Clin Chem Lab Med 2015; 53(3): 349–355
Mini Review Emanuela Galliera*, Lorenza Tacchini and Massimiliano M. Corsi Romanelli
Matrix metalloproteinases as biomarkers of disease: updates and new insights DOI 10.1515/cclm-2014-0520 Received May 16, 2014; accepted July 18, 2014; previously published online August 7, 2014
growth factors, and regulating cell movement, intercellular interactions and communication [1]. ECM formation and degradation is a crucial event in biological processes involved in maintaining tissue homeostasis and regeneraAbstract: Matrix metalloproteinases (MMPs) play a pivtion, such as for wound healing and fibrosis. These events otal role in remodeling the extracellular matrix (ECM) and are under the control of a family of 28 endopeptidases are therefore of interest for new diagnostic tools for the known as matrix metalloproteinases (MMPs), multidoclinical management of diseases involving ECM disrupmain zinc metalloproteinases specialized in degradation tion. This setting ranges from the classical areas of MMP of the different components of the ECM [2]. The ECM itself studies, such as vascular disease, cancer progression or consists mainly of polysaccharides, such as hyaluronic bone disorders, to new emerging fields of application, acid, collagen and elastin fibers for tissue flexibility, prosuch as neurodegenerative disease or sepsis. Increasing teoglycans, such as chondroitin sulfate or heparan sulfate, the knowledge about the role of MMPs in the pathogenesis binding growth factors and drawing in water in order to of diseases where a clear diagnostic panel is still lacking keep the microenvironment hydrated. The different ECM could provide new insight and improve the identification components are specific targets of different MMPs. On and the clinical treatment of these human diseases. This the basis of the substrate specificity, MMPs can be clasreview focuses on the latest descriptions of the clinical sified in five main groups: collagenases (MMP-1, -8, -13), use of MMP as biomarkers in the diagnosis, prognosis and gelatinases (MMP-2, -9), stromyelysins (MMP-3, -10, -11), monitoring of different diseases, such as diabetes, cardiomatrilysins (MMP-7 and -26), and membrane-type MMPs vascular diseases, cancer and metastasis, neurodegenera(MP MMP-14, 15, 16 and -24) [3]. Others include MMP-12, tive disorders and sepsis. -20 and -27 which have a structure similar to stromyelysin; MMP-19, which is a basement membrane-degrading Keywords: diagnosis; metallorproteasis; serum biomarkers. protease involved in tissue remodeling, healing and cell migration; MMP-20, a tooth-specific protease; MMP-21, a gelatinase expressed in several carcinomas; MMP-23, Introduction which is expressed in ovary, prostate and testis and plays a role in reproduction; MMP-28 (epilysin), mainly expressed The extracellular matrix (ECM) is the extracellular comin rheumatoid arthritis and osteoarthritis [1]. ponent of tissues, providing support for cells, storage for The activity of the MMPs is closely regulated on several levels, starting from gene transcription, to transla*Corresponding author: Dr. Emanuela Galliera, PhD, Assistant tion, pro-enzyme activation and inhibition by regulatory Professor of Clinical Pathology, Department of Biomedical, Surgical proteins, such as specific tissue inhibitors of metalloand Oral Science, Università degli Studi di Milano, I Via Luigi proteinases (TIMPs), which can neutralize the proteases Mangiagalli 31, 20133 Milano, Italy, Phone: +39 02 50315354, thereby preventing excessive matrix degradation. Fax: +39 02 50315338, E-mail: [email protected]; and MMPs are required in biological functions, such as IRCCS Galeazzi Orthopedic Institute, Milan, Italy Lorenza Tacchini: Department of Biomedical Sciences for Health, cell proliferation, differentiation, apoptosis, immune Università degli Studi di Milano, Milan, Italy function, tissue healing, and angiogenesis. Therefore, Massimiliano M. Corsi Romanelli: Department of Biomedical they are also involved in multiple pathological condiSciences for Health, Università degli Studi di Milano, Milan, Italy; tions [4], where they play a role in tissue destruction and Service of Laboratory Medicine1-Clinical Pathology, Department (such as in cancer invasion and metastasis, arthritis, difof Health Service of Diagnosis and Treatment-Laboratory Medicine, ferent types of ulcers, inflammatory diseases), fibrosis IRCCS Policlinico San Donato, San Donato (Milan), Italy
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350 Galliera et al.: Matrix metalloproteinases as biomarkers of disease (such as liver cirrhosis, multiple sclerosis, atherosclerosis) and matrix degradation (such as cardiomyopathy, aortic aneurysm), and in various bone and joint diseases [5, 6]. MMPs and their inhibitors, the TIMPs, have consequently been proposed as biomarkers for certain pathologies [7]. They can be examined at different levels, by measuring tissue gene expression, protein amount, and plasma levels, as well as proteolytic activity, by gel zymography. Since MMPs are sensitive enzymes, extreme care must be taken in blood sampling and handling, because these procedures can alter the concentration ratios between plasma and serum, particularly for MMP-9 [8, 9]. Therefore, the plasma or serum sample must be selected appropriately. However, in some cases, such as MMP-2 and MMP-9, there is a positive correlation between plasma and serum levels [10]. This review focuses on the latest evidence on the use of MMP as circulating biomarkers in the diagnosis, prognosis and monitoring of different human diseases, as summarized in Table 1.
MMPs and diabetes Dysregulation of MMP activity has been involved in the pathogenesis of several diabetic co-morbidities, from diabetic retinopathy to diabetic nephropathy and chronic non-healing ulcers. Diabetic nephropathy is due to progressive, abnormal degradation and turnover of ECM in the glomerulus, where MMPs are major players so changes in their expression or activation can contribute to renal hypertrophy and affect ECM deposition, leading to renal injury in diabetic nephropathy [11]. Abnormal ECM deposition is the hallmark of diabetic nephropathy, and this is mainly due to alteration of MMPs expressed in the nephron. Specific MMPs and the relative TIMPs are expressed in kidney tissue; these include MMP-2, -3, -9, -10, -14, -15, and TIMP-2 and -4 [12]. Besides their role in ECM turnover, MMPs are also involved in the release and activation of several factors present in the ECM and associated with diabetic nephropathy, such as TGFβ, TNFα, insulin-like growth factors and heparin-binding epidermal growth factor, thus contributing indirectly to tubular cell
Table 1 Summary of the different MMPs as biomarkers of human disease discussed in this review. Human disease
MMPs involved
Serum/plasma level
Biomarker of
Increased
Diabetic nephropathy
MMP-1,-2,-3,-7,-9 (plasma), MT-MMP-14, -24 TIMP-1,-2,-3 MMP-2,-9 (plasma) MMP-8,-9 (plasma) MMP-9 (plasma) MMP-1,-3,-8 MMP-2
Increased Increased Increased Increased Increased
MMP-2
Decreased
MMP-8,-9 (plasma) MMP-9 (plasma), TIMP-1 MMP-1 MMP-2,-9 (plasma) TIMP-1,-2 MMP-2,-8,-9 (plasma) MMP-9 (plasma):TIMP-1 ratio, MMP-10 MMP-9 (plasma) MMP-9 (plasma) MMP-8,-9 (plasma) TIMP-1 MMP-9 (plasma):TIMP-1 ratio MMP-3 MMP-3,-2,-9 (plasma) MMP-1,-2,-9 (plasma), TIMP-1 MMP-3
Increased Increased Increased Increased
Diabetic retinopathy Diabetic chronic wound healing inability Diabetic cardiovascular risk Atherosclerosis progression Abdominal aortic aneurysm with primary abdominal hernias Abdominal aortic aneurysm without primary abdominal hernias Plaque rupture risk Stroke mortality risk in metabolic syndrome Poor prognosis Poor prognosis
Diabetes mellitus
Cardiovascular diseases
Sepsis
Neurological diseases
Increased Increased Increased Increased Increased Decreased Decreased Increased Increased Increased Increased
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Severe outcome Severity and mortality Tissue damage in MODS Ischemia, infarct size in acute ischemic stroke Multiple sclerosis (MS) progression Decreased MS activity Higher in relapse than remission of MS Progression of myasthenia gravis Progression of amyotrophic lateral sclerosis Progression of Alzheimer’s disease
Galliera et al.: Matrix metalloproteinases as biomarkers of disease 351
proliferation and renal fibrosis, leading to renal damage. A robust association with diabetic nephropathy has been reported for gelatinases MMP-2 and -9, matrilysins MMP-7, membrane-type MT-MMP-14, -24, stromyelysin MMP-3 and collagenase MMP-1, TIMP-1, -2, and -3 [11]. We still need a better understanding of the precise role of MMPs in the pathogenesis of diabetic nephropathy, to target MMPs therapeutically, so as to prevent or reverse diabetic renal disease. MMPs are involved in other diabetic co-morbidities too, such as diabetes-related retinopathy. MMP-2 and -9 have a part in diabetes-induced mitochondrial dysfunction and consequent retinal damage [13]. Similarly, TIMP-1, together with TGF-β2, has a role in the angiogenic phase of proliferative diabetic retinopathy. Levels of TIMP-1, MMP-2 and -9 have been reported to be related to the degree of neovascularization [14]. One evident consequence of diabetes is the chronic inability of wounds to heal, due to activated MMPs, which remodel tissue, resulting in apoptosis. The recent identification of the main MMPs involved, MMP-9 and -8, in murine models of diabetic wounds, is valuable for targeting new pharmacological interventions. Another major co-morbidity of diabetes is cardiovascular disorders. Myeloperoxidase, as well as MMP-9, are reported to be early biomarkers of cardiovascular risk in diabetic patients [15]. Given the close relation between MMPs and their inhibitors with the pathogenesis of diabetes and related disorders, there is growing agreement that these molecules might be useful biomarkers for clinical use in diagnosis. Genetic study of MMPs has pointed to a relation between specific polymorphisms and type 2 diabetes and diabetic foot ulcers [16]. Circulating levels of MMPs and TIMPs can be used as diagnostic and prognostic biomarkers. For example, serum MMP-9 and TIMP-1 predict poor wound healing in diabetic foot ulcers, while serum MMP-9 is related with progressive diabetic vitrectomy [17].
MMPs and cardiovascular disease Abnormal angiogenesis and vascular remodeling contribute to the pathogenesis of several disorders, such as atherosclerosis, re-stenosis, and hypertension, increasing cardiovascular risk. MMPs are important in vascular remodeling and in several processes, such as angiogenesis, collateral artery formation and thrombus resolution, but also in the pathogenesis of atherosclerosis, plaque rupture, aortic aneurysm and varicose veins [18]. Vascular
tissue expresses high levels of MMP-1, -2, -3, -7, -8, -9, -12 and MT-MMPs [18]. Therefore various MMPs have been studied as potential investigative tools in the diagnosis and monitoring of vascular diseases. Collagenases MMP-1, -3, and -8 are proposed as indicators of atherosclerosis progression, while high MMP-2 serum levels can distinguish patients with abdominal aortic aneurysm associated with primary abdominal hernias, who have high serum MMP-2, from those who have abdominal aortic aneurysm alone, and lower levels of serum MMP-2 [19]. One of the principal factors of cardiovascular risk is hypertensive cardiac disease, caused not only by stress, genetic predisposition, lipid metabolism disorders, or diabetes, but also by increased vessel fibrosis, which raises systemic blood pressure. Among the MMPs acting in concert with hypertension-promoting mediators, MMP-2 and -7 are the most important and have been proposed as therapeutic targets in patients with hypertensive cardiac disease [20]. The main adverse event with hypertension and atherosclerosis is plaque rupture. Since MMPs are involved in carotid atherosclerotic plaque remodeling, monitoring MMPs, such as MMP-8 and -9 can provide useful information about plaque progression and the risk of rupture. A new imaging approach has been developed [21] to visualize the inflammatory process in the vessel wall and plaque, so intra-plaque levels of MMPs might be helpful in assessing the risk of plaque rupture. The importance of MMPs in plaque vulnerability is confirmed by the relations between MMP-9 serum levels and markers of carotid atherosclerosis and lesion risk, such as fibrous cap thickness, lipid core collagen content and intra-plaque hemorrhage [22]. High cardiovascular risk is one of the main clinical features of metabolic syndrome, defined as the combination of obesity, arterial hypertension, diabetes mellitus and dyslipidemia. Patterns of serum MMPs are altered in patients with metabolic syndrome. In particular, high serum MMP-9 and TIMP-1 are associated with cardiovascular risk and stroke mortality. Hypertension is one of the main cardiovascular diseases, and is an important complication in pregnancy and a severe risk factor for cardiac adverse events. Therefore, it is essential to monitor hypertension for the prevention of cardiovascular disorders. MMP-2 is important in this context, as the target of different strategies based on MMP inhibitors, aimed at improving hypertensioninduced cardiovascular alterations [23–25]. Similarly, many drugs already used by patients can affect MMP concentrations, particularly MMP-9 [26]. Therefore, particular care must be taken in taking the patient’s history and clinical evaluation in order to avoid misinterpretation of MMP results.
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352 Galliera et al.: Matrix metalloproteinases as biomarkers of disease Given the prominent role of MMPs in vasculature remodeling, they are also important therapeutic targets in the prevention and treatment of atherosclerosis and coronary artery diseases [27]. Several pharmacological approaches have been studied to inhibit MMP activity, using tissue MMP inhibitors – TIMPs, pleiotropic mediators, such as tetracycline, doxocyclins, small-molecule MMP inhibitors (MMPis) and inhibitory antibodies, but they still have broad specificity, because they prevent the ECM degradation function of different MMPs. Therefore, further studies are still needed to find MMP is with narrower specificity, targeting the action of a specific MMP.
MMPs and sepsis Sepsis is the result of an extreme inflammatory response to pathogen invasion, that can lead to organ failure (severe sepsis), persistent blood pressure loss (septic shock) and, ultimately, a fatal outcome. Therefore early diagnosis and prompt therapeutic intervention are vital in the management of septic patients, which is often complicated by different clinical presentation and responses to therapy. Sepsis is also associated with alterations in coagulation and endothelial response, but the pathogenic mechanism of these effects is still poorly understood, limiting the development of therapeutic strategies. MMPs are important in tissue remodeling and the host’s inflammatory response to pathogen invasion; therefore they could serve as a diagnostic tool and therapeutic target in sepsis. Early evidence came from Tressel et al., who found a positive correlation with circulating MMP-1 and poor prognosis in septic patients [28]. MMP-1 is released by endothelial cells under stress conditions, such as sepsis, leading to a loss of endothelial integrity, a consequent increase of vessel permeability and, ultimately, an alteration of the coagulation process. Similarly, Muhl et al., in a prospective study describing the time course of MMP-2, -9 and TIMP-1 and -2 in patients with severe sepsis, noted the importance of the MMP:TIMP ratio in the correlation with sepsis progression [29]. Another prospective study, by Gaddnas et al., found high levels of MMP-2, -8 and -9 in skin blisters and serum of severe sepsis patients, and noted that higher MMP-2 in particular correlated with more severe organ failure [30]. Different studies have described the important prognostic value of MMPs as non-invasive tools for determining the outcome in septic patients. The MMP-9:TIMP-1 ratios and increased MMP-10 strongly correlate with severity and mortality [30].
A systemic inflammation response syndrome can also occur after trauma and infection, leading to multiple organ dysfunction syndromes (MODS), an important cause of morbidity and mortality in intensive care units. Animal model studies have indicated a high level of MMP-9 in the early stage of MODS, which also correlated with the degree of renal, hepatic and pulmonary injury [31], suggesting that MMP-9 might hold out promise as a biomarker to predict the severity of tissue damage in human MODS.
MMPs and neurological disease MMPs are involved in ECM remodeling not only in the vascular and musculoskeletal systems but also in the central nervous system (CNS). ECM degradation due to MMP activity is fundamental for blood brain barrier (BBB) leakage, leading to edema and ultimately to cerebral ischemia and stroke [32]. MMP-9 was recently reported to be higher in acute ischemic stroke (AIS) patients and it correlated with ongoing brain ischemia and infarct size, severity of stroke and worse functional outcome. MMP-9 is also described as a predictor of hemorrhage in patients receiving thrombolytic therapy [33]. Little is known about the exact cellular source of the MMPs expressed in brain, but it appears that astrocytes, brain endothelium, neurons, and inflammatory activated cells release different MMPs such as MMP-2, -3, -8, -9, -10 and -13. Therefore, these MMPs and their inhibitors are preferential targets of therapeutic strategies aimed at stroke prevention, such as anti-inflammatory drugs to reduce MMP activity and improve the acute treatment of human brain ischemia. Since vascular perturbations occur in the brain in several neurodegenerative diseases, MMPs are involved in the pathogenesis of various CNS disorders. Multiple sclerosis (MS) is an inflammatory autoimmune CNS disease with acute inflammation of axons and glia, followed by a phase of recovery and structural repair, post-inflammatory gliosis and neurodegeneration. MMPs are involved in BBB disruption and MS lesion formation. Animal models of experimental autoimmune encephalomyelitis and clinical trials of MS have brought to light the importance of MMP-8 and -9 and TIMP-1, with an increase of the two MMPs and a decrease of the inhibitor, in the loss of BBB integrity [34]. MMP-9 in particular has been proposed as a useful biomarker for monitoring MS treatment, as a drop in the serum MMP-9:TIMP-1 ratio correlates with lower disease activity. Another important MMP in MS monitoring is serum MMP-3, which is higher during MS relapse
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Galliera et al.: Matrix metalloproteinases as biomarkers of disease 353
than remission. Therefore a decrease could be a useful indicator of disease activation. Considering the pivotal roles of MMPs in MS disease pathogenesis and progression, different therapeutic approaches to control the disease are aiming at the different MMPs. In a MS clinical trial, erythropoietin raised TIMP-1 expression, helping maintain BBB integrity, while MS patients treated with IFN β 1 showed lower levels of MMP-8 and -9 [35]. Similarly, tetracycline (which inhibits MMP-2 and -9) and selective MMP-8 inhibitors are currently being tested in clinical trials to reduce relapse rates. Myasthenia gravis (MG) is an autoimmune disease involving auto-antibodies against the acetylcholine receptor (AChR) which impair neuromuscular transmission, leading to progressive muscle weakness. MMPs are involved in the pathogenesis and progression of MG, but little is known about their specific role. The first evidence from animal and human studies indicated MMP-9 as a target of MG auto-antibodies and serum MMP-2 and -9 are higher in MG patients than in healthy controls. Similarly, MG patients had high serum levels of MMP-3 and, interestingly, there was a significant sex difference, with higher MMP-3 in MG males than females.
With agrin as a substrate, MMP-3 has been suggested as controlling the neuromuscular junction through regulation of agrin levels. Therefore, high serum levels of MMPs such as MMP-3 and its regulators MMP-9 and -2 might be involved in autoimmunity and in the progression of MG [36]. MMPs can be useful biomarkers in other neurodegenerative diseases too, such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s dementia (AD). In ALS, serum MMP-1, -2, -9 and TIMP-1 in ALS patients was higher than in healthy controls, while another study correlated MMP-2 with disease progression. Animal models of ALS therapy based on MMP inhibitors have indicated beneficial results and longer life expectancy and neuronal survival. In a recent study on AD, MMP-3 was higher in serum and cerebrospinal fluid (CSF), while MMP-2 was low in CSF and unaltered in serum. Similarly, MMP-9 was low and MMP-10 was unaltered in serum, while both were undetectable in CSF. MMPs are also involved in other neurodegenerative disorders, such as epilepsy and migraine, but their pathogenic roles and their diagnostic potential still need to be clarified.
Table 2 MMPs as biomarkers of cancer progression. Human cancer
MMPs involved
Biomarker function
Cervical cancer
MMP-10 MMP-2,-9 MMP-2,-9
MMP-14 MMP-3 MMP-2,-9
MMP-10 expression positively correlates with cancer invasiveness Cancer lesion progression MMP-9 and MMP-2 correlates with histology grade and lymph vascular invasion MMP-9 correlates with pulmonary metastatic invasion Correlates with malignant progression Involved in cancer tumorigenesis and progression Lymphatic metastasis formation
MMP-1
MMP-3 MMP-1,14, TIMP-2
MMP-2,-24,-25 MMP-14
Breast cancer
Pancreatic adenocarcinoma Maxillofacial squamous cell carcinoma Prostate cancer
Gastric carcinomas
Non-small cell lung cancer (NSCLC) Colorectal carcinoma Renal cell carcinoma Brain tumor Ovarian carcinoma Head and neck squamous cell carcinoma Esophageal cancer Osteosarcoma Oropharyngeal squamous cell carcinoma
MMP-2,-7,-9 MMP-9/TIMP-2 ratio MMP-14 MT1-MMP MMP-1,-3,-7,-10,-12,- 14,-19 MMP-2,-9 MMP-9 MMP-2,-9, TIMP-1 and TIMP-2
Combined with over-expression of PAR-1, contributes to the malignant progression Correlates with malignant invasion MMP-1,14, TIMP-2 expression on mononuclear cells correlates with progression of gastric cancer Prognostic molecular markers of gastric cancer progression High expression of MMP-14 correlates with cancer progression MMP-2,-9 correlates with cancer progression Correlates with progression-free survival in patients receiving therapy Correlates with glioma progression High expression of MMT-1-MMP correlates with lower cancer progression Head and neck cancer invasion and angiogenesis Esophageal cancer progression Predictor of survival of patients with osteosarcoma Predictors of lymph node metastasis
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354 Galliera et al.: Matrix metalloproteinases as biomarkers of disease
MMPs and cancer ECM remodeling by MMPs is one of several aspects of cancer development, from angiogenesis to metastasis. MMPs have consequently been widely studied in cancer pathogenesis, metastasis progression and as biomarkers for clinical monitoring. Given the multiple aspects of cancer involving MMPs, it is still hard to clearly establish the diagnostic and prognostic value of a single MMP as a cancer biomarker [37]. On the one hand MMPs are involved in pathogenic ECM remodeling and are therefore considered tumor-promoting enzymes, but on the other there is evidence that some MMPs may act as tumor suppressors [38]. MMPs certainly play a large part in cancer progression and metastatic invasion, since they can promote cancer growth, development and metastatic diffusion, particularly in the ‘pre-metastatic niche’, the site where the early changes occur in cancer cells, in order to start metastatic diffusion [39]. Given their pivotal role in ECM remodeling, MMPs are involved in the malignant progression of various forms of cancer. Several recent studies, summarized in Table 2, report a strong correlation between MMP levels and the cancer stage, the severity of the disease and, in some cases, the poor or good prognosis. Therefore, the evaluation of MMPs as biomarkers of cancer progression and metastatic invasion is an important tool in the clinical monitoring of a variety of cancers. In view of their importance in cancer progression, MMPs have also been studied as potential therapeutic targets for solid and hematological malignancies.
Conclusions MMPs play a pivotal role in ECM remodeling and are therefore of interest in the development of new diagnostic tools for the clinical evaluation and management of a variety of diseases involving ECM. This setting ranges from the classical areas of MMP studies, such as vascular disease, cancer progression or bone disorders, to new emerging fields of application, such as neurodegenerative diseases or sepsis. Current studies focus not only on improving the diagnostic and prognostic value of the MMPs involved in the pathogenic process, but also on increasing the therapeutic potential, identifying MMPs as new pharmacological targets. Therefore, MMPs may be pivotal in pathologies where clear diagnostic tools are still lacking, improving the identification, monitoring and clinical treatment of these human diseases.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Financial support: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Mini Review Emanuela Galliera*, Lorenza Tacchini and Massimiliano M. Corsi Romanelli
Matrix metalloproteinases as biomarkers of disease: updates and new insights DOI 10.1515/cclm-2014-0520 Received May 16, 2014; accepted July 18, 2014; previously published online August 7, 2014
growth factors, and regulating cell movement, intercellular interactions and communication [1]. ECM formation and degradation is a crucial event in biological processes involved in maintaining tissue homeostasis and regeneraAbstract: Matrix metalloproteinases (MMPs) play a pivtion, such as for wound healing and fibrosis. These events otal role in remodeling the extracellular matrix (ECM) and are under the control of a family of 28 endopeptidases are therefore of interest for new diagnostic tools for the known as matrix metalloproteinases (MMPs), multidoclinical management of diseases involving ECM disrupmain zinc metalloproteinases specialized in degradation tion. This setting ranges from the classical areas of MMP of the different components of the ECM [2]. The ECM itself studies, such as vascular disease, cancer progression or consists mainly of polysaccharides, such as hyaluronic bone disorders, to new emerging fields of application, acid, collagen and elastin fibers for tissue flexibility, prosuch as neurodegenerative disease or sepsis. Increasing teoglycans, such as chondroitin sulfate or heparan sulfate, the knowledge about the role of MMPs in the pathogenesis binding growth factors and drawing in water in order to of diseases where a clear diagnostic panel is still lacking keep the microenvironment hydrated. The different ECM could provide new insight and improve the identification components are specific targets of different MMPs. On and the clinical treatment of these human diseases. This the basis of the substrate specificity, MMPs can be clasreview focuses on the latest descriptions of the clinical sified in five main groups: collagenases (MMP-1, -8, -13), use of MMP as biomarkers in the diagnosis, prognosis and gelatinases (MMP-2, -9), stromyelysins (MMP-3, -10, -11), monitoring of different diseases, such as diabetes, cardiomatrilysins (MMP-7 and -26), and membrane-type MMPs vascular diseases, cancer and metastasis, neurodegenera(MP MMP-14, 15, 16 and -24) [3]. Others include MMP-12, tive disorders and sepsis. -20 and -27 which have a structure similar to stromyelysin; MMP-19, which is a basement membrane-degrading Keywords: diagnosis; metallorproteasis; serum biomarkers. protease involved in tissue remodeling, healing and cell migration; MMP-20, a tooth-specific protease; MMP-21, a gelatinase expressed in several carcinomas; MMP-23, Introduction which is expressed in ovary, prostate and testis and plays a role in reproduction; MMP-28 (epilysin), mainly expressed The extracellular matrix (ECM) is the extracellular comin rheumatoid arthritis and osteoarthritis [1]. ponent of tissues, providing support for cells, storage for The activity of the MMPs is closely regulated on several levels, starting from gene transcription, to transla*Corresponding author: Dr. Emanuela Galliera, PhD, Assistant tion, pro-enzyme activation and inhibition by regulatory Professor of Clinical Pathology, Department of Biomedical, Surgical proteins, such as specific tissue inhibitors of metalloand Oral Science, Università degli Studi di Milano, I Via Luigi proteinases (TIMPs), which can neutralize the proteases Mangiagalli 31, 20133 Milano, Italy, Phone: +39 02 50315354, thereby preventing excessive matrix degradation. Fax: +39 02 50315338, E-mail: [email protected]; and MMPs are required in biological functions, such as IRCCS Galeazzi Orthopedic Institute, Milan, Italy Lorenza Tacchini: Department of Biomedical Sciences for Health, cell proliferation, differentiation, apoptosis, immune Università degli Studi di Milano, Milan, Italy function, tissue healing, and angiogenesis. Therefore, Massimiliano M. Corsi Romanelli: Department of Biomedical they are also involved in multiple pathological condiSciences for Health, Università degli Studi di Milano, Milan, Italy; tions [4], where they play a role in tissue destruction and Service of Laboratory Medicine1-Clinical Pathology, Department (such as in cancer invasion and metastasis, arthritis, difof Health Service of Diagnosis and Treatment-Laboratory Medicine, ferent types of ulcers, inflammatory diseases), fibrosis IRCCS Policlinico San Donato, San Donato (Milan), Italy
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350 Galliera et al.: Matrix metalloproteinases as biomarkers of disease (such as liver cirrhosis, multiple sclerosis, atherosclerosis) and matrix degradation (such as cardiomyopathy, aortic aneurysm), and in various bone and joint diseases [5, 6]. MMPs and their inhibitors, the TIMPs, have consequently been proposed as biomarkers for certain pathologies [7]. They can be examined at different levels, by measuring tissue gene expression, protein amount, and plasma levels, as well as proteolytic activity, by gel zymography. Since MMPs are sensitive enzymes, extreme care must be taken in blood sampling and handling, because these procedures can alter the concentration ratios between plasma and serum, particularly for MMP-9 [8, 9]. Therefore, the plasma or serum sample must be selected appropriately. However, in some cases, such as MMP-2 and MMP-9, there is a positive correlation between plasma and serum levels [10]. This review focuses on the latest evidence on the use of MMP as circulating biomarkers in the diagnosis, prognosis and monitoring of different human diseases, as summarized in Table 1.
MMPs and diabetes Dysregulation of MMP activity has been involved in the pathogenesis of several diabetic co-morbidities, from diabetic retinopathy to diabetic nephropathy and chronic non-healing ulcers. Diabetic nephropathy is due to progressive, abnormal degradation and turnover of ECM in the glomerulus, where MMPs are major players so changes in their expression or activation can contribute to renal hypertrophy and affect ECM deposition, leading to renal injury in diabetic nephropathy [11]. Abnormal ECM deposition is the hallmark of diabetic nephropathy, and this is mainly due to alteration of MMPs expressed in the nephron. Specific MMPs and the relative TIMPs are expressed in kidney tissue; these include MMP-2, -3, -9, -10, -14, -15, and TIMP-2 and -4 [12]. Besides their role in ECM turnover, MMPs are also involved in the release and activation of several factors present in the ECM and associated with diabetic nephropathy, such as TGFβ, TNFα, insulin-like growth factors and heparin-binding epidermal growth factor, thus contributing indirectly to tubular cell
Table 1 Summary of the different MMPs as biomarkers of human disease discussed in this review. Human disease
MMPs involved
Serum/plasma level
Biomarker of
Increased
Diabetic nephropathy
MMP-1,-2,-3,-7,-9 (plasma), MT-MMP-14, -24 TIMP-1,-2,-3 MMP-2,-9 (plasma) MMP-8,-9 (plasma) MMP-9 (plasma) MMP-1,-3,-8 MMP-2
Increased Increased Increased Increased Increased
MMP-2
Decreased
MMP-8,-9 (plasma) MMP-9 (plasma), TIMP-1 MMP-1 MMP-2,-9 (plasma) TIMP-1,-2 MMP-2,-8,-9 (plasma) MMP-9 (plasma):TIMP-1 ratio, MMP-10 MMP-9 (plasma) MMP-9 (plasma) MMP-8,-9 (plasma) TIMP-1 MMP-9 (plasma):TIMP-1 ratio MMP-3 MMP-3,-2,-9 (plasma) MMP-1,-2,-9 (plasma), TIMP-1 MMP-3
Increased Increased Increased Increased
Diabetic retinopathy Diabetic chronic wound healing inability Diabetic cardiovascular risk Atherosclerosis progression Abdominal aortic aneurysm with primary abdominal hernias Abdominal aortic aneurysm without primary abdominal hernias Plaque rupture risk Stroke mortality risk in metabolic syndrome Poor prognosis Poor prognosis
Diabetes mellitus
Cardiovascular diseases
Sepsis
Neurological diseases
Increased Increased Increased Increased Increased Decreased Decreased Increased Increased Increased Increased
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Severe outcome Severity and mortality Tissue damage in MODS Ischemia, infarct size in acute ischemic stroke Multiple sclerosis (MS) progression Decreased MS activity Higher in relapse than remission of MS Progression of myasthenia gravis Progression of amyotrophic lateral sclerosis Progression of Alzheimer’s disease
Galliera et al.: Matrix metalloproteinases as biomarkers of disease 351
proliferation and renal fibrosis, leading to renal damage. A robust association with diabetic nephropathy has been reported for gelatinases MMP-2 and -9, matrilysins MMP-7, membrane-type MT-MMP-14, -24, stromyelysin MMP-3 and collagenase MMP-1, TIMP-1, -2, and -3 [11]. We still need a better understanding of the precise role of MMPs in the pathogenesis of diabetic nephropathy, to target MMPs therapeutically, so as to prevent or reverse diabetic renal disease. MMPs are involved in other diabetic co-morbidities too, such as diabetes-related retinopathy. MMP-2 and -9 have a part in diabetes-induced mitochondrial dysfunction and consequent retinal damage [13]. Similarly, TIMP-1, together with TGF-β2, has a role in the angiogenic phase of proliferative diabetic retinopathy. Levels of TIMP-1, MMP-2 and -9 have been reported to be related to the degree of neovascularization [14]. One evident consequence of diabetes is the chronic inability of wounds to heal, due to activated MMPs, which remodel tissue, resulting in apoptosis. The recent identification of the main MMPs involved, MMP-9 and -8, in murine models of diabetic wounds, is valuable for targeting new pharmacological interventions. Another major co-morbidity of diabetes is cardiovascular disorders. Myeloperoxidase, as well as MMP-9, are reported to be early biomarkers of cardiovascular risk in diabetic patients [15]. Given the close relation between MMPs and their inhibitors with the pathogenesis of diabetes and related disorders, there is growing agreement that these molecules might be useful biomarkers for clinical use in diagnosis. Genetic study of MMPs has pointed to a relation between specific polymorphisms and type 2 diabetes and diabetic foot ulcers [16]. Circulating levels of MMPs and TIMPs can be used as diagnostic and prognostic biomarkers. For example, serum MMP-9 and TIMP-1 predict poor wound healing in diabetic foot ulcers, while serum MMP-9 is related with progressive diabetic vitrectomy [17].
MMPs and cardiovascular disease Abnormal angiogenesis and vascular remodeling contribute to the pathogenesis of several disorders, such as atherosclerosis, re-stenosis, and hypertension, increasing cardiovascular risk. MMPs are important in vascular remodeling and in several processes, such as angiogenesis, collateral artery formation and thrombus resolution, but also in the pathogenesis of atherosclerosis, plaque rupture, aortic aneurysm and varicose veins [18]. Vascular
tissue expresses high levels of MMP-1, -2, -3, -7, -8, -9, -12 and MT-MMPs [18]. Therefore various MMPs have been studied as potential investigative tools in the diagnosis and monitoring of vascular diseases. Collagenases MMP-1, -3, and -8 are proposed as indicators of atherosclerosis progression, while high MMP-2 serum levels can distinguish patients with abdominal aortic aneurysm associated with primary abdominal hernias, who have high serum MMP-2, from those who have abdominal aortic aneurysm alone, and lower levels of serum MMP-2 [19]. One of the principal factors of cardiovascular risk is hypertensive cardiac disease, caused not only by stress, genetic predisposition, lipid metabolism disorders, or diabetes, but also by increased vessel fibrosis, which raises systemic blood pressure. Among the MMPs acting in concert with hypertension-promoting mediators, MMP-2 and -7 are the most important and have been proposed as therapeutic targets in patients with hypertensive cardiac disease [20]. The main adverse event with hypertension and atherosclerosis is plaque rupture. Since MMPs are involved in carotid atherosclerotic plaque remodeling, monitoring MMPs, such as MMP-8 and -9 can provide useful information about plaque progression and the risk of rupture. A new imaging approach has been developed [21] to visualize the inflammatory process in the vessel wall and plaque, so intra-plaque levels of MMPs might be helpful in assessing the risk of plaque rupture. The importance of MMPs in plaque vulnerability is confirmed by the relations between MMP-9 serum levels and markers of carotid atherosclerosis and lesion risk, such as fibrous cap thickness, lipid core collagen content and intra-plaque hemorrhage [22]. High cardiovascular risk is one of the main clinical features of metabolic syndrome, defined as the combination of obesity, arterial hypertension, diabetes mellitus and dyslipidemia. Patterns of serum MMPs are altered in patients with metabolic syndrome. In particular, high serum MMP-9 and TIMP-1 are associated with cardiovascular risk and stroke mortality. Hypertension is one of the main cardiovascular diseases, and is an important complication in pregnancy and a severe risk factor for cardiac adverse events. Therefore, it is essential to monitor hypertension for the prevention of cardiovascular disorders. MMP-2 is important in this context, as the target of different strategies based on MMP inhibitors, aimed at improving hypertensioninduced cardiovascular alterations [23–25]. Similarly, many drugs already used by patients can affect MMP concentrations, particularly MMP-9 [26]. Therefore, particular care must be taken in taking the patient’s history and clinical evaluation in order to avoid misinterpretation of MMP results.
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352 Galliera et al.: Matrix metalloproteinases as biomarkers of disease Given the prominent role of MMPs in vasculature remodeling, they are also important therapeutic targets in the prevention and treatment of atherosclerosis and coronary artery diseases [27]. Several pharmacological approaches have been studied to inhibit MMP activity, using tissue MMP inhibitors – TIMPs, pleiotropic mediators, such as tetracycline, doxocyclins, small-molecule MMP inhibitors (MMPis) and inhibitory antibodies, but they still have broad specificity, because they prevent the ECM degradation function of different MMPs. Therefore, further studies are still needed to find MMP is with narrower specificity, targeting the action of a specific MMP.
MMPs and sepsis Sepsis is the result of an extreme inflammatory response to pathogen invasion, that can lead to organ failure (severe sepsis), persistent blood pressure loss (septic shock) and, ultimately, a fatal outcome. Therefore early diagnosis and prompt therapeutic intervention are vital in the management of septic patients, which is often complicated by different clinical presentation and responses to therapy. Sepsis is also associated with alterations in coagulation and endothelial response, but the pathogenic mechanism of these effects is still poorly understood, limiting the development of therapeutic strategies. MMPs are important in tissue remodeling and the host’s inflammatory response to pathogen invasion; therefore they could serve as a diagnostic tool and therapeutic target in sepsis. Early evidence came from Tressel et al., who found a positive correlation with circulating MMP-1 and poor prognosis in septic patients [28]. MMP-1 is released by endothelial cells under stress conditions, such as sepsis, leading to a loss of endothelial integrity, a consequent increase of vessel permeability and, ultimately, an alteration of the coagulation process. Similarly, Muhl et al., in a prospective study describing the time course of MMP-2, -9 and TIMP-1 and -2 in patients with severe sepsis, noted the importance of the MMP:TIMP ratio in the correlation with sepsis progression [29]. Another prospective study, by Gaddnas et al., found high levels of MMP-2, -8 and -9 in skin blisters and serum of severe sepsis patients, and noted that higher MMP-2 in particular correlated with more severe organ failure [30]. Different studies have described the important prognostic value of MMPs as non-invasive tools for determining the outcome in septic patients. The MMP-9:TIMP-1 ratios and increased MMP-10 strongly correlate with severity and mortality [30].
A systemic inflammation response syndrome can also occur after trauma and infection, leading to multiple organ dysfunction syndromes (MODS), an important cause of morbidity and mortality in intensive care units. Animal model studies have indicated a high level of MMP-9 in the early stage of MODS, which also correlated with the degree of renal, hepatic and pulmonary injury [31], suggesting that MMP-9 might hold out promise as a biomarker to predict the severity of tissue damage in human MODS.
MMPs and neurological disease MMPs are involved in ECM remodeling not only in the vascular and musculoskeletal systems but also in the central nervous system (CNS). ECM degradation due to MMP activity is fundamental for blood brain barrier (BBB) leakage, leading to edema and ultimately to cerebral ischemia and stroke [32]. MMP-9 was recently reported to be higher in acute ischemic stroke (AIS) patients and it correlated with ongoing brain ischemia and infarct size, severity of stroke and worse functional outcome. MMP-9 is also described as a predictor of hemorrhage in patients receiving thrombolytic therapy [33]. Little is known about the exact cellular source of the MMPs expressed in brain, but it appears that astrocytes, brain endothelium, neurons, and inflammatory activated cells release different MMPs such as MMP-2, -3, -8, -9, -10 and -13. Therefore, these MMPs and their inhibitors are preferential targets of therapeutic strategies aimed at stroke prevention, such as anti-inflammatory drugs to reduce MMP activity and improve the acute treatment of human brain ischemia. Since vascular perturbations occur in the brain in several neurodegenerative diseases, MMPs are involved in the pathogenesis of various CNS disorders. Multiple sclerosis (MS) is an inflammatory autoimmune CNS disease with acute inflammation of axons and glia, followed by a phase of recovery and structural repair, post-inflammatory gliosis and neurodegeneration. MMPs are involved in BBB disruption and MS lesion formation. Animal models of experimental autoimmune encephalomyelitis and clinical trials of MS have brought to light the importance of MMP-8 and -9 and TIMP-1, with an increase of the two MMPs and a decrease of the inhibitor, in the loss of BBB integrity [34]. MMP-9 in particular has been proposed as a useful biomarker for monitoring MS treatment, as a drop in the serum MMP-9:TIMP-1 ratio correlates with lower disease activity. Another important MMP in MS monitoring is serum MMP-3, which is higher during MS relapse
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Galliera et al.: Matrix metalloproteinases as biomarkers of disease 353
than remission. Therefore a decrease could be a useful indicator of disease activation. Considering the pivotal roles of MMPs in MS disease pathogenesis and progression, different therapeutic approaches to control the disease are aiming at the different MMPs. In a MS clinical trial, erythropoietin raised TIMP-1 expression, helping maintain BBB integrity, while MS patients treated with IFN β 1 showed lower levels of MMP-8 and -9 [35]. Similarly, tetracycline (which inhibits MMP-2 and -9) and selective MMP-8 inhibitors are currently being tested in clinical trials to reduce relapse rates. Myasthenia gravis (MG) is an autoimmune disease involving auto-antibodies against the acetylcholine receptor (AChR) which impair neuromuscular transmission, leading to progressive muscle weakness. MMPs are involved in the pathogenesis and progression of MG, but little is known about their specific role. The first evidence from animal and human studies indicated MMP-9 as a target of MG auto-antibodies and serum MMP-2 and -9 are higher in MG patients than in healthy controls. Similarly, MG patients had high serum levels of MMP-3 and, interestingly, there was a significant sex difference, with higher MMP-3 in MG males than females.
With agrin as a substrate, MMP-3 has been suggested as controlling the neuromuscular junction through regulation of agrin levels. Therefore, high serum levels of MMPs such as MMP-3 and its regulators MMP-9 and -2 might be involved in autoimmunity and in the progression of MG [36]. MMPs can be useful biomarkers in other neurodegenerative diseases too, such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s dementia (AD). In ALS, serum MMP-1, -2, -9 and TIMP-1 in ALS patients was higher than in healthy controls, while another study correlated MMP-2 with disease progression. Animal models of ALS therapy based on MMP inhibitors have indicated beneficial results and longer life expectancy and neuronal survival. In a recent study on AD, MMP-3 was higher in serum and cerebrospinal fluid (CSF), while MMP-2 was low in CSF and unaltered in serum. Similarly, MMP-9 was low and MMP-10 was unaltered in serum, while both were undetectable in CSF. MMPs are also involved in other neurodegenerative disorders, such as epilepsy and migraine, but their pathogenic roles and their diagnostic potential still need to be clarified.
Table 2 MMPs as biomarkers of cancer progression. Human cancer
MMPs involved
Biomarker function
Cervical cancer
MMP-10 MMP-2,-9 MMP-2,-9
MMP-14 MMP-3 MMP-2,-9
MMP-10 expression positively correlates with cancer invasiveness Cancer lesion progression MMP-9 and MMP-2 correlates with histology grade and lymph vascular invasion MMP-9 correlates with pulmonary metastatic invasion Correlates with malignant progression Involved in cancer tumorigenesis and progression Lymphatic metastasis formation
MMP-1
MMP-3 MMP-1,14, TIMP-2
MMP-2,-24,-25 MMP-14
Breast cancer
Pancreatic adenocarcinoma Maxillofacial squamous cell carcinoma Prostate cancer
Gastric carcinomas
Non-small cell lung cancer (NSCLC) Colorectal carcinoma Renal cell carcinoma Brain tumor Ovarian carcinoma Head and neck squamous cell carcinoma Esophageal cancer Osteosarcoma Oropharyngeal squamous cell carcinoma
MMP-2,-7,-9 MMP-9/TIMP-2 ratio MMP-14 MT1-MMP MMP-1,-3,-7,-10,-12,- 14,-19 MMP-2,-9 MMP-9 MMP-2,-9, TIMP-1 and TIMP-2
Combined with over-expression of PAR-1, contributes to the malignant progression Correlates with malignant invasion MMP-1,14, TIMP-2 expression on mononuclear cells correlates with progression of gastric cancer Prognostic molecular markers of gastric cancer progression High expression of MMP-14 correlates with cancer progression MMP-2,-9 correlates with cancer progression Correlates with progression-free survival in patients receiving therapy Correlates with glioma progression High expression of MMT-1-MMP correlates with lower cancer progression Head and neck cancer invasion and angiogenesis Esophageal cancer progression Predictor of survival of patients with osteosarcoma Predictors of lymph node metastasis
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354 Galliera et al.: Matrix metalloproteinases as biomarkers of disease
MMPs and cancer ECM remodeling by MMPs is one of several aspects of cancer development, from angiogenesis to metastasis. MMPs have consequently been widely studied in cancer pathogenesis, metastasis progression and as biomarkers for clinical monitoring. Given the multiple aspects of cancer involving MMPs, it is still hard to clearly establish the diagnostic and prognostic value of a single MMP as a cancer biomarker [37]. On the one hand MMPs are involved in pathogenic ECM remodeling and are therefore considered tumor-promoting enzymes, but on the other there is evidence that some MMPs may act as tumor suppressors [38]. MMPs certainly play a large part in cancer progression and metastatic invasion, since they can promote cancer growth, development and metastatic diffusion, particularly in the ‘pre-metastatic niche’, the site where the early changes occur in cancer cells, in order to start metastatic diffusion [39]. Given their pivotal role in ECM remodeling, MMPs are involved in the malignant progression of various forms of cancer. Several recent studies, summarized in Table 2, report a strong correlation between MMP levels and the cancer stage, the severity of the disease and, in some cases, the poor or good prognosis. Therefore, the evaluation of MMPs as biomarkers of cancer progression and metastatic invasion is an important tool in the clinical monitoring of a variety of cancers. In view of their importance in cancer progression, MMPs have also been studied as potential therapeutic targets for solid and hematological malignancies.
Conclusions MMPs play a pivotal role in ECM remodeling and are therefore of interest in the development of new diagnostic tools for the clinical evaluation and management of a variety of diseases involving ECM. This setting ranges from the classical areas of MMP studies, such as vascular disease, cancer progression or bone disorders, to new emerging fields of application, such as neurodegenerative diseases or sepsis. Current studies focus not only on improving the diagnostic and prognostic value of the MMPs involved in the pathogenic process, but also on increasing the therapeutic potential, identifying MMPs as new pharmacological targets. Therefore, MMPs may be pivotal in pathologies where clear diagnostic tools are still lacking, improving the identification, monitoring and clinical treatment of these human diseases.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Financial support: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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