We used an innovative SOMAscan proteomic assay technology
12 to determine the proteins from a blood sample in the first week of life that place an infant at a higher risk of developing clinically significant ROP. We studied over 1000 analytes simultaneously across many protein pathways.
5 It is of interest that we found that lower levels of many proteins distinguished infants with and without clinically significant ROP. An advantage of using this aptamer-based technology is that concentrations of low-abundant proteins can be measured.
5 Our study has initiated a new area of exploration of biomarkers for ROP. Indeed, we uncovered proteins early in the neonatal period that were novel in their association with clinically significant ROP (MnSOD, CRDL1, PCSK9), proteins with links to established signaling pathways for ROP (IGFBP-7),
1,2 and some proteins such as MnSOD that may be a target for a future therapeutic interventions. We also noted different patterns in the trend of concentrations of proteins across the clinically significant, low-grade, and no ROP groups (
Fig. 2). Aligned with the results of previous studies (reviewed in Ref. 2), we also found that infants at the lower extreme of gestational age and birth weight were at the highest risk of developing ROP.
Superoxide dismutase, mitochondrial (MnSOD), an antioxidant found in the mitochondria, was clearly distinguished from other proteins displayed in
Figure 1: MnSOD has a role in scavenging oxygen radicals that result from oxidative stress and provides the cell with a powerful defense against the deleterious effect of elevated levels of reactive oxygen species (ROS) (reviewed in Ref.
13). An increased susceptibility to oxidative mitochondrial injury specifically in central nervous system neurons, cardiac myocytes, and other metabolically active tissues has been reported in a murine model of MnSOD deficiency, after postnatal exposure to ambient oxygen concentrations.
14 Moreover, a role for oxidative stress has been proposed in ROP (reviewed in Ref.
1). Indeed, it has been demonstrated that an increase in SOD ameliorates oxygen-induced retinopathy in transgenic mice.
15 Furthermore, supplementation with liposome-encapsulated SOD significantly increased retinal SOD activity and reduced oxygen-induced retinopathy in a rat model.
16 The results of our study suggest that low levels of MnSOD may be an early neonatal marker of immaturity of the oxygen defense system that places the infant at risk for ROP.
Chordin-like protein 1, a bone morphogenic protein-4 (BMP-4) antagonist, is also a prominent protein as shown in
Figure 1. Kane et al.
17 investigated hypoxia inducible factor-1α-driven expression of CRDL1 in human pericytes and found that expression of CRDL1 and vascular endothelial growth factor (VEGF) was upregulated by hypoxia. The hypoxic state resulted in secretion of CRDL1 from the human pericytes. Chordin-like protein 1 complexed with BMP-4 to antagonize the antiangiogenic effects of BMP-4 on endothelial cells. This tipped the balance in favor of an angiogenic environment.
17 We suggest that the low levels of CRDL1 observed in our study in the first week of life may contribute to an antiangiogenic state. Our suggestion would be consistent with the avascular state of the retina observed during the first phase of ROP. Indeed, ROP is understood to have two phases, possibly preceded by a prephase of antenatal sensitization via inflammation.
2,3 These phases have links with dysregulation of both neuronal and vascular development of the retina.
18,2,3 In phase 1 of ROP, there is a cessation in growth of the retinal blood vessels secondary to high oxygen levels that contribute to a downregulation in oxygen-regulated growth factors such as VEGF.
2 In contrast, phase 2 of ROP is characterized by retinal neovascularization induced by hypoxia.
2,19 During phase 2 of ROP, the compromised retinal blood vessels cannot supply enough oxygen to the developing retina. The metabolic demands of the retina increase, leading to increased hypoxia-driven local VEGF production and retinal neovascularization.
2
In addition to hyperoxia, phase I of ROP is precipitated after a preterm delivery by the loss of factors normally provided by the mother in utero, including IGF-1, which promotes VEGF-mediated development of retinal blood vessels.
2 In our study we found that lower levels of IGF-1 and VEGF placed an infant at risk for ROP, but neither of these relationships with ROP was statistically significant (
Supplementary Table S1). However, another less well studied member of the IGF family of proteins, namely, IGFBP-7, emerged as important in our analysis. We found that levels of IGFBP-7 were lower in infants who subsequently developed ROP compared to infants with no ROP. Aligned with the results of our study, other authors have studied a murine model of oxygen-induced retinopathy and found that the IGFBP-7 gene was downregulated in expression in retinas removed from hyperoxia chambers when compared to retinas exposed to states of normal oxygen concentrations.
20 Furthermore, other investigators have found ties between mutations of the IGFBP-7 gene with a human disease characterized by familial retinal artery macroaneurysms (FRAM) and supravalvular pulmonic stenosis, suggesting a role for IGFBP-7 in vasculogenesis and angiogenesis.
21 Indeed, it is biologically plausible that the immaturity in levels of IGFBP-7 seen in our study may contribute to the abnormal vascular development seen in infants with ROP.
Other potentially interesting protein targets (
Fig. 1) included PRL, a polypeptide hormone produced by the anterior pituitary that stimulates mammary gland development and lactation and has a role in angiogenesis and tumorigenesis.
22 We also found lower levels of some proteins with ties to the inflammatory pathway, for example, HCC-1
23 and eotoxin.
24
Several proteins found at elevated levels were associated with an increased likelihood of developing ROP (
Fig. 1). Fibroblast growth factor 19, a member of the fibroblast growth factor family, is a key regulator of energy metabolism.
25 Infants with elevated levels of MSP, a protein with links to inflammation, were over eight times more likely to develop ROP.
26 Indeed, it is suggested that the presence of infection or inflammation may modify the risk of ROP.
3 Lutenizing hormone, a hormone that stimulates the testes and ovaries to synthesize steroids, was also elevated in infants who developed clinically significant ROP.
27 The significance of this finding is uncertain but perhaps represents an alteration in function of the hypothalamic–pituitary–gonadal axis. Other proteins identified in this group included cystatin-M, a cysteine protease inhibitor, which has a role in the process of desquamation or cell shedding,
28 and plasminogen, the inactive precursor of the enzyme plasmin.
29 Proprotein convertase subtilisin/kexin type 9 was discovered in 2003 and is distinguished as a target for pharmaceutical intervention to reduce low-density lipoprotein-cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 mutations are related to higher levels of LDL-C and an increased risk for cardiovascular disease.
30 With definite links to vascular disease, this protein could potentially have a role in ROP.
There are some limitations to our study. The main limitation was the small sample size, which reduced our ability to conduct a more extensive analysis such as adequate adjustment for confounding variables, adjustment for multiple comparisons, and stratification by gestational age at delivery. The original study was powered for a different outcome (BPD) and utilized infants from additional sites for which we did not have ROP outcomes. As expected, given the small sample size, adjustment for gestational age resulted in the loss of statistical significance of our highly ranked proteins with ROP. We view gestational age as an outcome of pregnancy that is not modifiable once the infant is born. In contrast, the novel proteins discovered in this study are potential modifiable targets for therapeutic interventions in infants at risk for ROP. This was the focus of our study, and it is important to note that even with the small sample size, we uncovered novel proteins early in the neonatal period with a large magnitude of association related to the subsequent development of ROP. Although adjustment for gestational age affected statistical significance of risk for ROP, the magnitude of risk remained meaningful even if it was not statistically significant (
Table 2). These observations are important and deserve future investigation in an adequately powered study of infants at risk for ROP to determine the independent relationship of gestational age and the individual proteins with ROP. An additional limitation was that we conducted the proteomic analysis only on a single sample from the early neonatal period. It will be important in future studies to characterize protein levels and profiles at the time of the ROP examination when the pathologic events in the retina are known to be different from those in the earlier phase of ROP.
2
Notwithstanding these limitations, the results from this state-of-the-art proteomic analysis are informative, and we suggest validation of the results in a larger cohort of preterm infants. The findings of our pilot study related to the protein target MnSOD were especially compelling and deserve further investigation. Infants with lower levels of this protein may not be able to handle oxidative stress in the neonatal period. This result suggests that it is important to consider clinical interventions early in the neonatal period to attenuate the effects of oxygen stress in this vulnerable group of infants.