Elevated aqueous endothelin-1 concentrations in advanced diabetic retinopathy.

PURPOSE: Endothelin-1 (ET-1) is a potent vasoconstrictor which seems to be involved in the pathogenesis of diabetic retinopathy (DR). However, studies on intraocular ET-1 in DR have been limited. Thus, we investigated aqueous ET-1 levels in patients with DR.
METHODS: A total 85 subjects were included in this prospective study. Those were classified into three groups: advanced DR group included those with severe nonproliferative DR or proliferative DR, whereas early DR group included those with mild nonproliferative DR or moderative nonproliferative DR. Those who underwent cataract surgery and had no concomitant ocular disease were included in the control group. Aqueous humor levels of ET-1 were obtained before intravitreal bevacizumab injection (IVB) and after 1 month for the DR patients, and at the time of cataract surgery for the control group.
RESULTS: Advanced DR group included 40 eyes (47.1%), whereas early DR group did 19 eyes (22.4%), and control group (26 eyes, 30.5%). Mean aqueous ET-1 level was 10.1±4.1 pg/mL (6.0-21.0 pg/mL) in advanced DR group, 1.9±0.7 pg/mL (0.6-2.8 pg/mL) in early DR group, and 2.1±1.0 pg/mL (0.7-3.9 pg/mL) in control group (P < 0.001). Advanced DR group was further subdivided into severe nonproliferative DR (15 eyes, 12.8%) and proliferative DR (25 eyes, 34.3%). Mean aqueous ET-1 level was 10.1±4.3 pg/mL (6.0-20.1 pg/mL) in patients with severe nonproliferative DR, and 10.0±4.0 pg/mL (6.0-21.0 pg/mL) in those with proliferative DR (P = 0.928) at baseline. Mean ET-1 level at 1 month after intravitreal injection was 2.5±1.0 pg/mL (0.3-4.8 pg/mL) in patients with severe proliferative DR and 2.9±1.7 pg/mL (1.0-7.0 pg/mL) in those with proliferative DR (P = 0.443). Mean aqueous ET-1 level was significantly reduced in both groups (P < 0.001, respectively).
CONCLUSION: The mean aqueous ET-1 level was significantly higher in the eyes with advanced DR than those with early DR and the control group. The mean aqueous ET-1 level was significantly reduced after intravitreal injections in the advanced DR group. Based on our results, future studies on the exact role of ET-1 in the pathogenesis of DR and future implication for intervention would be helpful for managing DR.

Introduction

Diabetic retinopathy (DR) is one of the most common retinal vascular diseases threatening vision in all age groups [1,2]. In Korea, 14% of the population has diabetes mellitus (DM), and 11–19% of those with DM have DR [3-6]. Because DR can lead to permanent vision loss, DR appears to have significant socioeconomic effects, especially when it affects younger, working-age individuals.

Microvascular alterations are key features of DR, including microvascular endothelial cell dysfunction, vascular permeability, increased tissue ischemia, and angiogenesis [7]. Selective loss of capillary pericytes, impairment of retinal vascular autoregulation, and disturbance of capillary circulation are the characteristic retinal vascular changes in early DR [8-10]. Low-grade inflammation due to hyperglycemia subsequently leads to up-regulation of various vasoactive cytokines such as endothelial cell adhesion molecules and vascular cell adhesion molecule-1 [7]. The persistent inflammation and subsequent vascular wall injury lead to up-regulation of vasoconstrictors such as endothelin-1 (ET-1) and vasodilators such as nitric oxide (NO) [11,12]. Because retinal blood flow is auto-regulated primarily by these local vasoactive factors, alteration of the responses to these local factors by pericytes and endothelial cells leads to abnormal vascular tone and alteration of retinal blood flow.

ET-1 is a potent vasoconstrictor peptide, and several studies indicate that ET-1 is involved in the pathogenesis of DR [13-17]. These studies found that the endothelin system, including ET-1, the endothelin system is of importance in mediating retinal changes in diabetes, although mechanisms of the endothelin system alteration as well as their effects might vary among individuals [13-17]. One experimental study suggests that retinal blood flow showed an increased resistivity index, an indicator of vasoconstriction, after 1 month of diabetes [15]. The retina from the diabetic animals showed increased mRNA expression for ET-1, ET-3 and ET A after one month, as well as increased ET B mRNA expression after 6 months [15]. They also found that ET-1 and ET-3 immunoreactivity and endothelin receptor concentrations were increased in the retina of diabetic rats [15]. It has been also shown that 15 minutes after intravitreal injection of ET-1, there were significant prolongation of retinal circulation times and retinal artery constriction in the animal model [16]. However, only a few studies investigated intraocular concentrations of ET-1 in patients with DR, and the results are inconsistent [18-20]. One study investigated vitreous ET-1 levels in patients who underwent pars plana vitrectomy. It found higher elevations in ET-1 levels in patients with proliferative DR (PDR) than in the control group and in those with nonproliferative DR [18]. The patients with nonproliferative DR and macular edema have lower vitreous ET-1 levels than those with proliferative DR [18]. They speculated that the reductions in ET-1 levels in the patients with nonproliferative DR reflects the increase in retinal blood flow that occurs in nonproliferative DR as it has minimal vasoconstrictive effects [18]. However, this study included a relatively small study population (i.e., 5 patients with proliferative DR and 15 patients with nonproliferative DR) [18]. Another study found significantly lower ET-1 levels in vitreous fluid of patients with PDR than in normal control group patients [19]. Another study found significantly higher levels of vitreous ET-1 in patients with proliferative DR than in those with nonproliferative DR or the control group [20].

In this study, we investigated aqueous ET-1 levels in patients with DR versus a control group. We also evaluated the change in mean aqueous ET-1 level after intravitreal anti-vascular endothelial growth factor (VEGF) injection in patients with advanced DR with macular edema.

Methods

Enrollment of study subjects

This prospective, comparative study was performed at the Catholic Kwandong University College of Medicine, International St. Mary’s Hospital. The study design and protocol were approved by the Institutional Review Board of International St. Mary’s Hospital, Catholic Kwandong University College of Medicine and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each participant in this study.

Study subjects were chosen as participants from May 2019 to October 2019. The study population was classified into three groups: advanced DR, early DR, and control group. The advanced DR group included the patients with severe non-proliferative DR or proliferative DR. These patients underwent intravitreal anti-VEGF injections for concomitant macular edema on pro re nata basis. At the time of the first intravitreal anti-VEGF injections, two consecutive aqueous samplings right before the injection and 1 month after the first intravitreal anti-VEGF injection were performed to compare if there would be any change in aqueous ET-1 level. For each intravitreal injection, 1.25 mg/0.05 mL of bevacizumab (Avastin®; Genentech Inc., San Francisco, CA, USA) was used. The early DR group included the patients with no gross DR, mild nonproliferative DR, or moderate nonproliferative DR. Classification of the stage of DR in each participant was based on the International Clinical Diabetic Retinopathy and Diabetic Macular Edema Severity Scales [21].

Treatment-naïve patients for DR were included in this study. The control group included the patients who underwent uncomplicated cataract surgery, had no other concomitant ocular disease, and had no history of diabetes mellitus. If both eyes met the inclusion and exclusion criteria, the right eye was chosen for analysis.

The primary outcome measure was comparison of differences in the mean aqueous ET-1 levels of three groups. The secondary outcome measures were the changes in mean aqueous ET-1 levels after the first intravitreal anti-VEGF injections in the patients with advanced DR with macular edema and its clinical effects on the 1-year prognosis.

Ocular examination

An ophthalmologic examination that included a slit-lamp examination, an intraocular pressure measurement using a non-contact tonometer, and a fundus examination after pupillary dilation was performed at each visit. The refractive error of each eye was measured using an autorefractor, and then converted to spherical equivalents [diopters (D)]. Best-corrected visual acuity (BCVA) of the affected eye(s) was checked using a decimal visual acuity chart; each value was converted to the logarithm of the minimum angle of resolution (logMAR). Spectral domain optical coherence tomography (SD OCT) (Spectralis; Heidelberg Engineering, Heidelberg, Germany) imaging was performed using enhanced depth imaging. Fluorescein angiography (FA) was performed at the first visit using the Heidelberg Retina Angiograph system (HRA-2; Heidelberg Engineering) with a confocal scanning laser ophthalmoscope. FA was used to evaluate the nonperfusion area and presence of neovascularization.

Central macular thickness (CMT) measured using SD OCT was defined as the mean retinal thickness in the central subfield, a region with a diameter of 1.0 mm around the fovea. The inner and the outer rings had diameters of 3.0 mm and 6.0 mm, respectively. CMT using Early Treatment Diabetic Retinopathy Study (ETDRS) macular grid subfields was automatically calculated using the Heidelberg software. The ETDRS grid was manually centered at the fovea if needed. All SD OCT images were reviewed for segmentation errors.

Sampling of aqueous humor in study subjects

All ocular procedures were performed by one retinal specialist (HM Kang) using a standardized approach. Each aqueous sampling was routinely performed at the beginning of the ocular procedure in our clinic, which enabled this study. Under sterile operating room conditions, topical anesthesia was achieved using 0.5% proparacaine hydrochloride (Alcain®; Alcon Inc., Fort Worth, TX, USA). A 10% povidone-iodine solution was used for periocular sterilization. The surgeon used a solution of povidone-iodine 10% to clean the patient’s skin around the eye and used povidone-iodine 5% for irrigation (Betadine®; Alcon, USA). An eyelid speculum was placed after the drape was applied. After further topical anesthesia using 4% lidocaine, anterior chamber paracentesis was performed to drain 0.1 mL aqueous humor.

Endothelin-1 concentration measurement

Laboratory procedures for aqueous ET-1 concentration was done as previously decribed in the previous study conducted by previous study [22], and our study on retinal vein occlusion [23]. Aqueous humor samples were immediately stored at -80°C until analysis. On the day of analysis, stored samples were thawed and centrifuged at 400 x g for 5 minutes at 4°C to remove cell contents and cell debris [22,23]. ET-1 was measured using a sandwich ELISA kit (R&D SYSTEMS, QUANTIKINE ELISA endothelin-1 kit, catalog number DET-100) according to the manufacturer’s protocol [22,23]. In brief, 75-μL aqueous humor samples were pipetted into 96-well plates pre-coated with a monoclonal antibody specific for human ET-1 and that did not cross-react with other isoforms or species. Same volumes of ET-1 standards (concentrations 25 pg/mL, 12.5 pg/mL, 6.25 pg/mL, 3.13 pg/mL, 1.56 pg/mL, 0.78 pg/mL, 0.39 pg/mL, and 0 pg/mL) were also pipetted into 96-well plates to make a standard curve. Color intensity was measured using a multi-well plate reader (Bio Tek Instruments, Inc., South Korea). The wavelengths for all samples and standards were corrected by subtracting OD540 values from OD450 values. After plotting the standard curve, the corrected OD450 values for samples were extrapolated on the Y-axis and the concentration in pg/mL was measured on the X-axis [22,23].

Statistical analysis

The data were presented as mean ± standard deviation (range) values. Baseline characteristics included age, sex, refractive error, and BCVA at the initial visit. Medical histories, including hypertension and end-stage renal disease, were obtained from the medical charts of the patients. IBM SPSS Statistics software for Windows, version 22.0 (IBM Corp., Somers, NY, USA) was used for the statistical analyses. For continuous variables, the Kruskal-Wallis test was used for comparisons among three groups, and Mann-Whitney U test was used for comparison between two groups. The chi-square test was used for categorical variables. Repeated-measures analysis of variance was used to compare values for mean BCVA, mean CMT, and mean aqueous ET-1 levels after intravitreal injections. Pearson’s correlation analysis was performed to evaluate for any significant correlations between aqueous ET-1 level with visual and anatomical outcomes in the patients with advanced DR after intravitreal injection. Multiple regression analysis by using stepwise approach was used to investigate the correlation between the baseline factors with the mean aqueous ET-1 level at baseline. Mauchly’s test of sphericity and Kolmogorov-Smirnov analyses were used to confirm statistical validity. A P value <0.05 was considered statistically significant.

Results

Baseline characteristics of the study population

Eighty-five eyes of 85 patients were included in this study. The advanced DR group included 40 eyes (47.1%), the early DR group included 19 eyes (22.4%), and the control group included 26 eyes (30.5%). The advanced DR group was further subdivided into severe nonproliferative DR (15 eyes, 12.8%) and proliferative DR (25 eyes, 34.3%). The early DR group included no DR (7 eyes, 8.2%), mild nonproliferative DR (10 eyes, 11.8%), and moderate nonproliferative DR (2 eyes, 2.4%). In the overall study population, mean age at the time of diagnosis was 64.4±10.9 (37.0–86.0) years. Mean aqueous ET-1 level was 10.1±4.1 pg/mL (6.0–21.0 pg/mL) in the advanced DR group, 1.9±0.7 pg/mL (0.6–2.8 pg/mL) in the early DR group, and 2.1±1.0 pg/mL (0.7–3.9 pg/mL) in the control group (P < 0.001). The results for comparison among the three subgroups are presented in Table 1.

Table 1

Baseline characteristics of the study population.

	Advanced DR group(N = 40)	Early DR group (N = 19)	Control group (N = 26)	P value
Mean age (years)	58.6±9.6(37–79)	69.5±7.9(52–82)	69.6±9.7(51–86)	0.028*
Sex (male)	14 (35.0%)	11 (57.9%)	14 (53.8%)	0.630†
Hypertension	15 (37.5%)	7 (36.8%)	12 (46.2%)	0.512†
ESRD	8 (20.0%)	0	0	0.004†
Mean fasting glucose (ml/dl)	198.2±63.6(96.0–333.0)	146.5±39.0(107.0–245.0)	111.2±16.7(87.0–132.0)	<0.001*
Mean HbA1c (%)	7.8±1.3(6.0–11.0)	7.8±1.1(5.8–12.0)	5.1±0.4(4.5–5.5)	0.863*
Mean creatinine (ml/dl)	2.8±3.6(0.52–15.0)	0.8±0.4(0.41–2.17)	0.7±0.2(0.37–1.03)	<0.001*
Nonperfusion area on FA	40 (100%)	0	N/A	<0.001†
NVE on FA	25 (62.5%)	0	N/A	<0.001†

*The Kruskal-Wallis test was used for continuous variables and

†the chi-square test was used for categorical variables.

Abbreviations: DR, diabetic retinopathy; ESRD, end-stage renal disease.

Changes after intravitreal anti-vascular endothelial growth factor injections for advanced diabetic retinopathy with macular edema

During the 12-month follow-up period, a mean number of 1.7±0.9 (range 1–4) intravitreal bevacizumab injections were performed for the advanced DR group. The mean BCVA value was 0.6±0.4 logMAR (0.1 to 1.3 logMAR) at baseline and 0.3±0.3 logMAR (0 to 1.3 logMAR) at 1 month after IVB (P < 0.001). Mean CMT was 415.5±104.5 (352.0 to 810.0 μm) at baseline and 313.0±70.8 μm (205.0 to 483.0 μm) at 1 month after intravitreal anti-VEGF injections (P < 0.001). Mean baseline aqueous ET-1 level was 10.1±4.1 pg/mL (6.0–21.0 pg/mL), which was significantly reduced to 2.8±1.4 pg/mL (0.3–7.0 pg/mL) (P < 0.001) after intravitreal anti-VEGF injections.

At 12 months after the first injection, mean BCVA was 0.3±0.3 logMAR (1.3 to 0 logMAR) and mean CMT was 323.9±103.4 μm (205.0 to 808.0 μm. Mean BCVA and mean CMT values at the 12-month follow-up point were significantly improved, compared with the baseline values (P < 0.001, respectively).

Comparison between severe nonproliferative diabetic retinopathy and proliferative diabetic retinopathy

Then, we compared various characteristics between the patients with severe nonproliferative DR and those with proliferative DR. Except the mean age, there were no significant differences in various baseline characteristics between two groups. Detailed comparison is shown in Table 2.

Table 2

Comparison between patients with severe nonproliferative diabetic retinopathy (NPDR) versus those with proliferative diabetic retinopathy (PDR).

	Severe NPDR(N = 15)	PDR(N = 25)	P value
Mean age (years)	63.3±8.4(48–79)	55.8±9.3(37–66)	0.015*
Sex (male)	10 (66.7%)	14 (56.0%)	0.372†
Hypertension	5 (33.3%)	10 (40.0%)	0.469†
ESRD	1 (6.7%)	7 (28.0%)	0.108†
Mean fasting glucose (ml/dl)	198.3±67.5(96.0–311)	198.0±70.1(107.0–333.0)	0.934*
Mean HbA1c (%)	7.8±0.9(6.3–9.8)	7.9±1.2(6.0–11.0)	0.956*
Mean creatinine (ml/dl)	1.7±2.3(0.52–9.6)	3.4±4.1(0.6–15.0)	0.295*
Mean number of intravitreal injections	1.7±0.7 (1–3)	1.7±1.1 (1–4)	0.966*

* Mann-Whitney U tests were used for continuous variables and

†chi-square tests were used for categorical variables.

Abbreviations: BCVA, best-corrected visual acuity; DR, diabetic retinopathy; ESRD, end-stage renal disease.

There were no significant differences in mean aqueous ET-1 levels, mean BCVAs, and mean CMTs between the severe nonproliferative DR group and the proliferative DR group (P = 0.928, P = 0.315, and P = 0.120, respectively; Figs 1 and 2). After the first intravitreal injection, mean aqueous ET-1 level was significantly reduced in both the patients with severe nonproliferative DR (P < 0.001) and those with proliferative DR (P < 0.001, Fig 1). After 1 month and 12 months after the first intravitreal bevacizumab injection, there were also significant improvement of mean BCVA and mean CMT after the first intravitreal injection in both groups as shown in Fig 2.

Fig 1

The changes of mean aqueous endothelin-1 (ET-1) after intravitreal anti-vascular endothelial growth factor (VEGF) injections in the patients with severe nonproliferative diabetic retinopathy (DR) and proliferative DR.

The mean baseline ET-1 level was 10.1±4.3 pg/mL (6.0–20.1 pg/mL) in the patients with severe nonproliferative DR and 10.0±4.0 pg/mL (6.0–21.0 pg/mL) in those with proliferative DR (P = 0.928). Mean aqueous ET-1 level was significantly reduced after intravitreal anti-VEGF injections in both the patients with severe nonproliferative DR (P < 0.001) and those with proliferative DR (P < 0.001).

Fig 2

The changes of mean best-corrected visual acuity (BCVA) and mean central macular thickness (CMT) the patients with severe nonproliferative diabetic retinopathy (DR) and proliferative DR.

(A) The mean BCVA was 0.5±0.4 (1.3~0.2) logMAR in the severe nonproliferative DR group, and 0.6±0.4 (1.3~0.1) logMAR in the proliferative DR group (P = 0.315). Mean BCVA was significantly improved at 1 month and 12 months after intravitreal injection, compared with baseline (P < 0.001 for both). (B) Mean CMT was also significantly reduced at 1 month and 12 months after intravitreal injection, compared with baseline (P < 0.001 for both). At each follow-up point, the mean BCVA (P = 0.667 at 1 month, P = 0.907 at 12 months) and the mean CMT (P = 0.432 at 1 month, P = 0.182 at 12 months) were not significantly different between the patients with severe nonproliferative DR and proliferative DR.

Correlation between aqueous endothelin-1 levels with visual and anatomical outcomes in patients with advanced diabetic retinopathy

We performed correlation analyses between baseline aqueous ET-1 levels and clinical outcomes in the patients with advanced DR. There was no significant correlation between baseline aqueous ET-1 levels and postoperative visual outcomes at 12 months (P = 0.606). There was also no significant correlation between baseline aqueous ET-1 level and postoperative CMT at 12 months (P = 0.910).

Possible predictive factors for the baseline aqueous endothelin-1 levels in the study population

We further investigated the possible factors correlated with baseline aqueous ET-1 levels in the study population. Baseline characteristics such as presence of hypertension, chronic renal disease/end-stage renal disease on dialysis, age, sex, fasting glucose level, HbA1c, and creatinine level were included. In addition, baseline BCVA, baseline CMT, and severity of DR (early DR and advanced DR) were also included, as shown in Table 3. Among these various factors, the severity of DR was significantly associated with the mean aqueous ET-1 level at baseline (B = 2.116, β = 0.691, P < 0.001; Table 3).

Table 3

The possible factors associated with the mean aqueous endothelin-1 level in the study population by multiple regression analysis.

Characteristics	β	P value
Age	-0.012	0.918
Sex	-0.208	0.290
Fasting glucose (mg/dl)	-0.020	0.843
Creatinine (mg/dl)	-0.045	0.665
Presence of hypertension	-0.018	0.581
Presence of end-stage renal disease	-0.135	0.194
HbA1c (%)	0.52	0.589
Severity of diabetic retinopathy	0.691	<0.001

P value < 0.001 is set for statistical significance.

Discussion

This study is the first to investigate aqueous ET-1 levels in patients with DR. The mean aqueous ET-1 level was significantly higher in the patients with advanced DR than in those with early DR and the control group. The mean aqueous ET-1 level was significantly reduced after intravitreal anti-VEGF injections for macular edema associated with advanced DR, with concomitant improvements in vision and CMT.

A few studies have investigated vitreous ET-1 levels in patients with DR; however, the results are inconsistent [18-20]. We speculate that the different results in intraocular ET-1 levels in these patients with advanced DR [18-20], including in our study, might be due to differences in patient status at the time of study enrollment. In those who undergo pars plana vitrectomy due to advanced DR may have prolonged, progressed disease status or different features than those with treatment-naïve patients such as those in our study. There is another possibility regarding the different intraocular ET-1 levels among the previous studies [18-20] versus our results. The intraocular ET-1 level may be different during each stage of DR, just as suggested in the previous study [15]. As our study and previous studies [13-20], ET-1 seems to involved in the pathogenesis of DR. Along with hypoxic insults in the pathogenesis of DR, increasing endothelin family including ET-1 may aggravate DR itself by constricting retinal vessels and delaying blood circulation time throughout retinal vascular system [13-20]. However, further studies should be followed to identify the exact role of ET-1 in the pathogenesis of DR, as well as possible interactions with diabetes itself and other cytokines involved in the pathogenesis of DR.

In our study, intravitreal bevacizumab injections significantly reduced the mean aqueous ET-1 level. Hypoxia increases hypoxia-inducible factor-1 alpha, leading to upregulation of genes such as erythropoietin, VEGF, and ET-1 [24]. Reduction in VEGF normally leads to reduction in ET-1; this relationship is explained by the stimulatory interaction between VEGF and ET-1 [25]. We speculate that intervention by using intravitreal anti-VEGF agent may reduce abnormally upregulated vasoactive and/or inflammatory cytokines, leading to reduced expression of ET-1. Timely intervention may prevent further vasoconstriction and protect blood-retinal barrier from further damage in patients with advanced DR. Similarly, a previous study found significant reductions in plasma VEGF, ET-1, and NO levels after pan-retinal photocoagulation in 40 patients with PDR [13]. We could not verify the exact mechanism and further impact of reduced ET-1 level by anti-VEGF agent in this current study because of relatively small study population. However, our results seem to suggest further research point regarding ET-1 level and the role of anti-VEGF agent. Further investigations on the clinical implication of reducing intraocular ET-1 level by anti-VEGF agents can help us understanding the role of ET-1 in DR.

We also investigated the possible factors associated with baseline aqueous ET-1 level in the patients with DR. Among various factors, the severity of DR at baseline was the only factor significantly associated with baseline aqueous ET-1 level among the patients with DM. Our results also support the previous findings that ET-1 is involved in the pathogenesis of DR.13-20 We also found that there were individual differences of aqueous ET-1 levels among the patients with advanced DR. The range of aqueous ET-1 levels in the patients with advanced DR was from 6.0 to 21.0 pg/mL. We could speculate that there may be other factors affecting aqueous ET-1 levels in the patients with DM. One possible factor can be plasma ET-1 level. Several studies have suggested that plasma ET-1 levels may be transmitted through the peripapillary vasculature [26-28], elevated plasma ET-1 may also contribute intraocular ET-1 levels in patients with DR. In our study, we found that the group of patients with advanced DR had significantly higher prevalence of end-stage renal disease on hemodialysis and older mean age when compared with those with early DR. Plasma ET-1 levels can increase due to mid repeated ischemia-reperfusion events somewhere in the body, concomitant micro- and/or macrovascular complications of DM may also affect plasma ET-1 level [29-31]. Resultant difference in plasma ET-1 levels among individuals with DM may contribute the difference in intraocular ET-1 levels. However, we did not investigate plasma ET-1 levels in this current study, so that we could not confirm the possible relationship between plasma ET-1 and aqueous ET-1. In addition, there may be other unknown factors contributing aqueous ET-1 level. We suggest further researches on the possible factors contributing individual variability of aqueous ET-1 level in the patients with DM, and these can enhance our understanding the role of ET-1 in the pathogenesis of DM.

This study had some limitations, including the relatively small number of patients in the study population. We lacked aqueous humor sampling of the eyes with advanced DR, but without ME, and of plasma ET-1 levels in the study subjects, because of its retrospective manner. In addition, the changes of aqueous ET-1 level during follow-up period may widen our understandings of ET-1 in the pathogenesis and treatment response in advanced DR. Further prospective study which overcomes these weaknesses is needed.

In conclusion, the mean aqueous humor ET-1 level was significantly higher in patients with advanced DR than in those with early DR and the control group. The severity of DR was significantly associated with mean aqueous ET-1 level at baseline in the patients with DM. Further investigations on the exact role of ET-1 in the pathogenesis of DR and its clinical implication can improve our understanding for DR, and may also contribute to the management of these patients.

8 Mar 2022

Elevated aqueous endothelin-1 concentrations in advanced diabetic retinopathy

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In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear author, I congratulate you for this excellent study. Although it has been known that endothelin-1 levels are upregulated in hypoxic states with concomitant vasoconstriction, this study conclusively shows that the levels are higher with severe grades of DR indicating extreme levels of hypoxia, which reduced after the intravitreal injection of Anti-VEGF agents.

However, please check the manuscript for grammatical errors.

Also, the abstract needs rephrasing: purpose section mentions this as a retrospective study, and the methods section mentions this as prospective.

I also suggest if you could revise the keywords. Having macular edema in this does not make sense. However, I would like to add aqueous endothelin-1, bevacizumab etc.

Overall, an excellent study.

Thank you

Reviewer #2: Authors present a novel work. Following suggestions would help improving the manuscript.

Major Comments

1. Purpose section of Abstract needs to provide a statement about the rationale of the study.

2. Definition of early diabetic retinopathy and advanced diabetic retinopathy needs to be provided in Methods section in Abstract

3. Conclusion in Abstract is just the reiteration of the data in the Results. Conclusion should provide interpretable message of the study along with possible future implications if any.

4. “These studies found that ET-1 has an important role in the pathogenesis and progression of DR.” The role of endothelin 1 in pathogenesis of diabetic retinopathy as indicated in this sentence in Introduction needs to be further elaborated.

5. How was proliferative diabetic retinopathy diagnosed?

6. What is the meaning of “two consecutive intravitreal injections” and how was this number achieved at?

7. “The early DR group included the patients with no gross DR,………..” What is the meaning of “gross DR”?

8. Results is lengthy and needs to be shortened. It discusses the data and provides comparisons. It should include only data and tables. There should not be any discussion of the data; which is part of Discussion section.

9. “A few studies have investigated vitreous ET-1 levels in patients with DR; however,

the results are inconsistent.18-20” Do authors believe that measuring endothelin level in vitreous would have been more appropriate than aqueous levels?

10. “This ‘certain point’ may exist between moderate and severe nonproliferative DR and/or development of macular edema, based on our study results. Advancement to severe nonproliferative DR and concomitant development of macular edema both may be involved in the increase of ET-1 inside the eye” Reviewer believes that this assertion would require different study design where same set of patients are followed up over a period of time to assess endothelin 1 levels alongside evolution of diabetic retinopathy. Or it could be concluded with present study design too, had the sample size been larger enough. Hence the present study should possibly refrain from providing a continuum of endothelin 1 levels alongside evolution of diabetic retinopathy and leave it to future studies.

11. A lot has been discussed about plasma endothelin 1 levels. Reviewer feels that this is speculative and beyond the scope of present study.

12. “If we can detect the critical ‘point’ in patients with DR, interventions antagonizing ET-1 may prevent further disease progression. Then we may prevent further vision loss in these patients with DR.” As stated in previous comment, present study design is not suitable for this assertion.

Minor Comments

1. “For the control group, those who underwent uncomplicated cataract surgery and had no other concomitant ocular diseases were included” This information has been written twice in Methods section.

2. “After reaching ‘certain threshold point’ and progression to advanced DR, then intraocular ET-1 level may be various with different in accordance with each eye status.” This sentence in Discussion needs to be reframed.

**********

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Reviewer #1: No

Reviewer #2: No

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30 Mar 2022

Thank you for the review for PONE-D-21-28261 “Elevated aqueous endothelin-1 concentrations in advanced diabetic retinopathy”, and giving the authors the chance to improve this article. We authors made our every efforts to improve this manuscript according to the comments, and hope that it really improved the quality of this manuscript for PLOS ONE.

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Answer: We authors reviewed the style requirements and the current manuscript, and ensured that this manuscript followed the requirements.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This research is supported by a grant of the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI21C1251).”

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Answer: Thank you for the comment, and we removed the funding statement from the Acknowledgments section, and rephrased in the Funding Statement section in online submission system.

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Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Answer: Thank you for the comment. We authors fully understand the data availability of PLOS ONE. However, we also had to follow the decision of the Institutional Review Board of International St. Mary’s Hospital, Catholic Kwandong University College of Medicine. Because Haemin Kang, the corresponding author, is the sole retinal specialist at the time of this study, so the IRB committee worried about any potential of identifying patient information, even by sex and age with specific diagnosis and treatment. Instead, the IRB committee is willing to provide the fully anonymized dataset upon request of investigators. If there is any request for the data, the IRB committee would undergo further discussion, and if possible, it would provide data. The investigators can reach to Prof. Jehoon Park, the head of IRB committee (via ceccil@ish.ac.kr) to request the data used in this study.

4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

Answer: Thank you for the comment, and in the first paragraph of the Method section, this is written as “This prospective, comparative study was performed at the Catholic Kwandong University College of Medicine, International St. Mary’s Hospital. The study design and protocol were approved by the Institutional Review Board of International St. Mary’s Hospital, Catholic Kwandong University College of Medicine and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each participant in this study.”

5. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252530

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Answer: Thank you for the comment, and we added this citation to the revised manuscript in the Method section.

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear author, I congratulate you for this excellent study. Although it has been known that endothelin-1 levels are upregulated in hypoxic states with concomitant vasoconstriction, this study conclusively shows that the levels are higher with severe grades of DR indicating extreme levels of hypoxia, which reduced after the intravitreal injection of Anti-VEGF agents.

However, please check the manuscript for grammatical errors.

Also, the abstract needs rephrasing: purpose section mentions this as a retrospective study, and the methods section mentions this as prospective.

I also suggest if you could revise the keywords. Having macular edema in this does not make sense. However, I would like to add aqueous endothelin-1, bevacizumab etc.

Overall, an excellent study.

Thank you

Answer: Thank you for the comments. As the reviewer’s recommendation, we authors corrected the abstract and grammatical errors throughout the manuscript. I also revised the keywords, including aqueous endothelin-1 and bevacizumab, omitting macular edema.

Reviewer #2: Authors present a novel work. Following suggestions would help improving the manuscript.

Major Comments

1.Purpose section of Abstract needs to provide a statement about the rationale of the study.

Answer: Thank you for the comment, and we added a statement about the rationale of the study to the purpose section of Abstracts as following: “Endothelin-1 (ET-1) is a potent vasoconstrictor which seems to be involved in the pathogenesis of diabetic retinopathy (DR). However, studies on intraocular ET-1 in DR have been limited.”

2. Definition of early diabetic retinopathy and advanced diabetic retinopathy needs to be provided in Methods section in Abstract.

Answer: Thank you for the comment, and we authors provided the definitions in the Abstract as following: “Those were classified into three groups: advanced DR group included those with severe nonproliferative DR or proliferative DR, whereas early DR group included those with mild nonproliferative DR or moderative nonproliferative DR.”

3. Conclusion in Abstract is just the reiteration of the data in the Results. Conclusion should provide interpretable message of the study along with possible future implications if any.

Answer: Thank you for the comment, and we revised the Conclusion in Abstract, as following “Based on our results, future studies on the exact role of ET-1 in the pathogenesis of DR and future implication for intervention would be helpful for managing DR.”

4. “These studies found that ET-1 has an important role in the pathogenesis and progression of DR.” The role of endothelin 1 in pathogenesis of diabetic retinopathy as indicated in this sentence in Introduction needs to be further elaborated.

Answer: Thank you for the comment, and we added further details in the Introduction section as commented.

5. How was proliferative diabetic retinopathy diagnosed?

Answer: Thank you for the comment. We authors usually previously diagnosed DR according to fundus findings by using the ETDRS criteria combined with FA and OCT. For this study, we adopted the Disease Severity Scales for Diabetic Retinopathy and Diabetic Macular Edema. We added this to the Method section.

6. What is the meaning of “two consecutive intravitreal injections” and how was this number achieved at?

Answer: Thank you for the comment, and we authors apologize for this confusion. We authors treat patients with diabetic macular edema on pro re nata basis in this study, and this sentence was meant to be ‘two consecutive aqueous samplings right before and 1 month after the first intravitreal anti-VEGF injection’. We revised the Method section as following: “At the time of the first intravitreal anti-VEGF injections, two consecutive aqueous samplings right before the injection and 1 month after the first intravitreal anti-VEGF injection were performed to compare if there would be any change in aqueous ET-1 level.” Thank you for the comment.

7. “The early DR group included the patients with no gross DR,………..” What is the meaning of “gross DR”?

Answer: Thank you for the comment. As we previously answered to the comment 5, we used the Disease Severity Scales for Diabetic Retinopathy and Diabetic Macular Edema, and no apparent DR is no visible abnormalities in fundus in the presence of diabetes mellitus.

8. Results is lengthy and needs to be shortened. It discusses the data and provides comparisons. It should include only data and tables. There should not be any discussion of the data; which is part of Discussion section.

Answer: Thank you for the comment, and as the comment, we revised the Results section much more briefly.

9. “A few studies have investigated vitreous ET-1 levels in patients with DR; however,

the results are inconsistent.18-20” Do authors believe that measuring endothelin level in vitreous would have been more appropriate than aqueous levels?

Answer: Thank you for the comment. Vitreous fluid can be more directly affected by retinal status, however, those changes are usually reflected to aqueous humor. Endothelin-1 level in vitreous fluid may be more direct indicator for retinal changes, however, vitreous sampling may be much difficult to perform when compared with aqueous sampling. When we consider all pros and cons, we authors think that aqueous can be used as good method to investigate intraocular changes.

10. “This ‘certain point’ may exist between moderate and severe nonproliferative DR and/or development of macular edema, based on our study results. Advancement to severe nonproliferative DR and concomitant development of macular edema both may be involved in the increase of ET-1 inside the eye” Reviewer believes that this assertion would require different study design where same set of patients are followed up over a period of time to assess endothelin 1 levels alongside evolution of diabetic retinopathy. Or it could be concluded with present study design too, had the sample size been larger enough. Hence the present study should possibly refrain from providing a continuum of endothelin 1 levels alongside evolution of diabetic retinopathy and leave it to future studies.

Answer: Thank you for the comment, and we authors discussed this in further details. We authors agree that we have several limitations to suggest this, and we revised Discussion section as recommended.

11. A lot has been discussed about plasma endothelin 1 levels. Reviewer feels that this is speculative and beyond the scope of present study.

Answer: We appreciate this comment, and we authors discussed this point. We authors agreed, and re revised this paragraph.

12. “If we can detect the critical ‘point’ in patients with DR, interventions antagonizing ET-1 may prevent further disease progression. Then we may prevent further vision loss in these patients with DR.” As stated in previous comment, present study design is not suitable for this assertion.

Answer: Thank you for the comment, and we also revised this as recommendation.

Minor Comments

1.“For the control group, those who underwent uncomplicated cataract surgery and had no other concomitant ocular diseases were included” This information has been written twice in Methods section.

Answer: Thank you for the comment, and we authors apologize this error. We corrected this error.

2. “After reaching ‘certain threshold point’ and progression to advanced DR, then intraocular ET-1 level may be various with different in accordance with each eye status.” This sentence in Discussion needs to be reframed.

Answer: Thank you for the comment, and we reframed this sentence as recommended.

Again, we authors really appreciate this chance to improve our manuscript, and if you have any further questions or request, we’ll be glad to hear that. Thank you for all of your efforts.

28 Apr 2022

Elevated aqueous endothelin-1 concentrations in advanced diabetic retinopathy

PONE-D-21-28261R1

Dear Dr. Kang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tomislav Bulum

Academic Editor

PLOS ONE

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for the revised manuscript. All the revisions are acceptable. I do not recommend any further revisions.

Reviewer #2: Authors have modified the manuscript as suggested. There are no more suggestions or modifications for the manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Kumar Saurabh

2 May 2022

PONE-D-21-28261R1

Elevated aqueous endothelin-1 concentrations in advanced diabetic retinopathy

Dear Dr. Kang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

Dr. Tomislav Bulum

Academic Editor

PLOS ONE