|Year : 2017 | Volume
| Issue : 2 | Page : 71-76
Evaluation of erythropoietin level in vitreous and serum with detection of erythropoietin gene polymorphism in Egyptian patients with proliferative diabetic retinopathy
Tarek Abdou Moustafa1, Mona Mohamad Aly1, Zakia Abu-Zahab2, Radwa S Shahien2
1 Department of Ophthalmology, Faculty of Medicine, Al-Azhar University, Nasr City 11754, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Nasr City 11754, Cairo, Egypt
|Date of Web Publication||17-Nov-2017|
Mona Mohamad Aly
Department of Ophthalmology, Faculty of Medicine, Al-Azhar University, Nasr City 11754, Cairo
Source of Support: None, Conflict of Interest: None
Purpose: To measure erythropoietin (EPO) level in vitreous and serum of patients with proliferative diabetic retinopathy (PDR) and to detect EPO single nucleotide polymorphism (SNP) genotyping expression in them. Materials and Methods: Concentrations of EPO in vitreous and serum of twenty diabetic patients with PDR undergoing vitrectomy (PDR group) were measured by chemiluminescent immunoassay and compared with those in nondiabetic (ND) patients undergoing vitrectomy for recent retinal detachment (ND group). Genotyping of EPO gene SNP (rs551238) was performed using real-time polymerase chain reaction technique. Results: There were a significant increase in serum EPO level in PDR group than in ND group and a highly significant increase in vitreous EPO level in PDR group than in ND group. Within the PDR group, vitreous EPO level was significantly higher than its serum level; there was up to 6-fold increase in the EPO vitreous level than its serum level. EPO gene SNP rs551238 was significantly associated with PDR in Egyptian patients. Conclusion: The results of the current study suggest the involvement of EPO in the pathogenesis of PDR. Hence, augmenting vascular endothelial growth factor inhibition with the use of EPO inhibitors may provide a better outcome in PDR treatment. The detection of EPO gene SNP rs551238 in Egyptian patients with PDR may help in the identification of the genetic predisposition to diabetic retinopathy in Egyptians and so may help in prevention and early detection of susceptible candidates to PDR.
Keywords: Erythropoietin, proliferative diabetic retinopathy, single nucleotide polymorphism
|How to cite this article:|
Moustafa TA, Aly MM, Abu-Zahab Z, Shahien RS. Evaluation of erythropoietin level in vitreous and serum with detection of erythropoietin gene polymorphism in Egyptian patients with proliferative diabetic retinopathy. Egypt Retina J 2017;4:71-6
|How to cite this URL:|
Moustafa TA, Aly MM, Abu-Zahab Z, Shahien RS. Evaluation of erythropoietin level in vitreous and serum with detection of erythropoietin gene polymorphism in Egyptian patients with proliferative diabetic retinopathy. Egypt Retina J [serial online] 2017 [cited 2020 Apr 5];4:71-6. Available from: http://www.egyptretinaj.com/text.asp?2017/4/2/71/218586
| Introduction|| |
Diabetes mellitus (DM) is a leading cause of blindness worldwide. It is associated with microvascular complications, such as diabetic retinopathy (DR). The pathogenesis of DR is complex with multifactorial biochemical causes influenced by genetic and environmental factors. The retina is the most metabolically active tissue in the human body and therefore is highly sensitive to reductions in oxygen tension. Damage to retinal blood vessels by long-standing hyperglycemia leads to retinal hypoxia which stimulates the DNA-binding activity of hypoxia-inducible factor (HIF). HIF is the primary hypoxic signaling protein in cells for regulating angiogenesis and is able to induce the transcription of >70 genes, including vascular endothelial growth factor (VEGF)., Many vasoactive factors stimulated by hyperglycemia or oxidative stress have been identified. In normal ocular tissues, angiogenic homeostasis is controlled by the balance between various angiogenic stimulators, such as VEGF and angiogenic inhibitors, such as pigment epithelium-derived factor. The onset of proliferative diabetic retinopathy (PDR) is thought to occur primarily after progressive retinal ischemia produces increased expression of the hypoxia-inducible VEGF leading to retinal vascular proliferation and permeability. Although nearly all patients with diabetes will develop some degree of retinopathy in their lifetimes, PDR occurs only in approximately the half. Although inhibition of VEGF reduces retinal neovascularization, it does not completely inhibit ischemia-driven retinal neovascularization. Thus, the involvement of other angiogenic factors in this process seems likely. Erythropoietin (EPO) is glycoprotein related to erythropoiesis synthesized by the fetal liver and the adult kidney. EPO is a multifunctional protein consisting of 165 amino acids. Its expression has also been demonstrated in the brain and the fetal and adult retina. EPO is expressed in amacrine and bipolar cells, and EPO receptor is detected in ganglion cells, amacrine cells, and astrocytes in the mammalian retina. The endogenous EPO serum concentration in humans varies diurnally, with the highest concentrations presented in the evening and at night. Hypoxia is strong stimulant of systemic and intraocular synthesis of EPO. Production of EPO takes place in the retina in response to hypoxic stimuli and its expression is mediated by HIF-1α, which also stimulates the secretion of VEGF. In advanced stages, when retinal hypoxia is predominant and when there are also high levels of VEGF, EPO contributes to neovascularization and consequently to the worsening of DR. The effects of EPO appear to be independent of VEGF despite having an identical effect on PDR, namely, stimulation of ischemia-induced retinal angiogenesis. EPO demonstrates stimulatory angiogenic activity in the microvasculature and red cell proliferation while preventing cell apoptosis, all actions contributory to neovascularization in PDR.
The duration of diabetes and the glycemic control are the two most important factors in the development of DR. However, these factors alone do not explain the occurrence of retinopathy. It may be absent in some patients with poor glycemic control even over a long period of time while others may develop retinopathy in a relatively short period despite good glycemic control. This raises the possibility of a genetic predisposition to retinopathy.
The present study was conducted:
- To measure EPO levels in the vitreous fluid and serum of patients with PDR, a condition in which the retinal ischemia is a predominate event, and to compare these with obtained samples from nondiabetic (ND) patients without systemic or ocular ischemic diseases
- To detect EPO single nucleotide polymorphism (SNP) genotyping expression in patients with PDR and to compare with its expression in the ND group.
| Materials and Methods|| |
This prospective comparative study was conducted on twenty patients with type 2 DM and in the stage of PDR requiring pars plana vitrectomy (PPV). Twenty ND participants requiring PPV for nonvascular eye diseases were included in the study as control group.
In the PDR group, only patients with the need for vitrectomy were included in the study. The indications for vitrectomy were PDR with tractional retinal detachment (RD) and nonclearing organized vitreous hemorrhage or PDR with persistent diabetic macular edema and vitreoretinal interface maculopathy. In the ND group; the indications for vitrectomy were recent RDs whether rhegmatogenous associated with macular hole or pseudophakic.
These were acute ocular infection, uveitis, long-standing RD and proliferative vitreoretinopathy, argon laser photocoagulation in the past 6 months before PPV, previous vitreoretinal surgery, previous intravitreal injections within the past 6 months, recent vitreous hemorrhage (<3 months before vitrectomy), renal impairment or failure, patients on EPO treatment, and presence of anemia or other causes of systemic hypoxia. Controls were free from systemic diseases and ocular vascular diseases.
The systemic condition of all included patients was under control (for diabetic patients, their blood glucose level was under control preoperatively with oral hypoglycemic drugs or insulin and had within normal range blood pressure). Each patient received a complete ophthalmic examination, which included best-corrected visual acuity, intraocular pressure measurement using Goldmann applanation tonometry, slit-lamp examination, and fundus examination with indirect ophthalmoscope and slit-lamp biomicroscopy with +90 D noncontact lens. Ophthalmic ultrasonic examination of the posterior segment (High-resolution Mentor-Advent™ A/B system [Mentor Corporation, Santa Barbara, CA, USA] equipped with 7.5–15 MHz real-time high-frequency probe with the contact method) was performed for all patients to delineate the posterior segment details. All patients were given a detailed explanation of the treatment and the potential risks and benefits. An informed consent was obtained from all patients before the interventions, and the study was approved by the local ethics committee.
The vitreous, serum, and blood samples were collected on the same day of the vitrectomy operation and in the morning for the circadian rhythm of EPO.
Serum and blood samples collection
Venous blood samples were collected aseptically from all patients in four separate test tubes. One sterile serum separator tube without anticoagulants was used for biochemical analysis. After coagulation, sample was centrifuged and serum was harvested, divided into aliquots, and stored at −20°C until analysis. Three sterile tubes containing EDTA were collected for hemoglobin A1c (HbA1c), complete blood count (CBC), and DNA extraction.
Vitreous sample collection
All patients had 23-gauge sutureless PPV. The undiluted vitreous sample was obtained at the beginning of vitrectomy through the aspiration port before turning on infusion. Limited core vitrectomy was performed first followed by aspiration of the vitreous sample (0.3–0.5 mL) by manual suction with a syringe, then proceeding with the vitrectomy operation. The collected samples were frozen at −20°C until assayed.
Laboratory investigations included CBC, biochemical analysis (kidney functions, cholesterol, triglyceride, fasting, and postprandial blood sugar), and HbA1c. EPO in vitreous and serum was measured by chemiluminescent immunoassay (Immulite 1000 Chemiluminescent Immunometric Assay, Diagnostic Products Corporation, Siemens, USA).
Genotyping of erythropoietingene single nucleotide polymorphism
The EPO gene SNP rs551238 was selected. SNP genotyping was conducted using the real-time polymerase chain reaction (PCR). Total genomic DNA was extracted from peripheral venous blood of each individual.
Data were analyzed using SPSS version 17.0 (Statistical Package for the Social Sciences Inc., Chicago, IL, USA) and Microsoft Excel 2010. Parametric data were expressed as mean ± standard deviation (SD) and nonparametric data were expressed as number and percentage. Student's t-test was done to compare between two groups. Chi-square test was used to examine the relationship between two qualitative variables. P > 0.05 was considered nonsignificant, P ≤ 0.05 was considered significant, and P < 0.01 was considered highly significant.
| Results|| |
Of the total 40 patients, 20 patients suffered from type 2 diabetes and were in the stage of PDR and 20 ND patients had recent RD of various etiologies. Patients in PDR group had a mean age of 56.25 ± 7.5 years (range, 48–75 years); 40% were male and 60% were female. Patients in ND group had a mean age of 47.9 ± 9.7 years (range, 24–67 years); 65% were male and 35% were female. The duration of the DM in PDR group was 14.5 ± 4.4 years (mean ± SD). The HbA1c level in PDR group (8.7 ± 0.92) was significantly higher than that in ND group (5.48 ± 0.56) (P < 0.01). The age, sex, duration of diabetes mellitus, and the indications of PPV are summarized in [Table 1].
The mean serum EPO (s-EPO) level was significantly increased in PDR group than in ND group while there was a highly significant increase in mean vitreous fluid EPO (VF-EPO) level in PDR group than in ND group [Table 2] and [Figure 1]. Within the PDR group, vitreous EPO concentrations were significantly elevated as compared with s-EPO. There was up to 6-fold increase in the vitreous EPO level than the s-EPO level [Table 3].
|Table 2: Comparison of erythropoietin in serum and vitreous fluid between the two groups (mean±standard deviation)|
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|Figure 1: (a) Comparison of erythropoietin levels in serum and (b) vitreous fluid between proliferative diabetic retinopathy and nondiabeitc groups|
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|Table 3: Comparison of mean erythropoietin in serum and vitreous fluid in proliferative diabetic retinopathy group|
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There was a highly significant increase in HbA1c and cholesterol while there were a significant decrease in platelets and a significant increase in triglyceride in PDR group as compared with ND group. There were no significant differences between both groups regarding the remaining parameters [Table 4].
|Table 4: Comparison of different laboratory parameters in the two groups|
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Receiver operating characteristic curve (ROC curve) analysis was performed to determine the diagnostic significance of EPO in serum and vitreous fluid in PDR group compared with ND group. The best cutoff value of EPO serum was 1.2 mIU/ml. The best cutoff value of EPO vitreous fluid was 6.2 mIU/ml. The area under ROC curve (AUC) was 0.735 for EPO serum and 0.965 for EPO vitreous fluid (AUC closer to 1 means higher diagnostic performance) [Table 5] and [Figure 2].
|Table 5: Sensitivity and specificity of the best-selected cutoff value to differentiate between proliferative diabetic retinopathy and nondiabetic cases|
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|Figure 2: Receiver operating characteristic curve of serum erythropoietin and vitreous fluid erythropoietin for discriminating proliferative diabetic retinopathy from nondiabetics|
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The frequency of SNP rs551238 in both groups showed that the homozygous mutant type (TT) was detected in 75% of PDR group and 20% of ND group while the heterozygous mutant type (GT) was detected in 25% of PDR group and 80% of ND group with highly significant differences between both groups [Table 6].
|Table 6: Frequency of single nucleotide polymorphism rs551238 in diabetic patients with proliferative diabetic retinopathy compared to nondiabetic group|
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The SNP rs551238 allele genotypes in both groups showed that the number of T alleles was increased in PDR group (35) as compared with (24) T alleles in ND group [Table 7].
|Table 7: Percentage of alleles of single nucleotide polymorphism rs551238 genotypes in proliferative diabetic retinopathy compared to nondiabetic group|
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| Discussion|| |
As inhibition of VEGF is not associated with total regression of retinal neovessels, angiogenic factors other than VEGF may play a role in this process. EPO was suggested as a potent retinal angiogenic factor, acting independent of VEGF, and has the capability of stimulating ischemia-induced retinal angiogenesis.
The present study investigated EPO serum level in PDR patients compared with that in ND patients. There was a significant increase of the s-EPO level in patients with PDR. In their study, Gholamhossein et al. reported a significant increase in plasma EPO level in PDR group. The elevated mean plasma EPO level demonstrated a significant correlation with PDR would be an indication of its role in PDR.
Furthermore, in the current study, comparison of EPO level in the vitreous fluid obtained from PDR patients with that obtained from ND patients revealed a highly significant increase in EPO level in the vitreous fluid in PDR group. These results are consistent with those of Asensio-Sánchez et al. who also studied the EPO concentrations in the vitreous body from patients with PDR and found that the concentration of EPO in the vitreous body was significantly higher in patients with PDR than in the control group. Furthermore, in their study, Katsura et al. found that vitreous EPO concentrations were markedly higher in PDR patients than in macular hole patients who had no retinopathy. Mohan et al. study had shown similar increase in EPO levels from PDR patients in both vitreous and plasma samples.
In the present study, vitreous EPO concentration was significantly elevated as compared with s-EPO within the PDR group; the mean vitreous level of EPO (36.49 mIU/L) was up to 6 folds higher than its serum level (6.01 mIU/L); this may point to a contribution of ocular tissue in local EPO production in PDR in response to retinal ischemia. In a study with 12 PDR patients, EPO concentrations in the vitreous were found 30 times higher than in serum. Therefore, it seems clear that EPO is produced locally in the retina. It was suggested that increased EPO levels in the vitreous fluid of PDR patients are due to increased local production in the retina but not in the proliferative membrane itself. EPO derived from the vitreous fluid in patients with PDR was bioactive and stimulated proliferation of bovine retinal microvascular endothelial cells in vitro. Chen et al. animal study had shown EPO inhibitors preventing neovascularization, further supporting the role of EPO in the development of PDR.
EPO and VEGF are both upregulated in the vitreous of patients with PDR and they appear to act independently. Localized EPO inhibition within the eye might have therapeutic potential for the treatment or prevention of PDR. Suppression of both EPO and VEGF leads to a greater inhibition of retinal neovascularization than when either is inhibited alone.
The present study showed that the mean platelet count was significantly lower in the PDR group than ND group (P < 0.05). Hekimsoy et al. had observed the same finding with lower platelet count in diabetic group compared to healthy participants. Altered platelet morphology and functions have been reported in diabetic patients and are linked with the pathological processes and high risk of vascular disease.
In the current study, there was a highly significant increase in HbA1c in PDR group as compared with ND group. Hyperglycemia is the main culprit in DR development; the high blood glucose levels in diabetic patients can stimulate the production of EPO and VEGF. In Mohan et al. study, the vitreous EPO and VEGF levels correlated significantly with the HbA1c in the patients with PDR.
In the current study, long-standing RD was excluded from the study. Retinal neovascularization can occur in the setting of a rhegmatogenous RD of long duration with elevation of EPO levels in these patients. Furthermore, anemia (as a systemic cause of hypoxia) was excluded in the current study. Anemia induces systemic hypoxia and may increase hypoxia in the retina and it is a poor prognostic factor for PDR. Furthermore, systemic EPO intake was excluded to restrict the evaluation of EPO levels on its endogenous sources only.
Several lines of evidence suggest a genetic contribution to the risk of DM microvascular complications. In particular, significant familial clustering of both diabetic nephropathy and severe DR has been reported after accounting for cross-sectional conventional risk factors. Familial aggregations as well as racial and ethnic differences in incidence suggest that genetic components play a role in the development of DR. Therefore, revealing the genetic markers for DR would make it possible to identify patients with inherited susceptibilities that may ultimately benefit from preventive treatment. EPO messenger RNA is expressed in the human retina and the human EPO gene is located on chromosome 7q21. Polymorphic variability in the genetic makeup of an individual can profoundly influence the expression of a gene and its response to environmental factors. EPO expression is influenced by SNPs in EPO. Three EPO gene variants, rs1617640, rs507392, and rs551238, were shown to be associated with increased risk of DR in Caucasian Type 2 DM participants. A genetically determined ability of increased EPO synthesis predisposes diabetic patients to the development of PDR. In the current study, genotyping the participants of both PDR and ND groups was done. DNA was extracted from the peripheral blood samples, and PCR was used to estimate the frequency of SNP rs551238 in both PDR and ND participants. The homozygous mutant type (TT)was detected in 75% of PDR and in 20% of ND patients while the heterozygous mutant type (GT) was detected in 25% of PDR and in 80% of ND patients with a highly significant differences between both groups; this indicates that SNP rs551238 was significantly associated with DR and its advanced form the PDR in Egyptian participants as the homozygous mutant (TT) type was detected in up to 75% of PDR cases.
The SNP rs551238 is located in the 3'-hypoxia-responsive element of the EPO gene close to the HIF-1-binding site. This SNP is one of the SNPs deemed to influence the expression of EPO or associated with DR.
In the current study, the number of T alleles was increased in patients with PDR (35) as compared with (24) T alleles in ND patients. The risk allele (T) is associated with elevated EPO in the vitreous body of the human eye and mouse models of diabetic eye and kidney complications. In the current study, the TT variant was present in 15 of PDR group compared with 5 in ND group. The TT variant in diabetic patients may convey increased EPO expression, resulting in an increased risk for the development of diabetic complications in the retina and kidney.
The genotype results suggest that caution may be warranted when maintaining higher hemoglobin concentrations using exogenous EPO treatment in diabetic patients because it might accelerate progression to PDR. The variations in reported observations on the correlation of EPO gene polymorphisms with DR may provide insights into the relative importance of genetic risk factors. Furthermore, ethnic differences in the prevalence of DR may also contribute to the disparate results between studies.
Limitations of the current study included the relatively small number of enrolled patients and the genotyping of only one SNP (SNP rs551238). Hence, further studies on larger number of patients are required to confirm the current findings and to detect the genetic susceptibility to DR in Egyptians.
| Conclusion|| |
The results of the current study suggest the involvement of EPO in the pathogenesis of PDR. Hence, augmenting VEGF inhibition with the use of EPO inhibitors may provide a better outcome in PDR treatment. The detection of EPO gene SNP rs551238 in Egyptian patients with PDR may help in the identification of the genetic predisposition to DR in Egyptians and so may help in prevention and early detection of susceptible candidates to PDR.
Financial support and sponsorship
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], [Table 6], [Table 7]