MMP-14, MMP-15, MMP-16, and MMP-17) have certain unique structural characteristics.

▪ They are not secreted into the extracellular matrix.

▪ Rather, they exist on cell membranes and appear to function by binding to other matrix metalloproteinases and activating them.

ACTIVATION AND DEACTIVATION

1)Gene level by transcriptional control

2)Molecular level by requiring factors to convert the proenzyme form to the active form.

3)Tissue level -Through Tissue Inhibitors of Metalloproteinases(TIMP)

TRANSCRIPTIONAL CONTROL

▪ Two extensively studied factors which influence the MMP transcription are: IL-1β and TGF – β

▪ IL-1 β stimulates, whereas TGF- β inhibits MMP gene expression .

▪ Transcription of many MMPs is promoted by inflammatory cytokines, growth factors, chemokines, oncogenes

MOLECULAR CONTROL▪ MMPs are synthesized as inactive zymogens and require activation.

▪ The cysteine in the propeptide interacts with a Zn2+ ion in the enzyme’s active site.

▪ This interaction, [Zn2+-cysteine], keeps the proMMP inactive

▪ Activation can occur by physical or chemical means by disturbing the cysteine –zinc interaction of the cysteine switch .

DEACTIVATION OF MMP▪ Tissue inhibitors of matrix metalloproteinases (TIMP) are major endogenous

regulators of MMP

▪ The critical points in MMP inhibition by TIMPs are centred on the disulfidebonds at Cys and Cys.

▪ TIMPs catalysis of disulfide bond formation between cysteines prevents autocatalytic MMPs activation through the cysteine switch mechanism.

▪ In addition, TIMPs in complexes with other molecules involve in the inhibition of already active MMP

PHYSIOLOGICAL ROLES

▪ Apoptosis

▪ Ovulation

▪ Inflammation

▪ Angiogenesis

▪ Bone remodeling

▪ Mammary gland development

▪ Embryogenic remodeling

▪ Organ morphogenesis

APOPTOSIS

▪ MMP-3 has pro-apoptotic actions on neighboring epithelial cells by loss of

β1-integrin engagement and epidermal growth factor receptor expression.

▪ Whereas MMP-7 is able to release membrane bound Fas ligand and

induces epithelial cell apoptosis.

OVULATION

▪ The preovulatory LH surge stimulates an increase in ovarian matrix metalloproteinases (MMPs) and their associated inhibitors, the TIMPs prior to follicular rupture .

▪ Proteolytic enzymes are locally activated that digest a small part of the dominant follicle’s wall and extracellular matrix, releasing the oocyte .

OVULATION

Pre Ovulatory phase-IHC-MMP2Early ovulatory phase-TIMPs

INFLAMMATION

▪ MMPs function as inflammatory cytokines during vascular formation or remodeling.

▪ In matured and quiescent vessels, active MMPs are absent or expressed at low levels.

▪ But in tissues undergoing abnormal angiogenesis and vascular remodeling, MMPs are markedly expressed, secreted, and activated.

▪ Macrophages and neutrophils are an important resource of MMPs in vascular tissue.

MMP IN INFLAMMATION▪ TNF-α and ILs MMP Stimulate

• Degrade ECM to facilitate migration and recruitment of cells.

• Cleave cell surface receptors and other non-ECM molecules to mediate adhesion, proliferation, and apoptosis of cells.

MMP IN WOUND HEALING▪ Healing of a skin wound requires several processes :

Cell migration

ECM degradation and

Tissue reorganization.

MMP-1 :Keratinocyte migration

MMP-3: Wound contraction

VASCULAR REMODELING

▪ Hemodynamic forces such as shear stress and arterial pressure regulate MMP expression and participate in vascular remodeling.

▪ Elevated transmural pressure Extracellular signal–regulated kinases (ERKs)

NF-κB

↑MMP 2 and 9

Remodeling due to hemodynamic adaptation of the vein to the arterial condition leads to saphenous vein graft bypass failure.

BONE REMODELLING▪ During long bone development, an initial cartilaginous template is formed and

replaced by bone in a coordinated and refined program.

▪ Chondrocyte proliferation and maturation, vascular invasion, recruitment of adult stem cells and intense remodeling of cartilage and bone matrix.

▪ Osteoclast express –MMP 9 and 13

MAMMARY GLAND DEVELOPMENT

▪ The epithelial ductal network of the mammary gland expands greatly during puberty in response to both local and systemic signals.

▪ This process requires degradation of the basement membrane and ECM, restructuring of the endogenous vascular network and large-scale epithelial morphogenesis

MAMMARY GLAND DEVELOPMENT▪ MMP 2-Primary invasion

▪ MMP 3-Secondary branching

EMBROGENESIS▪ Embryonic growth and tissue morphogenesis are fundamental events that require

disruption of ECM barriers to allow cell migration and matrix microenvironment remodeling.

▪ Trophoblasts express high levels of MMP-9

▪ MMP2 and 9 enable trophoblast cells to penetrate uterine

vasculature during Implantation

▪ Development of the placenta starts with the invasion and migration of trophoblast cells into the maternal tissue to establish connection with the maternal circulation .

MMPS IN PATHOLOGICAL CONDITIONS

▪ Diseases of CNS

▪ Cardiovascular diseases

▪ Lung fibroblastic disease

▪ Liver fibrosis

▪ Diseases of bone

▪ Tumor development and metastasis

MMP IN DISEASES OF CNS▪ MMPs are largely absent from the normal CNS and their upregulation has been

reported in several neurological disorders and after injury.

▪ Metalloproteinases in neuroinflammation and Multiple sclerosis: MS is an immune disorder characterized by demyelination and axonal loss.

▪ The presence of proteinases(MMP 9) in the cerebrospinal fluid (CSF) of patients with MS has been known for over 20 years.

▪ MS patients show increased MMP9 messenger RNA in leukocytes and elevated MMP9 levels in serum.

MMP IN DISEASES OF CNS▪ Leukocytes use MMPs to disrupt the basement membrane of the blood–brain

barrier.

▪ MMPs can disrupt myelin and cause

demyelination.

▪ MS patients treated with interferon-β

show a decreased number of MMP9

expressing leukocytes.

MMP IN DISEASES OF CNS▪ A role for metalloproteinases in stroke is indicated by the finding that MMP2 and -

9 are rapidly upregulated after focal cerebral ischaemia .

▪ The elevated MMP expression might contribute to the tissue destruction in stroke.

▪ Viral infections of the CNS have been increasingly associated with the production of MMPs.

▪ Elevated expression of MMP9 has been detected in the CSF of HIV-infected patients.

MMP IN CARDIOVASCULAR DISEASES

▪ Atherosclerosis:

MMP 9-Adherence of circulating monocytes to the vascular endothelium, through which they gain entry to the sub intimal tissue.

Plaque ruptures are associated with increased MMP-9 proteolytic activity.

Increased activity of TIMPs-Vascular calcification

MMP IN CARDIOVASCULAR DISEASES

Aneurysm :Consists of destruction and functional loss of elastin in the aortic medium.

▪ MMP plays a significant role in weakening the ECM components (elastin, collagen, fibronectin and proteoglycans), thus harming the aortic wall.

Varicose veins: Interstitial collagen III is important for the stretch resistance of the connective tissue of veins.

▪ MMP-3 may contribute to vein wall weakness and varicose vein formation via excessive degradation of collagen III.

MMPS IN ACUTE MYOCARDIAL INFARCTION

▪ Myocardial collagens maintain the structural integrity of adjoining myocytes and cardiac pump function.

▪ Myocardialinfarction (MI) leads to complex architectural alterations involving both the infarcted and noninfarcted myocardium.

▪ Patients exhibiting extensive infarct expansion after MI are more likely to experience complications:

Congestive heart failure

Aneurysm formation and

Myocardial rupture.

MMPS IN ACUTE MYOCARDIAL INFARCTION

▪ One of the determinants of left ventricular remodeling is damage to and loss of the myocardial extracellular matrix (ECM) during the healing process after MI.

▪ Reduction in LV dilatation after MI that can be achieved with MMP inhibitor treatment.

MMPS IN LUNG FIBROBLASTIC DISEASE

▪ Lung fibrosis is the final result of a large and heterogeneous group of lung disorders, known as interstitial lung diseases.

▪ Scar formation, the accumulation of excess fibrous

connective tissue leads to thickening of the walls,

and causes reduced oxygen supply in the blood.

▪ MMP1/Collagenase1 :Expressed primarily by epithelial

cells –Cystic spaces (Honey comb pattern)

▪ MMP2- Tissue migration of fibrocytes

MMPS IN EMPHYSEMA▪ The lung is a sophisticated matrix scaffold on which lung epithelium and

endothelium reside.

▪ When the wall of an alveolus is destroyed, the air sacs coalesce to form larger ones.

▪ These enlarged airspaces empty more slowly, resulting in airflow obstruction, the hallmark of COPD.

MMPS IN EMPHYSEMA▪ Of all the MMPs, the evidence supporting a role for macrophage elastase

(MMP12) in the pathogenesis of emphysema is the strongest.

▪ Mmp12 expression is greatly increased in response to cigarette smoke exposure, due to a number of overlapping mechanisms.

▪ Activation of the plasmin/thrombin–proteinase activated receptor (PAR-1) cascade leads to the expression of Mmp12.

▪ Inhibited by A1AT and TGF-β .

MMPS IN LIVER FIBROSIS▪ Liver fibrosis represents chronic wound repair following diverse insults.

▪ MMPs as bad role: In liver fibrogenesis hepatic stellate cells undergo dramatic morphological and functional changes –star shaped HSC are converted to myofibroblastic cells .

▪ Proteolytic degradation of the normal ECM helping inmobilization, migration and proliferation of cells during repair and regeneneration.

▪ MMPs as good role: The MMP-mediated resolution of tissue fibrosis may act through ECM degradation as well as by induction of HSC apoptosis.

MMPS IN BONE DISEASES▪ The irreversible destruction of the cartilage, tendon, and bone that comprise synovial

joints is the hallmark of both rheumatoid arthritis (RA) and osteoarthritis (OA).

▪ Cartilage is made up of proteoglycans and type II collagen

▪ Tendon and bone are composed primarily of type I collagen

▪ RA is an autoimmune disease affecting numerous joints throughout the body

▪ OA develops in a small number of joints, usually resulting from chronic overuse or injury.

MMPS IN BONE DISEASES▪ In both RA and OA:

Inflammatory cytokines:IL-1 βand TNF-α MMPs that can degrade all components of the extracellular matrix.

▪ MMP-1 synovial cells that line the joints

▪ MMP-13 is a product of the chondrocytes that reside in the cartilage

In addition to collagen, MMP-13 also degrades the proteoglycan molecule, aggrecan, giving it a dual role in matrix destruction.

stimulate

MMPS IN TUMOR GROWTHMMPs can regulate the growth of tumour cells by the following mechanisms :

▪ Release of cell membrane bound precursors of some growth factors

▪ Modulating the bioavailability of growth factors that are sequestrated by ECM proteins.

▪ Indirectly regulating proliferative signals through integrins

▪ Because of the shedding of the E cadherin, β catenin translocates to the nucleus and leads to proliferation

MMPs can inhibit growth by

▪ Activation of TGF-β

▪ Generation of proapoptotic molecules. e.g. Fas ligand or TNF α

REGULATION OF INVASION AND METASTASIS ▪ Tumour invasion is a multistep process in which cell motility is coupled

with proteolysis and involves interaction of cells with ECM .

▪ Cadherin are cell adhesion molecules that mediate cell-cell adhesion in

normal mucosal cells and maintain epithelial integrity and its deregulation is

associated with cancer progression .

REGULATION OF INVASION AND METASTASIS ▪ Decreased expression of E-cadherin causes loss of cell adhesion and

contributes to cell dissociation, increased motility and invasion .

▪ E-cadherin is cleaved by MMP3 and 7

Detachment of cells by loss of intercellular junction

Epithelial to mesenchymal transition(EMT)

Migration

Invasion

SUMMARY▪ MMPs

▪ Physiological roles Pathological roles

CollagenasesGelatinasesStromelysins and Membrane-type metalloproteinases.

ApoptosisOvulationInflammationAngiogenesisBone remodelingMammary gland developmentEmbryogenic remodelingOrgan morphogenesis

Diseases of CNSCardiovascular diseasesLung fibroblastic diseaseLiver fibrosisDiseases of boneTumor development and metastasis

REFERENCES▪ Kumar, Vinay., Robbins and Cotran Pathologic Basis of Disease.9th edition. Reed Elsevier

India Private Limited, New Delhi.2015

▪ Brinckerhoff CE, Matrisian LM. Matrix metalloproteinases: a tail of a frog that became a prince. Nature Rev Mol Cell Biol 2002;3:207–214.

▪ Birkedal-Hansen H, et al. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 1993;4:197–250.

▪ Stickens D, et al. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 2004;131:5883–5895.

▪ Ducharme A, et al. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 2000;106:55–62

▪ Yu Q, Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-β and promotes tumor invasion and angiogenesis. Genes Dev 2000;14:163–176.

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