G.M. Albaiceta and A. Fueyo
Introduction
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases belonging to the metzincin superfamily of metalloproteinases. MMPs can degrade most of the components of the extracellular matrix and basement membrane. In addition, these enzymes can cleave some inflammatory mediators. This variety of substrates gives MMPs a wide number of functions during physiologic and patho- logic processes. In this sense, many of the MMPs are not expressed in normal tis- sues, but expression and activity increases dramatically during matrix turnover, inflammation and repair.
Acute lung injury (ALI) is a devastating condition that leads to an acute inflam- mation of the lungs. In some cases, mechanical ventilation is needed, thus causing an additional stress to the lung with potential for the so-called ventilator-induced lung injury (VILI). The repair process after this injury can lead to near-normal reso- lution or, in other cases, an increase in the collagen content of the lung interstitium and, thus, pulmonary fibrosis. MMPs may be involved in this sequence of events, from the initial events until the resolution of the disease. In this chapter, we will review the functional role of MMPs in ALI and discuss the derived therapeutic implications.
Matrix Metalloproteinases
There are 24 human genes encoding 23 different MMPs. The typical structure of a MMP consists of a propeptide region (about 80 amino acids), a catalytic domain (170 amino acids, with a zinc ion bounded to histidine residues), and a ‘hinge region’ that links a hemopexin domain (200 amino acids). MMP-7, -23 and -26 are exceptions to this model, lacking the linker and hemopexin domains. Addition of other domains leads to a diversity of proteins with different substrates and activities.
Based on their structure and preferential substrates, MMPs can be divided into dif- ferent groups (Table 1).
Most of the MMPs are not expressed in quiescent cells, but MMP transcription occurs in tissues in response to different stimuli. No single factor responsible for the expression of MMPs has been identified. Instead, a variety of cytokines, growth fac- tors, and oncogene products induce MMP synthesis and release (e.g., tumor necrosis factor [TNF]-[ , interleukin [IL]-1). The pathways responsible for this expression are also diverse (see [1] for a review). Cell deformation or mechanical stress can lead to MMP expression through a pathway dependent on nuclear factor-κB (NF-κB), one of
Table 1. Matrix metalloproteinases.
Extracellular matrix substrates
Other substrates
Collagenases MMP-1
(interstitial collagenase)
Native collagen MCP-1, IL-1, pro-TNF-[ MMP-8
(neutrophil collagenase) MMP-13 (collagenase-3) Gelatinases
MMP-2 (gelatinase A) Gelatin (denatured collagen) MCP-3, pro-IL-8, pro-TNF-[ , pro-TGFq , pro-IL-1, plasminogen MMP-9 (gelatinase B)
Stromelysins
MMP-3 Laminin, fibronectin, gelatin MCP (1 – 4), pro-TNF-[
MMP-10 Pro-MMP-1, pro-MMP-8, pro-MMP-10
MMP-11 E-cadherin, cell surface bound Fas-L
Membrane-type MMPs
MMP-14 (MT1-MMP) Native collagen, gelatin MCP-3, CD44 MMP-15 (MT2-MMP) Proteoglycan
MMP-16 (MT3-MMP) Type III collagen, fibronectin MMP-17 (MT4-MMP) Gelatin, fibrin
MMP-24 (MT5-MMP) Fibronectin, proteoglycans MMP-25 (MT6-MMP) Type IV collagen, gelatin Matrilysins
MMP-7 Fibronectin, laminin, type IV collagen, gelatin
Pro-TNF-[ , E-cadherin, cell surface bound Fas-L
MMP-26 Other
MMP-12
(macrophage elastase)
Elastin, fibronectin, laminin Plasminogen, pro-TNF-[ MMP-19 Type IV collagen, gelatin,
laminin
MMP-20 Amelogenin
MMP-23 Gelatin
MMP-27 MMP-28
TNF: tumor necrosis factor; IL: interleukin; TGF: transforming growth factor; MCP: monocyte chemoattrac- tant protein
the transcription factors related to the inflammatory response [2]. Other molecules, like transforming growth factor (TGF)q , interferon (IFN) * and glucocorticoids can block MMP expression. MMPs are usually secreted as a proenzyme, thus requiring activation to be fully functional. This process can be mediated by reactive oxygen spe- cies (ROS), endogeneous proteases (including MMPs) or even bacterial proteases [3].
Classically, the main function of MMPs was thought to be the degradation and turnover of the extracellular matrix. However, this paradigm has changed, in view of the variety of substrates and the effects caused by their enzymatic activity [3]. Cur- rently, MMPs are considered to regulate the cell-cell and cell-matrix interactions, through the cleavage of inflammatory mediators (TNF-[ , IL-8, insulin-like growth factor [IGF]-1, among others) [4], or through breakdown of components of the extracellular matrix and the release of growth factors bound to the matrix. Cleavage
of collagen and elastin is required for cell migration, and it has been demonstrated that expression of epitopes of the extracellular matrix after proteolysis can induce the chemotaxis of inflammatory cells. Supporting this view, MMPs have been shown to be involved in cell migration, apoptosis, morphogenesis, inflammation, neovascu- larization, etc.
The main MMP plasma inhibitor is [ 2-macroglobulin. However, there is a family of more selective inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).
There are four TIMPs; although all of them inhibit virtually all MMPs, there are dif- ferences in their activity. For example, mice lacking TIMP-3 develop emphysema-like lung injury, while mice lacking TIMP-1 or -2 have no severe abnormalities. These findings emphasize the relevance of TIMP-3 in vivo.
Matrix Metalloproteinases in Acute Lung Injury
ALI and its more severe form, the acute respiratory distress syndrome (ARDS), can be viewed as being the result of an inflammatory process within the lungs, caused by pulmonary or extrapulmonary diseases. The lung epithelium and endothelium respond to this aggression with an increase in permeability, thus affecting the inter- stitium and the alveolar spaces. Inflammatory cells release cytokines and chemoki- nes, responsible for cell chemotaxis and the propagation of the response. As dis- cussed in the previous section, some of the inflammatory mediators involved in the acute phase response, as well as bacterial products or ROS, can precipitate the expression of MMPs. A variety of cells in the respiratory system can synthesize MMPs (Table 2) [5].
After the acute phase, repair can lead to chronic inflammation and deposition of collagen in the interstitium, thus causing pulmonary fibrosis. All these processes can be mediated by MMPs (Fig. 1). In some patients, mechanical ventilation is needed. If this is the case, high positive pressures in the lung can cause further damage, the so- called VILI. The implications of MMPs in this process will be discussed separately.
The role of MMPs in ALI has been studied in different animal models and patients. An upregulation of MMPs in bronchoalveolar lavage (BAL) fluid has been documented in different models of ALI. Following exposure of rats to 100 % oxygen, there is an increase in BAL fluid levels of MMP-9, and MMP-2, -7, -8 and -9 in lung tissue [6]. Similar results were found in a model using intratracheal endotoxin [7].
The expression of TIMPs also increases after a challenge, in order to maintain the normal equilibrium between the enzymes and their inhibitors [8]. The critical importance of this relationship is demonstrated in models of endotoxin-induced lung injury in mice deficient in TIMP-3 [9]. In this case, the increased activity of MMPs has no natural inhibitor. These animals develop severe airway enlargement
Table 2. Sources of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMPs) in the respiratory system
Cells MMPs released
Fibroblasts MMP-2, MMP-1, TIMP-1
Bronchial epithelial cells MMP-2, MMP-9, TIMP-1 Alveolar epithelial cells MMP-1, TIMP-2
Alveolar macrophages MMP-1, MMP-9, MMP-12, TIMP-1
Neutrophils MMP-8, MMP-9, TIMP-1
Fig. 1. Effects of matrix metallo- proteinases (MMPs) in acute lung injury.
and a decrease in the collagen content of the lungs. Similarly, blocking TIMP-2 in a model of immunocomplex-mediated alveolitis worsens lung injury [8].
MMP-9 may also be involved in lung injury from an extrapulmonary origin. In a pancreatitis model [10], high levels of MMP-9 were found in lung parenchyma, orig- inating from primed neutrophils. Treatment with an MMP inhibitor decreased neu- trophil concentration and capillary leakage in the lung. In a similar manner, gelati- nase and elastase activities increase in lung injury after abdominal sepsis [11] or cardiopulmonary bypass [12]. Blockade of gelatinases using COL-3 (a chemically modified tetracycline that inhibits gelatinases) decreased the degree of lung injury.
These results suggest a possible role of MMPs (and specifically MMP-9) in the loss of compartimentalization of the inflammatory response seen in different patholo- gies.
Neutrophils and macrophages can be a source for these enzymes. Neutrophils are rapidly recruited within the lungs during ALI. These cells contain MMP-8 and MMP- 9 in their secondary granules, and both enzymes are expressed on the surface (mainly at the active pole of the cell) and secreted [13]. In models of ALI, there is a correlation between the number of neutrophils and MMP levels in the BAL fluid [14].
Alveolar macrophages are also important contributors to the development of the lung inflammatory response. In a macrophage-dependent lung injury model (IgA immune complex-mediated alveolitis), there is an increase in BAL fluid gelatinases, parallel to neutrophil recruitment. Moreover, lung injury can be attenuated by treat- ment with TIMP-2 [15]. These results suggest that alveolar macrophages release gelatinases that can favor the migration of neutrophils.
In contrast, mice lacking MMP-8 have more neutrophils in BAL fluid and an increase in lung permeability after endotoxin challenge, suggesting an anti-inflam- matory role of this enzyme [16]. MMP-8 deficiency has been related to a slightly delayed, but persistent, inflammatory response in a model of carcinogenesis [17].
This could explain the protective effects of MMP-8 deficiency in hyperacute inflam- mation, but also its harmful effects in the long-term. These important properties of MMP-8 will be discussed later.
The implication of MMPs in ALI in patients has been addressed by different stud- ies. The levels and activity of MMP-8 and -9 are elevated in BAL fluid from children with ALI, compared to healthy controls, with no differences in MMP-2 levels [18].
MMP-9 levels in BAL fluid were also elevated in a sample of adult patients with ARDS at different stages (early versus late), and in patients with a risk factor for developing lung injury (but normal lung function) [19]. TIMP levels were also increased in this cohort. Interestingly, the ratio MMP-9/TIMP was above 1 in patients with risk factors and in those at a late stage of the syndrome. However, patients with early ARDS had a ratio lower than 1 [20]. It has been suggested that ratios higher than 1 could facilitate lung repair [19]. A recent study by Fligiel et al.
[21] confirmed an increase in MMP-2, -8 and -9 in patients with ALI. In addition, these authors showed that a subgroup of these patients with increased levels of MMP-1 and MMP-3 have an increased morbidity and mortality.
The elevation of MMP-2 in BAL fluid from patients with ARDS receiving mechan- ical ventilation has been correlated to the levels of type III procollagen and to an impairment on the mechanical properties of the respiratory system (i.e., the compli- ance measured on an inspiratory pressure-volume curve) [22]. This relationship fol- lows a logarithmic pattern, with an abrupt increase in MMP-2 and procollagen in patients with a compliance less than a critical value (28 ml/cmH2O). This suggests that collagen turnover, matrix remodeling, and, therefore, MMP activity, play a criti- cal role in the maintenance of the mechanical properties of the respiratory system during lung injury.
Matrix Metalloproteinases in Ventilator-induced Lung Injury
Application of relatively high pressures and volumes to the lungs during mechanical ventilation can cause or augment lung damage. Although the initial pathogenetic mechanism may be mechanical (including cell overstretching and changes in the cytoskeleton), it is clear that there can be a release of mediators that not only per- petuates, but also disseminates, the inflammatory response. The lung matrix sup- ports part of this initial mechanical stress and can contribute to this form of injury.
As in other lung diseases, MMPs are thought to play an important role.
Mechanical stress and cell deformation can release MMPs from various types of cells. Using endothelial cells exposed to stretch, Haseneen et al. [23] showed an increase in MMP-1, -2 and membrane type-1 MMP (which could be related to cell migration). Disruption of the cytoskeleton of fibroblasts increased MMP-1 expres- sion [2]. The pathway responsible for this process is NF-κB dependent. It is notewor- thy that this and other pathways related to MMP expression (for example, those related to p38 mitogen activated protein kinase [MAPK]) have also been implicated in the pathogenesis of VILI [24].
Different models of high pressure ventilation in animals have shown an increase in the expression of gelatinases in lung tissue and BAL fluid (MMP-9 and MMP-2, respectively) (Fig. 2). Moreover, this increase and the degree of lung injury can be attenuated by treatment with MMP inhibitors (either Prinomastat, a broad spectrum MMP inhibitor, or COL-3) [25, 26]. This protective effect was related to a decrease in neutrophil recruitment within the lungs, supporting the role of MMPs in cell migra- tion.
Fig. 2. Gelatin zymographies of lung tissue (left) and bronchoalveolar lavage (BAL) fluid (right) from mice ventilated using either high inspiratory pressures with zero PEEP (25/0 cmH2O) or low inspiratory pressures and PEEP (15/2 cmH2O). White bands, which represent gelatinolytic activity correspond to MMP-9 and MMP-2.
Note the significant increase of MMP-9 in lung tissue and MMP-2 in BAL fluid after injurious ventilation.
Therapeutic Possibilities
Based on their effects in ALI, MMPs could be an interesting therapeutic target.
There are different MMP inhibitors and some of them have been tested in clinical trials in cancer patients, but none has achieved a significant benefit. Although one can argue that cancer and ALI are different diseases, there could be some similari- ties in the reasons for this lack of benefit.
There are an increasing number of papers documenting a benefit of MMP inhibi- tion (either using genetically modified animals or pharmacologic inhibitors) in ALI and VILI. Mice lacking MMP-9 have less lung injury in different models, ranging from endotoxin [27] to immune complexes [28], and a higher survival after high doses of endotoxin [29]. In a similar way, inhibition of gelatinases with drugs decreased lung injury after sepsis [11], pancreatitis [10], cardiopulmonary bypass [12], and high tidal volume ventilation [25, 26]. These results would support a trial using a MMP inhibitor (more specifically, against MMP-9) in patients with ALI.
However, MMP inhibitors are poorly selective. Results using MMP-8 deficient mice show that this MMP may have a dual effect. MMP-8 may inhibit hyperacute inflammation, as MMP-8 knockout mice have less neutrophil infiltration and enhanced survival after acute liver injury [30], and a delay in skin inflammation after injection of a mutagen [17]. However, these animals have a sustained inflam- matory response over time, with an increase in BAL fluid neutrophils 24 hours after endotoxin administration [16] and, in the case of carcinogenesis, persistent skin inflammation and development of tumors [17]. It should be noted that MMP-8 and -9, which seem to have opposite effects (anti-inflammatory and pro-inflammatory, respectively), are both released from activated neutrophils.
These data show that we need to take into account the targets (specific enzymes) and the time frame when planning a therapeutic strategy with MMP inhibitors. It is possible that early inhibition of one MMP results in one effect, while late inhibition results in the opposite. Likewise, it seems unlikely that non-selective inhibition of different enzymes with antagonistic functions would result in a net beneficial effect.
Before planning this kind of study, we need to acquire a deeper knowledge of the role and implications of MMPs in ALI.
Conclusion
MMPs are a family of enzymes involved in many of the pathophysiological responses after ALI and VILI, including modulation of the inflammatory response, cell chemo- taxis, and turnover of the extracellular matrix. MMPs are also a therapeutic target in these diseases. Although non-selective inhibition of these enzymes has yielded inter- esting results in animal models, the diversity of enzymes, and the contrasting roles of some of them make this strategy difficult in the clinical arena. A deeper knowl- edge of their functions is needed, in order to define the precise therapeutic targets.
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