Plasma Ephrin-A1 level in a cohort of diabetic retinopathy patients

It is estimated that there are 382 million diabetic patients worldwide, and about one third of these patients have diabetic retinopathy (DR), part of them have diabetic macular edema. DR causes visual impairment in about 37 million people around the world [1]. About 60% of type 2 diabetes patients present DR of varying degrees 20 years after being diagnosed with diabetes. DR is induced by hyperglycemia, which leads to retinal vascular endothelial injury, retinal vascular loss, ischemia, and changes of leukocyte adhesion [2, 3]. Consequently, production of precursor substances of various angiogenic factors and inflammatory cytokines increased which induced abnormal neovascularization [4] and microvascular dysfunction.

In the pathogenesis process of DR, endothelial-derived growth factor (VEGF) is a major cytokine that promotes neovascularization, which leads the destruction of blood retinal barrier and indirectly promoting the progress of DR. Several spliced isoforms of VEGF present in human body with 121, 145, 165, 183,189, and 206 amino acids in length [5]. VEGF₁₆₅ appears to be the most abundant and potent isoform in inducing neovascularization, followed by VEGF₁₂₁ and VEGF₁₈₉ [5, 6]. In vivo and in vitro experiments have shown that ischemia and hypoxia induces VEGF expression, which causes retinal neovascularization [7]. In addition, it has been shown that VEGF levels in the vitreous of DR patients were higher than those in the control group [8]. Intravitreal injection of anti-VEGF drugs has a certain therapeutic effect on PDR patients [9, 10]. However, there are disadvantages that anti-VEGF drugs require multiple intravitreal injections for the treatment of DR, that maybe causing preretinal tissue shrinkage and retinal detachment [11]. In addition, anti-VEGF drugs have adverse effects such a mild cerebrovascular accident and myocardial infarction. Therefore, it is necessary to find more appropriate approaches or new therapeutic targets.

Ephrins and the Eph receptors have been identified as key regulators of angiogenesis [12]. Class A Eph receptors have been shown to regulate postnatal angiogenesis in adults. Earlier studies have shown that ephrin-A1 stimulates the migration of cultured endothelial cells and induces corneal angiogenesis [13]. Expression of ephrin-A1 was found in the process of embryonic vascular development [12] and tumor growth [14]. These evidences indicate that ephrin-A1 plays an important role in angiogenesis. We became interested whether ephrin-A1 is expressed in human plasma, and whether the plasma ephrin-A1 expression was altered in diabetic retinopathy patients.

Clinical and laboratory data were shown in Table 1. There was no statistical difference in age, sex, VEGF₁₆₅, serum creatinine and total cholesterol among the three groups of subjects. No relationship between Ephrin-A1 and VEGF₁₆₅ in human plasma had been found. There were differences in fasting blood glucose and HbA1c among the three groups. Compared to the non-DM subjects, DM patients and DR patients had higher fasting blood glucose and HbA1c (P < 0.05).

Ephrin-A1 could be measured in human plasma, the averaged concentration of ephrin-A1 was 1.52 ± 0.43 (mean ± SEM) ng/ml. The concentration of ephrin-A1 in the DR subjects was significantly higher than that of the other two groups (P < 0.05)(Fig. 1). This difference of the ephrin-A1 concentration was not found between the non-DM subjects and the DM subjects.

Plasma ephrin-A1 was correlated with the duration of diabetes (r = 0.513,P < 0.05) and with Serum creatinine (r = 0.338,P < 0.05), but was not correlated with age, BMI, serum VEGF₁₆₅, total cholesterol, fasting blood glucose and HbA1c in the DR patients, the DM subjects, and the non-DM subjects or all subjects.

Interaction between EphA receptor tyrosine kinases (RTKs) and ephrinA ligands is necessary for inducing of maximal neovascularization by VEGF [18]. EphA2 RTK is activated by VEGF through induction of ephrinA1 ligand [18]. Thus, we speculated that ephrin-A1 may be involved in the pathogenesis of DR and the ephrin-A1concentrations in DR patients may be changed. To the best of our knowledge, no such research has been conducted before. Meanwhile, 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 this study, ephrin-A1 concentrations in the plasma of non-diabetic, diabetic and DR patients were measured in a cohort of subjects. The averaged ephrin-A1 concentration in human plasma was 1.52 ± 0.43 (mean ± SEM) ng/ml, indicating ephrin-A1 was expressed in human plasma. We found ephrin-A1 concentration in the DR group was higher than that in the non-diabetic and the diabetic groups, suggesting ephrin-A1 may be involved in the development of DR. There is evidence showed that ephrin-A1 inhibits VEGF-induced intracellular signaling and suppresses retinal neovascularization and blood-retinal barrier breakdown [19]. Previous studies reported that ephrin-A1 has also been shown to be expressed in the developing vasculature during embryogenesis [14] and in nenvascular cells during tumor growth [12]. However, where does the increased ephrin-A1 come from in diabetic retinopathy remains unknown, it may require further studies including multiple systems and organs.

We also measured the plasma concentrations of VEGF₁₆₅ in these patients. We did not find difference in plasma VEGF₁₆₅ among the three groups. This result was inconsistent with a previous report that the plasma VEGF₁₆₅ concentration elevated in DR patients compared to non-DR controls [20]. Further studies are needed to confirm the changes of VEGF₁₆₅ in the systemic circulation in DR patients.

This study is the first report that ephrin-A1 is presented in circulation. Furthermore, ephrin-A1 expression is elevated in DR patients, suggesting it may be involved in the pathogenesis of DR. Whether it may become a biomarker for the disease deserves further studies. Where does the increase ephrin-A1 come from and the mechanism of ephrin-A1 upregulation requires further research.