This study was approved by institutional ethics committee in the Fenghua Hospital of Traditional Chinese Medicine. Informed consents in written format to publish these date were collected from each patient, who agreed and signed the consent to participate statement. Primary angle-closure suspect was inclusion criteria, because there were no reports about the association between ephrin-A1 and primary angle-closure suspects, or VEGF and primary angle-closure suspects in the literature. In addition, there is no vascular changes or cytokine changes that are associated with angiogenesis in primary angle-closure suspects. The recruited patients were enrolled between August 1, 2018 and October 31, 2019 with the following inclusion criteria:
- Over 40 years of age.
- Clinical diagnosis of primary angle-closure suspect, diabetes mellitus and DR.
- Patients agreed to provide informed consent.
The definition of primary angle-closure suspect was in accordance with the International Society of Geographical and Epidemiologic Ophthalmology criteria (15). Diabetes mellitus was diagnosed according to the international standards (16). Diagnosis of DR was made based on an international standard (17).
The subjects with any diseases that presented neovascularization such as kidney disease and cancer, or any of those subjects received anti-VEGF therapy or operations were excluded.
A total of fifty-five patients from Fenghua District were recruited. Nineteen patients with primary angle-closure but without diabetes were enrolled in the non-DM group. Sixteen patients with primary angle-closure and diabetes were enrolled in DM group. Twenty patients with DR were enrolled in DR group, about 35% of patients with proliferative diabetic retinopathy.
After routine physical and eye examinations, electrocardiogram, clinical laboratory tests including liver and kidney function tests, routine ophthalmic examinations, fundus photograph and fluorescein angiography were performed.
The averaged duration time of type 2 DM in the diabetic group was 46 ± 18.5 months. The averaged duration time of type 2 DM with DR was 118 ± 49 months.
Collection of Blood Samples
The blood samples were collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant from the peripheral vein of each patient and transferred to Department of Clinical Laboratory, which is a section of Fenghua Hospital of Traditional Chinese Medicine. Samples were centrifuged immediately for 15 minutes at 1000g. Then, plasma was collected and stored at -80℃ before enzyme-linked immunosorbent assay (ELISA) measurement.
All individuals were informed of the purpose of the study and their informed consent was obtained. This study followed the tenets of the Declaration of Helsinki and was approved by the ethics committee of the institutional Fenghua Hospital of Traditional Chinese Medicine.
Measurement of plasma Ephrin-A1 and VEGF165
Ephrin-A1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (LSBio, Seattle, WA, USA) and VEGF165 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN, USA), following the instructions. Results were obtained by a multifunction microplate reader (Molecular Devices Inc., Sunnyvale, CA, USA). In addition, 6 ml venous blood of subjects was taken for measurement of fasting blood glucose, HbA1c, serum creatinine, and total cholesterol levels.
Statistical analyses were performed using SPSS 18.0 (SPSS Inc. Chicago, IL, USA). The data were shown as mean ± SD. After inspecting the distribution of the data, we assessed statistical significances with One-way ANOVA , Kruskal-Wallis Test, Mann-Whitney U Test corrected by Bonferroni. Correlations between any two of parameters including age, BMI, plasma ephrin-A1, VEGF165, fasting blood glucose levels, HbA1c, serum creatinine and total cholesterol were examined by Spearman’s Correlation Analysis. Correlation between ephrin-A1 and duration of diabetes was performed by Pearson Correlation Analysis. P<0.05 was considered statistically significant.