Clinical implications of matrix metalloproteinases
Malay Mandal, Amritlal Mandal, Sudip Das, Tapati Chakraborti andSajal ChakrabortiDepartment of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India
Received 20 September 2002; accepted 14 April 2003
Abstract
Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are important for normal development, wound heal-ing, and a wide variety of pathological processes, including the spread of metastatic cancer cells, arthritic destruction of joints,atherosclerosis, pulmonary fibrosis, emphysema and neuroinflammation. In the central nervous system (CNS), MMPs have beenshown to degrade components of the basal lamina, leading to disruption of the blood brain barrier and to contribute to theneuroinflammatory responses in many neurological diseases. Inhibition of MMPs have been shown to prevent progression ofthese diseases. Currently, certain MMP inhibitors have entered into clinical trials. A goal to the future should be to design selec-tive synthetic inhibitors of MMPs that have minimum side effects. MMP inhibitors are designed in such a way that these can notonly bind at the active site of the proteinases but also to have the characteristics to bind to other sites of MMPs which might bea promising route for therapy. To name a few: catechins, a component isolated from green tea; and Novastal, derived from ex-tracts of shark cartilage are currently in clinical trials for the treatment of MMP-mediated diseases. (Mol Cell Biochem 252:305–329, 2003)
Four decades ago Cross and Lapiere [1], first described theinvolvement of collagenolytic activity involved in tail re-sorption of tadpole. Since then a plethora of information,arrived from researchers of standing in this field and the roleof MMPs in human health and diseases have now been widelyappreciated. The interaction of cells with extracellular ma-
trix (ECM) are critical for the normal development and func-tion of organisms. Modulation of cell-matrix interactionsoccurs through the action of unique proteolytic systems re-sponsible for hydrolysis of a variety of ECM components.By regulating the integrity and composition of the ECMstructure, MMPs play a pivotal role in the control of signalselicited by matrix molecules, which regulate growth and de-velopment of cells. The turnover and remodeling of ECM
Address for offprints: S. Chakraborti, Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India(E-mail: [email protected])
must be tightly regulated since uncontrolled proteolysis con-tributes to abnormal development and to the generation ofmany pathological conditions characterized by either exces-sive degradation or a lack of degradation of ECM components[2–4].
The matrix metalloproteinases (MMPs) are zinc-depend-ent endopeptidases known for their ability to cleave one orseveral ECM constituents, as well as non-matrix proteins.They comprise a large family of proteinases that share com-mon structural and functional elements and products of dif-ferent gene [4] (Table 1).
The MMPs are homogeneous enzymes, however, theirstructures vary depending upon which domains are present[5–7]. All members of this family contain a propeptide and acatalytic domain. The catalytic domain (about 170 aminoacids) contains the catalytic machinery including the zinc bind-ing site and a conserved methionine. This domain containsadditional zinc and calcium ions, which maintain the three di-mensional structure of MMPs required for their stability andenzymatic activities. Stromelysin-1, stromelysin-2 and intersti-tial collagenase has an added hemopexin-like domain on theC-terminal end. Gelatinases A and B have the C-terminalhemopexin-like domain between the active enzyme and the Znbinding site. Gelatinase B also has a type V collagen-like do-main between the Zn binding domain and the hemopexin do-main. Substrate specificity differs among these enzymes [8].Domain structure of MMPs is illustrated in Fig. 1.
Most cells synthesize and immediately secrete MMPs intothe extracellular matrix [5]. Inflammatory cells, however,store MMPs such as neutrophil collagenase and gelatinaseB. Tissue distribution of these proteinases varies widely.Some are constitutively synthesized (e.g. 72 kDa gelatinase)by many cells, while others are synthesized mainly upon stimu-lation (e.g. collagenase) [9, 10]. Matrix metalloproteinases areinvolved in a variety of biological functions such as tissueremodeling during development, wound healing, involutionof organs, and in the invasion of metastatic cells across base-ment membranes [5, 11, 12].
Pharmacological and genetic modulation of MMP expres-sion have been demonstrated to alter the course of MMP-mediated diseases. In recent years, certain MMP inhibitorshave entered into clinical trials. A goal to the future shouldbe to see whether any relationship exists between the levelsand the profile of MMPs in different diseases and responsesto the newly developed synthetic MMP inhibitors.
Biological functions of MMPs
General considerations
Growth and development are associated with rapid cell move-ment and with restructuring and reshaping of the extra-cellu-
lar matrix. A number of studies have provided evidence forthe involvement of MMPs in a variety of physiological proc-esses [6]. Evidence in support of the involvement of theMMPs in normal physiology has come largely from theirassociation with the expression of a particular enzyme to aparticular process. It appears difficult to identify the naturalsubstrates for most of the MMPs because of their relativelybroad and overlapping substrate specificities (Table 1).
The existing body of evidence suggests that MMPs are mo-bilized by cells in at least three different ways: (i) by initiat-ing transcription of growth factor-responsive MMP genes{(collagenase, stromelysin-1 (SL-1), 92 kDa gelatinase, andputative metalloproteinases (PUMP-1) by fibroblasts, en-dothelial cells, macrophages, and keratinocytes)}; (ii) byconstitutive expression of MMPs that are largely unrespon-sive to growth factors and cytokines (72 kDa gelatinase inmost cell types), and (iii) by the triggered release of pre-pack-aged MMP from granule storage sites (PMN-CL, 92 kDagelatinase in PMN leukocytes) [13].
Human endometrium
Menstruation is primarily an event of tissue destruction thatresults from partial breakdown of the functional layer ofendometrium at the onset of a normal reproductive cycle inwomen. Menstruation shares a number of features with in-flammatory responses which includes leukocyte infiltration,proliferation and activation that occur in the endometriumprior to menstruation. It has been proposed that the leukocyterelease MMPs during this stage. Interactions between leu-kocytes and the stromal and epithelial cells of the endometriuminduce and activate MMPs resulting in tissue breakdown [14].
The mRNA for the MMPs, the interstitial collagenase,stromelysin-1, -2, and -3, matrilysin, gelatinase A, and gel-atinase B are all expressed during the menstrual phase in thehuman endometrium [14, 16]. Matrilysin is expressed in theepithelium and glandular epithelial cells throughout the tis-sue. Interstitial collagenase and gelatinase B are in stromalcells, but concentrated in the luminal region of the tissue. Thetranscripts for stromelysin-3 and gelatinase A are localizedthroughout the stromal component of the tissue. The wide-spread expression of MMPs in all compartments of this tis-sue suggests that the coordinated effort of several MMPs mayplay a pivotal role in the breakdown and release of endome-trial tissue during menstruation [17].
The mRNA for the 72 kDa gelatinase A is the only MMPtranscript observed throughout the menstrual cycle, althoughthere is some variation of the intensity of signal [16]. Thelevel of gelatinase A mRNA is somewhat reduced during thesecretory phase of the cycle when progesterone levels areelevated. The mRNA of all other MMPs are absent during thisphase of the cycle, suggesting an inhibitory effect of proges-
terone on the expression of each of these genes. It has beensuggested that repression of MMP expression may be a criti-cal determinant for maintaining an appropriate endometrialenvironment for embryo implantation and development [17].
Wound repair
Wound repair is a physiological event, in which tissue injuryresults in a repair process, which finally leads to restorationof structure and function of the tissue. Cutaneous woundrepair can be divided in to three overlapping phases: (i) for-mation of fibrin clot followed by inflammation (early or late);(ii) re-epithelialization and granulation of tissue formation;and (iii) matrix formation and remodeling [8].
In the first phase of wound repair, a fibrin clot is formedas a result of platelet aggregation and blood coagulation. Vari-ous growth factors and chemotactic factors released from acti-vated coagulation pathways, injured cells, and platelets attractinflammatory cells to the wound area.
Proteolytic degradation of ECM is required in many stagesof wound repair, such as degradation of the provisional ma-trix, angiogenesis and keratinocyte migration. Followinginjury, MMP-1 is expressed by basal keratinocytes at themigration front of epidermis in several types of cutaneouswounds including incision wounds and blistering skin dis-eases [8]. The expression of MMP-1 in basal keratinocytesis rapidly induced after dermal injury, persists during heal-ing, and subsides at re-epithelialization [8]. MMP-1 expres-sion is most abundant at the very edge of the wound, and itdiminishes progressively away from the wound edge [18, 19].It has been shown that the MMP-1 activity is essential forkeratinocyte migration on native type I collagen and that thisinduction was found to be mediated via α
vβ
3-integrin [20].
However, transgenic mice over-expressing human MMP-1in the epidermis elicit hyperproliferative, hyperkeratinoic epi-dermal phenotype and delayed wound closure, suggestingthat proper regulation of MMP-1 expression is essential forre-epithelialization during wound healing [21]. Besides epi-thelium, MMP-1 is expressed by stromal dermal fibroblastsin both acute and chronic dermal wounds, as well as in hu-man burn wounds, indicating that MMP-1 plays a role inremodeling of the granulation tissue ECM [8, 22].
Cellular fibrinolytic activity
Interactions between MMP and the plasminogen/plasmin(fibrinolytic) system may affect cellular fibrinolysis. MMP-3 (stromelysin-1) specifically hydrolyzes urokinase (u-PA)yielding a 17 kDa NH
2 terminal fragment containing the
functionally intact receptor (u-PAR)-binding sequence anda 72 kDa COOH terminal domain. MMP generates an angi-
otensin like fragment from plasminogen. MMP-3 specifi-cally hydrolyzes human α
2-antiplasmin (α
2-AP), the main
physiological plasmin inhibitor. α2-AP cleaved by MMP-3 no
longer forms a stable complex with plasmin and no longerinteracts with plasminogen [23]. Cleavage and inactivationof α
2-AP by MMP-3 may constitute a mechanism favouring
local plasmin mediated proteolysis. Furthermore, MMP-3specifically hydrolyzes and inactivates human plasmino-gen activator inhibitor-1 (PAI-1). Stable PAI-1 bound tovitronectin is cleaved and inactivated by MMP-3 in a com-parable manner as free PAI-1, the cleaved protein, however,does not bind to vitronectin. Cleavage and inactivation ofPAI-1 by MMP-3 may thus constitute a mechanism of de-creasing the antiproteolytic activity of PAI-1 and impairingthe potential inhibitory effect of vitronectin bound PAI-1 oncell adhesion and/or migration. These molecular interactionsof MMP-3 with enzymes, substrates and inhibitors of thefibrinolytic system may thus play a role in the regulation ofcellular fibrinolysis [23].
Epithelial remodeling
Matrilysin is the smallest (28 kDa) of the known MMPs, isexpressed by non-injured, non-inflamed exocrine and mu-cosal epithelium in most tissues, for example, in the lung. Theexpression of matrilysin in normal epithelium suggests thatthis enzyme serves a common homeostatic function amongepithelia, and several observations implicate a role for mat-rilysin in innate immunity among epithelia. All tissues inwhich matrilysin is ‘constitutively’ expressed are open to theenvironment and, therefore, are vulnerable to bacterial ex-posure. Matrilysin is prominently upregulated in tissues witha heavy bacterial load, such as in lungs with cystic fibrosis(CF). It has been suggested that matrilysin is responsible forthe activation of prodefensins, the precursor form of defensinswhich kills bacteria by membrane disruption [24].
A common role of the mucosal epithelium is to functionas an active barrier against the external environment, and thesecretion of antimicrobial peptides by epithelial cells appearsto be an important component of innate immunity. The α-andβ-defensins comprise a family of cationic peptides that killbacteria by membrane disruption [25]. The pro-segment ofα-defensin precursors maintain them in an inactive state, andproteolysis at some point in the secretion pathway is neededto remove the pro-domain. Paneth cells are specialized epi-thelial cells that secrete defensins and other antimicrobialmolecules, and matrilysin activates these peptides in the se-cretion pathway. In matrilysin knockout mice, pro-α-defensinsare not processed to their active forms by matrilysin, and de-ficiency of this enzyme results in impaired bacteriocidal ac-tivity in vitro and in vivo [24]. Because of a lack of defensinactivation, matrilysin null mice cannot effectively kill patho-
genic Escherichia coli and are themselves killed by doses ofSalmonella typhimurium that are not lethal to wild-type mice.Matrilysin thus functions in mucosal immunity by regulatingthe activity of anti-microbial peptides indicating that MMPmay also play a role in lung physiology [26].
A functional role for matrilysin in airway re-epithelializa-tion was demonstrated by repair of injured tracheas fromgene-targeted mice [27]. As in human tissue, matrilysin isexpressed in airway epithelial cells that had migrated overthe cut edges of dissected mouse trachea [27]. In tracheasfrom wild-type mice, re-epithelialization progresses rapidly;however, wounds in tracheas from matrilysin null mice showno evidence of epithelial migration. Matrilysin-deficient micehave been shown to elicit the most severe wound-repair de-fect among the MMP knockout mice generated to date. How-ever, the mechanism by which matrilysin facilitates woundrepair is not clearly known. It may be required to loosen cell-matrix and cell-cell contacts, as has been suggested for otherMMPs in other epithelial repair/migration models [20, 28].Matrilysin may not be the only MMP involved in repair ofairway epithelial wounds. Gelatinase-B is also expressed uponinjury, for example, by bleomycin instillation in epithelial cellsin distal airways and deficiency of this MMP leads to exces-sive bronchiolization [29]. This suggests a role of gelatinaseB in either cell migration or differentiation. Legrand et al. [30]have also demonstrated that the activity of gelatinase-B is
required for the migration of isolated airway epithelial cellsover matrix substratum [30]. Several MMPs may, therefore,act in situ on different substrates to facilitate repair [26].
Aging
Aging is the major risk factor for the development of vascu-lar diseases, such as hypertension and atherosclerosis [31].There is considerable interest observed among scientists inunderstanding the underlying mechanisms of the vascularremodeling that occurs in advanced age.
Aged aorta has been shown to exhibit a significant increasein intimal width compared with that in young rats which ismainly composed of molecules, including collagen, proteo-glycan, and actin. It contains markedly higher levels of MMP-2, fibronectin, TGF-β, and ICAM-1. In contrast, the aorticintima of young rats consist of only single layer of endothe-lial cells. It appears, therefore, that actin-positive smoothmuscle cells in the older intima may have migrated from themedia [32].
Intimal growth during aging in the absence of experimen-tal injury in some ways resembles neointimal formation inresponse to injury [33]. It has been shown that neointimalgrowth in response to endothelial injury is markedly en-hanced in old versus young rats and is due to factors intrin-
Fig. 1. Schematic representation of the domain structure of MMPs. S – signal peptide; P – propeptide; C – catalytic domain; F – fibronectin type II do-main; CP – cysteine proline rich and IL-1 receptor like domain; L – linkage domain; H – hemopexin like domain, FR – furin recognition site; T – trans-membrane domain; V – vitronectin like domain. Numbers in the figure indicate domain structures.
sic to the vessel wall [34] and that may be attributable in partto enhanced smooth muscle cells (SMCs) chemotaxis or pro-liferation in response to growth factors, for example, plate-let derived growth factor (PDGF) [35].
Recent studies have demonstrated that chemotactic inva-sion of a reconstituted basement requires MMP-2 activity insmooth muscle cells [36]. Additionally, the expression andactivity of MT-MMP (MMP-14), MMP-2, and MMP-9 in-crease during mechanical injury to arteries [37]. MMP-2 hasalso been shown to be present within atherosclerotic lesions.The proteolytic activity, conceivably, weaken the fibrous cap,resulting in its rupture [38, 39]. Age-associated disorganiza-tion of the internal elastic lamina has been observed in theaorta in the absence of externally imposed experimental in-jury [32]. Both MMP-2 and MMP-9 exhibit elastase activity[40], as does a metalloelastase of macrophages [41]. It hasbeen demonstrated that both latent and activated forms ofMMP-2 are greater in the aortas of old than in young rats [32].A significant amount of MMP-2 activity has been found tobe localized to the intima and elastic lamellae. MMP-2 hasbeen shown to accumulate in the area surrounding SMC lo-cated just beneath the broken internal elastic lamina and alongelastic laminae throughout the media indicating that MMP-2 may have role in the fragmentation of the elastic laminaewith aging [32].
Importantly, SMCs are potentially a source of age-associ-ated increase in MMP-2 in the aortic wall in situ, as earlypassage SMC from aged aorta secrete more MMP than thosefrom young aorta. MMP-2 production in SMCs of aged aortahas been shown to be triggered by stimulation with cytokines,including interleukin-1, TNF-α and TGF-β which suggestthat enhanced MMP-2 levels in the thickened intima of aor-tas from aged rats may reflect a chronically enhanced levelof cytokine stimuli in vivo [32].
The novel findings that increased MMP-2, TGF-β, andICAM-1 levels are chronically elevated and localized to thethickened intima of aged rats not only provide insights intopossible mechanisms of age-associated vascular remodelingbut also illuminate new links between senescence markers invitro and in cell senescence in vivo. These molecular changesduring vascular senescence in vivo may be targets for novelstrategies for the prevention and treatment of age-associatedvascular disorders [42].
Pathological consequences
Angiogenesis
Angiogenesis or neovascularization can occur throughoutone’s adult life span. During angiogenesis, capillary EC ofparent venule are stimulated by an angiogenic stimulus, suchas fibroblast growth factor (FGF), vascular endothelial cell
growth factor (VEGF) and vascular permeability factor (VPF).An increasing body of evidence has documented the differen-tial expression and production of MMPs by EC. The perivas-cular ECM is composed predominantly of type I collagen, andtwo specific MMPs, interstitial collagenase (MMP-1) andneutrophil collagenase (MMP-8), are capable of degradingtype I collagen [43]. In a recent study, it has been demon-strated that MMP-1 activity appears to be required for ang-iogenesis [44].
MMPs facilitate EC release via the degradation of the ven-ules basement membrane. Proteolytic activity is also requiredfor the migration of EC into the perivascular stroma. Theseevents are followed by sprout extension and subsequent lu-men formation [45, 46].
The role of thrombin as a regulator of EC function and as apotential physiologic regulator of angiogenesis has receivedrecent attention. Of particular interest is the report that proteo-lytically active thrombin can induce the activation of MMP-2in human umbilical vein endothelial cells (HUVECs) by a proc-ess probably involving the endothelial plasma membrane[47]. This finding represents a novel activation mechanismfor MMPs by EC and smooth muscle cells. It has been sug-gested that the remodeling of the capillary endothelium thatrequires during angiogenesis can be induced by thrombin [47].
Tumor cell invasion and angiogenesis share a number offunctional similarities. Initiation of cellular invasion in bothprocesses requires attachment to a basement membrane, fol-lowed by creation of a proteolytic defect in the basementmembrane and migration through this defect. After invadingcells cross this connective tissue barrier, cell proliferation andcontinued invasive behaviour results in production of eithera new vessel lumen or metastatic foci. In addition to sharingthese functional similarities, angiogenesis and tumorigenesismay be mutually stimulating. Formation of new blood ves-sels permits expansion of tumor foci in three dimensions [48].Prior to vascularization, tumor foci exist as small, asympto-matic lesions restricted by the limitation of passive oxygenand nutrient diffusion. Following vascularization, the tumorfoci undergo rapid local expansion and that in turn acquireenhanced metastatic potential that correlate directly with thedegree of vasularization of the primary tumor [49]. Thus,tumor invasion and metastasis formation are closely linkedto tumor induced neoangiogenesis.
Evidence for the role of MMPs and TIMPs in angiogen-esis come from a number of studies. Nanomolar concentra-tion of TIMP-2 has been shown to block the angiogenesisproduced by cytokines [50]. Cytokines are produced by vas-cularized tumors [50]. TIMP-1 has also been shown to inhibitendothelial cell invasion of human amniotic membranes invitro [51]. Schnaper et al. [52] have demonstrated the criti-cal nature of the balance of MMPs and TIMPs in an in vitromodel of angiogenesis. Addition of excess activated gelatinaseA beyond a critical level resulted in a decrease in tube for-
mation that was reversed by addition of exogenous TIMP-2.Thus, early stages of endothelial tube formation are depend-ent on a critical balance of active protease, gelatinase A andinhibitor, TIMP-2. Excess protease activity although initiallystimulatory, becomes inhibitory in higher concentrations, andTIMP-2 can reverse this effect. Thus, MMP inhibitors, espe-cially the gelatinase A specific inhibitors may have dual po-tential for clinical prevention of tumor cell dissemination andtumor associated neovascularization [50].
Chronic ulcers
Chronic ulcers are defined as the wounds, which does not healwithin the normal biological time range. Examples of chronicwounds include ulcers, superficial surgical wounds, chemi-cal burns and burn wounds. The mechanisms of impairedwound healing have been widely studied. Differences in in-flammatory response, in fibroblast phenotype and in proteoly-sis have been documented. Increased amounts of inflammatorycells are detected in human chronic ulcers. Fibroblasts fromchronic ulcers have a reduced growth and reduced mitogenicresponse to PDGF, compared to fibroblasts from acute wounds.In addition, these fibroblasts have impaired ability to synthe-size collagen in culture and fail to stimulate type I collagenproduction in response to TGFβ1 associated with a decreasedTGFβ1 type II receptor expression [8]. It has been suggestedthat the over expression and activation of MMPs and reducedexpression of TIMPs may lead to excessive degradation ofconnective tissue and formation of non healing chronic ulcer.The expression of MMP-1 and -2 are increased and the ex-pression of TIMP-2 decreased in vivo in lipodermatosclerosis,a skin induration preceding dermal ulcer [8, 53].
In dermal ulcers, the expression of several MMPs havebeen detected in vivo. In general, the expression of MMPs isincreased in chronic ulcers, compared to acute wounds. Astriking difference between acute and chronic dermal woundsis that MMP-13 is expressed by fibroblasts in the ulcer bed[8, 54].
Compared to the normal healing process of acute wounds,the expression levels of MMP-2, MMP-9 and their activatedforms are increased in wound fluids of chronic ulcers [55, 56].Levels of activated u-PA and MMP-9 are elevated similarlyin chronic wound fluids. It has been suggested that u-PA mayactivate proMMP-9 [57]. A recent study shows that woundfluids of chronic ulcers contain elevated levels of activatedMMP-1 and MMP-8, the predominant collagenase present inwounds [8, 58].
Liver fibrosis
Liver fibrosis is traditionally viewed as a progressive patho-
logical process involving multiple cellular and molecularevents that lead ultimately to deposition of excess matrixproteins in the extracellular space. When this process is com-bined with ineffective regeneration and repair, there is increas-ing distortion of the normal liver architecture, and the endresult is cirrhosis. Studies with hepatic stellate cells (HSCs)suggested that liver fibrosis is dynamic and can be bi-direc-tional. In addition to an increase in matrix synthesis, thispathological process involves major changes in the regulationof matrix degradation [59].
There is some evidence to suggest that MMPs capable ofdegrading normal liver matrix are expressed in liver injuryand fibrosis. There is a long-standing assumption that deg-radation of fibrillar matrix in liver (and the type I and IIIcollagens that this contains) is mediated by the interstitialcollagenases (MMP-1 in humans and MMP-13 in rats). Re-cent studies suggest that the true picture that prevails underin vivo conditions may be significantly more complex andcould involve other MMPs, especially, MT1-MMP (MMP-14) [59].
In contrast to MMP-1 and MMP-13, gelatinase A andMT1-MMP are both expressed in stimulated HSCs, and ex-pression of both is increased in liver fibrosis. These enzymesare both associated with the cell surface of HSCs in theiractive forms, situating them in an ideal location to play a sig-nificant role in pericellular degradation of fibrotic liver ma-trix. During regression of liver fibrosis, expression of bothgelatinase A and MT1-MMP gradually returns to base linecontrol values in a similar manner to that of TIMP-1 andTIMP-2 [59].
Lung diseases
Asthma
Airway smooth muscle has important secretory functions andparticipates in pro- and anti-inflammatory responses in ad-dition to its contractile role [60]. Production of MMP-2 byairway smooth muscle suggested that it contributes to extra-cellular matrix turnover and airway remodeling and in inflam-matory diseases such as asthma. MMP-2 may be producedby other airway cells including fibroblasts and macrophages;thus these cells may interact to support airway smooth mus-cle proliferation [60].
Acute respiratory distress syndrome (ARDS)
The involvement of MMPs in tissue remodeling have beenreported in a variety of diseases, including pulmonary fibro-sis [61]. 92 kDa gelatinase activity in broncho alveolar lav-age fluid (BALF) of patients having acute respiratory distress
syndrome (ARDS) was significantly increased. Both neu-trophils and macrophages are potent sources of 92 kDa gel-atinase. The close correlation with the number of neutrophilsin BALF, but not with the number of macrophages, suggeststhat 92 kDa gelatinase originates from neutrophils in thealveolar space. The high level of 92 kDa gelatinase in BALFcould lead to matrix degradation that occur in ARDS [62].
Clinically, the early stage of ARDS is exemplified by amarked inflammation with edema and fibrin deposition,whereas tissue remodeling is a hallmark of late phases. The92 kDa gelatinase might be deleterious as well as beneficialbecause it not only degrades matrix, but it could also preventfibroproliferation. The 92 kDa gelatinase level of normal andARDS patients are similar during the early phases. Therefore,the sole and direct involvement of 92 kDa gelatinase in thedevelopment of ARDS is questionable. By contrast, its roleseems important in late phases when it could allow the clear-ance of the abnormal interstitial matrix, to facilitate recon-stitution of the normal structure of lung [62].
MMPs alone are unable to determine the clinical courseof ARDS. However, there is growing evidence that cytokinesand MMP interact closely to produce ARDS. While IL-1 andTNF-α enhance matrix degradation by stimulating proteaseproduction [63], the other cytokines, for example, TGF-β re-press MMP expression and also increase production of tissueinhibitors of metalloproteinases (TIMPs) [64]. Until recently,too much attention has been paid on inflammatory cells, espe-cially alveolar macrophages, whereas cells of the lung paren-chyma e.g. endothelial cells, epithelial cells, and fibroblastsmay also participate actively in this scenario. Importantly,direct interaction between activated leukocytes and connec-tive tissue cells appears to play a pivotal role for the forma-tion of ARDS [65].
Chronic obstructive pulmonary disease (COPD)
The progressive structural damage associated with emphy-sema and other forms of chronic obstructive pulmonarydisease (COPD) appears to be due to degradation of se-lective extra-cellular matrix components. The proteinasesthat can degrade elastin are involved in the developmentof COPD. The elastin content of the lung parenchyma isdecreased in emphysema, while the collagen content is in-creased [66].
Several MMPs are produced in human emphysematouslung [67]. For example, induced over expression of inter-leukins results in the production of several MMPs, leading toemphysema [26]. Furthermore, several studies using transgenicand gene-targeted mice have supported the role of MMPsin emphysema [68].
Exposing proteinase null mice to cigarette smoke providesa highly controlled model to assess the role of specific MMPs
in emphysema. Long-term exposure of wild-type mice to ciga-rette smoke leads to inflammatory cell recruitment followedby alveolar space enlargement that is quite similar to the le-sions that develop in humans. Mice deficient in metallo-elastase, however, are markedly protected from developmentof emphysema due to long-term smoke exposure [69].
Metalloelastase is not the sole proteinase responsible forthe human disease. There are probably several proteinasesand inflammatory cells involved in the development of em-physema in humans. Human macrophages probably have abroader spectrum of MMPs (including metalloelastase). Col-lagenase-1, -2 and -3 also undoubtedly contribute to loss ofthe airspace and an increase in collagen deposition leadingto airway obstruction [26].
Importantly, different enzymes from multiple families mayact in concert in tissue remodeling. For instance, metallo-elastase, as well as other MMPs, degrades α
1-proteinase in-
hibitor; and neutrophil elastase, a serine proteinase, degradesTIMPs. These enzymes, by neutralizing each other’s naturalinhibitors, can accentuate overall proteolytic activity [70].
Cardiovascular diseases
Atherosclerosis
Atherosclerosis is an inflammatory process in which plaquesare formed in the intimal layer of the vessel wall as a resultof accumulation of lipid-rich macrophages, smooth musclecells, and lipids, and deposition of extracellular matrix. Clini-cal complications of atherosclerosis are often triggered byrupture of unstable plaques, triggering intravascular throm-bosis and tissue ischemia [71–74]. Alternatively, thinning ofthe atherosclerotic vessel wall due to elastin and collagendegradation and matrix necrosis may result in aneurysm for-mation and bleeding [75, 76]. The MMP system has beenimplicated in the pathogenesis of atherosclerosis and aneu-rysm formation [73, 77, 78]. In animal models, over expres-sion of TIMP-1 prevented aortic aneurysm formation andrupture. Human atherosclerosis appears to be an importantexample of the potentially harmful effects of 92 kDa gelatinaseproduction. Immunostaining has shown that in both stable andunstable angina, 92 kDa gelatinase production in arteries isincreased, whereas in normal arteries 92 kDa gelatinase is notexpressed [79]. Production of 92 kDa gelatinase by macro-phages in human aortic aneurysm has recently been observed,giving this macrophage enzyme a potentially important rolealso in atherosclerosis [80]. Thus, increased production of 92kDa gelatinase in atherosclerosis may contribute to the ma-trix destruction and subsequently leads to plaque rupture.Interstitial collagenase and stromelysin have also been iden-tified in atherosclerotic coronary arteries [39]. The factorsregulating the production of metalloproteinases in atheroscle-
rotic lesions are currently not clearly known. TNF-α and IL-1β have been shown to increase 92 kDa gelatinase produc-tion by human monocyte-derived macrophages in vitro. Theseproinflammatory cytokines have also been reported to stimu-late 92 kDa gelatinase production in human smooth musclecells [81]. Coronary arteries, macrophages and smooth mus-cle cells have been determined to be the major source of 92 kDagelatinase [39]. Thus, increased expression of TNF-α [82, 83]and IL-1β [83] by the inflammatory cells of the arterial wallcould dramatically increase production of 92 kDa gelatinasein the lesions. Cells within the vessel walls produce and se-crete MMPs. Expression of various latent (pro-)MMPs isincreased in atherosclerotic lesions. The spectrum of MMPsis diversified through the presence of inflammatory cells,stimulation by soluble factors, cell-cell, and cell-matrix in-teractions. Degradation of matrix by activated MMPs, detect-able in vessels undergoing remodeling, is thought to facilitatecell migration and general reorganization of vascular tissue.Ultimately, MMPs are thought to weaken the arterial wall,thus contributing to the stabilization and rupture of athero-sclerotic plaques [84, 85].
One mechanism for the growth of atherosclerotic lesionsis through the recruitment of circulating inflammatory cellsmediated via interaction with adhesion molecules expressedby the activated endothelium [86]. The mechanisms allow-ing for the subsequent infiltration of leukocytes through theendothelial layer and its associated basement membrane af-ter the adhesion event remain largely unknown. Recent ex-periments suggests that MMP action may facilitate this step[86].
In vitro cellular interactions between T lymphocytes andEC monolayers was shown to trigger secretion of MMP-2from T cells [87]. The release of MMP-2 was dependent onthe expression of VCAM-1 by the ECs. MMP degradationof EC basement membrane during diapedesis of inflamma-tory cells could contribute to a decreased endothelial barrierfunction [88] with increased influx of plasma proteins includ-ing lipoproteins. Once inside the vessel wall, infiltrating cellsinteract with ECM, oxidized lipids, and with each other. Allof these interactions have been shown to increase productionof MMPs in macrophages [89–91]. Macrophages also pro-vide stimuli for MMPs production in neighbouring cells andmechanisms for activation of secreted MMP zymogens [92].The increase in MMPs activities in developing atheroscleroticlesions may facilitate further structural changes and enabletheir growth.
Alternative or complimentary systems for activation oflatent MMPs in atherosclerotic plaques have been suggested.Thrombin has been shown to proteolytically activate purifiedpro-MMP2 in vitro and thus could provide cell independentMMP activation at sites of vascular injury [93]. In compli-cated atherosclerotic plaques, thrombin could promote plaqueinstability in episodes of intraplaque hemorrhage of super-
imposed plaque thrombosis by increasing the local matrixdegrading activity of MMPs. The mutually activating MMP/thrombin system may serve as an important positive feed-back loop in acute coronary syndrome [94]. An acute plaquedisruption leads to local thrombin production at the site ofvascular injury, this may facilitate proteolytic activation ofMMP-2, shown to be able to platelet aggregation [95], thusfurther generation of thrombin and, respectively, more MMP-2 activation.
Pericellular activation of proMMP-2 can be achieved byMT-MMPs, expressed by vascular ECs and SMCs in re-sponse to cytokines and oxidized lipoproteins [86, 96]. Theplasminogen cascade represents another proteolytic activat-ing mechanisms of MMP zymogens [86]. Proinflammatorymolecules IL-1α, TNF-α and ox-LDL augment MT1-MMPexpression, leading to increased activation of pro-MMP2 insmooth muscle cell mediated vascular remodeling in normaland atherosclerotic human arteries. Ox-LDL directly or byinducing activators such as cytokines may influence remodel-ing of the ECM in atherosclerosis. It was reported that thereactive oxygen species can promote activation of MMPswhich argue in favour of the concept that proinflammatorycytokines or ox-LDL-mediate the activation of MT1-MMPby generating highly reactive oxygen species [97].
Inflammatory myopathy
MMPs have been suggested to play a role in inflammatorymyopathies. These immune mediated disorders are character-ized by the invasion of mononuclear phagocytes and T-lym-phocytes and loss of muscle fibres. Muscle biopsies obtainedfrom patients diagnosed as having polymyositis, sporadicinclusion body myositis and, for example, from cases of vari-ous muscular dystrophies were examined. MMP-1 and MMP-9 levels were elevated in polymyositis and dermatomyositisand to a lesser extent in inclusion body myosites, whereas thelevel of expression of TIMP remained unchanged in compari-son with controls. These observations indicate a pathogenicrole for specific MMPs in the genesis of inflammatory my-opathies, and open new strategies for therapeutic intervention[98].
Congestive heart failure
MMPs are expressed at very low levels in normal myocar-dium, such as collagenase-3 (MMP-13) and membrane type-1MMPs (MT1-MMP) are substantially upregulated in conges-tive heart failure (CHF). However, MMP species are notuniformly increased in patients with the end stage CHF,suggesting that a specific portfolio of MMPs are expressedin the failing myocardium. With the use of animal models of
CHF, a mechanistic relationship has been demonstrated withrespect to myocardial MMP expression and the left ventricu-lar (LV) remodeling process. The disparity between MMPand TIMP levels favours a persistent MMP activation statewithin the myocardium and likely contributes to the LV re-modeling process in the setting of developing CHF. The elu-cidation of upstream signaling mechanism that contribute tothe selective inhibition of MMP species within the myocar-dium as well as strategies to normalize the balance betweenMMPs and TIMPs may yield some therapeutic strategieswhich control myocardial extracellular remodeling andthereby show the progression of the CHF process [99].
A milestone in the progression of congestive heart failure(CHF) is myocardial remodeling. Left ventricular (LV) re-modeling during the progression of CHF is accompanied bychanges in the structure of the myocardial extracellular ma-trix. Recent clinical and experimental studies have noted thatan increase in the release of tumor necrosis factor-α can con-tribute to LV myocardial remodeling. Experimental studieshave noted that the induction of TNF-α can result in LV di-lation and proceed to LV pump dysfunction. The biologicaleffects of TNF-α are mediated through TNF-α receptor thatare present on all nucleated cells in the heart. TNF receptoractivation can induce a number of cellular and molecularevents which contribute to LV remodeling in CHF and in-clude changes in myocyte size and viability and alterationsin myocardial structure/composition. In vitro studies havedemonstrated that TNF-α receptor activation can cause theinduction of MMPs. The MMPs are upregulated in modelsof LV dysfunction and possesses the capacity to degrade awide variety of extracellular matrix components. Therefore,one pathway by which TNF-α can influence LV myocardialremodeling is through the induction of a specific portfolio ofMMP species [100, 101].
It has been clearly demonstrated that the development ofLV failure and myocardial remodeling is accompanied byincreased levels of certain MMP species [102, 103]. For ex-ample, myocardial MMP-9 levels have been shown to beincreased in both animal and human models of LV remodel-ing [104–106]. Moreover, recent studies in transgenic micesuggest that this MMP species plays a role in post infarctionmyocardial remodeling [101]. Clinical studies [107, 108]have demonstrated the emergence of the interstitial colla-genase MMP-13 in pathological remodeling states such ashuman breast cell carcinoma and osteoarthritis. More impor-tantly, in a recent study [109] elevated MMP-13 levels wereobserved in end-stage human heart failure. Increased myo-cardial MMP-13 levels were shown to occur with chronicpacing which has been suggested to be associated with themyocardial remodeling process [101].
Role of TNF-α blocking protein on MMP levels in an evolv-ing heart failure model has been established [101]. In transgenicmice, myocardial over expression of TNF-α has been shown
to produce LV dilation [110]. It has been shown that admin-istration of TNF-α blocking protein during chronic pacingnormalized myocardial MMP-9 and MMP-13 levels. Inter-estingly, myocardial MMP-2 levels appeared unaffected byeither chronic pacing or the TNF-α blocking strategy. A po-tential mechanism for the differential myocardial MMP lev-els may lie in the fact that TNF-α receptor activation resultsin the formation of specific DNA transcription factor such asthose binding to activating protein-1 (AP-1) [111]. Notably,AP-1 response elements are present on the genes of MMP-9and MMP-13 but absent from the MMP-2 gene promoter[112]. MMP-9 and MMP-13 expression, therefore, may po-tentially be induced by TNF-α stimulation and inhibited byTNF-α blockade whereas MMP-2 induction may not occurvia the same regulatory pathway. The present study suggeststhat MMP-2 may not be induced by cytokine activation butrather is constitutively expressed in the myocardium. TNF-αmay, therefore, influence myocardial remodeling by the se-lective induction of a specific portfolio of MMPs such asMMP-9 and MMP-13, which, in turn, facilitate myocardialremodeling. It should be noted that there are other mecha-nisms, for example, angiotensin II which can potentially in-duce MMP expression and are operative in the heart failure[13, 101].
Pacing induced atrial cardiomyopathy
Rapid atrial pacing produces atrial systolic and diastolic fail-ure characterized by absent of atrial booster pump function,increased atrial chamber stiffness, enhanced atrial conduitfunctions, and atrial enlargement. However, the process un-derlying these abnormalities are poorly understood. Left atrialmyocardium from dogs with rapid pacing-induced atrial fail-ure was compared with that from control dogs. The activityof MMP-9 was found to be selectively increased and the levelof complexed TIMP-4 protein was decreased in samples fromdogs with atrial failure. Thus, rapid pacing induced atrialfailure is associated with differential changes in MMP activ-ity [113].
Cardiac fibrosis
MMPs not only play a role in degrading matrix components,but also modulate collagen synthesis. An increase in the levelof MMPs is accompanied with increased fibrosis as seen infailing heart. MMPs may participate in the fibrosis and re-modeling process through direct digestion of matrix compo-nents, and regulation of the formation of matrikines [114].Imbalance of extracellular matrix turnover (synthesis anddegradation) is the cause and not the effect of structural dis-
ease in the heart. A balance between ECM, MMP and TIMPsconcentrations are required for normal structure remodeling[115].
It has been demonstrated in many animal models and inpatients with various etiological background with congestiveheart failure that cardiac remodeling includes not only ven-tricular dilation but also the presence of interstitial cardiacfibrosis in both RV and LV [116]. Indeed in infarcted hearts,cardiac fibrosis may occur at sites distal to the region of in-farction and in right ventricle [117]. The destruction amongevents leading to development in altered cardiac collagenconcentration (interstitial fibrosis) and cardiac remodeling(dilation i.e., geometric chamber expansion) of ventricularchamber may be related to the stage of heart failure [115,118].
Recent studies have directed the interest at modeling theheart failure process through inhibition of activated MMPs.A loss of MMP inhibitory control by TIMP-1 deficiency hasbeen shown to alter the course of post infarction remodelingand induced chronic myocardial infarction in wild-type andTIMP-1 knockout mice [119].
LV pressure volume loops obtained from wild type andTIMP-1 knockout mice elicited that LV-end diastole volumesand LV-end diastole pressure were significantly increased inthe TIMP-1 knockout mice during latter stages of post myo-cardial infarction (MI). LV contractility was reduced to asimilar degree in both the wild-type and TIMP-1 knockoutgroups after MI, as indicated by a significant fall in preloadstroke volume. Ventricular weight and cross sectional areas ofLV-myocytes were significantly increased in TIMP-1 knock-out mice indicating that the hypertrophic response was morepronounced. The observed significant loss of fibrillar colla-gen in the TIMP-1 knockout controls may have an impor-tant contributory factor for the observed LV alterations inthe TIMP-1 knockout mice after MI. These findings demon-strate that TIMP-1 deficiency amplified adverse LV-remodel-ing following MI in mice and emphasizes the importance oflocal endogenous control of cardiac MMP activity by TIMP-1 [119].
The fibroblasts of infarcted heart express receptors forTGFβ1, angiotensin II, endothelin and proinflamatory cyto-kines. Angiotensin II generated de novo within the infarctedheart has autocrine and paracrine properties that influence theturn over of connective tissue [120]. In infarcted rat heart,locally generated angiotensin II is connected to TGFβ1 ex-pression and synthesis [121]. It is proposed that early induc-tion of TGFβ1 via the angiotensin II type I receptor plays amajor role in the development of cardiac fibrosis [122].TGFβ1 treatment of cardiac fibroblasts increases the abun-dance of pro α2 (I) and pro α2 (III) mRNA type I and typeIII collagens [120]. Similarly recombinant adenovirus medi-ated over expression of TGFβ1 results in elevated levels of
type II collagen gene expression in vascular smooth musclecells and fibroblasts [123]. It has been found that TGFβ1 lev-els can correlate with the deposition of collagens in the hu-man heart indicating a role of TGFβ1 in the regulation ofmyocardial fibrosis [114].
Genetic diversity of the MMPs in disease progression
Molecular genetic strategies have been adopted to identifyassociation between genetic polymorphism in specific mem-bers of MMP families and a range of phenotypes representa-tive of atherosclerotic lesions progression and rupture [124].Interestingly, a recent study has reported an association be-tween the MMP-3 5A allele and myocardial infarction [125].In vitro transfection studies showed that this allele drovehigher levels of transcription than the 6A allele [126]. Inseems apparent that an over representation of the 5A allelein cases of plaque rupture, resulting in myocardial infarction,is consistent with the fact that this higher expressing allelecould contribute to excess local matrix degradation.
With MMP-9, substantially more information was avail-able on the organization of the gene and its promoter thanMMP-3 [126]. This gene, in aortic aneurysm, has a (CA)
n
repeat polymorphism in the promoter [127]. It is necessaryto investigate the potential impact on disease of this relativelycommon polymorphism apparently affecting promoter func-tion.
Research in the recent past have focused on the macrophageelastase MMP-12 which plays an important role in aorticaneurysm in mouse model of atherosclerosis. This enzymeis known primarily to degrade elastin, but it also degradesubstrates such as type IV collagen and fibronectin, and it isknown to be expressed widely by macrophages in atheroscle-rotic lesions [129]. Genetic analysis resulted in the identifi-cation of a promoter variant, arising from a single nucleotidesubstitution and having an estimated population frequency of0.16. Interestingly, this polymorphism is located very closeto the consensus sequence for one of this cis-elements in theMMP-12 promoter critical to the gene regulation apparatus.The presence of a relatively frequent variable base so closeto such an element may have a significant impact on the bind-ing of the nuclear proteins and the subsequent transcriptionof the gene. Experimental evidence suggested that nuclearproteins do bind in this region and that appears to be a differ-ence in binding affinity between the two alleles. In vitro tran-scription assays in macrophage cell lines also demonstrateallele specific differences [130]. Further work on the char-acterization of this polymorphism, as well as the completionof the screening for variants in the coding region and 3′-untranslated region of the gene warrants immediate atten-tion.
Upregulation of angiotensin II receptor may be involved inthe initiation and progression of atherosclerosis. The contri-bution of ATI receptor in the expression of MMP-1 and itsinhibitor TIMP-2 in lipid deposited arterial tissues have beenexamined. It has been found that ATI receptor blockade withlosartan reduces lipid deposition and exerts potent inhibitoryeffects of NF-κB activation and modulates the expression ofMMP-1 and TIMP-2 in hypercholesterolemic rabbits [131].
Angiotensin II has direct cellular effects on the pathobiologyof vascular diseases through growth promoting activity. Bind-ing of angiotensin II to its target type I (ATI) receptor in thevascular tissues activates PLC, raises intracellular Ca2+ [132],activates nuclear elements that affect gene expression, andstimulate protein synthesis, mitogenesis and hypertrophy[133–135]. Angiotensin II also mediates vascular smoothmuscle cell growth and migration, monocyte/ macrophageactivation and platelet activation and stimulates atherogen-esis [136]. It has been suggested that ATI receptor activationstimulates leukocyte chemotaxis [137], release of reactiveoxygen species, uptake of oxidized low density lipoprotein(ox-LDL) by endothelial cells [138], and expression of thetranscription factor κB (NF-κB) [139].
NF-κB, an oxidative stress response transcription factor ispresent in cytosol as a heterodimer composed of NF-κB (p50)and Rel (p65) subunits bound to an inhibitor protein, IκB.After activation, NF-κB translocates from the cytosol to thenucleus of the cells, bind to specific DNA sequences, andinitiates transcription. Proteolytic degradation of IκB playsa pivotal role in NF-κB activation in endothelial cells [131].Brand et al. [140] demonstrated NF-κB activity in endothe-lial cells in atherosclerotic lesions. Hermandez-Presa et al.[141] confirmed that activation of NF-κB in atherosclerosisin rabbits. Maziere et al. [142] have suggested that oxidativestress activates NF-κB in fibroblasts, endothelial cells andsmooth muscle cells.
Evidence from both clinical and experimental studies in-dicate that angiotensin II expression is upregulated in theatherosclerotic tissue [143, 144]. Angiotensin II has been re-ported to regulate the expression of MMPs and TIMPs inseveral pathophysiological processes such as hypertensionand myocardial ischemia as well as cardiac and renal failure,and both angiotensin converting enzyme inhibitors and ATIblockers can modulate this regulation of MMPs and TIMPsby angiotensin II [145, 146].
NF-κB activation can be attributed to the enhanced expres-sion of ATI receptors and oxidative state in hyperlipidemia[131]. Ronet-Benzinels et al. [147] showed that angiotensinII activates NF-κB by the protein kinase C pathway. How-ever, several other studies suggested that angiotensin II couldalso induce the degradation of κBα, the cytoplasmic inhibi-tor of NF-κB [148, 149].
Angiotensin II blocker losartan (LS) reduced expressionof MMP-1 and TIMP-2. The data on the expression of TIMP-2 with LS are important and may relate to tissue protectioneffects of ATI blockade. The overall decrease in MMP-2 inthe intima and adventitia with losartan therapy suggests thatMMP-2 up regulation in atherosclerosis occurs as a result ofATI receptor activation and that inhibition of TIMP-2 expres-sion may represent a regulatory step as the expression ofMMP-2 is diminished [131]. A detailed examination revealedthat TIMP-2 expression was very intense in the intima. Thecompact nature of TIMP-2 in the intima may, at least, theo-retically be considered tissue protective as it would result inthe formation of fibrous cap rich in collagen and preventrupture of the plaque. The inhibition of reactive oxygen spe-cies with losartan may be the basis for inhibition of NF-κBactivation and other factors responsible for collagen degra-dation and MMP-1 [131].
Rheumatoid arthritis
One of the strongest predictors of long-term outcome in rheu-matoid arthritis (RA) and osteoarthritis (OA) is progressivestructural joint damage. The rheumatic diseases continue torepresent a significant health care burden in the 21st century.However, despite the best standards of care and recent thera-peutic advances it is still not possible to consistently preventthe progressive joint destruction that leads to chronic disabil-ity. In rheumatoid arthritis and osteoarthritis this progressivecartilage and bone destruction is considered to be driven byan excess of MMP enzymes [150].
In rheumatoid arthritis (RA), MMP contribute to joint de-struction in at least two ways. First, they can directly degradethe cartilage and bone. The major MMPs implicated in thisprocess include stromelysin-1, collagenase-1, collagenase-3,gelatinase-A and gelatinase-B. Second, MMPs are importantduring angiogenesis (the formation of new blood vessels)which is a prominent feature of rheumatoid arthritis. Duringangiogenesis, endothelaial cells degrade at least two distinctbarriers, the microvascular basement membrane and the in-terstitium. The gelatinases play a pivotal role during thesestages [151].
The continuous or intermittent destruction of rheumatoidjoints has been the subject of several investigations aimed atestablishing a link between disease progression and MMPexpression. Several studies have shown an increase in colla-genase activity in rheumatoid synovial fluids [152] and inculture media from rheumatoid synovial tissues and cells[153]. Collagenase and SL-1 have been identified near theareas of destruction at the cartilage-pannus junction and inthe synovial lining cells [154]. An examination in MMP ex-pression in cartilage from patients with rheumatoid arthritisdemonstrated an increase in the expression of stromelysin and
collagenase mRNA and protein in the synovial lining, hyper-proliferative pannus cells overlaying destroyed cartilage, andin the chondrocytes themselves [17]. McCracken [155] hasdetermined that the mRNA for stromelysin-1 is localized tocells positive for a monocyte/macrophage marker as well ascells of fibroblast lineage. In a comparative study, Case et al.[156] found higher levels of SL-1 mRNA and protein in hu-man synovium from patients with rheumatoid arthritis than inpatients with osteoarthritis. SL-1 transcripts and SL-1 proteinwere detected in lining cells, and in the underlying stroma,and in chondrocytes and osteoclasts of the joints of rats [157].The expression of stromelysin and interstitial collagenase insynovial tissue and cells, and their correlation with diseaseprogression support the concept that MMPs play a crucial rolein RA [13].
Human periodontal diseases
Collagenase that appears to be predominantly of the PMN-type is frequently detected in gingival crevicular fluid (GCF)in natural human periodontitis and in experimental periodon-titis in dogs and monkeys [158]. Enzyme activity generallyincreases with disease severity [159].
Although different mechanisms have been suggested to ex-plain the important pathogenic elements of human periodon-tal diseases yet it has not been possible to unequivocallyidentify those microbial host cell interactions that lead toattachment loss. A body of evidence has established thatexpression or activity of MMP may be stimulated in a vari-ety of cultured cells derived from the human periodontal tis-sue [13]. In addition to PMNs, these include fibroblasts,macrophages, and keratinocytes [13]. MMP activity may bestimulated either directly by microbial products from thebacterial plaques that colonize the teeth and their surround-ings [160] or indirectly by inflammatory mediators generatedin response to oral microorganism [161]. Candidate micro-bial products that are likely to play a role in the transcriptionalactivation of endogenous degradative pathways include bac-terial lipopolysaccharide (LPS), proteinases and perhapslectins [13]. Analysis of GCF have provided evidence thatmediators such as IL-1α, β and TNF-α which are potentiallycapable of inducing MMP expression, are present in GCF inphysiologically meaningful concentrations [162].
Cancer
MMPs mediate ECM and basement membrane degradationduring the early stages of tumorigenesis contributing to theformation of a microenvironment that promote tumor growth.MMPs are also active in the latter stages of cancer develop-ment in that they promote metastasis as well as other aspectsof tumor growth. During tumor invasion, malignantly trans-
formed cells detach from the primary tumor, migrate andcross structural barriers, such as basement membranes andsurrounding stromal collagenous ECM. Degradation of stro-mal ECM is also considered essential in tumor-induced ang-iogenesis. The role of MMPs in tumor invasion is mainlybased on the observations that invasive malignant tumorsexpress high levels of MMPs [163]. It has been shown thatMMP activity is required for increased motility of the epi-thelial cells and for growth of metastasized tumor cells.MMPs have also been shown to play an essential role in an-giogenesis and tumor cell intravasation, both of which arerequired for tumor cell growth and metastasis [42]. Additionalevidence for the role of MMPs in invasion and growth oftumors has recently been provided by MMP knockout mice.Mice lacking MMP-7 showed reduction in intestinal tum-origenesis, while MMP-2-deficient mice show reduced an-giogenesis and tumor progression. MMP-11 knockout miceshow reduced tumorigenesis in response to chemical muta-genesis [42].
MMPs promote the initiation and sustained growth of bothprimary tumors and metastatic foci by activating growth fac-tor, by activating growth factors binding protein, or by re-leasing mitogenic molecules from matrix proteins that aresequestered in the peritumor ECM. Thus, MMP activatedgrowth factors directly induce tumor cell proliferation, orindirectly regulate the behaviour of fibroblasts or endothelialcells that support tumor growth. MMPs also process cell ad-hesion molecules [164]. MMP-7 allows tumor cells to becomeresistant to apoptotic signals [165]. By destroying chemokinegradients that are laid down in the peritumor stroma to attractimmune cells, tumor derived MMPs assist in circumventingthe host anti tumor defence system [166]. MMPs also promotetumor angiogenesis by mobilizing or activating angiogenicfactors such as basic fibroblast growth factor (βFGF), vascularendothelial growth factor (VEGF) or transforming growthfactor β (TGF β) [164, 167]. They also negatively regulateangiogenesis by cleaving precursors of angiostatin and endo-statin, to generate active angiogenesis inhibitors [166].
Tumor invasion is a multistep process and there is recentdirect evidence that MMPs play an important role in tumorinvasion and progression. The expression of MMPs in tumorsis regulated in a paracrine manner by growth factors andcytokines secreted by tumor infiltrating cells as well as bytumor or stromal cells; recent studies have suggested continu-ous cross-talk between tumor cells, stromal cells and inflam-matory cells during the invasion process [42, 163, 168, 169].Tumor cell derived factors that increase the expression ofseveral MMPs in stromal cells have led to the identificationof EMMPRIN (extracellular matrix metalloproteinase in-ducer) was originally identified from the cell membrane ofLx-1 carcinoma cells has been shown to induce the expres-sion of MMP-1, MMP-3 and MMP-2 by fibroblasts [170].Furthermore, the ability of MMP to degrade and inactivate
interleukine 1β- (IL-1β) [171] and cleave the tumor necrosisfactor α (TNF-α) precursor to a biologically active form [172]as well as the capacity of TIMP-3 to inhibit activation of TNF-α [173], indicate that besides degrading ECM components,MMPs and TIMPs may also regulate the availability and ac-tivity of inflammatory cytokines at the site of tumor invasion.
In vivo expression of MMPs is localized to both tumor andstromal cells at the invading margin of tumor; providing amechanism for highly concerted degradation of ECM [42].Increased expression of MMP-1 has been observed in lungcarcinomas [174, 175], squamous cell carcinomas of the headand neck [176] and colorectal tumors [42]. Both gelatinasesMMP-2 and MMP-9 are abundantly expressed in variousmalignant tumors. MMP-9 not only expressed by malignantcells, but also by inflammatory cells including tissue macro-phages and eosinophils. Immunohisochemical analysis of theexpression of MMP-2 has shown an increase in the immuno-reactive enzyme at the neoplastic epithelium of breast, colonand gastric adenocarcinomas [42, 177, 178]. Both MMP-2 andMT1-MMP mRNA are also expressed by stromal fibroblastsof human vulva, breast, lung and head and neck carcinomassuggest that MT1-MMP binds to and activates MMP-2 at thecell surface of fibroblast [169, 179, 180]. In squamous carci-noma cells (SCCs), MMP-2 and MMP-9 may also be activatedby MMP-13 which, in turn, may be activated by MT1-MMP[169, 181]. These kinds of activation cascades may be re-quired to ensure cooperation between tumor and stromal cells;they greatly enhance cell surface localized proteolytic activ-ity in vivo and indicate that selective inhibition of one MMPmay be sufficient to block activation cascade and ECM deg-radation during tumor invasion [42].
There is increasing evidence that expression profiles forMMPs observed in cultured neoplastic cells are not repre-sentative of in vivo situations. Neoplastic cells in culture ex-press most member of the MMP family either constitutively orfollowing induction by oncogenes, growth factors or cytokines[182]. These expression profiles have led investigators tospeculate that expression of proteolytic enzymes by tumorcells was a critical step in the transformation process. In situhybridization studies have revealed, however, that expressionof MMPs in tumors in vivo is not limited to neoplastic cells,but frequently originates from associated stromal cells. Forexample, in carcinomas collagenase-1, gelatinase A, gelatinaseB, metalloelastase, stromelysin-2 and stromelysin-3 are ex-pressed by various stromal cells (activated fibroblasts, macro-phages, neutrophils, endothelial cells). On the other hand,expression of collagenase-3 has only been observed in breastcarcinoma cells. This enzyme was initially identified from abreast tumor derived cDNA library [179]. The normal expres-sion profile for collagenase-3 in human is still undefined. Inrodents, it is widely expressed particularly in brain. In carci-nomas of the colon, prostate and lung, matrilysin mRNA hasbeen found to be expressed only in tumor cells. In breast car-
cinomas, however, matrilysin expression was observed in bothtumor and tumor associated fibroblasts [179,182]. There hasbeen a recent report of matrilysin expression in an osteo-carcinoma confirming that its expression is not to cells ofepithelial origin [183]. Stromelysin-1 expression has also beenobserved in both epithelial and mesenchymal cells, the expres-sion being dependent on the stage and/or grade of the tumor.Stromelysin-1 is expressed in stromal fibroblasts in squamouscell carcioma although in the later stages of the disease, spin-dle cell carcinomas its expression shifts to tumor cells [184].Thus, increased expression of MMP family members doescorrelate with ECM remodeling during tumorigenesis andwith malignant phenotype.
Neurodegenerative diseases
In the central nervous system (CNS), MMPs have been shownto degrade components of the basal lamina, leading to disrup-tion of the blood brain barrier, and contribute to the neuro-inflammatory response in many neurological diseases. Braincell express both constitutive and inducible MMPs in responseto cellular stress. MMPs are tightly regulated to avoid un-wanted proteolysis. Secreted as inactive enzymes, the MMPsrequire activation by other proteases and free radicals. At thecell surface, they act as sheddases and release growth factors,which are important in cell survival and death [185].
The motor neurons in amyotrophic lateral sclerosis patientsexpress significantly higher levels of MMP-9 as evidencedby its increased levels in cerebrospinal fluid, suggesting a rolein neurodegeneration [186]. In serum, however, MMP-9 wassignificantly increased in amyotrophic lateral sclerosis (ALS)patients which is similar to that viral meningoencephalitis(VM) or bacterial meningitis (BM) patients [187].
It has recently been shown that axonal damage occurs with-in active plaques (localized area of myelin sheath destructionwithin the central nervous system) as a consistent consequenceof demyelination in multiple sclerosis [188]. Demyelinationnot only damages neural transmission directly, but also in-creases axonal vulnerability to the proteolytic enzymes andcytokines produced by activated immune and glial cells. Inthe animal model of multiple sclerosis, and experimental auto-immune encephalomyelities (EAE), MMP activity was foundto be dramatically increased in the cerebrospinal fluid of dis-eased animals as symptoms appeared, peaking at maximaldisease severity [189]. Increased MMP expression has alsobeen correlated with the pathologies of Alzheimer’s disease,malignant gliomas and amyotrophic lateral sclerosis [190].
Cerebral aneurysm
Cerebral aneurysm usually remain asymptomatic until rup-ture occurs. Despite modern therapy, aneurosmal subarach-
noid hemmorrhage remains one of the most severe forms ofcerebrovascular disease, with a mortality approaching 50%has found about 3-fold increase in a 72 kDa serum gelatinasein a sub-group of aneurysm patients [191].
Some conditions either directly or indirectly related to an-eurysm formation may influence circulating pro-MMP-2 lev-els by acting on sites distant from the cerebral aneurysm.Alternatively, it is possible that proMMP-2 may shed from theregion of the cerebral aneurysm representing a circulatorymarker of molecular events occurring with the aneurysm wall.MMP-2 has been suggested to play an active role in the ma-trix degradation that either initiated or propagated the forma-tion of intracranial aneurysms. Although the precise methodof pro-MMP-2 activation in vivo has not been characterized,recent research have identified a unique multistep activationpathway that involves members of both ECM degrading fami-lies. In both pathways, activation occurs on the cell surfacetargeting activation of pro-MMP2 to specific anatomic sites.The transmembrane MMPs (MT1-MMP and MT2-MMP) havebeen shown to activate proMMP-2 in some cases forming a64 kDa intermediate [192, 193]. The plasmin activator-plas-min system has been implicated was implicated in the secondstage of MMP-2 activation to the final active 62 kDa form[194]. Other studies show the urokinase-plasmin system play-ing a larger role in the control of gelatinase activity [195]. Thisis of interest since we recently localized both plasmin and MT1-MMP in cerebral aneurysm tissue, raising the possibility oflocalized activation of MMP-2 within the aneurysm [191, 196].
Parkinson’s disease
MMP-2 levels in post mortem brain tissue from Parkinson’sdisease (PD) cases in substantia nigra were investigated.Levels of MMP-2 were not significantly changes in the cor-tex and in hippocampus MMP-9 levels were unchanged inthe investigated brain regions. Immunohistochemically waslocalized primarily in astrocytes and microglia cells whereasMMP-9 was predominantly neuronal. Levels of TIMP-1, anendogenous tissue inhibitor of MMPs were significantly el-evated in the substantia nigra, but not in the cortex and hip-pocampus. TIMP-2 levels were unchanged in PD. MMP-1levels were unchanged in PD cases compared to controls.Together, these data show alterations of MMP-2 and TIMP-1 in the substantia nigra, consistent with the possibility thatalterations in MMPs/TIMPs may contribute to disease path-ogenesis [197].
Alzheimer’s disease
The neurones in the Alzheimer disease brain expressed MMP-9 and uPAR. MMP-9 may be produced for the degradationof amyloid beta protein [198].
MT5-MMP (MMP-24) has been found to be the predomi-nant species among the nongelatinase type isoforms in brain.MT5-MMP has most strongly expressed in cerebellum andwas localized in the membranous structure of expressing neu-rones, as assessed biochemically and immunohistochemically.In cerebellum, its expression was regulated developmentallyand was closely associated with dendritic tree formation ofPurkinje cells, suggesting that MT5-MMP may contribute toneuronal development. Furthermore, its stable post develop-mental expression and colocalization with senile plaques inAlzheimer brain indicates possible roles in neuronal remodel-ing naturally occurring in adulthood and in regulating patho-physiological processes associated with advanced age [199].
It has been suggested that there may be an inflammatorycomponent to the pathology of Alzheimer’s disease (AD), themajor form of degenerative dementia in the elderly. Activityof inflammatory cells and the elaboration of toxic moleculesby such cells may be a significant factor in disease progres-sion. In peripheral inflammatory states, the increased activi-ties of MMPs are a major cause of tissue breakdown andsecondary damage in diseases such as rheumatoid arthritis.MMP-1 levels were significantly elevated by approximately50% in AD in all cortical areas. It is, therefore, possible thatMMP-1 activity in AD may contribute to the blood brain bar-rier dysfunction seen in AD [200].
A growing body of evidence indicates that MMPs may playan important role in the pathogenesis of Alzheimer’s disease(AD). Stromelysin-1 (MMP-3) plays a central role in acti-vating latent type MMPs, which were originally secreted asproenzymes. The interstitium between myelinated axons andastrocytes in the white matter of all brain tissues and senileplaques in the grey matter of the patients with AD were stainedwith a monoclonal antibody to MMP-3. Comparison of thenumber of senile plaques stained with the antibody againstMMP-3 in the parietal cortex cells with that in the hippoc-ampus showed that fewer plaques were stained in the hippoc-ampus. The selective distribution of MMP-3 in the humanbrain suggests that MMP-3 might play an important role inthe pathogenesis of AD, especially in the degradation of β-amyloid protein [201].
Matrix metalloproteinase inhibitors
Tissue inhibitors of metalloproteinases
Matrix metalloproteinases are inhibited by two types of pro-teinases inhibitor. Tissue inhibitors of metalloproteinases(TIMPs) and inhibitors of metalloproteinases (IMPs) specifi-cally inhibit this class of enzymes.
All active forms of matrix metalloproteinases are inhibitedby TIMP-1 and TIMP-2 [13]. TIMP-1 is a 28.5 kDa glyco-protein that is synthesized by most connective tissue cells and
macrophages [13]. TIMP-2 is a 23 kDa unglycosylated pro-tein and is found at lower concentrations than TIMP-1 in tis-sue [13]. The homology of the amino acid sequences for thetwo inhibitors is only 40%, but the key 12 cysteines that formdisulfide bonds are conserved. Degradation products of theseinhibitors are not inhibitory [13].
TIMP-1 and TIMP-2 rapidly inhibit active gelatinase A.These inhibitors interact with the active site plus a site in thecarboxyl terminal hemopexin-like region. The C-terminalregion of the TIMPs interacts with the C-terminal region ofthe enzyme [190]. The C-terminal hemopexin-like regioncontributes stability to complexes of TIMP-1, with othermatrix metalloproteinases. Stromelysin binds to intact TIMP-1. The binding of TIMP-1 to collagenase is noncompetitive,but inhibition with the active site inhibitor or 1,10-phenan-throline prevents binding of TIMP-1. Active collagenase andstromelysin bind to TIMP-1 very slowly. These enzymes bindat a faster rate to α2-macroglobulin [7, 13, 190].
In addition to the inhibition of active matrix metallo-proteinases, TIMP-1 can regulate the activation of progelatinaseB while TIMP-2 is an effective regulator of progelatinase A[13].
Two other classes of matrix metalloproteinase inhibitors(MMPIs) have also been identified. These are the smallerinhibitors of metalloproteinase (IMPs) and the large inhibi-tor of metalloproteinases (LIMP). IMPs are smaller thanTIMPs [7]. The molecular masses of IMP-1, IMP-2 and IMP-3 are 26, 21 and 18 kDa, respectively. They do not cross re-act with TIMPs and are found in many cells of different species.LIMP is a complex composed of TIMP-2 and progelatianse A[7]. This complex inhibits collagenase, gelatinase A, andstromelysin. The ability of the TIMP-2-progelatinase complexto inhibit these enzymes indicates that the inhibitory site isexposed in the TIMP-2 molecule.
TIMPs are important in the control of numerous physiologi-cal processes. These include tumor cell invasion, angiogen-esis, degradation of join cartilage, trophoblast implantation,mammary gland involution, and wound healing [7, 13, 190].
α2-Macroglobulin
α2-Macroglobulin is a ubiquitous inhibitor of all MMPsknown to date. It inactivates susceptible proteinases by en-trapment following cleavage of the bait region [13]. Theproteinase cleaves one or more bonds in the 40 residue baitregion and thereby initiates a conformational change thatleads to entrapment of the proteinase [13, 202]. In almost allα2-macroglobulins. This conformational change leads tohydrolysis of one internal thiol ester bond [-C(=O)-S-] persubunit and to generation of a highly reactive glutamyl resi-due [203]. The nascent glutamyl residue reacts with a lysylside chain exposed on the surface of the attacking protein-
ase to covalently cross-link the proteinase to the inhibitor byan ε-lysyl-γ-glutamyl bond.
Synthetic inhibitors
Nearly all of the synthetic inhibitors analyzed so far in MMPcomplexes contain a chelating group (such as a hydroxamicacid, a carboxylate, or a thiol group) for zinc ion ligation.Chelating agents that interacts with (or remove) Zn2+ at theactive site such as 1,10-phenanthroline and EDTA are potentinhibitors of MMP but are of limited therapeutic potential [13,204].
Tetracyclines and certain synthetic analogues without an-tibiotic activity have been shown to inhibit PMN-CL.Themechanism of inhibition is not known, but it is suspected thatit depends on the chelating properties of the compounds.Especially chelates Ca2+ that is required for activation ofMMPs. Tetracyclines are considerably less effective againstFIB-CL, but the reason for this selectivity is not known [13,205].
Next generation inhibitors
Limited bioavailability and a lack of enzyme selectivity havehindered the development of MMPIs and present major chal-lenges for the next generation of compounds. Early inhibi-tors were designed based on sites of substrate cleavage werepeptidomimetic in nature with a hydroxamic acid moietyreplacing the terminal carboxylic acid of the correspondingpeptide cleavage product. Although they were very potentMMP inhibitors, they demonstrated poor bioavailability andshowed little specificity for individual MMPs. In addition,hydroxamic acid inhibitors are rapidly metabolized by theliver and require frequent administration to maintain thera-peutic plasma drug levels. Detailed structures of the enzymeinhibitor complexes have allowed the design of nonpeptidicinhibitors with improved pharmacokinetics profiles and in-crease selectivity [190, 203].
Glycomed has completed the initial trials for the peptidehydroxamate inhibitor galardin (GM-6001) a couple of yearsago and the drug showed a statistically significant benefit asa topical treatment of corneal ulcers [190, 206]. The MMPIwith the most advanced clinical development at this time isBritish Biotech’s Marimastat (BB-2536, Oxford, UK), abroad-spectrum peptide inhibitor effective for the advanced-stage refractory cancers. Recently, more selective compoundshave entered for clinical development. Compounds from Bayer(BAY 12-9566; West Haven, CT, USA) and Roche (RO 32-3555; Nutley, NJ, USA) effective for osteoarthritis and rheu-matoid arthritis are being evaluated in clinical trials. A MMPIfrom Agouron (AG3340; La Jolla, CA, USA) effective for
advanced lung and prostate cancers has also being studied forclinical trials. Several other companies are also evaluatingtheir compounds for clinical trials [206]. The results fromthese studies should eventually help define the safety andclinical use of the MMPIs.
To design more selective inhibitors that are devoid of ad-verse reactions detected with broad spectrum inhibitors, it isessential to increase the number of three dimensional struc-tures that are available for these enzymes [207]. New classof MMPIs are designed not only by binding at the active siteof enzyme but also either sites which might be a promisingresult for therapy. MMPIs extracted from different naturalproducts are also being increasingly explored as antitumoragents and, in some cases, these compounds are in someclinical trials, for example, green tea components such ascatechins that inhibit both MMP production and activity [208,209]. Similarly, Neovastal, which is derived from extracts ofshark cartilage and possesses potent MMP inhibitory andantiangiogenic properties is in active clinical trials for treat-ment of advanced non small cell lung cancer, metastatic re-nal cell carcinoma and multiple myeloma [164, 210].
Conclusion and future directions
The extracellular matrix, particularly type I fibrillar collagensurrounding the cardiomyocytes helps the cardiac muscle tosynchronize contraction and diastole, respectively [211]. Tocompensate for the increase in work load and to reduce thewall stress, the cardiac muscle undergoes hypertrophy. Thisleads to remodeling of the ECM. Remodeling implies synthe-sis and degradation of the ECM by MMPs where MMPs playsa crucial role [211]. In the normal myocardium, most of theMMP resides in latent form and activated in chronic heartfailure [211]. The mechanism of MMP activation in chronicheart failure is not well understood. The MMP is regulatedprimarily at three stages: (i) at the transcription level bymultiple cytokines, growth factors, and neurohormones,and oxidative stress; (ii) by their target tissue inhibitor ofmetalloproteinases (TIMPs); and (iii) by direct activation withoxidative stress and/or by proteolytic cleavage [211]. Altera-tions at any of these stages can lead to an imbalance in thecomposition and concentration of MMP and TIMP and maylead to development of a disease state [211].
The myocardial ECM is under constant remodeling byMMPs that in turn is regulated by various factors. Althoughsignificant advancement has been made in understanding theroles of MMPs, TIMPs and their regulators, there is still muchto be learned about the interaction of MMPs and their regula-tors in the development of diseases such as atherosclerosis, ar-thritis and cancer. Modulation of MMPs in these diseases mayalter the ECM remodeling process and that may eventuallyaffect progression of these diseases. Thus, a detailed infor-
mation about structural interaction between MMPs and TIMPsmay provide important therapeutic clues for the discovery ofnew drugs for treating these diseases.
The process of angiogenesis, like that of tumorogenesisand metastasis, has been described as being particularly sen-sitive to three biological events: a loss of growth control, de-regulation of proteolysis and an unbalanced regulation ofcellular mortality [212]. It has been documented that the de-regulation of MMPs can have a profound effect on the for-mation of new capillaries. Important questions that couldarise: are there specific cell surface receptors for MMPs anddo these receptors play a role in modulating MMPs in tumorinvasion and angiogenesis? Both type IV collagenases, the 72and 92 kDa, show multi-substrate activities including type IVcollagen, type V collagen, type VII collagen and gelatin invitro. What is the physiologic substrate for these enzymes invivo? What are the relative contribution of the 72 and 92 kDatype IV collagenase enzymes to normal physiologic processesof basement membrane turnover and to pathologic processessuch as tumor cell invasion and tumor angiogenesis? Thesequestions need to be addressed in future research.
Enhanced expression of different MMPs in cancer tissueand experimental in vitro data suggested an essential role forMMPs in tumor cell invasion. Although the expression ofMMPs in malignancies has been studied widely, the specificrole of distinct MMPs in the progression of cancer may bemore complex than has been assumed. Additional studies onthe regulation of MMP gene expression and activity for invivo malignancies are clearly needed to understand the roleand regulation of MMPs in tumor cell invasion.
TIMP-1 and TIMP-2 were originally identified as inhibi-tors of MMP-9 and MMP-2, respectively. However, TIMP-1 was later shown to be a growth factor for some cell types,including some cancers, and thus, resulted in worse progno-sis of the patients with high level of expression of TIMP-1[213–218]. Recently, TIMP-4 was reported to be an efficientinhibitor of MT1-MMP, and thus, an ideal inhibitor of MMP-2 activation [218, 219]. Synthetic MMP inhibitors (MMPIs),which inactivate a broad range of MMPs, have also been evalu-ated for their prognostic value in clinical trials [220–222].Clinical trials initiated in 1997–98 using Marimastat (BB-2516), prinomastat (AG 3340) and BAY 12-9566 alone or incombination with standard chemotherapy in patients with ad-vanced cancers (lung, prostate, pancreas, brain, GI tract) indi-cated no clinical efficacy of these agents [223, 224]. The onlyexception was the trial of marimastat in gastric adenocarci-noma patients [213]. The failure of clinical trials of MMPIsmight be attributed to the importance of MMPs in early as-pects of cancer progression such as local invasion and micro-metastasis and then may no longer be required once metastaticlesions have been established. Thus, future MMPIs trialsshould focus on patients with early stage cancer. Moreover,it seems necessary for successful clinical trials to identify
patients who are more likely to respond to MMPI therapy andbiomarkers that reflect activation of inhibition of MMPs invivo [213].
Acknowledgements
Financial assistance from the Indian Council of Medical Res-earch (ICMR, New Delhi, India), Department of Biotechnol-ogy (DBT, New Delhi, India) and the Life Science ResearchBoard (LSRB, Ministry of Defence, Government of India)are gratefully acknowledged. Thanks are due to ProfessorKasturi Datta (School of Environmental Science, JawaharlalNehru University, New Delhi, India) and Professor GerryShaw (Department of Neurosciences, University of Florida,Gainesville, FL) and Dr. W. Selvamurthy (Advisor, DefenceInstitute of Physiology and Allied Sciences, Delhi, India) fortheir interest in this work.
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