Aqueous and Vitreous Levels of Scube-1 in Patients With Proliferative Diabetic Retinopathy: A Pilot Study

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Aqueous and Vitreous Levels of Scube-1 in Patients With Proliferative Diabetic Retinopathy: A Pilot Study

https://doi.org/10.21203/rs.3.rs-2494239/v1

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Background

To determine the aqueous humor and vitreous levels of SCUBE-1 in patients with proliferative diabetic retinopathy (PDR).

Methods

A total of 21 patients with PDR and 11 patients with idiopathic epiretinal membrane (control) were included in this case-control study. The patients with PDR were divided into two groups: patients with vitreous hemorrhage (VH) and patients with tractional retinal detachment (TRD) due to an active fibrovascular membrane. Aqueous and vitreous samples obtained at the beginning of surgery were analyzed for SCUBE-1 levels.

Results

The mean aqueous humor SCUBE-1 value (ng/mL) was 0.82 ± 0.18 in the VH group, 0.87 ± 0.36 in the TRD group and 0.75 ± 0.29 in the control group. There was no significant differences between groups in terms of aqueous humor SCUBE-1 levels (p > 0.05, for all values). The mean vitreous SCUBE-1 value (ng/mL) was 1.95 ± 0.52 in the VH group, 1.17 ± 0.29 in the TRD group and 0.89 ± 0.21 in the control group. Vitreous SCUBE-1 values in the VH group were significantly higher than in the TRD and control groups (p = 0.034 and p = 0.027, respectively). The difference between the TRD group and control group was also significant in terms of vitreous SCUBE-1 values (p = 0.044).

Conclusion

Higher vitreous SCUBE-1 levels in patients with VH indicate that SCUBE-1 can be a valuable marker of retinal ischemia in diabetic patients. Further, the lower vitreous levels of SCUBE-1 in patients with TRD may suggest that SCUBE-1 is specifically involved in the angiogenesis phase. Further studies are needed to define the role of SCUBE-1 in the course of PDR.

Health sciences/Medical research/Biomarkers/Prognostic markers

Health sciences/Diseases/Eye diseases/Retinal diseases

Aqueous humor

proliferative diabetic retinopathy

SCUBE-1

tractional retinal detachment

vitreous

vitreous hemorrhage

Proliferative diabetic retinopathy (PDR) leads to severe vision loss by causing vitreous hemorrhage (VH) and tractional retinal detachment (TRD). In PDR, neovascular vessels formed due to ischemia may progress to vitreoretinal fibrosis over time [1, 2]. Angiogenesis is the most important aspect of the multifactorial pathogenesis of PDR [3]. Vascular endothelial growth factor (VEGF) plays a major role in the underlying angiogenesis process in PDR, although placental growth factor (PGF), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) are also considered as potential biomarkers of angiogenesis in PDR [4, 6].

The newly identified cell surface protein, signal peptide-CUB-EGF domain-containing protein (SCUBE) family consists of cell surface proteins with three different isoforms [7]. The isoforms are named according to the order in which they were discovered, and thus SCUBE-1 is the first member of this family. It has been shown that SCUBE-1 is secreted from the vascular endothelium and also released from platelets [8]. SCUBE-1 can be detected in diseases in which inflammation and hypoxia play a role in the pathogenesis. Moreover, laboratory and clinical studies have demonstrated a relation between SCUBE-1 and the angiogenesis process [9–12].

One study on patients with diabetic retinopathy (DR) demonstrated that serum SCUBE-1 levels were significantly higher in patients with DR and suggested that SCUBE-1 could be a marker in the early diagnosis of DR [13]. However, SCUBE-1 levels in the intraocular fluids of patients with DR have not previously been investigated.

In the present study, to determine the possible role of SCUBE-1 in the pathogenesis of PDR, aqueous humor and vitreous SCUBE-1 levels were analyzed in a series of patients with PDR and non-diabetic patients with idiopathic epiretinal membranes (ERMs). Further, the association of SCUBE-1 levels with clinical presentations of PDR with VH and with TRD was evaluated separately.

This case-control study was performed in a tertiary reference center, the Ulucanlar Eye Training and Research Hospital, University of Health Sciences, Turkey, between December 2020 and October 2021. The study was performed in accordance with the ethical principles stated in the Declaration of Helsinki and was approved by the institutional ethics committee of the Ankara Training and Research Hospital (No. E-21-643). Signed informed consent was obtained from each subject following a detailed explanation of the study objectives and protocol.

Diabetic patients who had undergone surgery for PDR due to nonclearing VH or TRD caused by active fibrovascular membranes were included. Age- and gender-matched non-diabetic patients who underwent surgery for an idiopathic ERM were enrolled as controls. The patients with PDR were classified into two groups: VH and TRD with active fibrovascular membranes. Age, gender, duration of diabetes, insulin use, fasting blood glucose levels, serum hemoglobin A1c and previous panretinal photocoagulation were documented.

Exclusion criteria were the presence of VH or TRD secondary to ocular disease other than PDR, previous vitrectomy, previous intravitreal injection of any drugs or laser photocoagulation within the last three months, previous ocular surgery within the last six months and a history of acute ischemic or inflammatory systemic disease within the last one year.

Sample collection

Aqueous humor samples (0.1 ml) were obtained through anterior chamber paracentesis with a 27-gauge needle prior to any surgical intervention. Undiluted vitreous samples (0.5 ml) were collected using a vitrectome at the onset of vitrectomy before opening the infusion pole. The specimens were immediately transferred to Eppendorf tubes and stored at − 80 C until the assay.

Measurement of SCUBE-1 levels

Both aqueous humor and vitreous concentrations of SCUBE-1 were determined using a human SCUBE-1 enzyme-linked immunosorbent assay kit (Elabscience, Shanghai, CHINA), following the manufacturer’s instructions. Aqueous humor and vitreous samples were added to micro ELISA plates coated with human SCUBE-1 specific antibodies without dilution and incubated at 37 ° C for 90 minutes. The samples were removed from the plates and then incubated at 37 ° C for 60 minutes by adding biotin-conjugated antibody specific for SCUBE-1 to the same plates. After washing, avidin-conjugated horseradish-peroxidase (HRP) was added to the plates and incubated at 37 ° C for 60 minutes. Then, unbound avidin-enzyme reagents were removed by washing. A substrate solution was added and incubated at 37 ° C for 15 minutes. After the solution turned blue, the reaction stop solution was added to the plates, and the yellow formed substrates were read using an ELISA reader at 450 nanometers (BioTek Elx800 Absorbance Microplate Reader). The results were calculated based on the calibration curve. The sensitivity of the ELISA kit was 0.38 ng/mL, the detection range was 0.63–40 ng/mL and the reproducibility (coefficient of variation) was < 10%.

Statistical Analysis

The data were analyzed with SPSS software (version 17.0, SPSS, inc., Chicago, IL, USA) The normality of the data was tested using analytical methods (Kolmogorov–Smirnov and Shapiro–Wilk tests). Sex was compared using the chi-square test. All data were normally distributed, and thus an independent t test was performed to compare two independent groups. To compare more than two independent groups, a one-way ANOVA was used. In addition, a paired t test was performed to compare the aqueous and vitreous levels in each group. The variables are presented as the mean ± standard deviation, minimum–maximum and percentage (%), where appropriate. A p-value of less than 0.05 was considered to be statistically significant.

A total of 21 patients with PDR and 11 patients with idiopathic ERM were included. Of the 21 patients with PDR, 10 were in the VH group and 11 were in the TRD group. All TRD patients and no VH patients had a panretinal laser photocoagulation (PRP) history.

The mean patient age (years) was 54.7 ± 9.2 in the VH group, 53.5 ± 10.2 in the TRD group and 58.2 ± 7.0 in the ERM (control) group, and 50% of the patients with VH, 45.5% of the patients with TRD and 45.5% of the control group were female. The groups were age and gender matched (p > 0.05) (Table 1).

Table 1

Comparison of demographic characteristics and laboratory and clinical data between groups
	PDR		Control (n:11)	p
	VH (n:10)	TRD (n:11)	Control (n:11)	p
Age (years)	54.7 ± 9.2	53.5 ± 10.2	58.2 ± 7.0	0.182^a
Gender (M/F)	5/5	6/5	6/5	0.934^b
DM period (years)	16.2 ± 2.8	14.8 ± 3.7		0.587^a
Duration of Insulin use (years)	6.21 ± 1.8	5.05 ± 1.7		0.659^a
Fasting blood glucose (mg/dl)	235.19 ± 7.32	248.25 ± 9.58		0.634^a
HbA1c (%)	9.7 ± 1.3	10.2 ± 1.9		0.815^a
Data are presented as mean ± standard deviation
DM diabetes mellitus, F female, Hbg A1c hemoglobin A1c, M male, PDR proliferative diabetic retinopathy, VH vitreous hemorrhage, TRD tractional retinal detachment
^a One-way ANOVA test
^b Chi-square test

The mean aqueous humor SCUBE-1 values (ng/mL) were 0.82 ± 0.18 in the VH group, 0.87 ± 0.36 in the TRD group and 0.75 ± 0.29 in the control group. There were no significant differences between groups in terms of aqueous humor SCUBE-1 levels (p > 0.05 for all values). The mean vitreous SCUBE-1 values (ng/mL) were 1.95 ± 0.52 in the VH group, 1.17 ± 0.29 in the TRD group and 0.89 ± 0.21 in the control group. The vitreous SCUBE-1 values in the VH group were significantly higher than in the TRD and control groups (p = 0.034 and p = 0.027, respectively). The difference between the TRD group and control group was also significant in terms of vitreous SCUBE-1 values (p = 0.044) (Table 2). The levels of SCUBE-1 in the vitreous were significantly higher than in the aqueous humor in the VH group (p = 0.033). There were no significant differences between the aqueous humor SCUBE-1 values and the vitreous SCUBE-1 values between the TRD and control groups (p > 0.05) (Table 2).

Table 2

Comparison of aqueous and vitreous SCUBE-1 levels in the control and proliferative diabetic retinopathy groups
		VH	TRD	Control	p^a	p^b	p^c
SCUBE-1 (ng/mL)	Aqueous	0.82 ± 0.18	0.87 ± 0.36	0.75 ± 0.29	0.842	0.342	0.254
SCUBE-1 (ng/mL)	Vitreous	1.95 ± 0.52	1.17 ± 0.29	0.89 ± 0.21	0.034	0.027	0.044
	p^d	0.033	0.235	0.756
Values in bold indicate statistically significant of p < 0.05 level
Data are presented as mean ± standard deviation
VH vitreous hemorrhage, TRD tractional retinal detachment
^a Between VH and TRD groups
^b Between VH and Control groups
^c Between TRD and Control groups
^d Between aqueous and vitreous in each group

In the present study, the vitreous SCUBE-1 levels of the PDR group were significantly higher than those of the non-diabetic control group. Moreover, among the PDR group, vitreous SCUBE-1 levels were significantly higher in patients with VH than in patients with TRD. To the best of our knowledge, this is the first study to investigate SCUBE-1 levels in the intraocular fluid of patients with PDR.

The SCUBE family is a novel vascular biomarker involved in the processes of ischemia, hypoxia, oxidative stress and pathological angiogenesis. SCUBE members have been proven to interact with angiogenesis-related molecules, such as TGF-β, platelet-derived growth factor D (PDGF-D) and interleukin (IL)-17F [7, 14]. Further, it has been demonstrated that SCUBE members are upregulated in the ischemic microenvironment and promote angiogenesis by acting as proangiogenic factors in angiogenesis-related molecule signaling networks [15, 16].

SCUBE-1 has been investigated in relation to various ischemic diseases, and it has been found to increase in the acute phase of ischemia. For example, Dai et al. demonstrated that plasma SCUBE-1 concentrations were significantly elevated in acute coronary syndrome and acute ischemic stroke [9]. SCUBE-1 levels were also found to be higher in acute mesenteric ischemia, and SCUBE-1 was proposed as a potential marker for the early diagnosis of acute vascular injury [17]. Moreover, Zhuang et al. evaluated SCUBE-1 levels in hypoxic kidney injury, finding that they increased during early injury and decreased in the late regeneration phase [16].

Proliferative diabetic retinopathy is a major cause of blindness, and ischemia and angiogenesis play important roles in its pathogenesis. The process starts with abnormal angioproliferation, followed by tractional fibrovascular bands [1–3]. VH in diabetic patients indicates the presence of active neovascularization of the disk (NVD) or neovascularization elsewhere (NVE) and expression of angiogenic factors, as retinal ischemia is still present. Tractional diabetic retinal detachment is an advanced form of PDR resulting from traction on the retina with newly formed vessels and an accompanying frame of fibrotic tissue and contractile elements [18]. Fibrosis increases in the late stages of PDR, especially following PRP, perhaps because of a decrease in VEGF levels and upregulation of connective tissue growth factors [19, 20]. Van Geest et al. defined this process as the “angio-fibrotic switch,” in which a shift in the balance between VEGF and growth factors causes the switch from angiogenesis to fibrosis in PDR [21]. In our study, patients with VH had higher vitreous levels of SCUBE-1, suggesting that SCUBE-1 could be a valuable marker or target molecule in retinal ischemia in diabetic patients. Further, the lower vitreous SCUBE-1 levels in patients with TRD may suggest that the level of SCUBE-1 in the vitreous decreases during the angio-fibrotic switch process and that SCUBE-1 is specifically involved in the angiogenesis phase.

We also compared the aqueous humor and vitreous SCUBE-1 values in both groups and found that the level of SCUBE-1 in the vitreous was significantly higher than that in the aqueous humor only in the VH group. Gradient-driven diffusion of SCUBE-1 from the vitreous to the aqueous in the VH group may explain this result.

There are several limitations of the present study that should be mentioned. First, the sample size of the groups was relatively small. Second, we only studied intraocular fluid levels of SCUBE-1; we did not analyze blood SCUBE-1 levels. However, this is the first study on the subject, and more comprehensive analyses are warranted.

In summary, our study indicates that SCUBE-1 appears to play a role in the angiogenic phase rather than in the later fibrotic phase of PDR. Further studies with larger sample sizes and more molecules are required to define the role of SCUBE-1 during the course of PDR.

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Aqueous and Vitreous Levels of Scube-1 in Patients With Proliferative Diabetic Retinopathy: A Pilot Study

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Abstract

Background

Methods

Results

Conclusion

Introduction

Materials And Methods

Sample collection

Measurement of SCUBE-1 levels

Statistical Analysis

Results

Discussion

References

Additional Declarations

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