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Evaluation of the relationship between serum and humor aqueous raftlin (Rftn1) levels and diabetic retinopathy

  • Selma Mesen ORCID logo EMAIL logo , Aysegul Comez ORCID logo , Ali Mesen ORCID logo , Abdullah Beyoglu ORCID logo and Muhammed Seyithanoglu ORCID logo
Published/Copyright: November 25, 2024
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 49 Issue 6

Abstract

Objectives

Comparing serum and humor aqueous (HA) raftlin levels in diabetic patients and control group.

Methods

In this prospective study, patients were divided into two groups diabetes mellitus (DM) and a control group. The DM group was divided into three subgroups. A total of 160 patients, including 35 without diabetic retinopathy (non-DR) (Group 1), 31 non-proliferative diabetic retinopathy (NPDR) (Group 2), 32 proliferative diabetic retinopathy (PDR) (Group 3), and 62 controls (Group 4), were included in the study. Venous blood and HA samples were taken from the patients and their raftlin levels were measured.

Results

Serum raftlin levels were decreased in the DM main group (p=0.046) but there was no significant difference in HA raftlin levels (p=0.838). There was no significant difference between the subgroups (Groups 1, 2, 3, and 4) in terms of serum and HA raftlin levels. Diabetic macular edema (DME), anti-vascular endothelial growth factor (anti-VEGF), and panretinal photocoagulation (PRP) application were not found to be associated with raftlin levels in Group 3. A moderate positive correlation was found between serum and HA raftlin levels (r=0.491). There was a negative correlation between serum raftlin levels and serum glucose levels (p=0.05). No correlation was found between serum and HA raftlin levels and serum leukocyte and lipid levels (p>0.05).

Conclusions

In our study, raftlin levels were examined for the first time in DM patients and it was observed that serum raftlin levels were negatively correlated with glucose levels. Raftlin molecule may be involved in the etiology of DM through VEGF.

Keywords: humor aqueous; raftlin; diabetic retinopathy; biomarker; serum

Introduction

Diabetic retinopathy (DR) is among the microvascular complications of diabetes mellitus (DM) and is the most common cause of preventable blindness worldwide [1]. The main known risk factors for the development of DR are hyperglycemia, duration of DM, hypertension, and genetic factors. Investigating the pathophysiological mechanisms that cause the development of DR will be of great benefit in the development of future alternative treatments. Although various biochemical, hemodynamic, and immunological mechanisms of vascular changes seen in DR have been reported, it is still not clearly understood. However, the importance of inflammation and angiogenesis in the pathogenesis of DR is gradually increasing. Vascular endothelial growth factor (VEGF) is known to play an important role in the development of DR and diabetic macular edema (DME). In hypoxic conditions, VEGF expression increases, endothelial cells (EH) are activated by vascular endothelial growth factor receptor-2 (VEGFR-2), and neovascularization development is induced [2].

Cell membranes contain specialized microregions called lipid piles (rafts). These regions are rich in cholesterol, sphingolipids, and phospholipids and also contain receptor proteins [3]. Lipid raft regions function in the vascular component of inflammation by regulating signal transduction [4]. Raftlin (RFTN-1) molecule is the major lipid raft protein first identified in B lymphocyte cells. It is involved in antigen receptor signal transduction [5]. In addition, it is known to play an important role in TLR3, and TLR4-mediated immune reactions, and autoimmune responses in various cell types 6], [7], [8. Raftlin is a new parameter involved in the pathophysiology of the vascular inflammatory response and associated with endothelial cell dysfunction. Bayliss AL et al. reported that there is raftlin protein in endothelial cells. This protein has been shown to be a component of the activated VEGFR-2 signaling complex and controls EH proliferation through the VEGF-VEGFR interaction [9].

Nowadays, anti-VEGF agents are widely used in the treatment of complications that develop due to VEGF stimulation of neovascularization in DR cases. Alternative proangiogenic pathways mediated by fibroblast growth factor, placental growth factor or interleukins are blamed for vascular pathologies resistant to anti-VEGF treatment. As a second perspective on the mechanism of resistance to anti-VEGF treatment, we thought that deficiency or blockade at any stage of the VEGF-VEGFR interaction pathways may be associated with a reduced effect. The fact that raftlin molecule is involved in the VEGF-VEGFR interaction pathways suggested that it may also play a role in the etiology of DR. If this relationship can be proven, the raftlin molecule may come to the fore as an alternative treatment agent for DR and DME in the future.

According to our research, there is no previous study examining raftlin levels in DR patients and humor aqueous (HA). The aim of this study is to have information about whether the raftlin molecule has a role in the etiology of DR. For this reason, we investigated serum and HA raftlin levels in DM patients and compared the data of DR patients grouped according to their stages and the control group.

Materials and methods

Clinical examination and study design

This research was designed as a prospective case-control study and it was approved by Kahramanmaras Sutcu Imam University Faculty of Medicine Clinical Research Ethics Committee (08.02.2021-101). In the study, the principles of the Declaration of Helsinki were adhered to and written consent was obtained from all participants.

The study was conducted by Kahramanmaras Sutcu Imam University Ophthalmology Department between March 2021 and November 2021. One hundred and sixty eyes of 160 patients were included in the study. The patients in the study were divided into two groups those with and without Type 2 DM. According to a detailed fundus examination of patients diagnosed with Type 2 DM; it is divided into three subgroups without diabetic retinopathy (non-DR), non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR). The control group consists of patients without DM. DR classification in our study was made according to the International Clinical Disease Severity Scale for DR, which was created based on the findings of the Wisconsin Diabetic Retinopathy Epidemiological Study (WESDR) and the Early Treatment of Diabetic Retinopathy Study (ETDRS) [10]. According to this classification, patients without significant diabetic fundus changes constitute the non-DR group. NPDR has three stages: mild, moderate and severe NPDR. Mild NPDR indicates the presence of a few microaneurysms. Medium NPDR indicates the presence of microaneurysms, intraretinal hemorrhage, or venous beading. Severe NPDR is based on the 4:2:1 rule. According to this rule, there are hemorrhages in four quadrants, venous beading in two or more quadrants, and intraretinal microvascular abnormalities in one or more quadrants. PDR is characterized by neovascularization of the optic disc, retina, iris or iridocorneal angle, vitreous hemorrhage or tractional retinal detachment.

Eye distribution in our research; 35 eyes are non-DR (Group 1), 31 eyes are NPDR (Group 2), 32 eyes are PDR (Group 3), and 62 eyes are control group (Group 4).

Inclusion criteria: 1. Patients between 40 and 80 years of age, 2. Control group patients not diagnosed with type 2 DM, patients without DR findings, NPDR and PDR patients.

Exclusion criteria: 1. Patients with the diagnosis of Type 1 DM, 2. Those with additional ophthalmological diseases such as glaucoma, uveitis, traumatic cataract, age-related macular degeneration, retinal artery or vein occlusion, retinal detachment, and degenerative myopia, 3. History of previous intraocular surgery or ocular trauma, 4. Patients with DR who underwent intravitreal dexamethasone implant in the last six months or intravitreal anti-VEGF in the last three months, 5. Patients with DR who underwent laser photocoagulation in the last three months, 6. History of systemic autoimmune and inflammatory disease.

Demographic data of all patients, detailed ophthalmological examination findings including ‘best-corrected visual acuity (BCVA in logMAR), biomicroscopic examination, intraocular pressure (IOP) measurement and detailed fundus examination’ and preoperative laboratory data were recorded.

The medical histories of the patients were examined by detailed file scanning. Patients who received any of the intravitreal anti-VEGF treatments such as ranibizumab (Lucentis®), bevacizumab (Avastin®), and aflibercept (Eylea®) were recorded. Cases in which a pan-retinal photocoagulation (PRP) scar was detected during detailed fundus examination were recorded as “PDR patients who underwent PRP”.

Data collection and analysis

Peripheral venous blood collection

Venous blood samples were taken from all patients by phlebotomy in yellow-capped tubes without anticoagulant on the day of the operation. The blood samples were centrifuged at 4,000 rpm for 10 min in Kahramanmaraş Sutcu Imam University Biochemistry Laboratory. The serum obtained after centrifugation was stored at −80 °C until the study day.

Removal of anterior chamber fluid

At the beginning of cataract surgery, a side port was opened with a 20 gauge microvitreoretinal (MVR) blade. Approximately 0.1–0.2 cc of HA fluid was taken from the anterior chamber with an insulin injector with a cannula. After the HA removal procedure, some balanced salt solution was administered to the anterior chamber, and the cataract surgery procedure was continued safely. HA samples were stored at −80 °C until the study day.

Determination of raftlin (RFTN1) by ELISA method

After the planned number of patients was reached, the samples were removed from −80 °C and allowed to dissolve slowly at room temperature. Raftlin (RFTN1) levels were analyzed by the ELISA method. Serum and HA raftlin levels were measured with MyBioSource Inc brand HUMAN RAFTLIN Elisa kits (San Diego, USA, Catalog No: MBS1600006). The procedure in the kit was used in the study. The kit detection range was 0.5–40 ng/mL and the kit sensitivity was 0.41 ng/mL. Sample results were calculated according to the standard curve and processed as ng/mL. The R2 values of their standard curves ranged from 0.989 to 0.999.

Laboratory test measurement methods

The levels of lipids and glucose were measured using original kits with a biochemical autoanalyzer (Roche Diagnostics GmbH, Mannheim, Germany). Complete blood count (CBC) parameters were analyzed with a Sysmex XN 1000 (Sysmex Corp., Kobe, Japan) hematology analyzer. Hemoglobin, erythrocytes, leukocytes, neutrophils, lymphocytes and platelet numbers were measured. HbA1c analysis was performed on the BioRad variant II device (Bio-Rad Laboratories, Hercules, CA, ABD) using HbA1c/thalassemia dual kits according to the manufacturer’s instructions. Measurement principle depended on cation exchange chromatography.

Statistical analysis

In the sample size calculation in G power analysis: one-way, effect size 0.5, alpha 0.05, beta 0.80, significance test of the difference between two means/independent Student’s t-test, the sample size is 51 for each group and a total of 102.

Statistical Package for the Social Sciences (SPSS) (ver:23) software program was used in the statistical analysis of the results obtained in the study. Descriptive statistics for continuous variables were expressed as mean ± standard deviation and median (min–max), while categorical variables were expressed as numbers and percentages. Kolmogorov-Smirnov or Shapiro-Wilk tests were applied to fit the normal distribution. Levene Test was performed for variance homogeneity. Statistical difference between two independent groups was evaluated with Mann-Whitney U Test and Student’s t-test. The statistical difference between more than two independent groups was evaluated with the Kruskal–Wallis Test and one-way ANOVA. Pearson or Spearman correlation coefficients were calculated. Chi-square test and ratio comparison were performed to determine the relationships between categorical variables. A logistic regression was applied to the multivariate analysis. Statistical significance level was accepted as p<0.05.

Results

Demographic data of the participants

Demographic data of the study groups are presented in Table 1. There was no significant difference between the groups in terms of age, sex, and body mass index (BMI) (p>0.05). In addition, no statistically significant difference was found between the participants in terms of preoperative BCVA and IOP (p=0.139 and p=0.873, respectively).

Table 1:

Demographic characteristics of the patients.

Diabetic patient Control p-Value
Group 1 (n=35) Group 2 (n=31) Group 3 (n=32) Group 4 (n=62)
Age, years
 Mean (SD) 65.08 (7.79) 67.64 (6.04) 64.40 (9.48) 64.54 (7.54) 0.283a
Gender, n (%)
 Female 25 (71.4) 23 (74.2) 18 (56.3) 34 (54.8) 0.166b
 Male 10 (28.6) 8 (25.8) 14 (43.8) 28 (45.2)
BMI
 Median (25–75 %) 29.40 (29.30–31.80) 29.30 (26.20–31.20) 28.55 (26.10–32.10) 27.60 (23.50–31.40) 0.159c
HT, n (%)
 Yes 23 (65.7) 19 (65.5) 23 (71,9) 23 (37.1) 0.002 b
 No 12 (34.3) 10 (34.5) 9 (28.1) 39 (62.9)
DM family history, n (%)
 Yes 21 (60) 26 (83.9) 26 (81.3) 23 (37.7) <0.001 b
 No 14 (40) 5 (16.1) 6 (18.8) 38 (62.3)
DM duration, years
 Median (25–75 %) 6.00 (2.00–15.00) 8.00 (5.00–10.00) 15.00 (10.00–20.00) <0.001 c
DM treatment, n (%)
 OAD 24 (68.6) 12 (38.7) 5 (15.6) <0.001 b
 İnsulin 8 (22.9) 17 (54.8) 22 (68.8)
 OAD+İnsulin 0 (0.0) 0 (0.0) 4 (12.5)
 Without treatment 3 (8.6) 2 (6.5) 1 (3.1)
BCVA (Log MAR)
 Median (25–75 %) 0.70 (0.70–1.00) 0.70 (0.52–1.00) 1.00 (0.70–1.30) 0.70 (0.52–1.00) 0.139c
IOP, mmHg
 Median (25–75 %) 14.00 (14.00–16.00) 14.00 (11.00–14.00) 15.00 (12.25–15.00) 14.00 (12.00–15.00) 0.873c

  1. Data in bold indicate statistically significant differences, i.e. p<0.05. aANOVA test, bPearson Chi-square test, cKruskal-Wallis H test, DM, diabetes mellitus; BMI, body mass index; HT, hypertension; OAD, oral anti-diabetic; BCVA, best corrected visual acuity; IOP, intraocular pressure.

Laboratory data of patients

In DM subgroups, it was observed that serum HbA1c and glucose levels increased statistically significantly as the severity of DR increased (p=0.017 and p=0.031, respectively). There was no statistical difference between the groups in terms of serum leukocyte levels. Likewise, no statistically significant difference was found between the groups in terms of serum lipid values (p=0.799 for VLDL, p=0.386 for LDL, p=0.184 for HDL, p=0.791 for triglyceride and p=0.051 for cholesterol) (Table 2). Considering Post Hoc pairwise comparison analyses among DM patient subgroups in terms of time to diagnosis, serum HbA1c, and glucose values; a significant difference was detected between Groups 1–3 and Groups 2–3 in terms of disease duration, between Groups 1–2 and Groups 1–3 in HbA1c values, and between Groups 1–2 and Groups 1–3 in serum glucose values (Table 3).

Table 2:

Laboratory results of the study groups.

DM Control p-Value
Group 1 Group 2 Group 3 Group 4
HbA1c, %
 Median (25–75 %) 7.15 (6.35–9.40) 9.10 (7.60–10.72) 9.30 (8.05–10.10) 0.017a
Glucose, mg/dL
 Median (25–75 %) 172.0 (109.0–227.0) 208.0 (145.0–324.0) 216.5 (161.5–254.7) 0.031a
Leukocyte values (109/L)
 LymphocyteMedian (25–75 %) 2.45 (1.96–3.27) 2.38 (1.75–2.79) 2.05 (1.69–2.40) 2.20 (1.93–2.67) 0.090a
 NeutrophilMedian (25–75 %) 5.07 (4.18–5.87) 5.50 (3.75–6.96) 4.82 (3.76–5.81) 4.32 (3.83–5.27) 0.147a
 EosinophilMedian (25–75 %) 0.16 (0.12–0.23) 0.15 (0.08–0.17) 0.14 (0.09–0.20) 0.15 (0.07–0.23) 0.537a
 MonocyteMedian (25–75 %) 0.64 (0.53–0.73) 0.64 (0.55–0.78) 0.52 (0.46–0.62) 0.57 (0.51–0.71) 0.070a
 BasophilMedian (25–75 %) 0.04 (0.03–0.07) 0.04 (0.02–0.05) 0.04 (0.03–0.06) 0.04 (0.03–0.06) 0.619a
Lipid profile, mg/dL
 VLDL-CMedian (25–75 %) 34.50 (23.00–46.25) 37.00 (24.75–42.00) 36.00 (26.00–52.00) 33.00 (23.00–50.50) 0.799a
 LDL-CMean (SD) 119.79 (36.03) 123.40 (33.04) 118.32 (36.84) 126.75 (29.14) 0.386b
 HDL-CMedian (25–75 %) 46.00 (38.00–53.50) 49.55 (41.00–56.00) 42.0 (37.00–50.50) 47.0 (39.50–53.00) 0.184a
 TriglycerideMedian (25–75 %) 173.50 (115.50–229.75) 186.50 (124.00–209.25) 181.00 (129.00–262.00) 164.00 (115.50–249.25) 0.791a
 CholesterolMean (SD) 185.91 (47.04) 187.92 (35.50) 187.62 (46.30) 192.95 (34.70) 0.051b

  1. Data in bold indicate statistically significant differences, i.e. p<0.05. aKruskal-Wallis H test, bOne-way-ANOVA test, VLDL, very-low density lipoprotein; LDL, low density lipoprotein; HDL, high-density lipoprotein.

Table 3:

Post hoc comparison analysis of DM duration, serum glucose and HbA1c values between groups.

Group 1–2 Group 1–3 Group 2–3
DM duration p=1.00 p≤0.01 p≤0.01
HbA1c p=0.04 p≤0.01 p=1.00
Glucose p=0.04 p=0.02 p=1.00

  1. DM, diabetes mellitus.

Serum and HA raftlin levels

There was a statistically significant difference in serum raftlin levels between DM patients and the control group (p=0.046). Serum raftlin levels were higher in the control group than in the DM group. HA raftlin levels were decreased in the DM group compared to the control group, but the difference was not statistically significant (p=0.838). No statistically significant difference was found between the DM subgroups and the control group in terms of serum and HA raftlin levels (p=0.071 and p=0.578, respectively) (Table 4).

Table 4:

Distribution of serum and HA raftlin (ng/mL) levels in DM patients and control group.

Serum raftlin, ng/mL HA raftlin, ng/mL
Diabetic main group
 Median (25–75 %) 3.45 (2.53–4.19) 7.93 (7.20–8.84)
Control
 Median (25–75 %) 3.69 (3.02–4.93) 8.03 (7.39–8.56)
p-Valuea (effect sizec) 0.046 (0.25) 0.838
Diabetic subgroups
 Median (25–75 %) Group 1 3.23 (2.32–4.06) 8.16 (7.49–8.79)
Group 2 3.35 (2.65–4.07) 7.97 (6.81–9.06)
Group 3 3.65 (3.07–4.56) 7.64 (6.75–8.75)
Control
 Median (25–75 %) Group 4 3.69 (3.02–4.93) 8.03 (7.39–8.56)
p-Valueb 0.071 0.578

  1. Data in bold indicate statistically significant differences, i.e. p<0.05. aMann-Whitney U Test, bKruskal-Wallis H Test, cCohen d calculation, HA, humor aqueousca.

None of the group 2 patients had DME and only 3 (9.6 %) patients had a previous history of intravitreal anti-VEGF injection. In group 3 patients, 10 (31.25 %) patients had DME, 19 (59.37 %) patients had a history of intravitreal injection, and 28 (87.5 %) patients had a history of PRP. Group 3 patients were divided according to clinical features such as DME, intravitreal injection, and PRP history. Serum and HA raftlin levels were compared according to these characteristics, but no statistically significant difference was found (Table 5).

Table 5:

The relationship between serum and HA raftlin levels and history of DME, intravitreal anti-VEGF and PRP in Group 3.

n Serum raftlin, ng/mL HA raftlin, ng/mL
DME

Mean (SD)		 Yes 10 3.53 (1.10) 7.26 (1.10)
No 22 4.02 (1.27) 7.93 (1.35)
p-Valuea 0.306 0.186
Anti-VEGF

Mean (SD)		 Yes 19 4.03 (1.23) 7.94 (1.16)
No 13 3.48 (1.08) 7.24 (1.44)
p-Valuea 0.219 0.197
PRP

Mean (SD)		 Yes 28 3.86 (1.21) 7.80 (1.29)
No 4 3.44 (1.06) 6.60 (0.92)
p-Valuea 0.507 0.134

  1. aIndependent Sample T-test, PRP, panretinal photocoagulation; SD, standard deviation; DME, diabetic macular edema; VEGF, vascular endothelial growth factor; PRP, pan-retınal photocoagulatıon; HA, humor aqueous.

Correlation analysis

When serum and HA raftlin levels were evaluated by Spearman’s correlation analysis, a moderate positive correlation was found between them (p<0.001, r=0.491) (Figure 1). No significant correlation was found between serum and HA raftlin levels and serum lipid levels and leukocyte levels.

Figure 1: Spearman’s correlation analysis graph of serum and humor aqueous raftlin values. The correlation value is moderate (0.491).
Figure 1:

Spearman’s correlation analysis graph of serum and humor aqueous raftlin values. The correlation value is moderate (0.491).

Linear regression analysis

According to the linear regression analysis, no correlation was found between serum raftlin levels and the patient’s age, BMI, duration of DM, and HbA1c values. It was determined that there was a negative correlation between serum glucose values and serum raftlin levels (p=0.05).

Logistic regression analysis

The results of multivariate logistic regression analysis of factors thought to be effective in the development of diabetes mellitus are shown in Table 6. Sensitivity and specificity were 89.0 % and 84.6 %, respectively.

Table 6:

Multivariate logistic regression analysis of factors thought to be effective in the development of diabetes mellitus.

Variable B Exp (B) 95 % C.I. for Exp (B) p-Value
BMI 0.008 1.008 0.886–1.147 0.903
HT 1.131 3.099 0.845–11.367 0.088
DM family history 0.907 2.477 0.672–9.123 0.173
Glucose −0.053 0.948 0.924–0.973 ≤0.001
Serum raftlin −0.093 0.911 0.642–1.294 0.604
HA raftlin 0.262 1.300 0.704–2.400 0.401

  1. Data in bold indicate statistically significant differences, i.e. p<0.05. BMI, body mass index; HT, hypertension; HA, humor aqueous.

Discussion

Our data showed a significant difference in serum raftlin levels between the DM and control groups. Serum raftlin levels of DM patients were lower than the control group. HA raftlin levels were decreased in DM patients compared to controls, however, the difference was not statistically significant. Although a decrease was observed in HA raftlin values in the DR subgroups as the severity of DR increased, the difference was not significant. It is known that the risk of DR increases in patients with high HbA1c values and poor glycemic control [11]. In previous studies, fasting plasma glucose and HbA1c values were generally used to measure plasma glucose levels. In our study, since it was not possible to measure fasting plasma glucose in outpatient settings, we randomly examined plasma glucose and HbA1c levels. Surprisingly, we observed that glucose levels also increased in proportion to the severity of DR, and the difference between groups was statistically significant. In addition, while HbA1c levels could not be correlated with serum raftlin levels, it was observed to be negatively correlated with glucose levels. Considering these results, we thought that high blood glucose levels might affect systemic raftlin levels through acute inflammation rather than chronic inflammation.

Our study was designed to examine raftlin levels only. Therefore, we did not have the chance to quantitatively show whether there is a relationship between raftlin-VEGF levels. However, there are many studies in the literature showing a positive correlation between increased serum and vitreous VEGF levels and DR severity 12], [13], [14. Considering the function of the raftlin protein in the VEGF-VEGFR-2 interaction in the study of Baylis et al., there may be a relationship between raftlin values and VEGF levels. In addition, serum and HA raftlin levels in diabetic patients were shown for the first time in this study. Therefore, our study may guide further comprehensive studies on whether raftlin levels are associated with systemic and local increased VEGF activation in DM patients.

DME is the main cause of decreased visual acuity in DR patients. It occurs as a result of extracellular fluid accumulation following internal blood-retinal barrier dysfunction caused by various pro-inflammatory mediators. DME can occur at any stage of DR, NPDR or PDR. One of the main treatment goals in DR cases is to eliminate DME. In our study, we examined whether there was any relationship between raftlin levels and DME. While working on this subject, we tried to avoid cataract surgery in the presence of DME. Because it is reported in the literature that there is an increase in the progression of DR after cataract surgery in patients with DM [15, 16]. However, surgery would be beneficial for cataracts that reduce the level of vision and prevent the follow-up and treatment of DR. Since Group 2 did not provide appropriate patient distribution in DME evaluation, this comparison was made in Group 3 cases. In Group 3, no statistically significant relationship was detected between the presence of DME and serum and HA raftlin levels. The main treatment of DR consists of intravitreal anti-VEGF injection and PRP application to the peripheral retina. Among the anti-VEGF agents, ranibizumab can bind VEGF-A, aflibercept binds VEGF-A and VEGF-B, and bevacizumab can bind all isoforms of VEGF. Laser treatment aims to cause thermal damage by absorbing the energy reaching the retina by the pigments in the tissue. It is thought that existing neovascular tissues regress due to increased oxygen transfer from the choroid in areas with laser scars and a general decrease in oxygen consumption as a result of retinal thermal damage. We thought that raftlin levels might be affected to these treatments. However, when we looked at Group 3, we saw that serum and humor aqueous raftlin values were not statistically affected. However, since the groups are not homogeneously distributed, this result may not reflect the truth. In addition, patients who underwent PRP in the last 3 months were not included in our study, considering that it may change the results. The majority of group 3 patients were PDR patients who were stable.

It has been reported that the protein content in HA consists mostly of glycoproteins secreted by the ciliary body and is of lower density compared to plasma [17]. However, it was interesting that the mean HA raftlin levels in our data were 2–3 times higher than the mean serum raftlin. Previous analyzes have reported increased or decreased expression of various proteins in HA content with the presence of cataracts, glaucoma, or previous vitrectomy surgery [18]. Although HA from healthy adults shows ideal results for such molecular-level studies, obtaining samples is ethically difficult. Participants with a history of previous ocular surgery or additional ocular pathology were not included in the study. The common feature of all participants was the presence of cataracts that required surgery. The presence of cataracts may have affected HA protein components and concentration, resulting in higher raftlin levels compared to plasma values.

Raftlin studies in ophthalmology are limited to genome-level analyzes only in glaucoma patients. Overexpression of the RFTN-1 gene was observed in the rat model with acute IOP elevation and in primary open-angle glaucoma (POAG) patients. It has been suggested that this may pose a risk for POAG through increased HA production or a change in HA content 19], [20], [21. Our study did not include any participant with a history or symptom of glaucoma. The IOP values of the participants were within the normal range and there was no statistically significant difference between the groups. According to our research, our study is the first study in which the raftlin molecule was shown in HA liquid.

In the literature review, serum and tissue levels of raftlin were investigated in various patient groups. Lee et al. determined the relationship between serum raftlin levels and the severity of the disease in sepsis model mice and sepsis patients. They suggested that raftlin may be a biomarker for endothelial dysfunction [22]. In addition, while raftlin levels were found to be high in wound tissue and obstructive sleep apnea syndrome (OSAS) patients, maternal serum and placental raftlin levels were found to be low in intrauterine growth restriction (IUGR) cases [23, 24]. We did not find a study evaluating raftlin levels in DM patients before, so we did not have the opportunity to compare our results. In our study, we showed that serum raftlin levels were statistically significantly decreased in DM patients compared to the control group.

The regulatory role of the raftlin molecule in signal transduction by T lymphocytes, dendritic cells, and macrophages in both innate and adaptive immune reactions has been emphasized in previous studies 6], [7], [8. To elucidate whether raftlin levels were affected by these individual differences, we also examined the patients’ serum leukocyte values. There was no significant relationship between serum and HA raftlin levels and serum lymphocyte levels. Monocytes values in the groups were at similar levels, and no statistically significant difference was found. It has also been suggested that serum lipid levels may alter immune responses by influencing the composition of lipid raft regions in the cell membrane structure [25]. Likewise, considering these effects, serum lipid values were examined. In our study, serum lipid values in the groups were at similar levels. In addition, the correlation coefficients between raftlin levels and serum lipids were also examined and no significant correlation was found.

When we look at the missing parts of our study, the patients in the NPDR and PDR groups were stable patients who did not need intravitreal anti-VEGF and PRP in the last three months. Cataract surgery was not performed, considering that patients with active DR may progress rapidly after surgery. Patients with active PDR (vitreous hemorrhage, tractional RD, etc.) and requiring vitrectomy were not included in the study because preoperative intravitreal anti-VEGF and PRP were applied. Our data are preliminary in this regard and may shed light on more comprehensive studies that can be done in the future by including these patient groups. Raftlin levels were not evaluated in vitreous samples in this study. However, according to previous studies, it has been proven that the biomarker levels in HA and vitreous are directly related, equally significant and reliable in evaluating the metabolic process in the retina [26].

Despite all these shortcomings, according to our research, this is the first study evaluating raftlin levels in DM patients. In addition, although raftlin levels have been investigated in various body fluids and tissues before, this study has revealed for the first time that it is detectable in the anterior chamber fluid. Raftlin may be effective in the etiology of DR via VEGF pathways. Our data are preliminary to this theory. In order to better understand this issue, studies with a larger participant population are needed in which vitreous or HA raftlin levels and other mediators associated with angiogenesis and inflammation are evaluated together in patients with active vitreous inflammation.

Corresponding author: Dr. Selma Mesen, MD, Eye Clinic, Turkoglu Dr. Kemal Beyazit Public Hospital, Turkoglu, Kahramanmaras, TR 46800, Türkiye, E-mail:

  1. Research ethics: The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

  2. Informed consent: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards.

  3. Author contributions: The authors has accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Use of Large Language Models, AI and Machine Learning Tools: None declared.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: This research was supported by Kahramanmaraş Sütçü İmam University Scientific Research Projects Management Unit as a 2021/2-26 D-coded Project.

  7. Data availability: The raw data can be obtained on request from the corresponding author.

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Received: 2023-08-10
Accepted: 2024-05-15
Published Online: 2024-11-25

© 2024 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

  1. Frontmatter
  2. Research Articles
  3. Can different formulae be used in the diagnosis and staging of chronic kidney disease?
  4. 25-Hydroxyvitamin-D levels in Sjögren’s syndrome: is it the right time to dismiss the case or not?
  5. Ektacytometric examination of red blood cells’ morphodynamical features in diabetic nephropathy patients
  6. Evaluation of the relationship between serum and humor aqueous raftlin (Rftn1) levels and diabetic retinopathy
  7. Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages
  8. Expression levels of some genes in the MAPK pathway (DUSP1, DUSP2, DUSP4, DUSP6 and DUSP10) in eyelid tumor tissue
  9. Decreased plasma gelsolin in the COVID-19-related acute respiratory distress syndrome
  10. Differential proinflammatory responses of colon epithelial cells to SARS-CoV-2 spike protein and Pseudomonas aeruginosa lipopolysaccharide
  11. Screening for creatine transporter deficiency in autism spectrum disorder: a pilot study
  12. An in vitro assessment of ionizing radiation impact on the efficacy of radiotherapy for breast cancer
  13. Possible protective effect of remifentanil against testicular ischemia-reperfusion injury
  14. Comparison effect of hyperglycaemia induced mixed meal tolerance and oral glucose tolerance test on body oxidative stress
  15. Is serum hornerin a potential biomarker in fibromyalgia? A pilot study
  16. Collaborative online international learning (COIL): an engaging strategy for narrowing learning distances between two continents
  17. Reviewer Acknowledgment
  18. Reviewer Acknowledgment
https://doi.org/10.1515/tjb-2023-0176
Keywords for this article
humor aqueous; raftlin; diabetic retinopathy; biomarker; serum
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Research Articles
  3. Can different formulae be used in the diagnosis and staging of chronic kidney disease?
  4. 25-Hydroxyvitamin-D levels in Sjögren’s syndrome: is it the right time to dismiss the case or not?
  5. Ektacytometric examination of red blood cells’ morphodynamical features in diabetic nephropathy patients
  6. Evaluation of the relationship between serum and humor aqueous raftlin (Rftn1) levels and diabetic retinopathy
  7. Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages
  8. Expression levels of some genes in the MAPK pathway (DUSP1, DUSP2, DUSP4, DUSP6 and DUSP10) in eyelid tumor tissue
  9. Decreased plasma gelsolin in the COVID-19-related acute respiratory distress syndrome
  10. Differential proinflammatory responses of colon epithelial cells to SARS-CoV-2 spike protein and Pseudomonas aeruginosa lipopolysaccharide
  11. Screening for creatine transporter deficiency in autism spectrum disorder: a pilot study
  12. An in vitro assessment of ionizing radiation impact on the efficacy of radiotherapy for breast cancer
  13. Possible protective effect of remifentanil against testicular ischemia-reperfusion injury
  14. Comparison effect of hyperglycaemia induced mixed meal tolerance and oral glucose tolerance test on body oxidative stress
  15. Is serum hornerin a potential biomarker in fibromyalgia? A pilot study
  16. Collaborative online international learning (COIL): an engaging strategy for narrowing learning distances between two continents
  17. Reviewer Acknowledgment
  18. Reviewer Acknowledgment
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