Correlation of mRNA expression of major MMPs and ADAMTSs to Pfirrmann MRI grades in patients undergoinglumbar spinal fusion

Sanjay S Aripaka1,3, R Bech-Azeddine2,3, LM Jørgensen1,2,3, SA Chughtai2, JH Mikkelsen,1,3,4

1Neurobiology Research Unit, University Hospital Copenhagen, Rigshospitalet, Copenhagen, Denmark2Copenhagen Spine Research Unit, Center for Rheumatology and Spine Diseases, Rigshospitalet, Glostrup, Denmark3Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark4Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

1

Contact: sunnysagar02@gmail.com

2

Relevance

• Low back pain (LBP) represents the top medical expense in the western societies,

and is a leading cause of disability worldwide1.

• LBP is age-related, and several lines of evidence show that the LBP is associated

with degenerative disc disease (DDD)2 even the etiology of DDD is not fully

understood.

• DDD is histologically characterized by cell number reduction, extracellular matrix

(ECM) loss and inflammation3,4

• LBP is associated with massive

degeneration of the intervertebral

disc (IVD)2 as visualized on MRI,

and lumbar spinal fusion of the

degenerated level may alleviate pain

for the patient.

Yi Feng et.al 20163

• Our hypothesis is that upregulation of MMP and ADAMTS expression implicated

in disc ECM destruction is modulated by inflammation and upregulated under in

chronic LBP

AIM:

The present study was aimed to gain better insight into the role of different MMP and

ADAMTS expression in IVD’s from patients and also study the relevance to

inflammation, We therefore determined levels of MMP transcript subtypes (1, 2, 3, 10,

and 13) and ADAMTS subtypes s (-1, -4, and -5) and the association to lumbar disc

degeneration in patients with chronic lumbar pain, as measured by the Pfirrmann

magnetic resonance imaging classification system (grade I-V) of lumbar DDD

4

Patients with prior lumbar surgery, other rheumatoid diseases, ongoing

treatments with steroids are excluded from study

Participants

5

RT-qPCR

MMP’s and ADAMTS’s mRNA expression was measured using RT-qPCR in a Light

Cycler 480 Real-Time PCR System using SYBR Green I Master mix(Roche

Diagnostics, IN). Expression of all the cytokines were measured relative to the

geometric mean of 3 housekeeping genes(SDHA, LDHA and β-Actin) 6

Findings

7

Expression of specific MMPs and ADAMTSs correlate strongly with Pfirrmann MRI

grades

2 3 4 5

0

2

4

6

MMP1

Grading

Fo

ld c

han

ge

2 3 4 5

0

2

4

6

MMP3

Grading

Fo

ld c

han

ge

2 3 4 5

0

2

4

6

MMP10

Grading

Fo

ld c

han

ge

2 3 4 5

0.0

1.5

3.0

4.5

MMP13

Grading

Fo

ld c

han

ge

2 3 4 5

0.0

1.5

3.0

4.5

ADAMTS 1

Grading

Fo

ld c

han

ge

2 3 4 5

0.0

0.5

1.0

1.5

2.0

ADAMTS 5

Grading

Fo

ld c

han

ge

2 3 4 5

0.0

0.2

0.4

0.6

0.8

TNF-

Grading

Fo

ld c

han

ge

2 3 4 5

0.00

0.05

0.10

0.15

0.20

0.25

IL-6

Grading

Fo

ld c

han

ge

r=0.67 p<0.001 r=0.61 p<0.001r=0.67 p<0.001 r=0.48 p=0.004

r=0.667p<0.001

r=0.53 p<0.001

r=0.37 p<0.03 r=0.35 p=0.04

a cb d

e hf g

A value of <0.05 is considered statistically significant for all comparisons. r= Spearmen's correlation coefficient.

A value of <0.05 is considered statistically significant for all comparisons. r= Spearmen's correlation coefficient.

8

Correlation between mRNA expression of MMPs and ADAMTSs

MMP1 1.00 0.79 0.94 0.88 0.87 0.85 0.58 0.58 0.25

MMP2 0.73 1.00 0.71 0.59 0.59 0.63 0.85 0.63 0.18

MMP3 0.89 0.69 1.00 0.89 0.91 0.87 0.48 0.51 0.21

MMP10 0.85 0.52 0.83 1.00 0.87 0.89 0.37 0.63 0.38

MMP13 0.77 0.54 0.82 0.88 1.00 0.82 0.31 0.38 0.27

ADAMTS 1 0.85 0.60 0.84 0.91 0.81 1.00 0.39 0.61 0.41

ADAMTS 4 0.57 0.83 0.53 0.45 0.47 0.45 1.00 0.63 -0.04

ADAMTS 5 0.64 0.54 0.58 0.69 0.49 0.68 0.63 1.00 0.27

MM

P1

MM

P2

MM

P3

MM

P10

MM

P13

AD

AM

TS

1

AD

AM

TS

4

AD

AM

TS

5

a

MMP 1 6.1E-08 4E-15 4.7E-11 9.4E-11 7.1E-10 0.00045 0.00045 0.16692

MMP 2 3.5E-06 5.3E-06 0.00039 0.00038 0.00012 8.3E-10 0.00011 0.33637

MMP 3 2.6E-11 0.00189 1.6E-11 9.5E-13 1.2E-10 0.00496 0.00283 0.25511

MMP 10 2.1E-09 0.0193 2.9E-09 1.1E-10 7E-12 0.03733 0.00013 0.03179

MMP 13 3.9E-07 0.00741 7.6E-09 2.6E-08 9.1E-09 0.08796 0.03244 0.1367

ADAMTS 1 3.5E-09 0.02114 1.1E-11 5E-09 2.3E-06 0.02575 0.0002 0.01969

ADAMTS 4 0.00074 6.4E-07 0.15603 0.06995 0.04982 0.17382 0.00011 0.84377

ADAMTS 5 0.0001 0.002 0.001 1E-05 0.00402 2.4E-05 9.5E-05 0.13126

MM

P 1

MM

P 2

MM

P 3

MM

P 1

0

MM

P 1

3

AD

AM

TS

1

AD

AM

TS

4

AD

AM

TS

5

b

A value of <0.05 is considered statistically significant for all comparisons. r= Spearmen's correlation coefficient.

9

Correlation of transcript levels of MMPs and ADAMTSs with pro-inflammatory cytokines.

0.0 0.2 0.4 0.6 0.8

0

1

2

3

4

TNF- Fold change

MM

P 2

Fo

ld c

han

ge

MMP 2

0.0 0.2 0.4 0.6 0.8

0.0

0.5

1.0

1.5

2.0

ADAMTS 5

TNF- Fold change

AD

AM

TS

5 F

old

ch

an

ge

0.0 0.2 0.4 0.6 0.8

0

1

2

3

4

ADAMTS 4

TNF- Fold change

AD

AM

TS

4 F

old

ch

an

ger=0.36 p=0.03 r=0.46 p=0.007 r=0.37 p=0.03

a b c

A value of <0.05 is considered statistically significant for all comparisons. r= Spearmen's correlation coefficient.. 10

0.00 0.05 0.10 0.15 0.20 0.25

0

2

4

6

MMP 1

IL-6 Fold changeM

MP

1 F

old

ch

an

ge

0.00 0.05 0.10 0.15 0.20 0.25

0

2

4

6

8

MMP 10

IL-6 Fold change

MM

P 1

0 F

old

ch

an

ge

0.00 0.05 0.10 0.15 0.20 0.25

0

1

2

3

4

5

MMP 13

IL-6 Fold change

MM

P 1

3 F

old

ch

an

ge

0.00 0.05 0.10 0.15 0.20 0.25

0

1

2

3

4

5

ADAMTS 1

IL-6 Fold change

AD

AM

TS

1 F

old

ch

an

ge

0.00 0.05 0.10 0.15 0.20 0.25

0

1

2

3

4

ADAMTS 4

IL-6 Fold change

AD

AM

TS

4 F

old

ch

an

ge

0.00 0.05 0.10 0.15 0.20 0.25

0.0

0.5

1.0

1.5

2.0

ADAMTS 5

IL-6 Fold change

AD

AM

TS

5 F

old

ch

an

ge

r=0.36 p=0.043 r=0.3

7p=0.035

r=0.34 p=0.05

r=0.35 p=0.040 r=0.42 p=0.01 r=0.39 p=0.025

a cb

d e f

Correlation of transcript levels of MMPs and ADAMTSs with pro-inflammatory cytokines.

• Our results showed an imbalance between catabolism and anabolism of IVD matrix

components

• We showed that most MMPs and ADAMTSs indeed express in NP, and their

expression levels increase with degeneration grade, which suggests the role of these

proteases in ECM breakdown and progression of degeneration.

• Our results also propose that stimuli coming from release of inflammatory cytokines

participate in the regulation of MMPs and ADAMTSs and dysregulation of their activity

can increase the disc's structural loss

Take home message

Conclusion :Our results contribute to the understanding of the role of different MMPs and other

aggrecanases in disc degeneration etiology with potential to integrate novel biomarkers in

diagnosis, therapy effects, and the prognosis for patients with DDD.

References:1. Vos T, Allen C, Arora M, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and

injuries, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. The Lancet. 2016;388(10053):1545-1602.

2. Hadjipavlou AG, Tzermiadianos MN, Bogduk N, Zindrick MR. The pathophysiology of disc degeneration. The Journal of Bone and Joint

Surgery British volume. 2008;90-B(10):1261-1270.

3. Risbud MV, Shapiro IM. Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol.

2014;10(1):44-56.

4. Weber KT, Jacobsen TD, Maidhof R, et al. Developments in intervertebral disc disease research: pathophysiology, mechanobiology,

and therapeutics. Curr Rev Musculoskelet Med. 2015;8(1):18-31. 11

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None of the authors has any potential conflict of interest