Increased activity of MMP-2 in hypertensive obese children is associated with hypoadiponectinemia
Funding agencies: This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP-Brazil).
Disclosure: The authors declared no conflict of interest.
Abstract
Objective
To compare the circulating levels of MMP-2 and TIMP-2 and the MMP-2/TIMP-2 ratio in control, obese, and obese hypertensive children and adolescents and to assess whether hypoadiponectinemia is associated with MMP-2 and TIMP-2 levels and MMP-2/TIMP-2 ratios.
Methods
Studies were carried out with 53 control, 73 obese, and 29 obese hypertensive children and adolescents in this cross-sectional study. Adiponectin and TIMP-2 concentrations were measured by ELISA. MMP-2 concentrations were measured by gelatin zymography. Multiple linear regression analysis was carried out to assess the effects of adiponectin on MMP-2 and TIMP-2 levels and MMP-2/TIMP-2 ratios.
Results
The obese hypertensive group had the lowest adiponectin levels among groups (P < 0.05) while obese subjects had lower adiponectin levels than control subjects (P < 0.05). Obese hypertensive subjects also had higher circulating MMP-2 concentrations than obese subjects (P < 0.05) and had the highest MMP-2/TIMP-2 ratios among groups (P < 0.05). Moreover, obese/hypertensive subjects had the lowest TIMP-2 levels among groups (P < 0.05). A multiple linear regression analysis showed that MMP-2 levels and MMP-2/TIMP-2 ratios are negatively associated with adiponectin levels (P = 0.034 and P = 0.011, respectively) while TIMP-2 levels is positively associated with adiponectin levels (P = 0.013).
Conclusions
Increased activity of MMP-2 (MMP-2/TIMP-2 ratio) and reduced TIMP-2 levels may play an important role in clinical hypertension of childhood obesity. Additionally, hypoadiponectinemia may contribute to increased activity of MMP-2 in obese/hypertensive children and adolescents.
Introduction
Childhood obesity has reached epidemic proportions and is frequently accompanied by insulin resistance, dyslipidemia, type 2 diabetes mellitus, and hypertension (1, 2). In obese subjects, adipose tissue is dysfunctional and releases abnormal amounts of several adipokines, which play important a role in the cardiovascular diseases pathogenesis, particularly hypertension (3, 4).
Adiponectin is an adipokine abundantly expressed in the adipose tissue, which is also found at high concentrations in the plasma (5). However, it production is dysregulated in obese subjects, and lower levels are found in obesity (6-8), atherosclerosis (9, 10), and hypertension, both in adults and children (8, 11, 12). Adiponectin protects against atherosclerosis and hypertension as a result of inhibitory effects on smooth muscle cell proliferation and monocyte adhesion to endothelial cells, in addition to increased nitric oxide (NO) bioavailability (8, 13, 14). As part of the inflammatory/angiogenic response observed in the hypertension, the extracellular matrix is remodeled by proteolytic enzymes known as matrix metalloproteinases (MMPs), among which the MMP-2 and its endogenous inhibitors, the tissue inhibitors (TIMP-2), play a vital role, and a critical equilibrium between MMP-2 and TIMP-2 must exist in order to maintain the integrity of the cardiovascular system (15, 16). Importantly, a study showed that adiponectin was also able to abrogate H2O2-induced MMP-2 activity in cardiomyocytes, and angiotensin II infusion increased the MMP-2/TIMP-2 ratio in left ventricular of adiponectin knockout mice (17).
Increased MMP-2 has been consistently implicated in vascular remodeling associated with hypertension in clinical and experimental hypertension (15, 18-21). Additionally to its involvement in vascular remodeling, evidence indicates that MMP-2 promotes vasoconstriction which could increase blood pressure (22). Indeed, our group found that mean blood pressure correlated positively with circulating MMP-2 levels in children and adolescents (23). Despite the relevance of TIMP-2 to the pathophysiology of cardiovascular diseases, little information is available so far with respect to the possible alterations in TIMP-2 levels as well as MMP-2/TIMP-2 ratios in obese children and adolescents with hypertension (23, 24). Additionally, no previous study has examined the possible correlation between plasma adiponectin and MMP-2, and TIMP-2 concentrations in obese children and adolescents with hypertension. We hypothesized that hypoadiponectinemia frequently observed in obesity may increase MMP-2 activity and decrease TIMP-2 levels, and predispose the development of cardiovascular diseases, especially hypertension, in obese children and adolescents.
In the present study, we aimed at comparing the plasma concentrations of TIMP-2, as well as the plasma MMP-2/TIMP-2 ratios in an obese/hypertensive group with those found in obese and control groups. Importantly, we investigated whether adiponectin levels are associated with MMP-2, TIMP-2, and MMP-2/TIMP-2 ratio levels in children and adolescents.
Methods
Subjects
This study was approved by the Institutional Review Board at the Federal University of Juiz de Fora, Juiz de Fora, Brazil. Parents and children were informed of the nature and purpose of the study. Parents gave their written consent and children gave their verbal consent. The study population consisted of 73 obese, and 29 obese/hypertensive subjects recruited as outpatients from the Endocrinology Ambulatory of the Adolescent and Child Institute at Juiz de Fora and from the Childhood Endocrinology Ambulatory of the IMEPEN Foundation at Juiz de Fora (MG, Brazil). The control group consisted of 53 healthy children and adolescents recruited from the local community and unrelated to the obese patients. All children underwent physical examination. Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer. Body weight was measured with a digital scale to the nearest 0.1 kg. Obesity was defined as a body mass index greater than the 95th percentile matched according to age and sex (25). Systolic (SBP) and diastolic (DBP) blood pressure were measured at least three times after at least 15 min of rest and hypertension was defined as SBP and/or DBP exceeding the 95th percentile (26).
At the time of clinical attendance, venous blood samples were collected after overnight (8-12 h) fasting, immediately centrifuged at 2,000g for 10 min at room temperature, and plasma samples were stored at −70°C until analyzed.
Enzyme immunoassays of adiponectin, TIMP-2, and insulin
Venous blood samples were collected after overnight (8-12 h) fasting, immediately centrifuged at 2,000g for 10 min at room temperature, and plasma samples were stored at −70°C until analyzed. Adiponectin and TIMP-2 concentrations were measured in EDTA-plasma using commercially available enzyme-linked immunosorbent (ELISA) assay kits (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Insulin concentrations were measured in EDTA-plasma using a kit (Genese Produtos Diagnosticos, Sao Paulo, Brazil). The estimate of insulin resistance obtained by homeostasis model assessment index (HOMA IR) was calculated as previously described (27).
SDS-polyacrilamide gel electrophoresis gelatin zymography of MMP-2
Gelatin zymography of MMP-2 from plasma samples was performed as previously described (28, 29). Briefly, plasma samples were subjected to electrophoresis on 7% SDS-polyacrilamide gel electrophoresis (PAGE) copolymerized with gelatin (1%) as the substrate. After electrophoresis was complete, the gel was incubated for 1 h at room temperature in a 2% Triton X-100 solution, and incubated at 37°C for 16 h in Tris–HCl buffer, pH 7.4, containing 10 mmol l−1 CaCl2. The gels were stained with 0.05% Coomassie Brilliant Blue G-250, and then destained with 30% methanol and 10% acetic acid. Gelatinolytic activities were detected as unstained bands against the background of Coomassie blue-stained gelatin. Enzyme activity was assayed by densitometry using ImageJ version 1.42q (Wayne Rasband National Institutes of Health, USA). The MMP-2 was identified as band at 72 kDa, by the relation of log Mr to the relative mobility of Sigma SDS-PAGE LMW marker proteins. As depicted, we always run a standard of bovine fetal serum in a separate lane. This standard is considered as an internal control, and a ratio is calculated between each of the quantified samples and the gel band correspondent to this standard. Therefore, we avoid interferences such as background gel staining by using this procedure, which could interfere with gel band analysis. The intra-assay and inter-assay coefficient of variation were 5.3 and 18.0%, respectively.
Biochemical analyses
Glucose concentrations and lipid parameters (triglycerides and high-density lipoprotein [HDL] cholesterol) were determined in plasma and serum, respectively, with routine enzymatic methods using commercial kits (Labtest Diagnostic, SA, Lagoa Santa, Brazil).
Statistical analysis
The clinical and biochemical characteristics were compared by ANOVA followed by Tukey test (normally distributed variables) or Kruskall–Wallis test followed by Dunn's Multiple Comparison test (not normally distributed variables). The categorical variables were compared between groups by χ2 tests. In addition, we carried out a multiple linear regression analyses to assess the effects the children or adolescent state, and sex on adiponectin, MMP-2, and TIMP-2 levels in the study groups. Again, multiple linear regression was performed to assess the relationship of adiponectin, MMP-2, TIMP-2, and MMP-2/TIMP-2 ratio concentrations with clinical variables that could influence their levels. Sex, age, waist-to-hip ratio, insulin, hypertension, obesity, and adiponectin were included as independent variables in a multiple linear regression model to explain changes in MMP-2, TIMP-2, and MMP-2/TIMP-2 ratio concentrations using JMP® software (SAS Institute, Cary, NC). The results were defined as statistically significant when P < 0.05.
Results
The clinical characteristics, anthropometric parameters, and biochemical data of subjects included in this study are shown in Table 1. As expected, significant differences were found among groups in terms of anthropometric measurements: body mass index and waist-to-hip ratio were higher in the obese and obese/hypertensive groups versus the control group (P < 0.05). Additionally, SBP, DBP, and MBP were higher in obese/hypertensive group when compared with obese, and control groups (P < 0.05). With regards to biochemical parameters, obese/hypertensive groups had higher glycaemia levels, and HOMA index than the control group (P < 0.05). The obese group presented with lower HDL cholesterol, and with higher HOMA index than control group (P < 0.05). No significant differences were found in the triglycerides levels among groups (P > 0.05).
| Control (n = 43) | Obese (n = 73) | Obese/hypertensive (n = 29) | |
|---|---|---|---|
| Gender (F/M) | 21/22 | 40/33 | 13/16 |
| Age (years) | 10.5 ± 2.9 | 10.3 ± 2.2 | 11.1 ± 3.2 |
| Anthropometry | |||
| BMI (kg m−2) | 17.7 ± 3.2 | 27.4 ± 3.7a | 29.6 ± 5.4a |
| Waist-to-hip ratio | 0.84 ± 0.06 | 0.95 ± 0.05a | 0.95 ± 0.04a |
| Traditional MRFs | |||
| SBP (mm Hg) | 103 ± 11 | 112 ± 9a | 139 ± 12ab |
| DBP (mm Hg) | 63 ± 10 | 72 ± 8a | 82 ± 8ab |
| MBP (mm Hg) | 77 ± 9 | 85 ± 9a | 101 ± 8ab |
| HDL cholesterol (mg dl−1) | 44.5 ± 11.9 | 36.8 ± 8.7a | 39.5 ± 12.8 |
| Triglycerides (mg dl−1) | 70.5 ± 27.6 | 88.7 ± 42.9 | 88.3 ± 46.7 |
| Glucose (mg dl−1) | 81.8 ± 10.9 | 87.0 ± 8.5 | 88.2 ± 10.6a |
| HOMA—IR | 1.8 ± 0.8 | 3.2 ± 1.9a | 3.2 ± 1.4a |
- F: female; M: male; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; MBP: media blood pressure; HOMA IR: homeostasis model assessment insulin resistance index.
- Values are the mean ± S.D.
- a P < 0.05 vs. control.
- b P < 0.05 vs. obese.
We found that the obese/hypertensive group had lower adiponectin levels when compared with control and obese groups (P < 0.05; Figure 1), and the obese group had lower adiponectin levels than the control group (P < 0.05; Figure 1). The obese/hypertensive group had higher circulating MMP-2 concentrations than the obese group (P < 0.05; Figure 2A). In addition, obese/hypertensive subjects had the lowest TIMP-2 levels among groups (P < 0.05; Figure 1B) while the obese group had the highest TIMP-2 levels, thus leading to higher MMP-2/TIMP-2 ratios in obese/hypertensive subjects when compared with obese and control groups (P < 0.05; Figure 2C). In addition, we carried out a multiple linear regression analyses to account for possible confounding factors, such as gender and child or adolescent state (Table 2). In accordance with the above results, we found that the control group was associated with higher adiponectin levels (P = 0.008) while the obese/hypertensive group state was associated with lower adiponectin levels (P = 0.02). Again, the obese/hypertensive state was associated with higher MMP-2 levels (P = 0.039), lower TIMP-2 levels (P < 0.001), and higher MMP-2/TIMP-2 ratios (P = 0.003). Importantly, in the present study, gender and child or adolescent status did not affect the adiponectin, MMP-2, TIMP-2, and MMP-2/TIMP-2 levels.
Plasma adiponectin concentrations in the study groups. The bars indicate median. *P < 0.05 vs. control group, and #P < 0.05 vs. obese group.
Plasma concentrations of (A) MMP-2, (B) TIMP-2, and (C) MMP-2/TIMP-2 ratio. The bars indicate median. *P < 0.05 vs. control, and #P < 0.05 vs. obese.
| Source | Adiponectin (µg ml−1) | MMP-2 (A.U) | TIMP-2 (ng ml−1) | MMP-2/TIMP-2 ratio | ||||
|---|---|---|---|---|---|---|---|---|
| Rsquare | RMSE | Rsquare | RMSE | Rsquare | RMSE | Rsquare | RMSE | |
| 0.074 | 0.216 | 0.055 | 0.177 | 0.201 | 0.091 | 0.124 | 0.006 | |
| Model | ||||||||
| Female (vs. male) | 0.015 | 0.408 | 0.010 | 0.515 | <−0.001 | 0.961 | <0.001 | 0.857 |
| Adolescents (vs. children) | −0.023 | 0.222 | 0.007 | 0.656 | −0.004 | 0.613 | 0.001 | 0.284 |
| Group (control) | 0.073 | 0.008a | −0.001 | 0.974 | −0.004 | 0.722 | <0.001 | 0.792 |
| Group (obese) | −0.001 | 0.963 | −0.052 | 0.011a | −0.058 | <0.001a | −0.003 | <0.001a |
| Group (ob/hyp) | −0.072 | 0.020a | 0.052 | 0.039a | −0.054 | <0.001a | 0.003 | 0.003a |
- MMP-2: matrix metalloproteinase 2; TIMP-2: tissue matrix metalloproteinase 2; Rsquare: proportion of the variation in the response around the mean that can be attributed to terms in the model rather than to random error; RMSE: root mean square error.
- a Statistically significant (P < 0.05).
To determine the influence of adiponectin concentrations and clinical variables (gender, age, waist-to-hip ratio, insulin, hypertension, and obesity) on plasma MMP-2, TIMP-2, and MMP-2/TIMP-2 ratio levels, we performed a multiple linear regression analyses with all children enrolled in this study (Table 3). After adjustment for these selected variables, we found that MMP-2 and MMP-2/TIMP-2 ratio concentrations were significantly and negatively associated with adiponectin levels (P = 0.044 and P = 0.019, respectively). On the other hand, TIMP-2 levels were significantly and positively associated with adiponectin levels (P = 0.050). Moreover, we found that MMP-2 and MMP-2/TIMP-2 ratio concentrations were significantly and positively associated with hypertension (P = 0.009 and P < 0.001, respectively) while TIMP-2 levels were significantly and negatively associated (P < 0.001). Additionally, MMP-2 and MMP-2/TIMP-2 ratio concentrations were significantly and negatively associated with obesity (P = 0.03 and P = 0.002, respectively).
| Source | MMP-2 (A.U) | TIMP-2 (ng ml−1) | MMP-2/TIMP-2 ratio | |||
|---|---|---|---|---|---|---|
| Rsquare | RMSE | Rsquare | RMSE | Rsquare | RMSE | |
| 0.103 | 0.172 | 0.229 | 0.094 | 0.177 | 0.006 | |
| Model | B | P | B | P | B | P |
| Female (vs. male) | −0.002 | 0.876 | −0.003 | 0.694 | <0.001 | 0.790 |
| Age (years) | −0.001 | 0.923 | −0.002 | 0.606 | 0.0001 | 0.634 |
| Waist-to-hip ratio | 0.489 | 0.095 | −0.034 | 0.808 | 0.017 | 0.091 |
| Hypertension (y) | 0.051 | 0.009a | −0.055 | <0.001a | 0.003 | <0.001a |
| Obesity (y) | −0.052 | 0.003a | 0.031 | 0.013 | −0.003 | 0.002a |
| Insulin (mUI ml−1) | −0.032 | 0.700 | 0.037 | 0.406 | <−0.001 | 0.753 |
| Adiponectin (ng ml−1) | −0.136 | 0.044a | 0.069 | 0.050a | −0.006 | 0.019a |
- MMP-2: matrix metalloproteinase 2; TIMP-2: tissue matrix metalloproteinase 2; Rsquare: proportion of the variation in the response around the mean that can be attributed to terms in the model rather than to random error; RMSE: root mean square error; B: parameter estimates; y: yes to hypertension or obesity.
- a Statistically significant (P < 0.05).
Discussion
The main findings of the present study are that: (i) obese/hypertensive children and adolescents have elevated MMP-2/TIMP-2 ratios and decreased circulating levels of TIMP-2 compared with control and obese groups, (ii) MMP-2 concentrations and MMP-2/TIMP-2 ratios were negatively associated with adiponectin levels, and (iii) TIMP-2 levels were positively associated with adiponectin levels in children and adolescents.
To our knowledge, this is the first study showing that the MMP-2/TIMP-2 ratios were the highest in the obese/hypertensive group. This result indicates that these children and adolescents have increased net MMP-2 activity since the MMP-2/TIMP-2 ratio is a better indicator of net MMP-2 activity than the circulating MMP-2 levels. Consistent with these results, previous clinical and experimental studies have reported increased expression and activity of MMP-2 in the plasma and vascular tissues of hypertensive adults, and in animal models of hypertension (15, 20, 30, 31). In addition to its involvement in vascular remodeling, growing evidence suggests that increased MMP-2 may promote increased arterial blood pressure through of cleavage of big endothelin-1 in a peptide with greater vasoconstrictor effect (22, 23, 30). Moreover, we found that MMP-2 levels were higher in the obese/hypertensive group when compared with obese group. However, we did not find differences in the MMP-2 levels between obese/hypertensive and control groups. There is much controversy with respect to MMP-2 levels in hypertension, and some studies showed lower (32) whereas other studies showed similar levels in hypertensive patients compared with normotensive controls (33, 34). These differences may be attributable to different criteria used in patient selection, variable severity of hypertension, use of drugs, and pre-analytical issues. Taken together, these findings suggest that imbalanced MMP-2/TIMP-2 ratio plays a key role in the vascular remodeling associated with hypertension in obese children and adolescents, and MMP-2/TIMP-2 ratio could be a better biomarker of hypertension than MMP-2 levels in obese children and adolescents. Importantly, MMP-2 and TIMP-2 were measured by different methods, zimography, and ELISA, respectively. Although the zimography is a semi-quantitative, this method is sensitive, specific, and well accepted as a standard technique at literature.
MMPs are inhibited by 1:1 stoichiometric interaction with their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs) (35). The TIMPs are a family of 4 members (TIMP-1 to TIMP-4) with different biochemical properties and MMP affinity (35). Inhibition with high affinity binding site has been described for TIMP-2:MMP-2 (36). In this regard, the regulation of MMP-2 activity is very complex, TIMP-2 plays dual role in regulating of the processing of pro-MMP-2, so that low TIMP-2 levels are required to cleave pro-MMP-2 in active MMP-2 and high TIMP-2 levels inhibit MMP-2 (36). In this context, under pathological conditions, changes of TIMP levels are considered to be important because they directly affect MMP activity (35). Few studies have investigated TIMP-2 levels on plasma in hypertension (30, 33, 34, 37), and none of those studies has investigated whether circulating TIMP-2 levels are altered in obese children and adolescents with hypertension. The present study is the first to report that obese/hypertensive children and adolescents have decreased circulating levels of TIMP-2 compared with control and obese groups. Giving support to this result, an experimental study showed decreased TIMP-2 mRNA levels in aortas from hypertensive rats (15). Moreover, clinical study showed that TIMP-2 expression, in both (subcutaneous and visceral cells), was reduced in hypertensive patients (38). As already shown previously by our laboratory, unlike what was observed in hypertensive obese children and adolescents, obese subjects had higher TIMP-2 levels. Although we have no precise explanation (mechanism), we suggest that TIMP-2 is involved in the development the adipose tissue in the obesity through biological activities that are independent of metalloproteinases such as the effects on cell growth and differentiation (39). Thus, TIMP-2 levels were higher in the obese group. However, decreased levels of TIMP-2 in obese/hypertensive were found in this study. Hypertension has been identified as possible regulator of TIMP-2 since decreased TIMP-2 has been implicated in vascular remodeling associated with hypertension (15). There is no mechanistic explanation for this finding, and further research is necessary to explain these results. The molecular mechanisms controlling TIMP-2 expression in obesity and hypertension may interact and downregulate its expression. The complexity of such interactions requires further investigation. In another study with adults, no significant difference on TIMP-2 levels was observed between the obese and control groups (40). Further studies are needed to confirm these findings and to explore whether childhood obesity differs from obesity in adults in terms of TIMP-2 levels.
The adipose tissue (AT) is a major organ endocrine, which releases soluble, very active factors, the adipokines, which act locally within the AT, but can also reach distant organs through the systemic circulation and exert a variety of role including the regulation of vascular function (3). Importantly, imbalanced adipokine production has been proposed to play a role in the pathogenesis of insulin resistance and cardiovascular diseases (3, 4). In this context, adiponectin exerts protective effects and its circulating levels are decreased in the obesity and comorbidities such as type 2 diabetes, hypertension, and atherosclerosis (6, 8-12). The protective effects of adiponectin on the vascular function are due to their anti-inflammatory functions and also its ability to stimulate the production of NO, and consequently, increase NO bioavailability (8, 13, 14). In line with these findings, we found that obese/hypertensive children and adolescents have lower adiponectin levels when compared with obese and control group, thus suggesting that these children are exposed at increased cardiovascular risk.
In addition to its ability to stimulate NO production, a recent study has shown that adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling in part by reducing MMP-2 activity (17). Moreover, a higher MMP-2/TIMP-2 ratio was shown in adiponectin knock out mice (17). In this regard, we carried out a multiple linear regression to assess whether adiponectin concentrations could influence MMP-2 and TIMP-2 levels and MMP-2/TIMP-2 ratios. Interestingly, our results showed that MMP-2 and MMP-2/TIMP-2 ratio concentrations were negatively associated with adiponectin levels. On the other hand, TIMP-2 levels were positively associated with adiponectin levels. Importantly, we found that increased MMP-2 and MMP-2/TIMP-2 ratio concentrations are associated with hypertension, while TIMP-2 levels are negatively associated with hypertension. These findings suggest that hypoadiponectinemia may contribute to the increased MMP-2 activity and to decreased TIMP-2 levels and therefore predispose to the development of cardiovascular diseases, especially hypertension, in obese children and adolescents.
Although our findings should be confirmed in a larger prospective study, they suggest that changes in the MMP-2/TIMP-2 balance may play an important role in the structural, functional, and clinical manifestations of hypertension of childhood obesity. Additionally, hypoadiponectinemia may contribute to imbalanced MMP-2/TIMP-2 ratios in obese/hypertensive children and adolescents. Further studies are necessary to understand the mechanisms by which adiponectin regulates MMP-2 and TIMP-2.
Acknowledgments
We thank all the Instituto Mineiro de Ensino e Pesquisa em Nefrologia (Imepen) research team members. In addition, we are grateful for study participants for their involvement.
