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“Association between platelet activating factor acetylhydrolase and diabetic retinopathy: Does inflammation affect the retinal status?”
Prostaglandins & other Lipid Mediators 122 (2016) 69–72
Contents lists available at ScienceDirect
Prostaglandins and Other Lipid Mediators
Original research article
“Association between platelet activating factor acetylhydrolase and diabetic retinopathy: Does inflammation affect the retinal status?” Marilita M. Moschos a,∗ , Panagiotis Pantazis a , Zisis Gatzioufas b , Georgios D. Panos c , Maria Gazouli d , Eirini Nitoda a , Dimitris Brouzas a a
University Eye Clinic, General Hospital of Athens G. Gennimatas, Greece Moorfields Eye Hospital, London, UK c Ipswich Hospital, University of Cambridge, UK d Department of Basic Medical Science, Laboratory of Biology, Medical School, National & Kapodistrian University of Athens, Greece b
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 14 October 2015 Received in revised form 27 December 2015 Accepted 6 January 2016 Available online 11 January 2016 Keywords: Diabetic retinopathy Inflammation PAF acetylhydrolase Platelet activating factor
Aim: To assess the role of plasma platelet activating factor acetylhydrolase (PAF-AH) in pathogenesis and progression of diabetic retinopathy (DR). Materials and methods: Sixty eight diabetics and 23 age-frequency-matched non-diabetic patients underwent blood sampling and the plasma PAF-AH activity was calculated. The diabetic patients were further classified into two groups, according to the Early Treatment Diabetic Retinopathy Study (ETDRS) classification, based on indirect fundoscopy and fluorescein angiography. Thirty seven patients with non-proliferative DR (NPDR) and 31 patients with proliferative DR (PDR) were finally included in the study. Results: The plasma PAF-AH activity was increased in diabetic patients with PDR (0.206 mol/min/ml) compared to control group (0.114 mol/min/ml, post-hoc Bonferroni comparison test: p < 0.0001) and to NPDR group (0.147 mol/min/ml, post-hoc Bonferroni comparison test: p = 0.012). Conclusions: The activity of PAF-AH in the plasma increases in parallel with DR severity. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is the leading cause of blindness worldwide and the most severe ocular complication in diabetic patients, who are estimated to be 439 million (7.7% among adults aged 20–79 years old) by the year 2030 [1]. The prevalence of DR worldwide is 34.6% and equals to 93 million patients, directly related to 97.5% of patients with type 1 diabetes mellitus (DM) and 77.8% of patients with type 2 DM, suffering from DR after 15 years of DM [2–4]. The pathogenesis of DR seems to be associated with the sorbitol pathway, the action of free radicals and nitric oxide, the accumulation of advanced glycation end products (AGEs) in the tissues, the changes induced in growth factors, and dehydroascorbate (uncharged form of Vitamin C) [5]. Moreover, the duration of DM, the levels of HbA1c and the blood pressure have proven to be major risk factors, implicating in the progression of DR [2].
∗ Corresponding author. Fax: +30 2104122319. E-mail addresses: [email protected] (M.M. Moschos), [email protected] (P. Pantazis), [email protected] (Z. Gatzioufas), [email protected] (G.D. Panos), [email protected] (M. Gazouli), [email protected] (E. Nitoda), [email protected] (D. Brouzas). http://dx.doi.org/10.1016/j.prostaglandins.2016.01.001 1098-8823/© 2016 Elsevier Inc. All rights reserved.
Platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3phosphocho-line) is a low molecular weight phospholipid which is involved in inflammation [6]. The affinity of PAF with its receptor (PAF-R, flt-1), a trans-membrane G-protein, along with the action of the PAF acetylhydrolases (PAF-AHs), seem to regulate the action of PAF [6,7]. The PAF-AHs represent a heterogeneous group of phospholipases (plasma/secreted and intracellular PAF-AHs), which exhibit high selectivity for phospholipids with short acyl chains at the sn-2 position, without affecting normal membrane phospholipids [7]. These phospholipases inactivate PAF through a hydrolytic cleavage of the sn-2 ester bond and the release of free acetate and biologically inactive lyso-PAF [7]. Beside its systemic action, PAF is released by the ocular tissues, including the iris, ciliary body, retina and vascular endothelium, implicating in ocular inflammation [8]. The retinal tissue responds to physiological and pathological stimuli, releasing metabolites from membrane phospholipids, eicosanoids (prostaglandins PGE2, PGF2, PGD2 and thromboxane A2) and PAF [9]. The activation of PAF results in the degeneration of small vessels and the death of endothelial cells, being related to the pathogenesis of ischemic retinopathies, such as diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity [9].
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Fig. 1. Box plot. The mean values of participants’ PAF-AH activity (in mmol/min/ml) for each group separately. The mean, the minimum, the maximum and the extreme values of PAF-AH activity are presented for each group (NPDR = Non-proliferative diabetic retinopathy, PAF-AH: platelet activating factor acetylhydrolase, PDR = proliferative diabetic retinopathy). A statistically significant increase in plasma PAF-AH activity was noted in PDR group compared to control and to NPDR groups (post-hoc Bonferroni comparison test: p < 0.0001 and p = 0.012, respectively).
The involvement of PAF in a variety of ocular diseases, including retinal ones and diabetic retinopathy, was the motivation to study the activity of plasma PAF-AH in diabetic patients. Investigating the role of the enzyme in pathogenesis and progression of diabetic retinopathy could contribute to therapeutic strategies.
2.3.1. Indirect fundoscopy The diagnosis of retinal lesions in non-diabetics and DR in diabetic groups was performed using slit lamp fundoscopy. Moreover, slit lamp was used to detect iris neovascularization and vitreous hemorrhage.
2. Materials and methods
2.3.2. Optical coherence tomography (OCT) All diabetic patients underwent OCT (Stratus, Carl Zeiss Meditec, USA) examination.
2.1. Patients This is a cross-sectional randomized study, conducted at the 1st University Eye Clinic of General Hospital of Athens G. Gennimatas (1st Department of Ophthalmology, Medical School, National & Kapodistrian University of Athens), from June 2012 to March 2013. Sixty eight Type 2 diabetic patients were classified into two groups, according to their retinal status and the Early Treatment Diabetic Retinopathy Study (ETDRS) classification scheme, as following: 37 patients with non-proliferative DR (NPDR), and 31 patients with proliferative DR (PDR). Twenty three sex- and age-frequencymatched non-diabetic patients, with no retinal alterations, were also included in the study and served as the control group. The study was performed in accordance to the tenets of the Declaration of Helsinki and the protocol used was approved by the ethics committee of the University Hospital. Written informed consent was obtained from all participants. The classification of the participants into three groups was performed according to their systemic and ocular history, as well as the ocular examination. The patients of NPDR group had at least one eye with lesions of this grade, provided that the other did not suffer from PDR. PDR alterations of at least one eye were the necessary criterion for the PDR group. The exclusion criteria of the study included retinal lesions in non-diabetic patients and vascular or other type of retinopathy in diabetic group. 2.2. Measurement of best corrected visual acuity The best corrected visual acuity was measured for all the participants and it was based on Snellen chart. 2.3. Evaluation of the retinal status It was performed as follow:
2.3.3. Fluorescein angiography (FA) FA was performed in all diabetics who were classified into two groups according to ETDRS system. 2.4. Blood sampling All participants were subjected to blood sampling, which was used in quantitative calculation of plasma PAF-AH activity. Blood samples (volume 2,5 ml) were stored in a sampling bottle, which contained EDTA (Ethylenediaminetetraacetic acid). EDTA is used extensively to bind metal ions and as an anticoagulant in blood samples used for a complete blood count (CBC). Centrifugation of the samples was followed immediately or within one hour, during which the samples were stored at temperature of 2–8◦ C (fridge). Centrifugation was performed at 1000 rpm for 10 min at 4◦ C. After centrifugation the collected plasma was stored in special vials at a temperature of −80◦ C until the quantitative analysis was performed. The method of collecting and storing samples secured the quality of the analysis, given that hemolysis affects the levels of PAF. 2.5. Quantitative calculation of PAF-AH activity The quantitative calculation of plasma PAF-AH activity was based on ELISA (Enzyme-linked Immunosorbent Assay) immunoassay technique for human PAF-AH (PAF Acetylhydrolase Assay Kit, Item no.760901, Cayman Chemical) and was performed at Department of Basic Medical Science, Laboratory of Biology of Medical School, according to the manufacturer instructions [10,11]. The change in absorbance (A414 /min) per minute was defined by plotting the average values as a function of time to obtain the slope (rate) of the linear portion of the curve. Afterwards, the rate of
M.M. Moschos et al. / Prostaglandins & other Lipid Mediators 122 (2016) 69–72
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Table 1 Distributions of participants’ gender, age and potency of PAF-AH within the groups. GROUP
GENDER (number and percentage within the group)
WOMEN MEN TOTALS
AGE(years) mean, (SD) PAF-AH (mol/min/ml) mean,(SD)
CONTROL
NPDR
PDR
13 (57%) 10(43%) 23 65.2 (10.1) 0.114 (0.050)
11 (30%) 26 (70%) 37 65.5 (7.8) 0.147 (0.102)
10(32%) 21(68%) 31 66.4 (8.9) 0.206 (0.074)
Mean: mean value, NPDR = Non-proliferative diabetic retinopathy, PAF-AH: platelet activating factor acetylhydrolase, PDR = proliferative diabetic retinopathy, SD: standard deviation.
A414 /min for the blank wells was determined and subtracted from that of the sample wells. The reaction rate at 414 nm can be specified using the DTNB [5,5 -dithio-bis-(2-nitrobenzoic acid), Ellman’s reagent] extinction coefficient of 10.66 mM. Finally, the plasma PAF-AH activity was calculated, using the following formula, given that one unit of enzyme hydrolyzes one mol of 2-thio PAF (the substrate for PAF-AH) per minute at 25 ◦ C: Plasma PAF − AH activity(mol/min/ml) = ×
A414 /min
The analysis highlighted that the plasma PAF-AH values exhibited statistically significant differences between the control group and diabetic patients with PDR (p < 0.0001) as well as between the two groups with diabetic retinopathy (p = 0.012). It is consequently clear that the activity of PAF-AH in the plasma increases in parallel with diabetic retinopathy level. Additional ANCOVA statistical analysis showed that the activity of PAF-AH was not affected by the age (p = 0.965, F1 = 0.002).
10.66mM−1
0.225ml × Sample dilution 0.01ml
2.6. Statistic analysis The statistical program IBM SPSS statistics 22.0 was used for the analysis. Descriptive analysis of all parameters, including the age, the gender and the activity of PAF-AH in patients’ plasma, was first carried out. Box plots of the quantitative variables were created. Non-parametric analysis Kolmogorov–Smirnov was used to check if the variables had a normal distribution. The possible gender differences among groups were estimated using the chi-squared test, whereas one-way ANOVA analysis was applied to identify the possible differences in means of age and PAF-AH among groups. The differences in means of plasma PAF-AH activity were additionally checked with post-hoc Bonferroni tests. Finally, ANCOVA statistical analysis was applied to detect the possible effect of patients’ gender and age on the PAF-AH activity. 3. Results 3.1. Demographics Ninety one sex-and age-frequency-matched persons were participated in this study, including 68 diabetic patients (37 with NPDR and 31 with PDR) and 23 participants, served as control group (Table 1). Fifty seven men (63%), clearly outnumbered in the diabetic groups, whereas thirty four women (37%), slightly predominated among control individuals (Table 1). However, Pearson Chi-Square test revealed no statistically significant differences in gender among groups (p = 0.087). Similar results came out from the statistical analysis One-way Anova [F (2,88) = 0.1, p = 0.867] as well as the post-hoc Bonferroni comparison test (p = 1.0 for all pairs of groups), estimating the mean values of the participants’ age. 3.2. Evaluation of PAF-AH activity The mean values of the plasma PAF-AH activity (Table 1, Fig. 1) were compared, using parametric analysis One-way ANOVA and statistically significant differences among the three groups came forth [F (2,88) = 10, p < 0.0001]. Post-hoc Bonferroni comparison test was applied in order to spot the differences among the groups.
4. Discussion In our study the activity of plasma PAF-AH was measured in sixty eight patients with diabetic retinopathy and compared with the activity in twenty three healthy participants. We noted that the activity of plasma PAF-AH is higher in the diabetic patients with proliferative retinal lesions compared to the healthy individuals and to diabetic patients with non-proloferative retinopathy. In addition, it is increased along with the severity of diabetic retinopathy and it is independent of participants’ age. Recent studies have revealed that high PAF-AH activity in type 1 and 2 diabetic patients is implicated in the chronic low-grade inflammation, endothelial dysfunction and atherosclerosis developed in DM [12–15]. These observations overturned Trapali et al. who noted that PAF concentration in the plasma of diabetic rats was elevated due to the decreased activity of plasma PAF-AH [16]. In addition, the high plasma PAF-AH has been correlated with age, glycated hemoglobin (HbA1c), uric acid, HDL-cholesterol (High Density Lipoprotein cholesterol), cholesterol and LDL-cholesterol (Low Density Lipoprotein cholesterol) levels, the cholesterol/HDLcholesterol and the LDL-cholesterol/HDL-cholesterol ratio [14,15]. Kujiraoka et al. studied the distribution of plasma PAF-AH between HDLs and other lipoproteins and revealed that HDL-associated and non-HDL-associated PAF-AHs were respectively elevated and decreased in diabetic and hyperlipidemic individuals compared to controls [17]. The enhanced platelet adhesion and aggregation in diabetic patients have been related to the raised levels of PAF-AH, resulting from the release of thromboxane A2 (TxA2) and 5hydroxytryptamine (5HT) [18,19]. The reduced production of reactive oxygen species by diabetic platelets and the alterations in the physico-chemical properties of their membranes explain their modified response to PAF stimulation [20]. Besides the alterations observed in platelets, polymorphonuclear leukocytes of streptozotocin-induced diabetic rats presented raised synthesis of PAF [21]. The high levels of activated phospholipase A2 and acetyltransferase along with the elevated cytosolic Ca2++ were responsible for the modified release of PAF [21]. On the other hand, PAF has been implicated in the retinal inflammation, promoting the binding of leukocytes in P-selectins of retinal vascular endothelium [22]. This mechanism contributes to the pathogenesis of retinal vascular endothelium damage and thrombosis in Behcet disease [23].
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Moreover, PAF has been associated with elevated vascular permeability observed in tumor necrosis factor-alpha (TNF-a) induced uveitis and in allergic conjunctivitis [24,25]. The selective expression of Vascular Endothelial Growth Factor (VEGF), the decrease in antio-angiogenic thrombospondin-1 (TSP-1) in corneal myofibroblasts and the promotion of endothelial cells migration in corneal basic membrane compose PAF action in corneal neovascularization [26,27]. In addition, PAF exhibits selective cytotoxicity in endothelial cells, rarely affecting retinal pericytes, perivascular astrocytes and smooth muscle cells [9]. The cytotoxic action of PAF results in the activation of specific phospholipases and proteases as well as in disturbance of mitochondrial permeability transition pores [9]. Lysophosphatidylcholine (LPC) Acyltransferase 1 (LPCAT1) seems to inactivate LPC and PAF through its catalytic effect, inhibiting the onset of atherosclerosis, coronary disease, DM and DR [28]. The retinal action of PAF induces functional disturbances, expressed with decreased b-wave in electroretinogram (ERG) [29,30]. Moreover, PAF has been implicated in the pathogenesis and progression of Age-Related Macular Degeneration (ARMD) [31]. Specifically, PAF levels in serum are reduced along with the increase in severity of macular lesions observed in ARMD [31]. The damage of the vascular endothelium, the enhanced vascular permeability and neovascularization represent the role of PAF in pathogenesis and progression of ischemic retinopathies, including DR.
5. Conclusion It is the first study to measure PAF-AH activity in the plasma of patients with diabetic retinopathy. In our study, we concluded that plasma PAF-AH activity is raised in diabetic patients exhibiting proliferative retinal lesions compared to the healthy individuals and to diabetic patients with non-proloferative retinopathy. Moreover, the activity of plasma PAF-AH is increased in parallel with the severity of diabetic retinopathy. Given that diabetic retinopathy is the leading cause of blindness and severe loss of vision worldwide, affecting 93 million patients, it is important to clarify its pathogenesis both for prevention and treatment of the disease.
Conflict of interest All authors have no conflict of interest to declare and no financial support was offered for the present study.
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Huseyinov, Platelet-activating factor and P-selectin activities in thrombotic and nonthrombotic Behc¸et’s patients, Rheumatol. Int. 25 (2005) 326–331. [24] L.N. Fleisher, J.B. Ferrell, M.G. Smith, et al., Lipid mediators of tumor necrosis factor alpha-induced uveitis, Invest. Ophthalmol. Vis. Sci. 32 (1991) 2393–2399. [25] O. Zinchuk, A. Fukushima, V. Zinchuk, et al., Direct action of platelet activating factor (PAF) induces eosinophil accumulation and enhances expression of PAF receptors in conjunctivitis, Mol. Vis. 11 (2005) 114–123. [26] X. Ma, P. Ottino, H.E. Bazan, N.G. Bazan, Platelet-activating factor (PAF) induces corneal neovascularization and upregulates VEGF expression in endothelial cells, Invest. Ophthalmol. Vis. Sci. 45 (2004) 2915–2921. [27] J. He, J.P. Eastlack, H.E. Bazan, The induction of an angiogenic response in corneal myofibroblasts by platelet-activating factor (PAF), Curr. Eye Res. 35 (2010) 1063–1071. [28] P. Hardy, M. Beauchamp, F. 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Contents lists available at ScienceDirect
Prostaglandins and Other Lipid Mediators
Original research article
“Association between platelet activating factor acetylhydrolase and diabetic retinopathy: Does inflammation affect the retinal status?” Marilita M. Moschos a,∗ , Panagiotis Pantazis a , Zisis Gatzioufas b , Georgios D. Panos c , Maria Gazouli d , Eirini Nitoda a , Dimitris Brouzas a a
University Eye Clinic, General Hospital of Athens G. Gennimatas, Greece Moorfields Eye Hospital, London, UK c Ipswich Hospital, University of Cambridge, UK d Department of Basic Medical Science, Laboratory of Biology, Medical School, National & Kapodistrian University of Athens, Greece b
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 14 October 2015 Received in revised form 27 December 2015 Accepted 6 January 2016 Available online 11 January 2016 Keywords: Diabetic retinopathy Inflammation PAF acetylhydrolase Platelet activating factor
Aim: To assess the role of plasma platelet activating factor acetylhydrolase (PAF-AH) in pathogenesis and progression of diabetic retinopathy (DR). Materials and methods: Sixty eight diabetics and 23 age-frequency-matched non-diabetic patients underwent blood sampling and the plasma PAF-AH activity was calculated. The diabetic patients were further classified into two groups, according to the Early Treatment Diabetic Retinopathy Study (ETDRS) classification, based on indirect fundoscopy and fluorescein angiography. Thirty seven patients with non-proliferative DR (NPDR) and 31 patients with proliferative DR (PDR) were finally included in the study. Results: The plasma PAF-AH activity was increased in diabetic patients with PDR (0.206 mol/min/ml) compared to control group (0.114 mol/min/ml, post-hoc Bonferroni comparison test: p < 0.0001) and to NPDR group (0.147 mol/min/ml, post-hoc Bonferroni comparison test: p = 0.012). Conclusions: The activity of PAF-AH in the plasma increases in parallel with DR severity. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is the leading cause of blindness worldwide and the most severe ocular complication in diabetic patients, who are estimated to be 439 million (7.7% among adults aged 20–79 years old) by the year 2030 [1]. The prevalence of DR worldwide is 34.6% and equals to 93 million patients, directly related to 97.5% of patients with type 1 diabetes mellitus (DM) and 77.8% of patients with type 2 DM, suffering from DR after 15 years of DM [2–4]. The pathogenesis of DR seems to be associated with the sorbitol pathway, the action of free radicals and nitric oxide, the accumulation of advanced glycation end products (AGEs) in the tissues, the changes induced in growth factors, and dehydroascorbate (uncharged form of Vitamin C) [5]. Moreover, the duration of DM, the levels of HbA1c and the blood pressure have proven to be major risk factors, implicating in the progression of DR [2].
∗ Corresponding author. Fax: +30 2104122319. E-mail addresses: [email protected] (M.M. Moschos), [email protected] (P. Pantazis), [email protected] (Z. Gatzioufas), [email protected] (G.D. Panos), [email protected] (M. Gazouli), [email protected] (E. Nitoda), [email protected] (D. Brouzas). http://dx.doi.org/10.1016/j.prostaglandins.2016.01.001 1098-8823/© 2016 Elsevier Inc. All rights reserved.
Platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3phosphocho-line) is a low molecular weight phospholipid which is involved in inflammation [6]. The affinity of PAF with its receptor (PAF-R, flt-1), a trans-membrane G-protein, along with the action of the PAF acetylhydrolases (PAF-AHs), seem to regulate the action of PAF [6,7]. The PAF-AHs represent a heterogeneous group of phospholipases (plasma/secreted and intracellular PAF-AHs), which exhibit high selectivity for phospholipids with short acyl chains at the sn-2 position, without affecting normal membrane phospholipids [7]. These phospholipases inactivate PAF through a hydrolytic cleavage of the sn-2 ester bond and the release of free acetate and biologically inactive lyso-PAF [7]. Beside its systemic action, PAF is released by the ocular tissues, including the iris, ciliary body, retina and vascular endothelium, implicating in ocular inflammation [8]. The retinal tissue responds to physiological and pathological stimuli, releasing metabolites from membrane phospholipids, eicosanoids (prostaglandins PGE2, PGF2, PGD2 and thromboxane A2) and PAF [9]. The activation of PAF results in the degeneration of small vessels and the death of endothelial cells, being related to the pathogenesis of ischemic retinopathies, such as diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity [9].
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M.M. Moschos et al. / Prostaglandins & other Lipid Mediators 122 (2016) 69–72
Fig. 1. Box plot. The mean values of participants’ PAF-AH activity (in mmol/min/ml) for each group separately. The mean, the minimum, the maximum and the extreme values of PAF-AH activity are presented for each group (NPDR = Non-proliferative diabetic retinopathy, PAF-AH: platelet activating factor acetylhydrolase, PDR = proliferative diabetic retinopathy). A statistically significant increase in plasma PAF-AH activity was noted in PDR group compared to control and to NPDR groups (post-hoc Bonferroni comparison test: p < 0.0001 and p = 0.012, respectively).
The involvement of PAF in a variety of ocular diseases, including retinal ones and diabetic retinopathy, was the motivation to study the activity of plasma PAF-AH in diabetic patients. Investigating the role of the enzyme in pathogenesis and progression of diabetic retinopathy could contribute to therapeutic strategies.
2.3.1. Indirect fundoscopy The diagnosis of retinal lesions in non-diabetics and DR in diabetic groups was performed using slit lamp fundoscopy. Moreover, slit lamp was used to detect iris neovascularization and vitreous hemorrhage.
2. Materials and methods
2.3.2. Optical coherence tomography (OCT) All diabetic patients underwent OCT (Stratus, Carl Zeiss Meditec, USA) examination.
2.1. Patients This is a cross-sectional randomized study, conducted at the 1st University Eye Clinic of General Hospital of Athens G. Gennimatas (1st Department of Ophthalmology, Medical School, National & Kapodistrian University of Athens), from June 2012 to March 2013. Sixty eight Type 2 diabetic patients were classified into two groups, according to their retinal status and the Early Treatment Diabetic Retinopathy Study (ETDRS) classification scheme, as following: 37 patients with non-proliferative DR (NPDR), and 31 patients with proliferative DR (PDR). Twenty three sex- and age-frequencymatched non-diabetic patients, with no retinal alterations, were also included in the study and served as the control group. The study was performed in accordance to the tenets of the Declaration of Helsinki and the protocol used was approved by the ethics committee of the University Hospital. Written informed consent was obtained from all participants. The classification of the participants into three groups was performed according to their systemic and ocular history, as well as the ocular examination. The patients of NPDR group had at least one eye with lesions of this grade, provided that the other did not suffer from PDR. PDR alterations of at least one eye were the necessary criterion for the PDR group. The exclusion criteria of the study included retinal lesions in non-diabetic patients and vascular or other type of retinopathy in diabetic group. 2.2. Measurement of best corrected visual acuity The best corrected visual acuity was measured for all the participants and it was based on Snellen chart. 2.3. Evaluation of the retinal status It was performed as follow:
2.3.3. Fluorescein angiography (FA) FA was performed in all diabetics who were classified into two groups according to ETDRS system. 2.4. Blood sampling All participants were subjected to blood sampling, which was used in quantitative calculation of plasma PAF-AH activity. Blood samples (volume 2,5 ml) were stored in a sampling bottle, which contained EDTA (Ethylenediaminetetraacetic acid). EDTA is used extensively to bind metal ions and as an anticoagulant in blood samples used for a complete blood count (CBC). Centrifugation of the samples was followed immediately or within one hour, during which the samples were stored at temperature of 2–8◦ C (fridge). Centrifugation was performed at 1000 rpm for 10 min at 4◦ C. After centrifugation the collected plasma was stored in special vials at a temperature of −80◦ C until the quantitative analysis was performed. The method of collecting and storing samples secured the quality of the analysis, given that hemolysis affects the levels of PAF. 2.5. Quantitative calculation of PAF-AH activity The quantitative calculation of plasma PAF-AH activity was based on ELISA (Enzyme-linked Immunosorbent Assay) immunoassay technique for human PAF-AH (PAF Acetylhydrolase Assay Kit, Item no.760901, Cayman Chemical) and was performed at Department of Basic Medical Science, Laboratory of Biology of Medical School, according to the manufacturer instructions [10,11]. The change in absorbance (A414 /min) per minute was defined by plotting the average values as a function of time to obtain the slope (rate) of the linear portion of the curve. Afterwards, the rate of
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Table 1 Distributions of participants’ gender, age and potency of PAF-AH within the groups. GROUP
GENDER (number and percentage within the group)
WOMEN MEN TOTALS
AGE(years) mean, (SD) PAF-AH (mol/min/ml) mean,(SD)
CONTROL
NPDR
PDR
13 (57%) 10(43%) 23 65.2 (10.1) 0.114 (0.050)
11 (30%) 26 (70%) 37 65.5 (7.8) 0.147 (0.102)
10(32%) 21(68%) 31 66.4 (8.9) 0.206 (0.074)
Mean: mean value, NPDR = Non-proliferative diabetic retinopathy, PAF-AH: platelet activating factor acetylhydrolase, PDR = proliferative diabetic retinopathy, SD: standard deviation.
A414 /min for the blank wells was determined and subtracted from that of the sample wells. The reaction rate at 414 nm can be specified using the DTNB [5,5 -dithio-bis-(2-nitrobenzoic acid), Ellman’s reagent] extinction coefficient of 10.66 mM. Finally, the plasma PAF-AH activity was calculated, using the following formula, given that one unit of enzyme hydrolyzes one mol of 2-thio PAF (the substrate for PAF-AH) per minute at 25 ◦ C: Plasma PAF − AH activity(mol/min/ml) = ×
A414 /min
The analysis highlighted that the plasma PAF-AH values exhibited statistically significant differences between the control group and diabetic patients with PDR (p < 0.0001) as well as between the two groups with diabetic retinopathy (p = 0.012). It is consequently clear that the activity of PAF-AH in the plasma increases in parallel with diabetic retinopathy level. Additional ANCOVA statistical analysis showed that the activity of PAF-AH was not affected by the age (p = 0.965, F1 = 0.002).
10.66mM−1
0.225ml × Sample dilution 0.01ml
2.6. Statistic analysis The statistical program IBM SPSS statistics 22.0 was used for the analysis. Descriptive analysis of all parameters, including the age, the gender and the activity of PAF-AH in patients’ plasma, was first carried out. Box plots of the quantitative variables were created. Non-parametric analysis Kolmogorov–Smirnov was used to check if the variables had a normal distribution. The possible gender differences among groups were estimated using the chi-squared test, whereas one-way ANOVA analysis was applied to identify the possible differences in means of age and PAF-AH among groups. The differences in means of plasma PAF-AH activity were additionally checked with post-hoc Bonferroni tests. Finally, ANCOVA statistical analysis was applied to detect the possible effect of patients’ gender and age on the PAF-AH activity. 3. Results 3.1. Demographics Ninety one sex-and age-frequency-matched persons were participated in this study, including 68 diabetic patients (37 with NPDR and 31 with PDR) and 23 participants, served as control group (Table 1). Fifty seven men (63%), clearly outnumbered in the diabetic groups, whereas thirty four women (37%), slightly predominated among control individuals (Table 1). However, Pearson Chi-Square test revealed no statistically significant differences in gender among groups (p = 0.087). Similar results came out from the statistical analysis One-way Anova [F (2,88) = 0.1, p = 0.867] as well as the post-hoc Bonferroni comparison test (p = 1.0 for all pairs of groups), estimating the mean values of the participants’ age. 3.2. Evaluation of PAF-AH activity The mean values of the plasma PAF-AH activity (Table 1, Fig. 1) were compared, using parametric analysis One-way ANOVA and statistically significant differences among the three groups came forth [F (2,88) = 10, p < 0.0001]. Post-hoc Bonferroni comparison test was applied in order to spot the differences among the groups.
4. Discussion In our study the activity of plasma PAF-AH was measured in sixty eight patients with diabetic retinopathy and compared with the activity in twenty three healthy participants. We noted that the activity of plasma PAF-AH is higher in the diabetic patients with proliferative retinal lesions compared to the healthy individuals and to diabetic patients with non-proloferative retinopathy. In addition, it is increased along with the severity of diabetic retinopathy and it is independent of participants’ age. Recent studies have revealed that high PAF-AH activity in type 1 and 2 diabetic patients is implicated in the chronic low-grade inflammation, endothelial dysfunction and atherosclerosis developed in DM [12–15]. These observations overturned Trapali et al. who noted that PAF concentration in the plasma of diabetic rats was elevated due to the decreased activity of plasma PAF-AH [16]. In addition, the high plasma PAF-AH has been correlated with age, glycated hemoglobin (HbA1c), uric acid, HDL-cholesterol (High Density Lipoprotein cholesterol), cholesterol and LDL-cholesterol (Low Density Lipoprotein cholesterol) levels, the cholesterol/HDLcholesterol and the LDL-cholesterol/HDL-cholesterol ratio [14,15]. Kujiraoka et al. studied the distribution of plasma PAF-AH between HDLs and other lipoproteins and revealed that HDL-associated and non-HDL-associated PAF-AHs were respectively elevated and decreased in diabetic and hyperlipidemic individuals compared to controls [17]. The enhanced platelet adhesion and aggregation in diabetic patients have been related to the raised levels of PAF-AH, resulting from the release of thromboxane A2 (TxA2) and 5hydroxytryptamine (5HT) [18,19]. The reduced production of reactive oxygen species by diabetic platelets and the alterations in the physico-chemical properties of their membranes explain their modified response to PAF stimulation [20]. Besides the alterations observed in platelets, polymorphonuclear leukocytes of streptozotocin-induced diabetic rats presented raised synthesis of PAF [21]. The high levels of activated phospholipase A2 and acetyltransferase along with the elevated cytosolic Ca2++ were responsible for the modified release of PAF [21]. On the other hand, PAF has been implicated in the retinal inflammation, promoting the binding of leukocytes in P-selectins of retinal vascular endothelium [22]. This mechanism contributes to the pathogenesis of retinal vascular endothelium damage and thrombosis in Behcet disease [23].
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Moreover, PAF has been associated with elevated vascular permeability observed in tumor necrosis factor-alpha (TNF-a) induced uveitis and in allergic conjunctivitis [24,25]. The selective expression of Vascular Endothelial Growth Factor (VEGF), the decrease in antio-angiogenic thrombospondin-1 (TSP-1) in corneal myofibroblasts and the promotion of endothelial cells migration in corneal basic membrane compose PAF action in corneal neovascularization [26,27]. In addition, PAF exhibits selective cytotoxicity in endothelial cells, rarely affecting retinal pericytes, perivascular astrocytes and smooth muscle cells [9]. The cytotoxic action of PAF results in the activation of specific phospholipases and proteases as well as in disturbance of mitochondrial permeability transition pores [9]. Lysophosphatidylcholine (LPC) Acyltransferase 1 (LPCAT1) seems to inactivate LPC and PAF through its catalytic effect, inhibiting the onset of atherosclerosis, coronary disease, DM and DR [28]. The retinal action of PAF induces functional disturbances, expressed with decreased b-wave in electroretinogram (ERG) [29,30]. Moreover, PAF has been implicated in the pathogenesis and progression of Age-Related Macular Degeneration (ARMD) [31]. Specifically, PAF levels in serum are reduced along with the increase in severity of macular lesions observed in ARMD [31]. The damage of the vascular endothelium, the enhanced vascular permeability and neovascularization represent the role of PAF in pathogenesis and progression of ischemic retinopathies, including DR.
5. Conclusion It is the first study to measure PAF-AH activity in the plasma of patients with diabetic retinopathy. In our study, we concluded that plasma PAF-AH activity is raised in diabetic patients exhibiting proliferative retinal lesions compared to the healthy individuals and to diabetic patients with non-proloferative retinopathy. Moreover, the activity of plasma PAF-AH is increased in parallel with the severity of diabetic retinopathy. Given that diabetic retinopathy is the leading cause of blindness and severe loss of vision worldwide, affecting 93 million patients, it is important to clarify its pathogenesis both for prevention and treatment of the disease.
Conflict of interest All authors have no conflict of interest to declare and no financial support was offered for the present study.
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