|Year : 2018 | Volume
| Issue : 1 | Page : 25-31
Association of cardiovascular complications with circulating levels of tribbles 3 human homolog and matrix metalloproteinases in Indian type 2 diabetic patients, with or without hypertension
Prashant Shirish Ratnaparkhi1, Namrata B Kulkarni2, Meghana U Ganu2, Sanjay G Godbole2, Sudha Shrikant Deo2
1 Sir H. N. Medical Research Society, Sir H. N. Reliance Foundation Hospital and Research Centre; Department of Life Science and Biochemistry, St. Xavier's College, Mumbai, Maharashtra, India
2 Sir H. N. Medical Research Society, Sir H. N. Reliance Foundation Hospital and Research Centre, Mumbai, Maharashtra, India
|Date of Web Publication||2-Apr-2018|
Dr. Sudha Shrikant Deo
Sir H.N. Medical Research Society, Sir H. N. Reliance Foundation Hospital and Research Centre, Raja Ram Mohan Roy Road, Prarthana Samaj, Girgaum, Mumbai - 400 004, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim and Objective: Matrix metalloproteinases (MMPs) and Tribbles 3 (Trb3) human homologue have been reported to induce atherosclerosis. We wanted to evaluate the association of circulating levels of Trb3 human homologue and MMPs (MMP2 and MMP9), with possible cardiovascular complications in Indian type 2 diabetic patients (type 2 diabetes mellitus [T2DM]), with or without hypertension (HT).
Materials and Methods: Serum from 144 individuals, classified as follows: Group A1= (DM + HT); T2DM >5 years + HT (n = 55); Group A2 = DM; T2DM <2 years, (n = 28); Group B1 = HT; (n = 31) and Group B2 = HC; (n = 30) age- and sex-matched healthy controls. Anthropometric measurements, biochemical profiles of sugar and lipids were established using auto analyser. MMP2, MMP9, Trb3, oxidised low-density lipoprotein cholesterol, and proinsulin were measured in the serum using ELISA.
Results: Using Bonferroni correction, we found that MMP2 levels were increased in (DM + HT), when compared to individuals with DM and HT (P = 0.006 and 0.000). HT group had reduced levels of MMP2, as compared to HC, (P = 0.000). The Mann–Whitney U-test for MMP9 revealed that DM group had elevated levels of MMP9 compared to (DM + HT), HT and HC group, (P = 0.011, 0.000, and 0.001). (DM + HT) had elevated levels of MMP9 when compared to HT group, (P = 0.012).). Levels of MMP9 in HT were lower than the HC group, although not significant. Levels of Trb3 were found to be elevated in (DM + HT) when compared to DM, (P = 0.032). The levels of Trb3 were higher in the HT, when compared to HC group, although not statistically significant. Multiple linear regression model for Framingham Risk Score, weighted with post prandial blood sugar yielded R2 = 0.338; F = 7.602 (df = 9), P = 0.000. Trb3 (β = −0.179, P = 0.019); MMP2 (β =0.021, P = 0.787) and MMP9 (β = −0.03, P = 0.684).
Conclusion: Trb3 is a useful marker for evaluating the association of cardiovascular risk in diabetic patients.
Keywords: Cardiovascular complications, matrix metalloproteinases, tribbles 3 homologue, type 2 diabetes
|How to cite this article:|
Ratnaparkhi PS, Kulkarni NB, Ganu MU, Godbole SG, Deo SS. Association of cardiovascular complications with circulating levels of tribbles 3 human homolog and matrix metalloproteinases in Indian type 2 diabetic patients, with or without hypertension. J Diabetol 2018;9:25-31
|How to cite this URL:|
Ratnaparkhi PS, Kulkarni NB, Ganu MU, Godbole SG, Deo SS. Association of cardiovascular complications with circulating levels of tribbles 3 human homolog and matrix metalloproteinases in Indian type 2 diabetic patients, with or without hypertension. J Diabetol [serial online] 2018 [cited 2020 Apr 5];9:25-31. Available from: http://www.journalofdiabetology.org/text.asp?2018/9/1/25/229009
| Introduction|| |
Type 2 diabetes mellitus (T2DM) has established itself as an important killer disease across the globe with number of cases rising from 4.7% in 1980 to 8.5% in 2014. There has been significant increase in cardiovascular complications associated with diabetes. India has the highest diabetic population of 62 million, as reported in the year 2014. Chronic diabetes has assumed the status of a social disease, with the high cost of maintenance for an average family.
T2DM patients have an increased load of reactive oxygen species (ROS) which induces insulin resistance, inflammation and dyslipidaemia., T2DM patients develop microvascular and macrovascular lesions that result in reduced blood flow and hypertension (HT). These result in atherogenic plaque formation and cardiovascular diseases. Changes in cell signalling pathways and gene expression are reported. Oxidative stress thus causes multilevel dysfunction at cellular and molecular level.
ROS causes oxidation of a variety of species, notably low-density lipoprotein (LDL) cholesterol and isoprostanes, which elicit cardiovascular complications in diabetic subjects., Oxidised LDL cholesterol (Ox LDL) mediate activities like activation of monocytes and macrophages and their adhesion to endothelium.,, Increased uptake of Ox LDL by monocytes result in foam cell formation and atherogenic plaque formation.
ROS influences the activity of matrix metalloproteinases (MMPs). MMP2 and MMP9 degrade type IV collagen, and cause atherosclerotic lesions by inflammation and vascular remodelling. The levels of MMP2 and MMP9 in hypertensive patients, when compared to HC group, have been reported to increase,, decrease,, or remain unchanged.,
ROS influences Tribbles 3 (Trib3) human homologue, a cytosolic pseudokinase, which has been reported to disrupt insulin signalling and activating mitogen activated protein kinases (MAPK).,, Thus, Trib3 gene over expression results in insulin resistance and increased cardiovascular risk.,
This cross-sectional study aimed at using the conventional Framingham risk score (FRS), to associate MMP2, MMP9 and Trib3 as risk factors of atherosclerosis and cardiovascular complications, in response to different tenure of diabetes in Indian subjects.
| Materials and Methods|| |
The study was conducted after seeking the approval of the Scientific Advisory Committee of the Sir H. N. Medical Research Society, Sir H. N. Hospital and Research Centre, Mumbai, India. The procedures used in this study were carried according to the guidelines set by the Indian Council of Medical Research for conducting research on human volunteers, in accordance to the Helsinki declaration, 2008.
Selection and description of participants
A total of 144 participants, age >40 years, were selected for this study. After obtaining the informed consent from these subjects, their details were entered in the clinical pro forma. The volunteers were divided into four groups; A1 (DM + HT) = diabetic >5 years with HT (n = 55); A2 (DM) = diabetic <2 years, without HT (n = 28); B1 (HT) = Hypertensive (n = 31), and B2 (HC) = Healthy controls (n = 30). The norms for designation of diabetes were followed according to American Diabetes Association guidelines, (fasting plasma glucose 25 100–125 mg/dl and glycated haemoglobin [HbA1c] =5.7%–6.4%). Joint National Committee 7 guidelines were followed for HT (systolic pressure of 120–139 mm Hg are pre-hypertensive and systolic pressure >140 mm Hg are hypertensive). Healthy controls were recruited as individuals, male or females, with values of FPG, HbA1c, and blood pressure within the normal ranges mentioned above. Oral glucose tolerance test was not conducted for the healthy controls.
Subjects below 40 years, and with major systemic illness, chronic inflammatory diseases and autoimmune diseases were excluded from the study.
Sample size and power
A total of 30 patients were considered in each group for this cross-sectional study and the power analysis was carried out with the data obtained, using online statistical power calculators.
Procurement of serum samples
Venous blood was obtained using standard venepuncture procedures and collected in the plain bulb. Serum thus obtained, was divided into set of five aliquots and stored at −80°C till further assay. Physical parameters such as BMI (weight in Kg divided by height in metres), Waist: Hip ratio (Waist measured at the level of umbilicus with the subjects in mid expiratory position and hip circumference measured at the widest point over the greater trochanter) and blood pressures (systolic and diastolic pressures taken using a mercury sphygmomanometer, when subjects were in a sitting position, with the right forearm placed on the desk, as recommended by the American Society of HT. The first appearance and disappearance of Korotkoff sound were used to define the systolic and diastolic blood pressures.), were measured. Biochemical parameters of the volunteers were estimated using standard ready-to-use kit protocols (blood sugar by glucose oxidase method; Spin React, Spain; triglycerides by GPO-POD method, Innoline-Merck India; total cholesterol by CHOD-POD method, High-density lipoprotein cholesterol by enzyme selective protection method, Agappe Diagnostics, India, and HbA1c by cation exchange resin method, Erba Diagnostics-Germany), and measured in Konelab 20 i, auto analyser (Thermo Electron Corporation, Waltham US).
Ready to use ELISA kits were purchased and used for the estimation of MMP2, MMP9 (R and D systems Minneapolis, US), Trb3 (WUHAN EIAab, China), Oxidised LDL cholesterol and Proinsulin (PI) (My Biosource, USA). All reagents and serum samples were brought to room temperature (freeze thaw cycles were avoided) and diluted as per kit instructions. Standards were prepared and ELISA assays were performed according to the instructions provided by the manufacturers. Serum samples were diluted (if required), to obtain absorbance values in agreement with the standard graph. Intra-assay and inter-assay controls were maintained in all ELISA experiments. The intra-assay CV (%) for MMP2, MMP9, and Trb3 were 2.3, 2.6 and 2.4, respectively and the CV (%) for inter-assay for MMP2, MMP9, and Trb3 were 7.2, 7.5 and 6.8, respectively.
Standard graphs were plotted using Curve Expert 1.4 software and concentrations in the unknown serum were determined using the 4th degree polynomial regression and taking the dilutions used into account. The results were analysed using IBM SPSS software version 21, Chicago IL, US. Mean ± standard deviation were reported for all parameters in the study groups. Normality of the data was confirmed using Kolmogorov–Smirnov test. Distribution of the mean values across the groups was confirmed using ANOVA and the Kruskal–Wallis test. Inter-group comparisons were performed using the independent t-test and Mann–Whitney U-test and significant deviations were identified with 95% confidence interval limits. Correlations between the parameters in question and the groups were established using the Pearson's correlation coefficient or the Kendall's rank correlation coefficient. Multiple linear regression was used to identify the parameters which possess greater prediction value.
| Results|| |
Results of anthropometric study of our subjects are listed in [Table 1]. We found a significant increase in blood pressures (systolic and diastolic), blood sugar (fasting and post-prandial), triglycerides; Total and LDL cholesterol as well as HbA1c among the diabetic groups (P < 0.05).
|Table 1: Anthropometric measurements and biochemical analysis in the study subjects|
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Oxidised LDL cholesterol and PI were found to be greatly increased among the diabetic groups (DM + HT, and DM), when compared to the HC (P < 0.05).
The biomolecules of interest, i.e., Trb3 and MMPs (MMP2 and MMP9) were greatly influenced due to diabetes and HT. When DM + HT and DM patients are compared, Trb3 (P = 0.032) and MMP2 (P = 0.006) were found to be decreased and MMP9 was increased (P = 0.011) in DM patients. A detailed group-wise analysis is represented in [Table 2] and in [Figure 1] and [Figure 2].
|Table 2: Results of different biomarkers in the serum using ELISA assay in the study subjects|
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|Figure 1: Graph representing the distribution of Tribbles 3 human homologue (mean ± 2 standard error)|
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|Figure 2: Graph representing the distribution of matrix metalloproteinases 2 and metalloproteinases 9, (mean ± standard error)|
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[Table 3] represents the data on Kendall's correlation analysis. Of the various parameters used in model prediction, only MMP9 showed no significant correlation with any other variables.
|Table 3: Correlation analysis between parameters used in regression study (values reported as Kendall's tau b)|
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[Table 4] represents two models (after adjusting for BMI, WHR and age), used for multiple linear regression analysis. Trb3, is seen to negatively influence FRS (t = −2.366, P = 0.019), but MMP2 (t = 0.271, P = 0.787), and MMP9 (t = −0.408, P = 0.684) are not statistically significant determinants for cardiovascular risk in diabetic patients.
| Discussions|| |
The aim, in this cross-sectional study, was to validate the use of circulating levels of MMP2, MMP9 and Trib3 as dependable biomarkers for identifying cardiovascular complications in Indian diabetic subjects. The study demonstrates significant variations in the levels of MMP2, MMP9 and Trb3, in the serum of (DM + HT), DM and HT groups, when compared to the HC group.
MMPs have been one of the most studied molecules in the serum and tissue biopsies of subjects with T2DM and in HT subjects for their association with cardiovascular complications. There are multiple results which show positive, negative and neutral associations of MMPs with diabetes. A study in the Caucasian population, as reported by Derosa et al. 2007, the concentration of MMP2 and MMP9 were significantly increased in the diabetic subjects as compared to the controls. We found that MMP2 was significantly lower in HT subjects when compared to the HC group. Also when compared to the HC, the levels of MMP2 were higher in (DM + HT) subjects, and lower in DM subjects, though both not statistically significant, and also reported in a similar studies., This observation for MMP2 could be probably due to the tenure of diabetes and the complications associated with HT, at molecular levels. Expression of MMP2 is known to be influenced by many intermediary molecules. The concentration of MMP2 was significantly higher in (DM + HT) subjects when compared to DM group and HT group. MMP2 appears to be elevated when the conditions of diabetes and HT exist simultaneously in subjects, though there was no statistical significance when compared to HC group. Thus reducing concentration of MMP2 could be an important cue for identifying the progression of T2DM related complications.
MMP9 was elevated in (DM + HT) patients, though not statistically significant, when compared with the HC. Concentration of MMP9 was significantly increased in both the diabetic groups, i.e., (DM + HT) and DM patients, when compared to the HT subjects. We also found that MMP9 levels were lower than the HC subjects, an observation contrary to that reported by Derosa et al., 2006. A striking contrast (with respect to MMP2) was seen as levels of MMP9 were reduced in (DM + HT) patients as compared to DM patients, probably a response to different tenure of diabetes. MMP2 and MMP9 were elevated in the (DM + HT) patients when compared to the HC group, although there was no statistical significance. The reciprocal trend of MMP2 and MMP9, seen in DM group when compared to the HC group could be due to different control mechanisms and molecular circuits for these biomolecules using different pathways, as reported by Abdel Wahab and Mason1996. The changes in the concentration of these MMPs (MMP2 and MMP9) could thus serve as valuable clinical indicators of the onset of cardiovascular complications in diabetic patients, and also possible therapeutic targets for diabetes management, as reported by Haffner et al.
Trib3 human homologue, a pseudokinase, influences the insulin pathway via the Akt and MAPK pathway. Increase in the levels of expression of Trib3 has been reported in T2DM as well as in cardiovascular remodelling., We did not find any significant difference in the overall distribution of Trib3 across the study groups. However, the concentration of Trib3 was significantly higher in (DM + HT) patients when compared with DM patients. This could be due to increased levels of oxidised LDL cholesterol levels in DM + HT patients, as reported similarly by Steverson et al. The lowered concentration of Trb3 in DM group is an interesting feature that we observed. We did not come across such similar observations and hence, it may be of potential importance for further enquiry. When the levels of Trb3 are compared with HT and (DM + HT) groups, we see an increasing trend. It could be possible that molecular circuits associated with HT, mediated by OxLDL could bring about an increase in the levels of Trb3, and hence further damage related to insulin resistance, which is a hallmark of T2DM. Duration-of-diabetes dependent increase in the concentrations of oxidised LDL cholesterol and PI in diabetic subjects connotes increased levels of oxidative stress. Oxidative stress influences molecules such as MMP2, MMP9 and Trib3, which may lead to atherosclerosis and cardiovascular diseases.
The limitation of our study is its cross-sectional nature and the modest sample size. Future studies with a larger recruited sample size would assist in establishing the importance and clinical significance of these molecules in routine diagnosis.
The strength of the study could be attributed to the quantification of MMP2 and MMP9 and Trb3 in the Indian population, with early and late diabetic stages, along with the assessment of the contribution of HT. Correlation of Trb3 in serum to FRS as a risk factor highlights the importance of this pseudokinase in indicating cardiovascular risk in the diabetic population.
| Conclusion|| |
From our study, we conclude that changing levels of MMP2 and MMP9 in early diabetic subjects may serve as indicators of the threat of atherosclerosis. Trb3 along with oxidised LDL cholesterol and systolic blood pressure are independent risk factors of cardiovascular complications in early diabetic subjects. As diabetes is a multi-factorial disease, a spectrum of such indicator biomolecules could add to the accuracy early estimation of the risk of cardio vascular complications associated with diabetes. They could be also assessed for their use as potential drug targets.
To consolidate these biomolecules as dependable biomarkers, a detailed prospective study could be conducted by recruiting diabetic subjects of different age groups, (i.e., from 30 to 40 years, 40–50 years, 50–60 years), along with a rigorous classification of the tenure of diabetes and HT (for estimating the impact of early and long-term stages of diabetes, with or without HT), along with diabetic patients who have undergone various levels of cardiovascular complications. Quantification of circulating levels MMP2, MMP9, and Trb3 in these subjects could be of great clinical significance, for the assessment of ensuing cardiovascular complications due to atherosclerosis. Such a prospective study will also assist in eliminating the current ambiguity associated with the use of these molecules as dependable biomarkers.
The authors wish to thank the Director, Sir H. N. Medical Research Society, the Scientific Advisory Committee of the Sir H. N. Reliance Foundation Hospital and Research Centre for approving our project. We also thank our participants for their time and blood samples..
Financial support and sponsorship
Sir H. N. Medical Research Society, Sir H. N. Reliance Foundation Hospital and Research Center, Mumbai, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]