Evaluation of Matrix Metalloproteinase-2 (MMP-2) and -9 (MMP-9) and Their Tissue Inhibitors (TIMP-1 and TIMP-2) in Plasma from Patients with Neurodegenerative Dementia

Abstract

Matrix metalloproteinases (MMPs) are substantial regulators of learning and memory and might be involved in neurodegeneration. It is known that MMPs are involved in pathogenesis of Alzheimer’s disease (AD) and are particularly involved in the amyloid-β processing pathway. However, information on circulating levels of these proteins and their tissue inhibitors (TIMPs) in AD and other neurodegenerative dementia (ND) diseases such as dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD) is not clear. Therefore, this study was directed toward finding out how plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 vary in AD, DLB, and FTD; and investigating the correlation of the levels of MMPs and their inhibitors with clinical parameters of the patients. MMP-2, MMP-9, TIMP-1, and TIMP-2 levels were measured by enzyme linked immunosorbent assay (ELISA). Plasma MMP-2 levels were significantly lower in all the patient groups than in the age-matched healthy controls (HCs) (p < 0.05). MMP-9 levels were significantly lower in the FTD patients than in the HCs (p < 0.05). Also, TIMP-1 levels were lower in the AD and FTD patients than in the HCs (p < 0.05). TIMP-2 levels were similar in all the groups. These findings highlight the importance of circulating MMPs in ND and suggest that MMPs and their inhibitors might play a role in impaired amyloid-β peptide metabolism which is responsible for the genesis and progression of ND. Furthermore, measurement of MMP-2 and MMP-9 and their inhibitors may be of great importance for large scale basic research and clinical studies of ND.

Keywords

ELISA matrix metalloproteinases neurodegenerative dementia tissue inhibitor of metalloproteinases

INTRODUCTION

Neurodegenerative dementia (ND) is a group of neurological disorders characterized by impairment of various cognitive domains where particular populations of neurons are selectively and progressively lost [1]. Alzheimer’s disease (AD) is the most common cause of dementia worldwide, accounting for more than 50% of all cases. Pathologically, AD is characterized by cortical atrophy, neuronal death, synaptic loss, glial proliferation, excessive formation of neurofibrillary tangles, and deposition of amyloid-β (Aβ) in neuritic plaques [2 –5]. The amyloid hypothesis suggests that AD is caused by an imbalance between generation and clearance of Aβ [6]. Dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD) are the less prevalent types of ND [7]. FTD is characterized by executive function deficit, language impairment, and behavioral disorders. It is considered as the most common ND with an onset usually before 65 years of age [8]. DLB is the second most common type of degenerative dementia in the elderly [9]. It is characterized by prominent visuoconstructive and frontosubcortical impairment, in association with core clinical neuropsychiatric features of fluctuating cognitive function, visual hallucinations, and spontaneous parkinsonian motor signs [10, 11]. DLB involves protein aggregates called Lewy bodies, balloon-like structures that form inside the nerve cells [7]. Although Aβ deposition is considered as a signature lesion for AD, it also occurs in certain cases of DLB [12].

Matrix metalloproteinases (MMPs), which are Zn^{2 +} and Ca^{2 +} dependent endopeptidases, degrade components of the extracellular matrix (ECM) in a variety of physiologic and pathophysiologic conditions [13]. MMP activity is regulated by interactions with tissue inhibitors of metalloproteinases (TIMPs), a family of multifunctional secreted proteins (TIMP-1–4) that promote growth and regulate the cell cycle in various cell types [14]. Physiological balance between MMPs and TIMPs is significant in many diseases. There is growing evidence that MMPs play an important role in inflammatory processes in the pathogenesis of AD. They may also be involved in the processing pathway of Aβ [14]. MMP-2 and MMP-9 play a significant role in brain Aβ degradation and clearance, and their expressions are increased in the AD brain, particularly in astrocytes surrounding the amyloid plaque [15 –17]. However, it is not known whether they play a protective or destructive role in the pathogenesis of AD. Thus, changes in MMP expression or function may affect the genesis and progression of ND and this change may be reflected in the plasma.

Given the emerging importance of MMP-9 and MMP-2 in various types of ND, it is noteworthy that clinical correlates of circulating MMP-2 and MMP-9 remain incompletely understood. Also, there is no clear view on the clinical utility of these proteins, as potential surrogate markers for various types of ND. In recent decades, several groups of researchers have reported an elevation of cerebrospinal fluid (CSF) or plasma MMPs in ND patients [13 , 19]. However, changes in circulating levels of MMPs and their inhibitors in AD, FTD, or DLB have not been consistent. For example, studies conducted by Lorenzl et al. [13, 18] concluded that patients with AD show higher concentrations of plasma MMP-9 than healthy controls (HCs). There was no significant difference in plasma MMP-2 and MMP-9 levels between patients with FTD and DLB and controls. In contrast, some reports have revealed that patients with AD have a lower level or activity of MMP-2 or MMP-9 in CSF or plasma than HCs [19 –21]. The inconsistent results among the studies could be attributed to the truly tiny changes in concentrations of MMPs in ND patients. There have been very few studies about plasma levels of MMP-2 and MMP-9 and their inhibitors in FTD and DLB patients. Although the role of MMPs in pathogenesis of ND has been revealed in the literature, it is not clear which MMP is more effective in neuronal degeneration in different types of ND. More studies might be needed to clearly discriminate plasma MMPs and TIMPs concentrations between patients with AD, FTD, and DLB and healthy individuals.

In this study, we investigated plasma levels of MMP-2, MMP-9, and their respective inhibitors, TIMP-2 and TIMP-1, in AD, DLB, and FTD patients and HCs. Thus, we tried to find out how circulating levels of MMPs and their inhibitors vary in different dementia types. We also analyzed correlations of MMPs and TIMPs levels with age and Mini-Mental State Examination (MMSE) parameters of the patients. We think that measurement of MMPs and their inhibitors in three different patient groups will shed light on the role of MMPs in differential diagnoses of the diseases.

MATERIALS AND METHODS

Subjects and specimens

Patients with AD (n = 30), FTD (n = 10), and DLB (n = 10) and HCs (n = 30) were enrolled in this study following local ethics committee approval (approval date: 06.06.2013; protocol no: 2013/21-21). Informed consent was obtained from all control subjects and family members of all patients with AD, FTD, and DLB. AD criteria were defined in accordance with NINCDS-ADRDA Work Group guidelines [22]. DLB and FTD were diagnosed according to DLB Consortium’s third report and Neary et al.’s criteria, respectively [11, 23]. All cases had mild to severe dementia based on their MMSE scores. Spouses of the patients and age-matched people living in Narlidere Rest Home, Izmir, served as the HCs. The MMSE scores of the HCs were higher than 26 points. None of the HCs had symptoms of dementia, or any other medical or neurological diseases, as judged by their clinical evaluation. Five milliliters of venous blood samples were collected from all the patients and the HCs in heparinized tubes. Samples were centrifuged at 3000 rpm for 10 min and obtained plasma was aliquoted and stored at –80°C until its analysis.

Measurements of MMP-2, MMP-9, TIMP-1, and TIMP-2 levels by ELISA

MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured with commercially available sandwich ELISA kits (RayBio-Norcross, GA, USA) in accordance with the manufacturers’ instructions. For TIMP-1, TIMP-2, and MMP-9 analyses, plasma samples were diluted at 1:300, 1:300, and 1:200, respectively. For MMP-2, plasma samples were studied without dilution. Standards and samples were pipetted into the pre-coated specific polyclonal antibody microplate. The MMP-2, MMP-9, TIMP-1, or TIMP-2 in standards and samples were sandwiched by the immobilized antibody, and biotinylated polyclonal antibody, which is recognized by a streptavidin peroxidase conjugate. All unbound materials were then washed away and TMB (perborate/3,3’,5,5’–tetramethylbenzidine) substrate solution was added. The color development was stopped, and the absorbance was measured at 450 nm using a microplate reader (Biotek, ELX 800, USA). The results were calculated in nanogram per milliliter (ng/mL) by using standard curves. The R² values of standard graphics were found to be greater than 0.98. The coefficient of variation (CV) of the intra-day precision of MMP2, MMP9, TIMP-1, and TIMP-2 was found to be 2.9%, 1.4%, 1.2%, and 1.2%, respectively, and the CV of the inter-day precision of these parameters was found to be 4.0%, 4.4%, 13.6%, and 4.9%, respectively, which were lower than the accepted criteria limits (15–20%). The ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1 were additionally calculated.

Statistical analysis

Statistical analyses were performed by using SPSS 22.0 (SPSS Inc., USA) and the graphs were generated with GraphPad Prism 7.0 (GraphPad Software Inc., USA). The values obtained from all measurements were presented as median and interquartile range (25–75%). Normality of data from each study group was tested with Shapiro-Wilk normality test. Based on the limited sample size and non-normality of continuous variables, data sets were assessed with Kruskal-Wallis test followed by Dunn’s multiple comparison test. Chi-Square test was used to examine categorical data. p < 0.05 was considered significant. Spearman correlation coefficients were used to analyze correlations between non-normally distributed variables.

RESULTS

The subjects’ demographic and clinical characteristics are summarized in Table 1. There were significant differences in MMSE scores between the study groups and the HCs (p < 0.05).

Table 1

Demographic features and MMSE scores of HCs and AD, FTD and DLB patients

	HCs	AD	FTD	DLB	p
	(n = 30)	(n = 30)	(n = 10)	(n = 10)
¹Gender (Number of females; %)	16; 53.3%	17; 56.7%	4; 40.0%	5; 50.0%	0.833
²Age (y)	75 (70–79)	77.5 (72–82)	60 (55–70)	78 (75–85)	1.000 ^# 0.002 ^* 0.422 ^$
²MMSE Scores	28 (28–28)	16 (10–21)	18 (8–24)	20.5 (17–25)	0.000 ^# 0.000 ^* 0.007 ^$

¹Chi-Square test, ²Kruskal-Wallis test followed by the Dunn’s multiple comparison test, ^# HCs compared with AD patients, ^*HCs compared with FTD patients, ^$HCs compared with DLB patients.

Table 2 and Fig. 1 show the levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 in plasma samples of the AD, DLB, and FTD patients and the HCs. The levels of MMP-2, MMP-9, and TIMP-1 were significantly lower in the FTD patients than those in the HCs (p = 0.031, p = 0.003 and p = 0.020, respectively). The AD patients had lower levels of MMP2 and TIMP1 compared to the HCs (p = 0.002, p = 0.015, respectively). MMP-2 levels were significantly lower in the DLB group than in the HCs group (p = 0.048). There were no statistically significant differences between the patient and the control groups regarding TIMP-2 levels (p > 0.05). The ratio of MMP2/TIMP2 was significantly lower in the FTD and AD patients than that in the HCs (p = 0.031 and p = 0.001, respectively). However, there was not a statistically significant difference between the patients and the HCs in terms of the MMP-9/TIMP-1 ratio (p = 0.072). Also, there were no statistically significant differences between the AD, FTD and DLB patients regarding MMP-2, MMP-9, TIMP-2, and TIMP-1 levels (p > 0.05). Plasma MMP-2 levels were significantly positively correlated with plasma TIMP-1, MMP-9, and TIMP-2 levels in the AD patients (r = 0.419, p = 0.021; r = 0.567, p = 0.001; and r = 0.625, p = 0.000, respectively) (Fig. 2). MMP-9 levels were positively correlated with TIMP-1 and TIMP-2 levels in the AD patients (r = 0.492, p = 0.006 and r = 0.483, p = 0.007, respectively) (Fig. 2). No significant relation was found between MMPs and TIMPs levels in the FTD and DLB patients. Also, MMPs and TIMPs levels were not correlated with MMSE scores and ages in all the groups. The median plasma MMP-2, MMP-9, TIMP-1, and TIMP-2 values of the patients and the HCs are summarized in Table 2.

Table 2

Plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 in patients with AD, DLB and FTD and HCs

	HCs	AD	FTD	DLB	p ¹
MMP-2 (ng/mL)	38.6	13.8	5.7	8.5	0.002 ^# 0.031 ^* 0.048 ^$
	(12.1–83.3)	(1.6–19.0)	(3.3–19.4)	(6.3–15.1)
MMP-9 (ng/mL)	2218	1877	480.1	1793	0.684 ^#0.003^* 1.000 ^$
	(1740–2853)	(891.3–2773)	(301.8–1837)	(695–2474)
TIMP-1 (ng/mL)	577.1	431.3	388.7	514.4	0.015^#0.020^* 0.913 ^$
	(493.5–707.3)	(335.2–565.9)	(294.4–515.9)	(420.9–561.4)
TIMP-2 (ng/mL)	178	166.3	158.7	144.6	1.000 ^# 0.354 ^* 0.066 ^$
	(153.9–202.5)	(151.3–191.6)	(134.3–177.2)	(125.8–174.9)
MMP-2/TIMP-2	0.224	0.062	0.037	0.058	0.001^#0.031^* 0.072 ^$
	(0.06–0.36)	(0.01–0.1)	(0.02–0.13)	(0.04–0.09)
MMP-9/TIMP-1	3.60	4.11	1.24	3.63	0.072²
	(2.62–4.6)	(1.9–5.4)	(0.97–3.18)	(1.81–4.41)

The results are expressed in median (range). ¹Data sets were assessed by using Kruskal-Wallis test followed by Dunn’s multiple comparison test, ²Kruskal-Wallis test, ^# HCs compared with AD patients; ^*HCs compared with FTD patients, ^$HCs compared with DLB patients.

Fig. 1.

Levels of MMP-2 (a), MMP-9 (b), TIMP-2 (c), and TIMP-1 (d) in healthy controls and patients.

Fig. 2.

Significant correlations of MMP-2 levels with TIMP-2 (a), TIMP-1 (b) and MMP-9 (c) levels in AD patients (r = 0.625, p = 0.000; r = 0.419, p = 0.021; and r = 0.567, p = 0.001, respectively) and significant correlations of MMP-9 levels with TIMP-1 (d) and TIMP-2 (e) levels in AD patients (r = 0.492, p = 0.006 and r = 0.483, p = 0.007, respectively).

DISCUSSION

AD is characterized with accumulation of Aβ in brain amyloid plaques. Genetic mutations in familial AD point to Aβ overproduction as a cause of the disease [24]; however, it has been suggested that the more prevalent sporadic form of the disease may be caused by impaired Aβ clearance. Aβ peptides produced from the amyloid-β protein precursor processing form oligomers that subsequently form amyloid deposits or plaques in the brain. Aβ oligomers activate inflammatory cells in the brain. When activated, microglia change their shape, migrate close to plaques, and release proinflammatory cytokines and MMPs. Secreted MMPs degrade Aβ and exacerbate inflammation in the brain, leading to death of neurons [25]. There are several proteases such as MMP-2 [26] and MMP-9 [27] playing a role in the mechanism of Aβ clearance. Yin et al. [17] found significant increases in the steady-state levels of Aβ in the brains of MMP-2 and MMP-9 knock-out mice compared with wild-type controls. They studied the roles of MMPs in astrocyte-mediated Aβ degradation. These MMPs also affect the endothelial tight junctions, change the pericyte phenotypes, and promote increased blood-brain barrier (BBB) permeability. Although it is still difficult to explain clearly the potential causes of neurodegenerative diseases, MMPs play a crucial role in the progression of ND such as AD, FTD, and DLB, and their function in these diseases seems to be much more complex than previously thought. Thus, we focused our study on MMPs and TIMPs because they have been shown to play an important role in the pathophysiology of neurodegenerative diseases [13 , 29].

Aβ levels in brain tissue are high in AD patients [30], whereas its plasma [31] and CSF [32] levels have been found to be low. It is thought that the reduction of Aβ₄₂ in CSF samples of AD is due to accumulation of Aβ in plaques and its diffusion to CSF at lower levels. Recent studies have shown strong correlations between low Aβ₄₂ in CSF and high numbers of plaques in the neocortex and hippocampus [32]. Similar to plasma Aβ levels reported in the literature, the present study showed that plasma MMP-2 levels in all the patient groups were significantly decreased compared to the HCs. Also, the MMP-9 level was significantly lower in the FTD patients than in the HCs. The abnormal changes or buildup of the tau protein in neurons lead to altered brain function, resulting in such symptoms as those seen in FTD and AD. Previous studies have demonstrated that tau protein is a substrate of MMP-9 and that this proteinase can be potent inducers of tau aggregation. In addition, some studies showed that MMP-2 and MMP-9 enzymes can contribute to neuronal cell death in FTD patients. Reduced plasma or CSF tau protein levels have been reported in AD and FTD patients [33]. In parallel with these results, the current study revealed that MMP-2 and MMP-9 levels in the FTD patients were lower than those in the healthy individuals. This is also consistent with the finding of several studies that Aβ levels in brain tissue were higher in patients with AD than in HCs, but that CSF and plasma Aβ levels were significantly lower in patients with AD compared to HCs [30 –32]. Plasma MMP-9 measurement can help to distinguish between AD and FTD. In the present study, TIMP-1 levels were lower in the AD and FTD patients than in the HCs. When taken into account together, these results implicate a potential role of MMPs and TIMPs in plasma of patients with ND. Likewise, Mlekusch and Humpel observed a significant decrease in MMP-2 and MMP-3 levels in AD patients and reduced Aβ levels in their CSF [21]. Kook et al. [34] studied the effects of Aβ on BBB integrity and MMP activity in cultured endothelial cells. They found that Aβ induced enhanced permeability, and increased MMP activity and that broad-spectrum MMP inhibition reversed the Aβ-induced BBB disruption.

Moreover, Lim et al. [20] found low MMP-2 activity in plasma in AD subjects in line with the findings of the present study. They determined MMP-2 and MMP-9 geltinolytic activities by using zymography and MMP-2 levels by using ELISA in plasma from patients with AD and mild cognitive impairment (MCI). However, they observed no significant difference in MMP-2 levels between the groups. They explained that low MMP-2 activity could cause higher Aβ levels, which could potentially contribute to Aβ deposits in the brain. Lorenzl et al. [13] also measured the activities of MMP-2, and MMP-9 in AD, MCI, DLB and FTD patients by zymographic analysis. They showed that MMP-9 activity was elevated in AD patients. They also measured MMP-2, MMP-9, MMP-1, TIMP-1, and TIMP-2 levels in patients and control groups. However, unlike the results of the current study, they showed that MMP-9 concentrations were significantly increased in patients with AD as compared to the other groups. There was no significant difference in plasma MMP-2 levels between patients with various types of dementia and HCs. TIMP-2 was significantly reduced in samples of FTD patients. In contrast to the findings reported by Lim et al. and obtained in the present study, Lorenzl et al. [18] showed that MMP-2 expression was not different between diseases and that MMP-9 was elevated in AD patients. MMP-9 levels were significantly elevated in the plasma of AD patients as compared to HCs. The plasma MMP-2 level was unchanged. However, they indicated that MMP-2 possesses alpha -secretase activity and was capable of cleaving the amyloid-β protein precursor [18 , 32]. Also, they suggested that increased circulating MMP-9 levels in AD might contribute to the endothelial pathology of AD patients [13]. Horstmann et al. [19] investigated levels of MMP-2, –3, –9, and -10 in plasma and CSF of AD patients by using zymography. In contrast to Lorenzl et al. [18], they reported that MMP-9 and MMP-10 could not be detected in CSF and that MMP-10 was unchanged in plasma, but that MMP-9 was significantly low. MMP-2 in CSF of AD patients was significantly decreased while plasma MMP-2 levels remained unchanged.

In conclusion, the present study showed significant changes in plasma MMPs and TIMPs levels in the AD, DLB, and FTD patients. It can be suggested that low plasma levels of MMP-2 and MMP-9 may reflect a dysregulated Aβ-MMPs interaction. These proteases might be assumed to have a protective role in AD, FTD, and DLB. On the other hand, MMPs may engage in the disease progression by degrading the brain matrix forming protective shields around the neurons. These diverse roles of metalloproteinases complicate efforts of treatment with broad-spectrum MMP inhibitors or activators. In addition, it can be concluded that MMP-2 and MMP-9 might be useful minimal-invasive biomarkers to provide clinical information about ND, or play a major role in the progression of this disease. However, as the results did not show any differences between the diseases analyzed (AD, DLB, and FTD), MMPs and TIMPs may not be biomarkers used to differentiate between dementias, but as a biomarker might be useful to identify between healthy individuals and individuals with dementia, which is also helpful in the clinic. Besides, the results obtained should be confirmed in a larger sample. The clinical utility of these proteins, as potential surrogate markers for ND remains to be established. More research is needed to understand the multiple roles of these proteases to design specific drugs and devise therapeutic strategies and accurate diagnostic measures for dementia.

Footnotes

ACKNOWLEDGMENTS

This research was supported by a grant from Dokuz Eylul University Scientific Research Projects Coordination Unit.

Authors’ disclosures available online ().

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