INTRODUCTION
Multiple
sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS)
predominantly supported by a T helper1 immune reaction .1Althoµgh
the etiology is predominantly unknown, MS is characterized pathologically by
demyelination and subsequent axonal degeneration.2
The
classification of process-specific biomarkers in multiple sclerosis has to be
based on careful evaluation of all contributing pathophysiological processes.
Biomarkers of pathophysiological mechanisms in multiple sclerosis were
classified into seven categories, one of these categories are biomarkers of
blood brain barrier (BBB) disruption as MMPs and their inhibitors (TIMP).
3,4
The
matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that have
important roles in development and physiology. The normal adult central nervous
system (CNS) contains low levels of most MMP members5,but several
MMPs are
significantly
upregulated in various neurological disorders of the CNS.6 The
elevated level of MMPs in the CNS is the result of increased expression of MMPs
by neural cells, and by leukocytes that infiltrate the CNS upon injury. The up-regulated MMPs have several potentially detrimental
roles, including the promotion of neuroinflammation, disruption of the
blood–brain barrier, demyelination and toxicity to axons and neurons.6
The
elevation of MMP-9 tends to be observed in MS patients with disease activity as
clinical relapse or gadolinium enhancement lesions in brain/spinal magnetic
resonance imaging (MRI).7 These findings correspond with the role of
elevated MMPs in disrupting the integrity of the blood–brain barrier, as
gadolinium enhancement is reflective of blood–brain barrier disruption.8
Aim of work: The
aim of the study is to determine the relation between the MMP9, MMP9/TIMP1
ratio and disease activity (relapse) in relapsing remitting multiple sclerosis.
SUBJECTS
AND METHODS
A
prospective study carried on thirty five relapsing remitting multiple sclerosis
patients and fifteen healthy volunteers, age and sex matched were recruited from Multiple sclerosis unit in Ain Shams
University Hospital, between January 2012 and June 2013. All subjects signed
informed consent.
Inclusion
criteria were; (a) patients aged 15-55
years, (b) relapsing remitting multiple sclerosis (RRMS) diagnosed according to
revised McDonald criteria 20109, and (c) within one month from onset
of relapse.
Exclusion criteria were; (a) patients age less than 15 years old and more than 55
years, (b) autoimmune, (c) chronic inflammatory (d) infectious diseases, (e)
any contraindication to undergo brain or spinal MRI, (f) other types of
multiple sclerosis, (g) patients after one month of relapse, and (h) patients
received steroids before collection of serum sample of MMP9 and TIMP1.
The study was designed to assess multiple sclerosis
patients during relapse and follow up
after 3 months, patients were subjected
to; (a) full medical history, (b) clinical examination with assessment
by Expanded Disability Status Scale (EDSS)10, (c) MRI brain, MRI spine (cervical or dorsal) done with 1.5
tesla Philips® MRI system with contrast(0.10mmol/kg) during relapse according
to patient clinical relapse and repeated after 3 months, (d) laboratory investigations which include
measurement of serum MMP-9 level,TIMP1 level and measurement of MMP9/TIMP1 ratio, and (e) the
healthy volunteers were subjected to measurement of MMP-9 level, TIMP1
level and MMP-9/TIMP1 Ratio.
Statistical Analysis
The
collected data was revised, coded, tabulated using Statistical package for
Social Science (SPSS 15.0.1 for windows; SPSS Inc, Chicago, IL,
2001). Descriptive statistics: mean,
standard deviation (±SD) and range for parametric numerical data, while Median
and Interquartile range (IQR) for non parametric numerical data, frequency and
percentage of non-numerical variables, correlation analysis, using Pearson's
method, to assess the strength of association between two quantitative
variables. The correlation coefficient denoted symbolically "r"
defines the strength and direction of the linear relationship between two
variables, Wilcoxon signed rank test was used assess the statistical
significance of the difference of a non parametric variable measured twice for
the same study group. ROC Curve (receiver operating characteristic) provides a
useful way to evaluate the Sensitivity and specificity for quantitative
Diagnostic measures that categorize cases into one of two groups. Qualitative Data presented in P-value
with P>0.05 being non-significant (NS), P< 0.05being significant (S), and
P<0.01 being highly significant (HS).
RESULTS
We
conducted the study on thirty-five relapsing remitting MS patients; 11 males
(31.4%) and 24 females (68.6%). The mean age of patients was 28±8.2 years. The mean duration of illness was
3.67 years, while mean number of relapses were 3.89 times. In our study 25 patients did not receive
disease modifying drugs (DMDs); receive 1gm methylprednisolone monthly, while
10 patients received DMDs in the form of interferon (3 patients),
Cyclophosphamide (4 patients) and Azathioprine (3patients). The mean baseline
EDSS score was 3.21, mean EDSS score 3 months later was 3.14, while mean change
in EDSS was 0.14±0.91 standard deviation. The mean level of MMP9 was 1529.29 µg/dl
during relapse, 456.25 µg/dl after 3 months, while change in MMP9 was 1093.75.
The mean level of TIMP1 was 10442.86 µg/dl during relapse, 6687.50µg/dl after 3
months, while change in MMP9 was 3556.25 µg/dl . The mean level of MMP9/TIMP1
ratio was 0.15 during relapse, 0.08 after 3 months.
Regarding
healthy control subjects mean baseline levels of MMP9, TIMP1, MMP9/TIMP1 ratio
were 29.67, 4903.33, 0.01 respectively. The mean baseline number of contrast
enhanced, restricted diffusion lesions among study cases were 3.37, 3.51
respectively. Meanwhile the mean number contrast enhanced lesions, restricted
diffusion lesions after 3 months were 1.82, 3.61 respectively. The mean change
in contrast enhanced, restricted diffusion lesion were 1.58, 0.00 respectively.
Receiver
operating characteristic (ROC) curve is used to determine a cut off value for a
clinical test and a graph of sensitivity (y-axis) versus specificity (x-axis).
ROC curve showed that MMP9 level could be used for
diagnosis of MS patients in relapse at a level of ≥850 µg/dl with 71%
sensitivity and 89.3%specificity (P=0.0001). Regarding MMP9/TIMP1, ROC curve
showed that MMP9/TIMP1 ratio could be used for diagnosis of MS patient in
relapse at a ratio of ≥ 0.102 with 64.5% sensitivity and 82.1% specificity
(P=0.001) (Figures 1 and 2).
In the
study group ROC curve showed that MMP9 level could be used for diagnosis of MS
patients not in relapse "follow up cases" at a level of ≥ 56.2 µg/dl with 100% sensitivity and 100%
specificity (P=0.0001) (Table
1).
ROC
curve showed that MMP9/TIMP1 could be used for diagnosis of MS patients
"not in relapse" at a ratio of ≥0.01 with 92.9% sensitivity and 100%
specificity (P=0.0001) (Table 2).
In the
study population patients showed statistically significant linear correlations
between change in number of contrast enhanced lesions with change in serum
level of MMP9 and change in serum level of MMP9/TIMP1 ratio (p=0.025, 0.05
respectively), meaning that decrease number of contrast enhanced lesions
correlate with decrease level of MMP9 and MMP9/TIMP1 ratio (Figures 3 and 4).
Comparing
patients with and without optic neuritis the result showed that patients having
optic neuritis during relapse (10 patients) showed statistically significant
higher baseline level of MMP9 and MMP9/TIMP1 ratio (p=0.014, 0.028
respectively) than patients without optic neuritis.
A
Statistically significant direct linear correlation between EDSS and serum
level of TIMP1 after 3months was found , meaning that decrease in EDSS score
had linear correlation with decrease in serum level of TIMP1 (Table 4).
Figure 1. ROC Curve for
diagnosis of active cases from follow up cases using
Matrix Metalloproteinase 9 level.
|
|
Figure
2. ROC Curve for diagnosis of active cases from follow up cases using
Matrix
Metalloproteinase 9 level /Tissue inhibitor matrix
metalloproteinase1ratio.
Table 1. ROC
Curve for diagnosis of follow up cases from control cases using MMP9 level.
|
Cut-off
|
AUC
|
95%
CI
|
Sensitivity
|
Specificity
|
PPV
|
NPV
|
P
|
MMP9
|
≥56.2
|
1
|
1-1
|
100%
|
100%
|
100%
|
100%
|
0.0001
|
AUC area
under the curve, MMP9 Matrix Metalloproteinase 9, NPV
negative predictive value, PPV positive predictive value
Table 2. ROC
Curve for diagnosis of follow up cases from control cases using MMP9/TIMP1
ratio.
|
Cutoff
|
AUC
|
95%
CI
|
Sensitivity
|
Specificity
|
PPV
|
NPV
|
P
|
MMP9/TIMP1
ratio
|
≥0.01
|
0.993
|
0.977-1.00
|
92.9%
|
100%
|
100%
|
88.2%
|
0.0001
|
AUC area
under the curve, MMP9 Matrix Metalloproteinase 9, NPV
negative predictive value, PPV positive predictive value, T1MP1 Tissue
inhibitor matrix metalloproteinase1
Table 3. Comparison
between cases with and without optic neuritis as regard baseline MMP9, TIMP1,
MMP9/TIMP1 ratio.
S
|
Optic
neuritis
|
P
|
Significance
|
No
|
Yes
|
Mean±SD
|
Median
|
Mean±SD
|
Median
|
Baseline
MMP9
|
1249±1018.74
|
875
|
2230±1154.51
|
1862.5
|
0.0144
|
Significant
|
Baseline
TIMP1
|
10422±3190.94
|
10200
|
10495±2038.58
|
10100
|
0.854
|
Non-significant
|
Baseline
MMP9/TIMP1 Ratio
|
0.13±0.11
|
0.11
|
0.22±0.13
|
0.17
|
0.028
|
Significant
|
MMP9 Matrix
Metalloproteinase 9, T1MP1 Tissue inhibitor matrix metalloproteinase1.
Table 4. Correlations
between EDSS 3 months later and each of MMP9, TIMP1 and MMP9/TIMP1 ratios 3 months
later.
|
MMP9
3
months later
|
TIMP1
3
months later
|
MMP9/TIMP1
Ratio
3
months Later
|
EDSS
Score 3 months
later
|
rho
|
0.309
|
0.389
|
0.211
|
P
|
0.117
|
0.045
|
0.291
|
Significance
|
Non-significant
|
Significant
|
Non-significant
|
EDSS
Expanded Disability Status scale, MMP9 Matrix Metalloproteinase 9, T1MP1 Tissue
inhibitor matrix metalloproteinase1
Figure
3. Correlation between change in Matrix Metalloproteinase 9 and change
in number of contrast-enhanced lesions after 3 months
Figure
4. Correlation between change in 4 Matrix Metalloproteinase 9 / Tissue
inhibitor matrix metalloproteinase1ratio and change in contrast enhanced
lesions after 3 months.
DISCUSSION
MS is a
progressive inflammatory disease of the central nervous system, affecting young
adults, so it has very expensive burden on patient life medically, socially and
economically.11 Biological markers provide a simple and less
expensive monitoring tool in clinical trials and routine patient
managementMMP-912, and its natural inhibitor TIMP-1 play a key role
in the pathogenesis of MS.13 MMP-9 may not only facilitate the
migration of inflammatory cells across membrane equivalents but may also play a
role in tissue damage observed in MS.14 MMP-9/TIMP-1 ratio has been suggested
as a comprehensive way of assessing the proteolytic balance in BBB destruction
in MS patients.15
As there is a wide range
of normal values for MMP9, TIMP1 and MMP9/TIMP1 ratio we measured these levels
in 15 healthy controls. In our study,
MMP9 level could be used for diagnosis of MS patients at a level of≥56.2 with
100% sensitivity and 100% specificity, and diagnosis of patients in relapse at
a level of 850 with 71% sensitivity and 89.3% specificity.
MMP9/TIMP1 ratio could be used for diagnosis of
MS patients at a ratio ≥ 0.01 with 92.9% sensitivity and 100% specificity
compared to healthy controls, and diagnosis of patients in relapse at a ratio ≥
0.0102 with 64.5% sensitivity and 82.1%specificity.
Using these cut off values we found that mean
baseline level during relapse of MMP9, TIMP1, and MMP9/TIMP1 ratio and after 3
months during remission period were higher than healthy controls. These results
were consistent with previous studies. The MS patients showed higher mean
baseline level of MMP9 (during relapse) than the levels after 3 months (in
remission period , these results are consistent with what was reported in
previous studies that MMP9 was elevated in the
serum of patients with relapsing remitting MS, particularly during
the acute phase of the disease.7,8,15-18
Meanwhile, Karabudak and colleagues19
accounted on decrease serum level of MMP-9 in RRMS patients than healthy controls, which
were against current study results. This might be due to small sample size (16
patients), different methodology used and no relapse detected one-year follow up
of the patients in his study.
In addition, the present
study reported that change in number of contrast enhanced lesions has linear
correlation with the change in level of MMP9 over three months meaning that
rate of decrease number of contrast enhanced lesions had linear correlation
with rate of decrease level of MMP9 over 3 months and this point is very
important indicator of disease activity, which was consistent with previous
studies.7,8,15,17
As
regard MMP9/TIMP1 ratio, mean baseline level of MMP9/TIMP1 during relapse was
higher than the level in MS patients during remission (after 3 months). These
results were consistent with what reported in previous studies that the serum
MMP- 9/TIMP-1 ratio was high in RRMS and might be of greater value in
monitoring disease activity.7,15,17,18,20,21
Moreover,
the present study found that change in number of contrast enhanced lesions
correlate with the change in level of MMP9/TIMP1 ratio over three months
meaning that rate of decrease number of contrast enhanced lesions had linear
correlation with rate of decrease level of MMP9/TIMP1 ratio over 3 months and
was important indicator of disease activity. These results were consistent with
results of previous studies that suggested increase serum MMP-9/TIMP-1 ratio as
a predictor of the development of new Gd+ lesion on MRI scans17,7
Karabudak
and colleagues19, showed decrease serum level of MMP-9/TIMP1 ratio
in relapsing remitting MS patients than healthy controls which is against our
study results. This might be due to low MMP9and high level of TIMP1 in his MS
patients than controls, which lead to decrease level of the ratio, small sample
size (16 patients) and due to different methodology used.
Conclusion
The
present study concluded that T1MP1 may be considered a predictor marker of
disease disability and severity during relapse as its level of change
correlates linear with decrease in EDSS score during remission period. This was
not described in previous studies. Also the study reported that patients
experienced optic neuritis relapses showed higher levels of MMP9 and MMP9/T1MP1
ratio.MMP9 and MMP9/TIMP1 ratio might be considered as a marker of disease
activity during relapsing remitting multiple sclerosis patients.
[Disclosure: Authors
report no conflict of interest]
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