Graefes Arch Clin Exp Ophthalmol (2013) 251:741–749 DOI 10.1007/s00417-012-2221-3

BASIC SCIENCE

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 & Eleni Papakonstantinou & Asimina Galli & Ioannis Tsinopoulos & Asimina Mataftsi & Spyridon Batzios & Stavros A. Dimitrakos

Received: 12 May 2012 / Revised: 6 November 2012 / Accepted: 20 November 2012 / Published online: 20 December 2012 # Springer-Verlag Berlin Heidelberg 2012

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 C. Symeonidis (*) : I. Tsinopoulos : A. Mataftsi : S. A. Dimitrakos 2nd Department of Ophthalmology, School of Medicine, Papageorgiou General Hospital, Aristotle University of Thessaloniki, Papageorgiou General Hospital, Thessaloniki Ring Road, Thessaloniki 564 03 Macedonia, Greece e-mail: [email protected] E. Papakonstantinou 2nd Department of Pharmacology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Macedonia, Greece A. Galli 4th Department of Paediatrics, School of Medicine, Aristotle University of Thessaloniki, Papageorgiou General Hospital, Thessaloniki, Macedonia, Greece S. Batzios 1st Department of Paediatrics, School of Medicine, Aristotle University of Thessaloniki, Hippokrateion General Hospital, Thessaloniki, Macedonia, Greece

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 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 whether 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

Introduction Diabetes mellitus (DM) has been identified as the cause of a number of ocular complications such as cataract, neovascular 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 recently been reported [5]. In that context, DR gravity has been shown to correlate

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with the severity of dry eye syndrome [6], while cytokine 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, 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) has 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, and keratoconus [19–21]. The role of these molecules as well as the MMP/TIMP imbalance has been studied in various aspects with regard to diabetes mellitus as well, and their implication in several aspects of the disease has already been 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 is the first study investigating the presence of MMP (MMP-2, -9) and TIMP (TIMP-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

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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. 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, Fremont, CA, USA), 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 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

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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 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 computerassisted 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 Concentrations 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: 0.16 ng/ml, MMP-9: 0.156 ng/ml, TIMP-1: 0.08 ng/ml, and TIMP2: 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.

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Statistic analysis Data obtained in this study are presented as mean ± standard error of the mean (SEM). Independent samples t-test (twosided) 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, USA).

Results 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 comigrated 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 00.155; MMP-9, p 0 0.018; TIMP-1, p00.039; TIMP-2, p00.0001, 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). Statistical analysis according to patient gender revealed significantly higher TIMP-2 levels in female patients (p0 0.033), while HBA1C was significantly higher in the female patients included in this study (p00.050). There was no significant difference between male and female patients regarding Schirmer test results (p00.457). Statistic analysis revealed a significant correlation between TIMP-2 and HBA1C values (r00.636, p00.035), while there were no significant correlations with MMP-2, MMP-9 and TIMP-1 (p 00.366, p00.356, p00.315, respectively, Table 2). A significant correlation was also observed between MMP-2 and MMP-9 (r00.552, p00.010). With regard to type 1 diabetes duration, there were no significant correlations with MMP or TIMP levels

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a

Arbitrary units

b

Fig. 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 ± standard error of the mean (SEM) of duplicate determinations for each sample. **p<0.01, ***p<0.001

revealed (MMP-2, p 00.908; MMP-9, p 00.170; TIMP1, p 00.106; TIMP-2, p 00.637). As far MMP/TIMP ratios were concerned, there were also no significant correlations (MMP-2/TIMP-1, p 00.986; MMP-2/TIMP-

Table 1 Patient age, glycosylated hemoglobin (HBA1C), Schirmer test values, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) levels and MMP/ TIMP ratios in tear samples from the patient and control groups

a

in years

b

in %

c

in millimeters

d

statistically significant

2, p 00.989; MMP-9/TIMP-1, p 00.193; MMP-9/TIMP2, p 00.788). 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 0−0.421, p 00.050, Table 2), while there was no significant correlation with patient age (p 0 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 00.442; MMP-9, p 00.840; TIMP-1, p 00.124; TIMP2, p 00.431). Regression analysis of all results for MMPs and TIMPs revealed no significant correlations with HBA1C (MMP-2, p00.387; MMP-9, p00.872; TIMP-1, p00.331; TIMP-2, p 00.191). 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 00.050 and p 00.005 respectively), while MMP-2/TIMP-1 and MMP-2/TIMP-2 ratios were nonsignificantly greater in the patient sample group (p 0 0.685 and p 00.698 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 00.616, p 00.003 and r 00.616, p 00.003 respectively).

Patients (n027) (mean ± standard error)

Controls (n017) (mean ± standard error)

P-value

Agea Disease durationa HBA1Cb Schirmer testingc MMP-2 (ng/ml) MMP-9 (ng/ml) TIMP-1 (ng/ml) TIMP-2 (ng/ml) MMP-2 (ng/mg protein) MMP-9 (ng/mg protein) TIMP-1 (ng/mg protein) TIMP-2 (ng/mg protein)

10.88±0.58 3.37±0.48 8.06±0.23 19.82±1.42 9.46±4.18 37.01±10.95 256.55±17.98 93.08±5.49 18.55±6.9 70.36±21.82 503.03±34.26 182.5±10.65

10.14±0.58 – – 21.50±1.72 2.85±1.21 7.59±4.76 180.90±11.06 66.56±0.79 7.12±3.02 18.97±11.90 450.22±27.65 166.4±1.97

0.377 – – 0.047d 0.155 0.018d 0.039d 0.0001d 0.146 0.022d 0.048d 0.0005d

MMP-2/TIMP-1 MMP-2/TIMP-2 MMP-9/TIMP-1 MMP-9/TIMP-2

0.03±0.01 0.11±0.06 0.14±0.05 0.39±0.17

0.01±0.009 0.03±0.026 0.05±0.031 0.01±0.001

0.685 0.698 0.050d 0.005d

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MMP-2 MMP-2 MMP-9 TIMP-1 TIMP-2 HBA1C Schirmer Age

a

statistically significant

r00.520 p00.032a r0−0.124 p00.687 r0−0.250 p00.486 r0−0.234 p00.366 r00.224 p00.442 r0−0.091 p00.728

MMP-9

TIMP-1

TIMP-2

HBA1C

Schirmer

Age

r00.520 p00.032a

r0−0.124 p00.687 r00.176 p00.566

r0−0.250 p00.486 r00.173 p00.612 r00.200 p00.555

r0−0.234 p00.366 r00.239 p00.356 r00.237 p00.315 r00.636 p00.035a

r00.224 p00.442 r0−0.060 p00.840 r0−0.376 p00.124 r0−0.301 p00.431 r0−0.421 p00.050a

r0−0.091 p00.728 r00.036 p00.892 r00.390 p00.089 r0−0.273 p00.416 r0−0.033 p00.875 r0−0.146 p00.517

r00.176 p00.566 r00.173 p00.612 r00.239 p00.356 r0−0.060 p00.840 r00.036 p00.892

r00.636 p00.035a r0−0.301 p00.431 r0−0.273 p00.416

r0−0.421 p00.050a r0−0.033 p00.875

r0−0.146 p00.517

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 cell groups, macrophages and endothelial cells [28]. In effect, elevated glucose levels significantly amplify 200

75

150

MMP-9 (ng/ml)

100

50

100 R-Square = 0,00

50

25 R-Square = 0,05

0

6

7

8

9

10

0

11

600

6

7

8

9

10

11

10

11

200

500

TMP-1 (ng/ml)

Fig. 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

MMP-2 (ng/ml)

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 HB A1C (MMP-2/TIMP-1, p 00.326; MMP-2/TIMP-2, p 00.352; MMP-9/TIMP-1, p 00.226; MMP-9/TIMP-2, p00.423).

r00.200 p00.555 r00.237 p00.315 r0−0.376 p00.124 r00.390 p00.089

150

R-Square = 0,05

400

TMP-2 (ng/ml)

Table 2 Correlation analysis between matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP), patient age, glycosylated hemoglobin and Schirmer test results

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300 200

R-Square = 0,18

100

50 100 0

6

7

8

9

10

11

0

6

7

8

Glycosylated hemoglobin (HBA1C)

9

746 100

100

75

MMP-2/TMP-2 ratio

MMP-2/TMP-1ratio

Fig. 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 between HBA1C (independent variable) and MMP/TIMP ratios (dependent variables) with a 95 % mean prediction interval

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50 R-Square = 0,04

25

0

6

7

8

9

10

R-Square = 0,04

25

7

8

9

10

11

200

200 150

MMP-9/TMP-2 ratio

MMP-9/TMP-1ratio

50

0

11

250

R-Square = 0,08

100 50 0

75

6

7

8

9

10

11

150 R-Square = 0,08

100

50

0

6

7

8

9

10

11

Glycosylated hemoglobin (HBA1C)

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 as 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]. Protein determination in our samples revealed a significant increase (p<0.05) of the protein content in tears of diabetic patients (0.51±0.04 mg/ml) as compared to controls (0.40±0.05 mg/ml), an observation that has been previously reported [31]. However, expressing the amount of MMP and TIMP as ng per mg of total protein in tear samples did not influence the statistical significance of our data. 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 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. With regard to 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

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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 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 (p00.170) and TIMP-1 (p00.106). 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 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 with regard to 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. 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

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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 with regard to 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 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.

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