Plasma Level of Apelin as a Promising Factor for Retinopathy of Prematurity

Retinopathy of prematurity (ROP) is a preventable cause of childhood blindness. This raises crucial considerations about ROP prevention efforts, which should include ensuring that all infants who are at risk are screened in screening programs [1‒3].

In China, the current ROP screening guidelines are based on perinatal variables such as low birth weight (BW) and gestational age at birth, which are common in most nations [4]. The existing screening criteria were sufficient to include all infants with severe ROP, allowing more children to receive treatment sooner [5]. However, the screening and diagnosis of ROP demands a high workforce load for ophthalmologists and is not universal owing to the lack of skilled personnel, especially in the rural areas. No laboratory test can aid in the early detection of ROP [6]. Therefore, any practical tool for predicting which infants may develop ROP, such as blood testing, would be extremely useful [7].

Vascular endothelial growth factor (VEGF) plays a key role in ROP, and one study suggests that VEGF may be a predictor of ROP onset [7]. In our previous study, we discovered that the apelin/APJ system may have a role in the development of ROP retinal neovascularization, which was linked to HIF-1 but not VEGF [8, 9]. A couple of studies and our previous studies have already found that apelin is increased in newborns with ROP [8, 10‒13]. Lofqvist et al. [14] suggested that babies be monitored using the WINROP algorithm, which takes insulin-like growth factor (IGF-1) levels into account. In addition to being a potent ischemia-induced angiogenic agent independent of VEGF, recombinant human erythropoietin (EPO) treatment has been linked to an increased risk of ROP [15]. All of the cytokines described have been proven to play key roles in the pathophysiology of ROP, although their potential value as a predictor of ROP development has yet to be determined. In this prospective study, we aimed to determine the levels of VEGF, apelin, EPO, and IGF-1 in newborn plasma and assess their prognostic value for the development of ROP.

The investigation was conducted in accordance with the Declaration of Helsinki and was approved by the Investigational Review Board of the PLA Rocket Force Characteristic Medical Center (KY2016017) and the Ethics Review Board of Peking University People’s Hospital (2015PHB108-01). All examinations and procedures required written informed consent from the parents or guardians, who submitted their written informed consent to participate.

Undiluted blood samples of fifty ROP patients (study group) and fifty non-ROP preterm patients (control group), with a gestational age (GA) of less than 35 weeks or weight less than 2,000 g at diagnosis, were collected. The exclusion criteria were as follows: (1) received either local (laser or anti-VEGF) or systemic (supplementary IGF-1 or EPO) treatment before the study, (2) congenital and chromosomal anomalies, or (3) absence of informed consent from the parents.

After pupillary dilation, the fundus of infants with ROP was evaluated using binocular indirect ophthalmoscopy under topical or general anesthetic. The medical histories of the patients were reviewed, and parents were asked for further medical and family history information. BW, GA, sex, postmenstrual age, and chronological age at hospital admission were all recorded. The infant’s ROP stage was documented at the time of the first procedure; if the infant’s eyes had distinct ROP stages, the infant’s stage was recorded as the more advanced stage. According to the international classification system, infants with ROP were divided into mild and severe groups. Infants treated for type 1 pre-threshold and threshold disease as well as those presenting with stages 4 and 5 ROP were included in the term “severe ROP” [16, 17]. Treatment is necessary for the severe group but not for the mild group.

The blood samples were taken in sterile tubes with EDTA and centrifuged for 10 min at 3,000 rpm at room temperature. The clear supernatant was immediately removed, frozen, and kept at −80°C until use in a sterile plastic corning (2 mL, Corning, NY). Within 6 months of collection, the samples were analyzed.

The plasma levels of apelin, EPO, IGF-1, and VEGF were quantified with enzyme-linked immunosorbent assay (ELISA), using kits for human apelin, EPO, IGF-1, and VEGF (human apelin ELISA Kit, Sigma-Aldrich, Inc., USA, and human EPO, IGF-1, and VEGF ELISA Kit; Quantikine EPO, IGF-1, and VEGF ELISA Kit, R&D Systems Inc., Minneapolis, MN, USA). Each test was carried out in accordance with the manufacturer’s instructions. There were a few missing values due to the limited volume of the plasma samples.

A commercially available statistical software tool (SPSS for Mac, version 22.0, SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis of the data. Data were presented as the mean ± standard deviation, medians (interquartile ranges), or as a number (%). A one-sample Kolmogorov-Smirnov test was used to determine whether the samples were normally distributed. Differences between two groups were estimated with a nonparametric Mann-Whitney rank sum test and t test when appropriate. To compare noncontinuous variables, the χ² test was utilized. With the skewed distribution data of cytokines, the nonparametric Spearman’s correlation test was used for ROP patients and full cohort. Statistical significance was defined as a two-tailed probability of less than 0.05.

A total of 100 newborns participated in the study, including 50 in the study group and 50 in the control group. The groups did not differ significantly in terms of gender, mean postmenstrual age, or mean chronological age. The mean BW and GA of babies with ROP were considerably lower than those without ROP (p < 0.001, p = 0.003, respectively, Table 1).

Among infants with ROP, the plasma levels of VEGF, EPO, and IGF-1 were significantly lower than among infants without ROP (p = 0.001, p < 0.001, and p < 0.001, respectively, Fig. 1). However, the plasma apelin level was higher in infants with ROP than among infants without ROP (p < 0.001, Fig. 1).

There was no significant difference in VEGF, EPO, and IGF-1 plasma levels in infants with mild or severe ROP (p = 0.488, p = 0.123, and p = 0.229, respectively, Fig. 2). However, in infants with severe ROP, the plasma apelin level was higher than in infants with mild ROP (p = 0.008, Fig. 2). All correlation tests showed that BW and GA were not significantly associated with cytokine levels in ROP infants (all p > 0.05), while plasma IGF-1 levels were significantly correlated with the BW and GA in the full cohort (p = 0.021, and p = 0.001, respectively, Table 2).

The receiver operating characteristic curve of the predictor in the ROP group was shown in Table 3. We compared the sensitivity and selected the best cut-offs while keeping the specificity constant (80.0%). Among all the criteria, plasma apelin levels had the best sensitivity (72%), with the cut-off of 21.08 pg/mL. The area under the curve (AUC) values for BW and GA were 0.65 and 0.52, respectively. The AUC values for apelin, VEGF, EPO, and IGF-1 were 0.70, 0.58, 0.63, and 0.61, respectively.

Multivariable logistic regression analyses showed that the plasma level of apelin was the only parameter associated with ROP (odds ratio 16.0; 95% CI, 1.54–166.53; p = 0.02, Table 4). The AUC of the regression model that comprised GA, BW alone was 0.67, while that of model that included apelin was 0.90 (Fig. 3).

The results showed higher apelin levels in ROP newborns’ plasma, which demonstrated good sensitivity and specificity with regard to the diagnosis of ROP, while the plasma levels of other cytokines decreased. Apelin has been reported as necessary for appropriate blood vessel expansion and endothelial cell proliferation [18, 19]. Some investigations in proliferative diabetic retinopathy have found greater apelin levels, particularly in the vitreous, and it has been postulated that this plays a role in the pathogenesis of proliferative retinopathy [20, 21]. In our previous study, we discovered that serum apelin levels were greater in infants with ROP, and apelin levels are a key factor in determining the likelihood of developing severe ROP, whereas infants with ROP had lower VEGF levels [12]. In this study, we demonstrated higher apelin levels in ROP newborns’ plasma and we evaluated its prognostic value in ROP.

The expression of apelin is increased during the creation of new vasculature and then downregulated once the vessels have stabilized [22]. In oxygen-induced retinopathy animal models, Kasai et al. [23] discovered that retinal apelin expression was greatly enhanced during the hypoxia phase. Furthermore, high apelin levels have been found in human placental tissue, indicating that this peptide plays an important role in fetal development through regulating placenta formation during pregnancy [24].

In China, there are many maternal hospitals and rural comprehensive hospitals that lack an ophthalmology department. Very few ophthalmologists can do the required screening, so a lack of properly trained ophthalmologists is common [25]. Harder still is that such premature babies are unable to withstand the long distance to be referred to a capable hospital to do the screening. Therefore, plasma levels in premature infants can be useful as an indicator in ROP screening, which can provide a preliminary risk notification to the recommendation for the screening, especially for the rural areas.

The regression analysis suggested that plasma apelin levels were the most associated factor with ROP. Through our analyses, we found that when the Youden index reaches the highest value, GA and IGF-1 had higher sensitivity, while VEGF and apelin levels had better specificity. In general, the cut-off value should be determined according to the specific requirement. When the disease is severe and treatable, sensitivity should be raised. Furthermore, if the disease is severe but untreatable, or if a false positive result has caused serious mental and economic trauma (such as an HIV antibody diagnosis), the specificity should be increased. However, in this study, we cared more about which baby definitely needed to be referred to a distant hospital to do the screening, even taking a risk. Thus, the best specificity should be more valuable. In this study, when the cut-off value of plasma apelin level was 21.08 pg/mL, the sensitivity and specificity reached 72–80%, respectively. In addition, the receiver operating characteristic curve of the plasma apelin level had strong discrimination capabilities in the context of diagnosing ROP.

In response to a variety of cytokines, vascular endothelial cells proliferate and migrate across the extracellular matrix during normal retinal vascular development [13]. In this investigation, we discovered that plasma levels of VEGF, EPO, and IGF-1 were significantly lower in newborns with ROP, which was consistent with previous research. Consecutive tests of VEGF in serum showed a progressive increase in normal infants, but the levels remained low in children with ROP who needed treatment, according to Kwinta et al. [26] On the 28th day after delivery, Yang et al. [27] found that the serum EPO level in the ROP group was considerably lower than in the non-ROP group, suggesting that serum EPO was an independent predictor of ROP. IGF-1 levels were clearly lower in the group that developed ROP at the third week postpartum, regardless of GA at birth (29.13 vs. 43.16 ng⁄mL, p < 0.05) [28]. Additionally, persistently low IGF-I serum concentrations following premature birth are linked to the development of ROP and other prematurity problems later on [29]. IGF-1 levels are lower in newborns with lower BW and VEGF levels are lower in infants with a higher chance of developing ROP, according to cord blood data [7].

Potential limitations of our study should be mentioned. First, this study enrolled a limited number of patients, and due to the small amount of plasma samples, there were a few missing values. Second, it is unfortunate that our center was not yet using Retcam technology; therefore, no electronic images can be saved. An experienced retinal disease ophthalmologist validated the accuracy of the diagnosis. Third, our findings should be regarded preliminary, screening criteria should be applied before the diagnosis, and cytokines should be collected prior to ROP diagnosis. Our findings merely suggest that plasma apelin levels could be a promising factor to include in screening criteria. To confirm, more research with cohort design and bigger number of samples would be beneficial. In conclusion, our study about the elevated plasma apelin level demonstrated good sensitivity and specificity for ROP, suggesting that it could be a potential factor to involve in the screening criteria and may decrease the examination burden.

The investigation was conducted in accordance with the Declaration of Helsinki and was approved by the Investigational Review Board of the PLA Rocket Force Characteristic Medical Center (KY2016017) and the Ethics Review Board of Peking University People’s Hospital (2015PHB108-01). All examinations and procedures required written informed consent from the parents or guardians, who submitted their written informed consent to participate.

The authors have no conflicts of interest to declare.

This study was supported by Beijing Hospitals Authority Youth Programme (No. QML20190303), National Natural Science Foundation of China (Nos. 82101142 and 82070948), Beijing Municipal Science & Technology Commission (No. Z171100001017229), Beijing Talent Project (No. 2020027), Shunyi District Beijing Science and technology achievements transformation coordination and service platform construction fund (SYGX202010), and Scientific Research Program of Beijing Municipal Commission of Education (KM202010025020).

Design and conduct of the study by Jing Feng, Xiaorui Zhang, and Yong Tao. Collection, management, analysis, and interpretation of the data by Jing Feng, Ge Liang, WeiPing Gao, Xiaoqin Li, Lin Wei, Hongyu Chang, Xiaorui Zhang, and Yong Tao. Preparation of the manuscript by Jing Feng, Xiaorui Zhang, and Yong Tao. Review and final approval of the manuscript by all the authors.

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding authors.