Matrix metalloproteinase (MMP-2, -9) and tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric type 1 diabetic patients

Matrix metalloproteinase (MMP-2, -9) and tissue inhibitor

(TIMP-1, -2) activity in tear samples of pediatric type 1

diabetic patients

MMPs in tear samples from type 1 diabetes

Chrysanthos Symeonidis 1, Eleni Papakonstantinou 2, Asimina Galli-

Tsinopoulou3, Ioannis Tsinopoulos 1, Asimina Mataftsi 1, Spyridon

Batzios 4, Stavros A. Dimitrakos 1

1 2nd Department of Ophthalmology, Aristotle University of

Thessaloniki, Thessaloniki, Macedonia, Greece

2 2nd Department of Pharmacology, Aristotle University of

Thessaloniki, Thessaloniki, Macedonia, Greece

3 4th Department of Paediatrics, Papageorgiou General Hospital,

Aristotle University of Thessaloniki, Macedonia, Greece

4 1st Department of Paediatrics, Hippokrateion General Hospital,

Aristotle University of Thessaloniki, Macedonia, Greece

Corresponding author: Chrysanthos Symeonidis, MD, 2nd Department of

Ophthalmology, School of Medicine, Aristotle University of

1

Thessaloniki, Papageorgiou General Hospital, Thessaloniki Ring Road,

564 03 Thessaloniki, Macedonia, Greece

Email address: [email protected]

Telephone no: +306944528127

Fax no.:+302310690417

Abstract

Background: The presence of matrix metalloproteinase (MMP-2, -9) and

tissue inhibitor (TIMP-1, -2) activity in tear samples of pediatric

type 1 diabetes mellitus (DM) patients and potential correlations

with clinical parameters (Schirmer testing, glycosylated hemoglobin-

HBA1C) were investigated.

Methods: Tear samples from the right eyes of 27 type 1 DM patients

and 17 healthy control subjects were included in this study. The MMP

gelatinolytic activity was determined by gelatin zymography analysis

using sodium dodecyl sulphate–polyacrylamide gel electrophoresis

(SDS-PAGE) while MMP and TIMP concentrations (in ng/ml) were

quantified in tears of type 1 diabetic patients and healthy

controls, with the use of enzyme-linked immunosorbent assay (ELISA).

Results: MMP-9, TIMP-1, -2 levels, MMP-9/TIMP-1 and MMP-9/TIMP-2

ratios in the patient group were significantly elevated. There was a

2

significant correlation between TIMP-2 and HBA1C values as well as

between MMP-2 and MMP-9.

Conclusions: Significant correlations between TIMP-2 and HBA1C and

between Schirmer test results and HBA1C were revealed. Significant

increase in tear MMP and TIMP levels in pediatric type 1 diabetic

patients may be suggestive of disease progression and localized

pathologic remodelling. Further studies are required in order to

ascertain weather MMPs and TIMPs could be employed as indicators of

early disease progression.

Keywords: Matrix metalloproteinases, tissue inhibitors of matrix

metalloproteinases, tears, pediatric type 1 diabetic patients,

pathophysiology

3

Introduction

Diabetes mellitus (DM) has been identified as the cause of a

number of ocular complications such as cataract, neovascular

4

glaucoma, diabetic retinopathy (DR) and occasionally significant

refraction fluctuations [1-4]. Even though DR pathogenesis is not

yet thoroughly understood, the implication of the inflammatory

process has been recently reported [5]. In that context, DR gravity

has been shown to correlate with the severity of dry eye syndrome

[6], while cytokines expression has been detected in the tears of

type II diabetic patients, providing additional evidence of the

involvement of inflammatory mechanisms in the DR pathophysiology

[5].

The matrix metalloproteinase (MMPs) family consists of a

heterogeneous group of proteolytic enzymes that are implicated, in

their active form, in extracellular matrix (ECM) homeostasis as well

as tissue repair and remodelling, wound contraction and

neovascularisation [7,8]. In addition, elevated MMP activity, apart

from influencing ECM homeostasis, modulating growth factor

bioavailability and thus regulating cellular proliferation, may be a

prerequisite for angiogenesis and tumorigenesis [9]. Gelatinase A

and B (MMP-2, -9), predominantly digest gelatins (denatured

collagens) but also collagens, fibronectin, elastin, and laminin

[10]. Most MMPs are secreted as zymogens and are activated through

proteolytic modification, whereas their transcription, translation

5

and pro-enzyme activity are regulated by numerous molecules, among

which tissue inhibitors of metalloproteinases (TIMPs) play an

important role [11].

MMPs have been implicated in numerous pathological processes,

both in adults and children, usually related to inflammation and

cell apoptosis [12-15]. In diabetic patients, expression of several

MMPs in tissues (aorta) have been shown to be significantly

increased [16]. On the other hand, TIMP expression has been reported

to be increased [17], unaltered [16] or even decreased [18] in a

number of tissues in diabetic subjects (human plasma, rat aortic and

skin tissue). In tear samples particularly, increased MMP and TIMP

activity has been reported in a broad variety of pathological

entities such as blepharitis, dry eye, ocular allergic disease,

keratitis, keratoconus [19-21]. The role of these molecules as well

as the MMP/TIMP imbalance has been studied in various aspects

concerning diabetes mellitus as well, and their implication in

several aspects of the disease has been already reported [16,22-25].

The aim of this study was the investigation of MMP/TIMP

implication in tear production and composition in the context of

pediatric type 1 diabetes mellitus. To our knowledge, this the first

study investigating the presence of MMP (MMP-2, -9) and TIMP (TIMP-

6

1, -2) activity in tear samples of pediatric type 1 diabetic

patients as well as potential correlations with clinical parameters

(Schirmer testing, glycosylated hemoglobin-HBA1C).

Materials and Methods

Patients

Tear samples from 27 consecutive type 1 diabetic patients who

attended the pediatric diabetes clinic were included in this study.

Mean patient age was 10.88±0.58 years (mean±standard error), ranging

from 6 to 15.

Exclusion criteria were observation of signs of ocular pathology,

medical history of autoimmune disease, use of ophthalmic drops or

systemic medications, or use of contact lenses within 14 days prior

to sample collection.

Samples from 17 healthy subjects were used as controls (mean age:

10.14±0.58, range: 4-13). Informed consent was obtained from all

patients and their parents who were included in the study. Patients

were thoroughly interviewed and their medical history was recorded,

including the approximate time of diabetes diagnosis. The study

conformed fully with the Declaration of Helsinki for biomedical

research on human subjects.

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Sample Collection

In this study, tear samples were collected by manual suction from

the right lateral canthus of each patient with the use of a

micropipette prior to Schirmer testing during regular follow-up

appointments in the outpatient clinics. Following collection, each

sample was placed in a 1 mL Eppendorf tube (Eppendorf, Freemont, CA)

and stored at –70°C until used.

Schirmer testing

During Schirmer testing, paper strips are inserted into the lower

conjuctival sac of each eye for several minutes in order to assess

tear production. A topical anesthetic is administered into the eye

before the filter paper is inserted to avoid tearing due to the

irritation from the paper. The use of anesthetic drops ensures that

only basal tear secretion is being measured. According to standard

Schirmer testing, eyes are closed for 5 minutes. The filter paper is

removed and the amount of moisture is measured in millimeters. A

test result of more than 15 mm of wetting is considered normal while

values 14-9, 8-4 and less than 4 are considered mild, moderate and

severe tear production dysfunction, respectively.

Glycosylated hemoglobin

8

Glycosylated hemoglobin (HBA1C) is formed as a result of

hemoglobin exposure to plasma glucose and is used as a marker for

average blood glucose level measurement over the months prior to the

most recent measurement. In the context of diabetes, the average

amount of plasma glucose increases and the fraction of glycated

hemoglobin increases accordingly. According to the International

Diabetes Federation and the American College of Endocrinology, HbA1c

values below 48 mmol/mol (6.5%) are recommended while according to

the American Diabetes Association, HBA1C values below 53 mmol/mol

(7.0%) are advisable for the majority of patients [26].

Gelatin zymography

The gelatinolytic activity of MMPs was determined by gelatin

zymography analysis using sodium dodecyl sulphate–polyacrylamide gel

electrophoresis (SDS-PAGE) under denaturing but non reducing

conditions. In brief, 5 μl of tears from diabetic patients and

healthy controls were applied on an 8% SDS/PAGE containing 0.1%

gelatin (25 mA, 2 h, at room temperature). Gels were then

equilibrated in 2.5% Triton X−100 buffer for 1 h and subsequently

incubated in 50 mM Tris-HCI, pH 7.3 buffer containing 200 mM NaCI, 5

mM CaCl2 and 0.1% Triton X−100 for 18 h, at 37°C. Bands of enzymatic

activity were visualized by negative staining with standard

9

Coomassie brilliant blue R−250 dye solution. Molecular size of bands

displaying enzymatic activity were estimated by comparison to

purified proMMP−2 (72 kDa), active MMP−2 (64 kDa), proMMP−9 (92 kDa)

and active MMP−9 (78 kDa) (Anawa Trading, Wangen). Prestained

standard protein molecular weight markers used were: myosin (250

kDa), phosphorylase (148 kDa), bovine serum albumin (98 kDa), L-

glutamic dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa),

carbonic anhydrase (36 kDa), myoglobin red (22 kDa), lysozyme (16

kDa), aprotinin (6 kDa) and insulin, B chain (4 kDa) (SeeBlue Plus2

Prestained, Invitrogen, USA). Gelatinolytic activity was quantified

using a computer-assisted image analysis program (1D Image Analysis

Software, Kodak Digital Science v.3.0, Eastman Kodak, Rochester, NY,

USA). All experiments were performed in duplicate.

Immunoassays

Concentration of MMP-2, MMP-9, TIMP-1 and TIMP-2 (ng/ml) were

quantified in tears of diabetic patients and healthy controls, using

an enzyme-linked immunosorbent assay kit (ELISA, R&D Systems Europe,

Abingdon, UK), performed according to manufacturer’s instructions.

The MMP-2 and MMP-9 assays measure total MMP-2 and MMP-9 (proenzymes

and activated forms). Sensitivity of the method employed was: MMP−2:

10

0.16 ng/ml, MMP−9: 0.156 ng/ml, TIMP−1: 0.08 ng/ml and TIMP-2: 0.011

ng/ml.

Protein determination

Protein content was determined in aliquots of tear samples by

the Bradford assay (Bio-Rad, Glattbrugg, Switzerland) using bovine

serum albumin (Sigma-Aldrich Chemie, Steinheim, Germany) as

standard.

Statistic Analysis

Data obtained in this study are presented as mean ±standard error

(SE). Independent samples t-test (two-sided) was used to compare

data between patient and control groups as well as between subgroups

of the patient group. In order to investigate the existence of

potential correlations between MMP levels and clinical parameters

(e.g. glycosylated hemoglobin), the Spearman rank correlation test

was employed. Regression analysis was used to introduce a possible

MMP model that correlated with glycosylated hemoglobin. Differences

were considered statistically significant when p≤ 0.05. Statistical

analysis was performed with the SPSS statistical package (SPSS Inc.,

Chicago, IL).

Results

11

Gelatin zymography analysis revealed that tears from pediatric

type 1 diabetic patients as well as from controls express gelatinase

activity of variable molecular mass (Fig. 1a). The gelatine lysis

band with the lower molecular mass co-migrated as purified MMP-2 (64

kDa) whereas the two gelatine lysis bands of higher molecular mass

correspond to proMMP-9 (92 kDa) and to activated MMP-9 (78 kDa).

Densitometric analysis of gelatine zymograms revealed that proMMP-9

and MMP-9 activity was significantly higher in type 1 diabetic

patients as compared to controls (p<0.01 and p<0.001, respectively)

(Fig. 1b). No significant differences in the activity of MMP-2

between type 1 diabetic patients and controls were evident.

MMP and TIMP values in the patient group were found to be

significantly greater compared to controls with the exception of

MMP-2 (MMP-2, p=0.155; MMP-9, p=0.018; TIMP-1, p=0.039; TIMP-2,

p=0.0001, Table 1). Similar results were also obtained when MMP and

TIMP values were expressed as ng/mg of total protein content in

tears (Table 1). With respect to patient age, there were no

significant correlations observed with MMP levels (Table 2).

Statistic analysis according to patient gender, revealed

significantly higher TIMP-2 levels in female patients (p=0.033),

while HBA1C was significantly higher in the female patients included

12

in this study (p=0.050). There was no significant difference between

male and female patients regarding Schirmer test results (p=0.457).

Statistic analysis revealed a significant correlation between TIMP-2

and HBA1C values (r=0.636, p=0.035), while there were no significant

correlations with MMP-2, MMP-9 and TIMP-1 (p=0.366, p=0.356,

p=0.315, respectively, Table 2). A significant correlation was also

observed between MMP-2 and MMP-9 (r=0.552, p=0.010).

Regarding type 1 diabetes duration, there were no significant

correlations with MMP or TIMP levels revealed (MMP-2, p=0.908; MMP-

9, p=0.170; TIMP-1, p=0.106; TIMP-2, p=0.637). As far MMP/TIMP

ratios were concerned, there were no significant correlations as

well (MMP-2/TIMP-1, p=0.986; MMP-2/TIMP-2, p=0.989; MMP-9/TIMP-1,

p=0.193; MMP-9/TIMP-2, p=0.788).

Regarding Schirmer testing, there was a significant difference

between the patient and control groups (Table 1). There was a

negative significant correlation with HBA1C (r=-0.421, p=0.050, Table

2), while there was no significant correlation with patient age

(p=0.517, Table 2). Furthermore, there were no significant

correlations observed between Schirmer testing and MMPs and TIMPs

included in this study (MMP-2, p=0.442; MMP-9, p=0.840; TIMP-1,

p=0.124; TIMP-2, p=0.431).

13

Regression analysis of all results for MMPs and TIMPs revealed no

significant correlations with HBA1C (MMP-2, p=0.387; MMP-9, p=0.872;

TIMP-1, p=0.331; TIMP-2, p=0.191). Linear regression analysis graphs

between HBA1C and MMPs/TIMPs with a 95% mean prediction interval are

presented in Fig. 2

Regarding MMP/TIMP ratios, MMP-9/TIMP-1 and MMP-9/TIMP-2 ratios

in the patient sample group were significantly greater compared to

the control group (p= 0.050 and p=0.005, respectively) while

MMP-2/TIMP-1 and MMP-2/TIMP-2 ratios were non-significantly greater

in the patient sample group (p= 0.685 and p=0.698, respectively).

Moreover, there were no significant correlations between the

MMP/TIMP ratios investigated and HBA1C. There were significant

correlations between the MMP-2/TIMP-1 and MMP-2/TIMP-2 and the MMP-

9/TIMP-1 and MMP-9/TIMP-2 ratios (r=0.616, p=0.003 and r=0.616,

p=0.003 respectively).

Scatterplots depicting MMP/TIMP ratios in relation to HBA1C values

and linear regression analysis between HBA1C (independent variable)

and MMP/TIMP ratios (dependent variables) with a 95% mean prediction

interval are presented in Fig. 3. Regression analysis of all results

for MMP/TIMP ratios revealed no significant correlations with HBA1C

14

(MMP-2/TIMP-1, p=0.326; MMP-2/TIMP-2, p=0.352; MMP-9/TIMP-1,

p=0.226; MMP-9/TIMP-2, p=0.423).

Discussion

Increased MMP levels have been associated with disease

progression as they have been shown to be involved in the

pathophysiology of several disorders [27]. Recent studies indicate

that elevated glucose levels induce dysregulation of the MMP/TIMP

balance in two key cells, macrophages and endothelial cells [28]. In

effect, elevated glucose levels significantly amplify MMP expression

and activity, resulting in an imbalance between ECM synthesis and

degradation [29].

In this study, we report a significant increase of MMP-9, TIMP-1

and TIMP-2 in tear samples of pediatric type 1 diabetic patients

compared to normal controls. Data were expressed in ng/ml since,

using fluorophotometry, which is a reliable method to measure tear

flow, it has been shown that the amount of tear secretion did not

differ between diabetic and non-diabetic individuals [30].

Furthermore, protein determination in our samples revealed a

significant increase (p<0.05) in the protein content in tears of

diabetic patients (0.51±0.04 mg/ml) as compared to controls

15

(0.40±0.05 mg/ml), an observation that has been previously reported

[31]. Therefore, expressing MMP/TIMP levels per mg of protein may

lead to unreliable conclusions.

Our findings are in accordance with previous studies reporting

elevated plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 in adult

diabetic patients [17]. To our knowledge, this is the first attempt

to comprehensively report values in ng/ml for MMP-2 and -9 as well

as TIMP-1, -2 in tear samples of pediatric type 1 diabetic patients

and to investigate possible correlations of these values with

clinical diabetes mellitus parameters.

Increased MMP-2 and MMP-9 levels in the systemic circulation have

been observed in pediatric patients with type 1 diabetes who

eventually developed microangiopathy over a 5-year period [32].

According to the relevant literature, MMPs are considered markers of

inflammation, and consequently increased MMP concentrations in the

circulation may be a result of the inflammatory process, and not a

potential cause of this process [15]. As elevated MMP-9 levels have

been associated with more acute pathologic circumstances [33], the

observed increase in MMP-9 concentrations may therefore be

indicative of a (relatively acute) active inflammatory process in

the lacrimal system in pediatric patients. Moreover, there was a

16

significant and previously not reported correlation observed between

MMP-2 and MMP-9 levels. This finding can be suggestive of the

complicated interactions between members of the MMP family in the

pathologic environment of type 1 diabetes.

With respect to TIMP levels in the context of type 1 diabetes,

the relevant literature is not conclusive [17,24]. In this study,

TIMP-1 and -2 levels were significantly increased in the tears of

type 1 diabetic patients. This increase in TIMP levels could not

negate the observed MMP-9 increase as both MMP-9/TIMP-1 and

MMP-9/TIMP-2 ratios were found to be significantly elevated.

Overall, the significant increase in tear MMP and TIMP levels in

pediatric type 1 diabetic patients may be indicative of disease

progression as well as localized pathologic remodelling.

Regarding patient gender, the finding that HBA1C values were

higher in female diabetic patients is in agreement with previous

similar reports. The finding that Schirmer test results did not

differ significantly between male and female patients is also in

accordance with the relevant literature [34]. Moreover, in this

study, MMP levels did not differ as a function of gender as

previously reported [24]. In contrast, TIMP-2 levels were

significantly higher in female patients, a novel observation in the

17

relevant literature. This finding, in combination with the

significant correlation between TIMP-2 and HBA1C may be further

indication of the potential role of TIMP-2 as a disease progression

marker, especially in female patients.

Duration of disease has been identified as an independent risk

factor for the development of complications such as diabetic

nephropathy [35]. Moreover, urine MMP-9 excretion positively

correlated with HbA1c and was higher in those type 1 diabetic patients

with a longer duration of disease [36]. The absence of significant

correlations of Schirmer test results with disease duration in this

study may be attributed to relatively short disease duration as dry

eye symptoms in diabetic patients have been associated with longer

disease duration [37]. Regarding MMPs/TIMPs and disease duration,

there was a trend for statistic significance for MMP-9 (p=0.170) and

TIMP-1 (p=0.106). Here, the absence of significant correlations may

be attributed to the relatively low study population size.

Previous studies have reported MMP-2, -9 and TIMP-1 level

correlation with HBA1C in biologic samples (peripheral blood and

urine) of type 1 diabetic patients, while TIMP-2 levels were not

investigated [24,38]. In the present study, TIMP-2 levels correlated

significantly with HBA1C in contrast to MMP-2, MMP-9 and TIMP-1

18

levels, an observation not previously reported. This novel finding

(a potentially localized variation of TIMP expression) could be

indicative of a degree of clinical significance for TIMP-2 in the

context of type 1 diabetes. If this correlation is verified by

further studies, it is conceivable that TIMP-2 could be used as a

marker for disease progression in the lacrimal system as its testing

is less invasive than HBA1C testing.

Lacrimal gland dysfunction in diabetic patients has been

previously reported [30]. In the patients included in this study,

decreased Schirmer test readings were found as compared with the

healthy control group. The Schirmer test is a rough screening tool

for the detection of tear hyposecretion but when performed in a

standardised procedure, it may provide significant information on

the tear secretion [30]. In the present study, there was a

marginally significant difference between patient and control groups

regarding Schirmer test results, a finding in accordance with the

relevant literature [37] but no significant correlation was found

with Schirmer test results. The observed significant inverse

correlation between Schirmer testing and HBA1C may be a more emphatic

indication of clinical significance in diabetes progression

monitoring for Schirmer testing.

19

With regard to inflammatory processes, a serum MMP-9 and

MMP-9/TIMP-1 imbalance has been identified as a potential risk

factor [39]. In previous studies, systemic MMPs have been utilized

for correlation analysis with disease severity parameters [40-42].

In this context, MMP/TIMP ratios appear to be of value in disease

diagnosis as well as prognosis [39]. In this study, MMP-9/TIMP-1 and

MMP-9/TIMP-2 ratios in the patient sample group were significantly

greater compared to the control group in contrast to MMP-2/TIMP-1

and MMP-2/TIMP-2 ratios. Even though TIMPs have been shown not to

have a high specificity for any specific MMP, relevant studies have

reported that TIMP-2 with MMP-2 and TIMP-1 with MMP-9 are

characterized by preferential binding [43]. Therefore, in pathologic

circumstances, the MMP-9/TIMP-1 ratio would be expected to be more

affected compared to the MMP-9/TIMP-2 ratio. However, in the present

study, the mean MMP-9/TIMP-2 ratio was higher in absolute values

compared to the MMP-9/TIMP-1 ratio while there was a notable

difference in the level of significance regarding their comparison

to healthy controls. This finding may render the MMP-9/TIMP-2 ratio

a more sensitive marker of localized inflammatory activity.

In the present study, elevated MMP-9 and TIMP-1, -2 levels in

pediatric type 1 diabetic patients are reported. Correlation

20

analysis revealed a significant correlation between TIMP-2 and HBA1C,

while there was a negative significant correlation between Schirmer

testing with HBA1C. MMP-9/TIMP-1 and MMP-9/TIMP-2 ratios in the

patient sample group were significantly elevated compared to the

control group. This novel finding could be an indication of a degree

of clinical significance for TIMP-2 in the context of type 1

diabetes.

The precise role of MMP/TIMP balance and its relation to

pathologic changes in the lacrimal system during type 1 diabetes

require further investigation. Further studies are required in order

to ascertain whether MMPs and TIMPs could be employed as indicators

of early disease progression.

Declarations

The authors wish to declare that there is no conflict of interest in

this manuscript and that there are no funding sources to declare.

21

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Figure legends

Figure 1. (A) Gelatin zymography of 5 μl of tears from pediatric

patients with type 1 diabetic patients and controls. Bands of

enzymatic activity were visualized by negative staining with

standard Coomassie brilliant blue dye solution. Arrows indicate

migration of commercially available MMP-2, proMMP-9 and MMP-9. (B)

Quantitation of the lysis bands using the computer-assisted image

analysis program EDAS of Kodak. Bars represent the mean ± SEM of

duplicate determinations for each sample. ** p<0.01, *** p<0.001.

Figure 2. HBA1C and MMP/TIMP linear regression analysis in

pediatric type 1 diabetic patients. Linear regression analysis

graphs between HBA1C and MMPs/TIMPs with a 95% mean prediction

interval.

Figure 3. HBA1C and MMP/TIMP ratios linear regression analysis in

pediatric type 1 diabetic patients. Scatterplots depicting MMP/TIMP

ratios in relation to HBA1C values and linear regression analysis

31

between HBA1C (independent variable) and MMP/TIMP ratios (dependent

variables) with a 95% mean prediction interval.

Figure 1

32

M M P-2 (64 kDa)

M M P-9 (78 kDa)proM M P-9 (92 kDa)

CONTROL Type 1 diabetic patients

M M P-2 (64 kDa)

M M P-9 (78 kDa)proM M P-9 (92 kDa)

CONTROL Type 1 diabetic patients

Figure 1. Gelatin zymography of 5 μl of tears from patients with type

1 diabetic patients and controls. Bands of enzymatic activity were

visualized by negative staining with standard Coomassie brilliant blue dye

solution. Arrows indicate migration of purified MMP-2, proMMP-9 and MMP-9.

33

Figure 2

Glycosylated hemoglobin (HBA1C)

34

6 7 8 9 10 110

25

50

75

100

ΜΜΡ-

2 ( ng

/ml)

R-Square = 0,05

6 7 8 9 10 110

50

100

150

200

ΜΜΡ-9 ( ng/ml )

R-Square = 0,00

6 7 8 9 10 110

100

200

300

400

500

600

ΤΙΜΡ-1 ( ng/ml)

R-Square = 0,05

6 7 8 9 10 110

50

100

150

200

ΤΙΜΡ-2 ( ng/ml) R-Square = 0,18

Figure 2. HBA1C and MMP/TIMP linear regression analysis in type 1 diabetic

patients. Linear regression analysis graphs between HBA1C and MMPs/TIMPs with a

95% mean prediction interval.

Figure 3

35

Linear Regression

6 7 8 9 10 110

25

50

75

100

MΜP-

2/ΤΙΜΡ-1 ratio

R-Square = 0,04

Linear Regression

6 7 8 9 10 110

50

100

150

200

250

MMΡ-9/ΤΙΜΡ-1 ratio

R-Square = 0,08

Linear Regression

6 7 8 9 10 110

50

100

150

200

MMP-9/TIMP-2 ratio

R-Square = 0,08

Linear Regression

6 7 8 9 10 110

25

50

75

100

MΜΡ-2/ΤΙΜΡ-2 ratio

R-Square = 0,04

Glycosylated hemoglobin (HBA1C)Figure 3. HBA1C and MMP/TIMP ratios linear regression analysis in type

1 diabetic patients. Scatterplots depicting MMP/TIMP ratios in relation to

HBA1C values and linear regression analysis between HBA1C (independent

variable) and MMP/TIMP ratios (dependent variables) with a 95% mean

prediction interval.

The original publication is available at www.springerlink.com

http://link.springer.com/article/10.1007%2Fs00417-012-2221-3

doi: 10.1007/s00417-012-2221-3

36