|Year : 2016 | Volume
| Issue : 4 | Page : 904-911
A study of changes in ET-1 gene expression in peripheral blood mononuclear cells in patients with diabetic retinopathy
Noha R Bayomy MD 1, Naglaa M Ghanayem1, Eman A Badr1, Rania M Azmy1, Nermeen M Badawi2
1 Medical Biochemistry Department, Menoufia University, Menoufia, Egypt
2 Ophthalmology Department, Menoufia University, Menoufia, Egypt
|Date of Submission||04-Apr-2015|
|Date of Acceptance||15-May-2015|
|Date of Web Publication||21-Mar-2017|
Noha R Bayomy
Medical Biochemistry Department, Menoufia University, Shebin Elkom, Menoufia, 32511
Source of Support: None, Conflict of Interest: None
The objective of this study was to assess the usefulness of relative quantification of endothelin-1 (ET-1) in peripheral blood mononuclear cells in diabetic retinopathy (DR).
DR is one of the most serious complications of diabetes mellitus (DM) that can lead to blindness. Alterations in activity of the ET system are believed to underlie the development of chronic complications of type 2 diabetes mellitus (T2DM).
Patients and methods
This is a clinical study carried out at Medical Biochemistry and Ophthalmology Departments. It included 50 patients divided into three groups: group I (included 14 diabetics without DR), group II (included 26 diabetics with DR), and group III (included 10 normal healthy controls). All studied patients were subjected to full history taking, clinical and ophthalmological examination, and laboratory investigations including fasting blood glucose, glycated hemoglobin (HbA1c), lipid profile, serum creatinine, urinary albumin, and creatinine and ET-1 mRNA level in peripheral blood mononuclear cells using real-time quantitative reverse transcription PCR.
The mRNA level of ET-1 was significantly higher in diabetics with DR than those without retinopathy and was higher in diabetics than in controls. In diabetics with DR, the blood mRNA level of ET-1 was significantly higher in patients with proliferative diabetic retinopathy than in those with nonproliferative diabetic retinopathy. Increased expression of ET-1 mRNA was associated with severity of DR, duration of DM, insulin therapy, and serum creatinine level. Decreased expression of ET-1 mRNA was associated with sulfonylureas treatment. Expression of ET-1 mRNA was not affected by age, sex, BMI, smoking, and control of blood sugar.
Expression of ET-1 is associated with severity of DR in T2DM. Expression of ET-1 is positively correlated with duration of DM, serum creatinine, and urinary albumin. A larger study is needed to confirm the role of ET-1 mRNA determination in the progression of retinal changes in patients with T2DM.
Keywords: diabetic retinopathy, endothelin-1, real-time PCR
|How to cite this article:|
Bayomy NR, Ghanayem NM, Badr EA, Azmy RM, Badawi NM. A study of changes in ET-1 gene expression in peripheral blood mononuclear cells in patients with diabetic retinopathy. Menoufia Med J 2016;29:904-11
|How to cite this URL:|
Bayomy NR, Ghanayem NM, Badr EA, Azmy RM, Badawi NM. A study of changes in ET-1 gene expression in peripheral blood mononuclear cells in patients with diabetic retinopathy. Menoufia Med J [serial online] 2016 [cited 2020 Jun 1];29:904-11. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/904/202494
| Introduction|| |
Diabetics are at an increased risk of developing long-term systemic microvascular and macrovascular complications. These typically develop after 10–20 years, but may be the first symptom especially in those with type 2 diabetes mellitus (T2DM). One of the most distressing complications is diabetic retinopathy (DR), which affects blood vessel formation in the retina of the eye, and it can lead to visual symptoms, reduced vision, and potentially blindness ,.
Ocular blood flow is autoregulated through non-nervous mechanisms, including endothelin-1 (ET-1). However, any imbalance in the ET-1 and other mediators contributes to retinal hemodynamic abnormality in DR. Pericytes of retinal vessels are the most important regulators of vascular tone in retinal capillaries. These cells include receptors of contractor proteins, among which ET-1 is of the utmost importance. In addition to its potent vasoconstricting feature, ET-1 acts as a powerful mitogen of smooth muscles . Alterations in activity of the ET system are believed to underlie the development of structural and functional lesions related to T2DM .
ET-1 is the most abundantly and widely expressed member of the endothelin family of proteins (ET-1, ET-2, and ET-3) . ET-1 is a peptide hormone that is a potent vasoconstrictor with proliferative, profibrotic, and proinfiammatory properties, and it may contribute to many facets of diabetic vascular disease. ET-1 mediates its diverse effects through two distinct G-protein-coupled receptor subtypes: ETA and ETB. ETA receptors, localized mainly on vascular smooth muscle cells of blood vessels, are responsible for the contractile and proliferative response to ET-1. ETB receptors located on endothelial cells mediate vasodilatation through the release of relaxing factors such as nitric oxide and prostacyclin (PGI2). This receptor subtype can also lead to vasoconstriction when the receptors are located on vascular smooth muscle cells in certain vascular beds .
The retinal pigment epithelium may be the source for ET-1 at the posterior pole of the eye. Pathologic increase in the locally secreted ET-1 produces vasoconstriction and affects cellular responses – cell migration and proliferation – that minimize damage to a blood–retinal barrier. In addition, released ET-1 may mediate vascular homeostasis as a mechanism to balance vasodilator influences; however, with excessive secretion, ET-1 may promote prolonged vasoconstriction and induce ischemic episodes in the retina. Such findings reveal the role of an inflammatory response during breakdown of the blood retinal barrier, as seen in proliferative vitreoretinopathy and DR .
The aim of this study is to assess the usefulness of relative quantitative (RQ) determination of ET-1 in peripheral blood mononuclear cells (PBMCs) for monitoring the severity of DR and showing its correlations with clinical and biochemical features in patients with T2DM.
| Patients and Methods|| |
This study was carried out at Medical Biochemistry and Ophthalmology Departments, Faculty of Medicine, Menoufia University. It included 50 patients divided into three groups: group I included 14 diabetic patients without DR (five male and nine female) with a mean age of 50.92 ± 11.86 years; group II included 26 diabetic patients with DR (13 male and 13 female) with a mean age of 57.0 ± 10.72 years – this group is further subdivided into group IIa, which included 13 patients with proliferative diabetic retinopathy (PDR), and group IIb, which included 13 patients with nonproliferative diabetic retinopathy (NPDR); and group III included 10 normal healthy controls (four male and six female) with a mean age of 53.70 ± 12.90 years. The patients were selected from the Ophthalmology Department, Menoufia University Hospitals. A written informed consent was taken from every patient who participated in this study, and the protocol was approved by the ethical committee of Menoufia Faculty of Medicine.
Each participant was subjected to the following: (a) full history taking including age, sex, and duration of diabetes mellitus (DM); (b) through medical examination; (c) ophthalmological examination; and (d) laboratory investigations including fasting blood glucose (FBG), HbA1c, lipid profile, serum creatinine, urinary albumin, and creatinine and ET-1 mRNA level in PBMCs.
Diagnosis of diabetes is based on fasting and 2-h postprandial blood glucose testing according to the American Diabetes Association diagnostic criteria (fasting plasma glucose ≥126 mg/dl or 2-h postprandial ≥200 mg/dl) . DR was determined on the basis of fundus examination by slit-lamp and fundus photography by fluorescence angiography.
Exclusion criteria were as follows: (a) patients with hypertensive retinopathy; (b) patients with glaucoma; (c) patients with optic nerve disorders; (d) patients with systemic hypertension; (e) patients with cardiac disease; (f) patients with diabetic nephropathy with macroalbuminuria; and (g) patients with diabetic neuropathy.
- Blood sample: 10 ml of venous blood was withdrawn from every overnight fasting subject (12 h). The samples were divided into three fractions: (a) 5 ml of blood was transferred into two EDTA tubes as an anticoagulant: one for estimation of blood HbA1c on i-CROMA reader (using kits supplied by Boditech Med Inc., Biggleswade, Bedfordshire, UK)  and the other for mRNA assay; (b) 2 ml of blood was transferred into a fluoride tube for FBG (by enzymatic colorimetric test, using Spinreact kit; Spinreact, Spain) ; and (c) 3 ml of blood was transferred into a plain tube, and the serum obtained was kept frozen at −20°C for assay of lipid profile [by enzymatic colorimetric test for serum total cholesterol (TC) and triglyceride (TG) using human kit supplied by Spinreact ,, whereas serum high-density lipoprotein-cholesterol (HDL-C) was determined by colorimetric method, using Human kit, Karlsruhe, Germany . Low-density lipoprotein-cholesterol (LDL-C) was calculated from TC, HDL-C, TG according to Friedewald et al.  and serum creatinine (using fixed-rate kinetic chemical method) 
- Urine sample: 10 ml of freshly voided morning urine samples were collected in a sterilized container. Each urine sample was subjected to immediate examination by urinary strips for urinary protein and glucose. One milliliter of urine was kept frozen at −20°C for measurement of urinary albumin (using enzyme-linked immunosorbent assay kit supplied by DRG Diagnostics, Springfield, NJ 07081 USA)  and creatinine (by fixed-rate kinetic chemical method) .
Assay of endothelin-1 mRNA
RNA extraction from PBMCs: Total RNA was extracted from specimens using a commercially available kit (QIAamp RNA Blood Mini Kit; Qiagen, USA)according to the manufacturer's instructions .
The purity of RNA was determined by measuring its absorbance at 260 nm (A260). Absorbance readings should be greater than 0.15 to ensure significance. The ratio between the absorbance value at 260 and 280 nm (A260/A280) gives an estimate of RNA purity. A260/A280 ratio greater than 1.6 was accepted. If the purity was lower than 1.5, it required re-extraction .
Total RNA concentration was determined using NanoDrop2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc., USA).
Real-time quantitative reverse transcription PCR assay: The mRNA level of ET-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were evaluated, using real-time RT-PCR and SYBR Green I chemistry (QuantiTect SYBR Green PCR Kit with readymade quantiTect Primer Assay; Qiagen, Applied Biosystems, USA) .
The thermal profile for two-step RT-PCR is as follows:
For reverse transcription, the cycle is incubation for 10 min at 42°C and then for 5 min at 4°C for genomic DNA elimination reaction, followed by incubation for 1 h at 42°C to achieve reverse-transcription reaction, and then incubation for 5 min at 95°C to inactivate Quantiscript reverse transcriptase, followed by incubation for 5 min at 4°C.
For cDNA amplification with SYBR Green I detection of ET-1, the cycle is 95°C for 15 min, 45 cycles at 94°C for 15 s, 55°C for 30 s, and 72°C for 34 s.
For RQ of the results obtained by RT-PCR, the comparative cycle threshold (Ct) method was used . Analysis was performed using Applied Biosystems 7500, software version 2.0.1. GAPDH was included to monitor RT-PCR efficiency for all samples. The point at which the PCR product is first detected above a fixed threshold – termed Ct – was determined for each sample. Each run was completed using melting curve analysis to confirm specificity of the amplification and absence of primer dimers.
Results were collected, tabulated, and statistically analyzed by SPSS (Statistical Package for the Social Science software) (SPSS Inc., Chicago, Illinois, USA) statistical package version 20 on an IBM-compatible computer. Quantitative data were expressed as mean ± SD (X ± SD) or median and range. Qualitative data were expressed as number and percentage [n (%)]. A P value less than 0.05 was considered statistically significant.
| Results|| |
The results of the present study were represented in [Table 1],[Table 2],[Table 3],[Table 4],[Table 5] and [Figure 1], [Figure 2].
|Table 2 Biochemical parameters and relative quantification of endothelin-1 mRNA expression of the three studied groups|
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|Table 3 Variables associated with changes in the endothelin-1 mRNA expression in the studied type 2 diabetes mellitus patients|
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|Table 4 Correlation coefficient between relative quantification of endothelin-1 mRNA and the studied clinical and biochemical parameters in studied groups|
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|Table 5 Correlation coefficient between relative quantification of endothelin-1 mRNA and the studied clinical and biochemical parameters in studied diabetic retinopathy subgroups|
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|Figure 1: Statistical comparison of relative quantification (RQ) of endothelin-1 mRNA between studied groups.|
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|Figure 2: Median and interquartile range (IQR) of relative quantification (RQ) of endothelin-1 mRNA regarding treatment in the diabetic group.|
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In this study, demographic and clinical data characteristics of the three groups were similar including age, sex, family history, smoking, systolic blood pressure, and diastolic blood pressure, whereas there was a significant increase in duration of diabetes in the patients' group with DR when compared with patients' group with T2DM ([Table 1]).
There was a significant statistical difference regarding all biochemical parameters except serum creatinine when patient groups were compared with controls. There was a significant statistical difference regarding HbA1c, HDL-C, TG, TC, and A/C ratio. There was a nonsignificant statistical difference regarding FBG, LDL-C, and serum creatinine when group I was compared with group II, whereas there was a nonsignificant statistical difference among the two subgroups of group II (PDR and NPDR) regarding all parameters except A/C ratio ([Table 2]).
There was a significant difference between the three studied groups regarding mRNA level of ET-1. It was significantly higher in patients with PDR (group IIa) than those with NPDR (group IIb) and higher in diabetic patients with DR (group II) than those without DR (group I). In addition, it was significantly higher in diabetic patients than in controls ([Table 2]).
There was a nonsignificant statistical difference among diabetic groups regarding RQ of ET-1 mRNA in relation to sex, family history, and smoking. There was a significant statistical increase in RQ of ET-1 mRNA in diabetic patients treated with insulin when compared with patients treated with sulfonylurea or diabetic patients treated with metformin, whereas there is no significant difference between diabetic patients treated with sulfonylurea when compared with patients treated with metformin ([Table 3]).
In group I, there was a significant positive correlation between RQ of ET-1 mRNA and each of BMI, LDL-C, TG, and TC, whereas no significant correlation exists regarding the other parameters ([Table 4]).
In group II, there was a significant positive correlation between RQ of ET-1 mRNA and each of duration of diabetes, LDL-C, TG, TC, serum creatinine, and urinary albumin, whereas no significant correlation exists regarding the other parameters ([Table 4]).
In group III, there was a nonsignificant correlation between RQ of ET-1 mRNA and all studied parameters.
In subgroup IIa (PDR), there was a significant positive correlation between RQ of ET-1 mRNA and each of LDL-C, TG, TC, and serum creatinine, whereas a nonsignificant correlation exists regarding the other parameters ([Table 5]).
In subgroup IIb (NPDR), there was a significant positive correlation between RQ of ET-1 mRNA and serum creatinine, whereas a nonsignificant correlation exists regarding the other parameters ([Table 5]).
[Figure 1] shows statistical comparison of RQ of ET-1 mRNA between studied groups.
[Figure 2] shows median and interquartile range of RQ of ET-1 mRNA regarding treatment in the diabetic group.
| Discussion|| |
DR is one of the most serious complications of T2DM, which affects blood vessel formation in the retina of the eye, that can lead to potentially blindness ,. DR results from the destructive effect of hyperglycemia on eye tissues. Thus, understanding the pathogenesis of vascular complications in T2DM is key for eventually reducing these deleterious sequelae .
ET-1 is the most abundantly and widely expressed member of the endothelin family of proteins (ET-1, ET-2, and ET-3). ET-1 is a peptide hormone with diverse biological actions. The ET-1 peptide has an important role in hypertension and other diabetic complications owing to its vasoactive and mitogenic properties . The source of this protein in DR is still unclear. PBMCs, as a source of the ET-1 protein, may significantly influence the molecular changes leading to microangiopathy in DM, independent of the organ studied, including retina .
In this study, the ET-1 mRNA level was significantly higher in diabetic patients (with and without DR) than in the control group, matching the results in many studies ,,.
This study showed that the greatest expression of ET-1 mRNA in the PBMCs was found in PDR patients, somewhat fewer in the NPDR patients, and the least level was in diabetic patients without DR. These results are in agreement with the work of Strzalka-Mrozik et al.  on ET-1 mRNA in the PBMCs and Zhu and Shi  on the plasma ET-1.
Similarly, Roldán-Pallarés et al.  reported that immunoreactive ET-1 levels in plasma and vitreous samples from diabetic patients were significantly higher than those in the control group. The levels for patients with PDR were significantly higher than those for patients with NPDR.
It was suggested that increases in ET-1 expression in PBMCs are associated with development of microangiopathy, which complicates the course of T2DM .
In this study, there was a positive correlation between ET-1 mRNA level and duration of DM in diabetic patients with DR. This is in agreement with the work of Strzalka-Mrozik et al. .
This may be explained by the fact that insulin stimulates synthesis and secretion of ET-1 from endothelial cells leading to increased levels of ET-1 in T2DM. ET-1 levels were found to be normal in T1DM . The relationship between plasma levels of ET-1 and insulin (both endogenous and exogenous) is suggested by the fact that plasma levels of ET-1 are higher in patients with T2DM than in those with T1DM, most likely because of higher insulin levels and associated insulin resistance .
In this study, treatment with sulfonylureas caused a decrease in ET-1 expression. The ET-1 mRNA level may be modulated by this agent, which suggests the potential protective effect of such therapy in DM. The potential pharmaceutical modulation of ET-1 release was previously described by Vingolo et al. who had shown that defibrotide had multisite activity, which reduced or delayed the need for laser treatment in NPDR patients. However, there are no data regarding the molecular mechanism by which the sulfonylureas can cause a decrease in ET-1 expression.
In this study, there was a significant increase in ET-1 mRNA level in diabetic patients treated with insulin than patients treated with sulfonylurea. This was in line with the work ofStrzalka-Mrozik et al.  and Ak et al. .
Prolonged duration of DM results in an increased mRNA level of ET-1, with subsequent increase of hyperglycemic incidents that lead to increased activity of kinase C protein , production of insoluble polymers (so-called advanced glycation end products) , and increased synthesis of diacylglycerol . All of the above result in increased generation of free radicals, which in turn activate proliferative changes and induce hyperglycemia-related injury to tissues, blood vessels, and the endothelium .
Duration of DM is a very important risk factor for development of DR as proved in many studies. Jenchitr et al. in a large case series that included 3049 patients concluded that the longer the duration of diabetes the higher the prevalence of DR. In NPDR, the retinopathy varied from 13.11 to 22.91% in persons having diabetes for less than 10 years and up to 42.86% in those with diabetes for up to 20 years. In the PDR group, the prevalence was 2.15 to 2.42% in persons with diabetes for less than 10 years and up to 10.20% for those with diabetes for up to 20 years.
Ak et al. found that ET-1 levels were elevated in diabetic patients with diabetes duration of more than 10 years when compared with patients less than 10 years.
This study showed a positive correlation between levels of ET-1 mRNA and serum creatinine levels; however, all currently studied patients had normal serum creatinine levels and had negative albumin strip test results. This suggests that the ET-1 mRNA level is a more sensitive marker of early epithelial damage within glomeruli, complicating the course of T2DM. Results of animal studies support this hypothesis ,.
This also supports the fact that diabetic nephropathy, as evidenced by proteinuria and elevated blood–urea nitrogen/creatinine levels, is an excellent predictor of retinopathy; both conditions are caused by DM-related microangiopathies, and the presence and severity of one reflects that of the other. Aggressive treatment of the nephropathy may slow progression of DR and neovascular glaucoma .
Bruno et al.  found that there was a significant positive correlation between plasma ET-1 levels and albumin excretion rate in diabetic patients.
The current study showed a positive correlation between ET-1 mRNA levels and TC and TGs; this is in agreement with the work ofGlowinska et al. .
In patients with DR, there was no correlation between levels of ET-1 mRNA with age, sex, BMI, smoking, fasting blood sugar, and HbA1c. This is consistent with the work of Ak et al. who stated that ET-1 levels were poorly correlated with metabolic control, age, sex, and smoking. Schneider et al.  and Bruno et al. found that there was weak association between plasma ET-1 levels and glycemic control. Strzalka-Mrozik et al.  stated that there was no correlation between plasma levels of ET-1 with smoking and age.
The present study proved a positive correlation between levels of ET-1 mRNA and duration of DM in contrast to a negative correlation with blood glucose levels, supporting the fact that development of DR is associated with duration rather than the severity of DM, as reported in other studies ,.
| Conclusion|| |
Expression of ET-1 in PBMCs is associated with severity of DR in patients with T2DM. Expression of ET-1 is positively correlated with duration of DM, serum creatinine, and urinary albumin. A larger study is needed to confirm the role of ET-1 mRNA determination in PBMCs in the progression of retinal changes in patients with T2DM.
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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], [Table 5]