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July2014 Vol.51 Issue:      3 (Supp.) Table of Contents
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Role of MMP9 and MMP9/TIMP1 Ratio in Assessment of Relapse in Multiple Sclerosis Patients

Samia Ashour1, Magd Zakaria1, Mahmoud Hemeda1, Nermin Helmy2,

Dina Abdelgawad1, Ahmed Elbassiony1, Ayman Abdelhady1

Departments of Neurology1, Clinical Pathology2; Ain Shams University; Egypt



ABSTRACT

Background: Multiple sclerosis (MS) is a progressive inflammatory disease of the central nervous system affecting young adults. The development of process-specific therapies is impossible without use of biological markers that can objectively reflect the targeted immunopathological process, and indicate patients’ responses to therapeutic interventions. One of these biomarkers are indicators of blood brain barrier (BBB) disruption as matrix metalloproteinases (MMPs) and their inhibitors; tissue inhibitor matrix metalloproteinase1 (TIMP). Objective: To determine the relation between the MMP9, MMP9/TIMP1 ratio, and disease activity (relapse) in relapsing remitting Multiple sclerosis. Methods: Thirty-five relapsing remitting multiple sclerosis patients and fifteen healthy volunteers were recruited. Patients submitted to clinical evaluation including Expanded Disability Status Scale (EDSS), laboratory investigations including serum measurement of MMP9, TIMP1 levels and MMP9/TIMP1 ratio, magnetic resonance imaging (MRI), while the healthy volunteers were subjected to measurement of MMP-9 level, TIMP1 level and MMP-9/TIMP1 Ratio. Results: MMP9 level in serum could be used for diagnosis of MS patients in relapse at a level of ≥850 µg/dl, while MMP9/TIMP1 ratio could be used for diagnosis of MS patient in relapse at a ratio of ≥0.102. The mean level of MMP9 was 1529.290µg/dl during relapse, 456.25µg/dl after 3 months, while change in MMP9 was 1093.750µg/dl. The mean level of MMP9/TIMP1 ratio was (0.15) during relapse, significantly elevated than level (0.08) after 3 months, this is consistent with MRI activity. Conclusion: MMP9 and MMP9/TIMP1 ratio may be considered a marker of multiple sclerosis disease activity. [Egypt J Neurol Psychiat Neurosurg.  2014; 51(3): 303-310]

 

Key Words: Matrix Metalloproteinase 9, Tissue inhibitor matrix metalloproteinase1, Multiple sclerosis

Correspondence to Dina Abdelgawad Zamzam, Department of Neurology, Ain Shams University, Egypt. Email: dina.zamzam@hotmail.com

 




 


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]

 

REFERENCES

 

1.          Bagnato F, Durastanti V, Finamore L, Voltane G, Milleflorini E. Beta2 microglobulin and neopterin as markers of disease activity in multiple sclerosis. Neurol Sci.2003;5:301-4.

2.          Calabresi PA. Diagnosis and management of MS. AM Fam Physician. 2004;70:1935-44.

3.          John GR, Shankar SL, Shafit-Zagardo B, Massimi A, Lee SC, Raine CS. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation. Nat Med. 2002; 8:1115-21.

4.          Lassmann H. Mechanisms of demyelination and tissue destruction in multiple sclerosis. Clin Neurol Neurosurg. 2002; 104: 168-71.

5.          Weaver A, Goncalves da Silva A, Nuttall RK, Edwards DR, Shapiro SD, Rivest S, et al. An elevated matrix metalloproteinase in experimental autoimmune encephalomyelitis is protective by affecting Th1/Th2 polarization. FASEB J. 2005; 19:1668-70.

6.          Yong VW, Power C, Forsyth P, Edwards DR. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci.2001; 2:502-11.

7.          Waubant E, Goodkin D, Bostrom A, Bacchetti P, Hietpas J, Lindberg R, et al. IFN beta lowers MMP-9/TIMP-1 ratio, which predicts new enhancing lesions in patients with SPMS.Neurology.2003;60:52-7.

8.          Lee M.A, Palace J, Stabler G, Ford J, Gearing A, Miller K. Serum gelatinase B TIMP-1 and TIMP-2 levels in multiple sclerosis. A longitudinal clinical and MRI study. Brain. 1999;122:191-7.

9.          Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 Revisions to the McDonald criteria. Ann Neurol.2011; 69(2):292-302.

10.       Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology, 1983 Nov 33(11):1444-52.

11.       Naci H, Fleurence R, Birt J, Duhig A. Economic burden of multiple sclerosis: a systemic review of literature. Pharmacoeconomics.2010;28(5):363-79.

12.       Bielekova B, Martin R. Development of biomarkers in multiple sclerosis. Brain. 2004; 127:1463-78.

13.       Rosenberg GA. Matrix metalloproteinases and neuroinflammation in multiple sclerosis. Neuroscientist.2002; 8:586-95.

14.       Leppert D, Ford J, Stabler G, Gryga C, Lienert C, Huber S, et al. Matrix metalloproteinase-9 (gelatinase B) is selectively elevated in CSF during relapses and stable phases of multiple sclerosis. Brain. 1998;121:2327-34.

15.       Waubant E, Goodkin DE, Gee L, Bacchetti P, Sloan R, Stewart T, et al. Serum MMP-9 and TIMP-1 levels are related to MRI activity in relapsing multiple sclerosis. Neurology.1999; 53:1397-1401.

16.       Hosokawa T, Nakajima H, Doi Y, Sugino M, Kimura F, Hanafusa T, et al. Serum MMP-2 and MMP-9 are elevated in neuromyelitis optica. J Neuroimmunol. 2011;236:81-6.

17.       Fainardi E, Castellazzi M, Bellini T, Manfrinato MC, Baldi E, Casetta I, et al. Cerebrospinal fluid and serum levels and intrathecal production of active matrix. metalloproteinase-9 (MMP-9) as markers of disease activity in patients with multiple sclerosis. Mult Scler. 2006; 12(3):294.-301.

18.       Sanli Turk A, Ozturk M, Soysal A, Doventas Y, Baybas S, editors. Assessing the level of MMP-9, TIMP-1 and MMP-9 TIMP-1 ratio in relapsing-remitting multiple sclerosis patients during relapse and remission periods. Proceedings of the 23rdEuropean Neurological SocietyConference; 2012 Jun 8-11; Barcelona, Spain.

19.       Karabudak R, Kurne A, Guc D, Sengelen M, Canpinar H, Kansu E. Effect of interferon beta-1a on serum matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase (TIMP-1) in relapsing remitting multiple sclerosis patients. One year follow-up results. J Neurol. 2004;251:279-83.

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الملخص العربي

 

دور الماتريكس ميتالوبروتيناز ومثبطاته في تقييم ألانتكاسه المرضية لمرضي التصلب المتناثر

               

تقسم الدلالات المبينة للانتكاسة المرضية وللتقدم المرضي في مرض التصلب المتناثر إلي سبعه أقسام منه دلالات اضطراب الحائل المخي الدموي مثل الماتريكس ميتالوبروتيناز ومثبطاته. يحتوي الجهاز العصبي المركز على مستوى ضئيل من عائلة الماتريكس ميتالوبروتينيز، ولكن كثيراً من تلك الأمراض مرض التصلب المتناثر حيث وجد بالأخص أن الماتريكس ميتالوبروتيناز رقم 9 يزداد في مرض التصلب المتناثر.

تعتبر الزيادة فى مستوى الماتريكس ميتالوبروتينيز رقم 9 فى مرض التصلب المتناثر لها دور بالغ الأهمية حيث ثبت أن الزيادة فى مستواه له علاقة بالانتكاسة المرضية ووجود إصابة صبغية بالرنين المغناطيسي وقد زادت هذه الأهمية وهذا الدور بقياس نسبة الماتريكس ميتالوبروتينيز رقم 9 إلى المثبط الخاص بها لمثبط النسيج ماتريكس ميتالوبروتينيز قم 1، حيث وجد أن النسبة بينهما تزداد مع حدوث الانتكاسة المرضية لمريض التصلب المتناثر.

أثبتت الدراسة أن مستوى الماتريكس ميتالوبروتينيز رقم 9 والنسبة بين الماتريكس ميتالوبروتينيز رقم 9 والمثبط النسيجي له رقم 1 كلاهما يرتفع ويزداد أثناء الانتكاسة المرضية وبعد ثلاثة أشهر من الانتكاسة مقارنة بالأشخاص الأصحاء، وأثبتت أيضًا أن مستوى الماتريكس ميتالوبروتينيز رقم 9 والنسبة بين الماتريكس ميتالوبروتينيز رقم 9 والمثبط النسيجي له رقم 1 كلاهما يزداد زيادة واضحة فى الانتكاسة المرضية قياسا بعد ثلاثة أشهر من الانتكاسة.

 



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