Retinopathy of prematurity (ROP) is a retinal vasoproliferative disorder that affects preterm infants (PTI) and represents an important cause of blindness and childhood visual impairment [1] in developed and developing countries [2, 3].

The development of the retinal vascularization begins around 16 weeks of gestation, proceeds centrifugally from the optical disc to the peripheral retina, and is completed approximately at term [4]. For this reason, the retina of PTI is incompletely vascularized, with a peripheral avascular area that depends on the immaturity of the newborn infant [4, 5].

Clinical findings in PTI and studies in animal models of oxygen-induced retinopathy (OIR), of which the mouse OIR model (5 days of exposure to 75% oxygen after birth, followed by room air) is one of the most used [6], have shown that ROP has two phases (Fig. 1) [7]. The exposition of the immature retinal blood vessels (BV) to a relatively hyperoxic environment due to premature birth interrupts the vascular development [8]. This leads to microvascular retinal degeneration with an arrest in the vascularization of the peripheral retina (phase 1) [9]. The resulting retinal ischemia triggers the release of growth factors responsible for pathological angiogenesis (phase 2) [7, 10]. The new BV lead to the formation of a fibrovascular scar that may cause retinal detachment and vision loss [11]. It remains unclear why some PTI have a severe and rapidly progressive form of ROP, designated aggressive ROP (A-ROP) [12].

Fig. 1
figure 1

Development of retinopathy of prematurity. During fetal growth, retinal vascularization normally develops from the optic nerve in a centrifugal direction toward the periphery of the retina. Low oxygen tension in utero is the main stimulus for retinal vascular development, which is interrupted due to the increased bioavailability of oxygen caused by premature birth. ROP pathogenesis develops in two phases. In the first phase retinal vessel growth stops and regresses because the hyperoxic environment necessary to maintain adequate levels of circulating oxygen for the survival of the preterm infant inhibits retinal vascularization (Phase 1). As the newborn grows and the metabolic requirements of retina increase, retina becomes hypoxic, inducing a compensatory, albeit devastating and aberrant, neovascularization driven by oxygen-regulated angiogenic factors (Phase 2). EPO erythropoietin; IGF-1 insulin-like growth factor 1; ROP retinopathy of prematurity; VEGF vascular endothelial growth factor

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Retinopathy of prematurity is classified in five stages according to its severity [12]. In stage 1 a fine demarcation line is visible between the vascular and the avascular area of the retina [12]. This flat line can progress to a ridge that defines stage 2 [12]. These two first stages are considered initial or mild ROP and can regress spontaneously. In stage 3 there is pathological vessel proliferation over the ridge and into the vitreous, a feature of severe ROP [13]. The new abnormal vessels can bleed into the vitreous chamber, causing fibrosis and traction and thereby lead to a partial detachment of the retina that defines phase 4 [13]. This can evolute to stage 5, in which retina is totally detached [13].

In most studies over time the incidence of ROP is approximately 60% and of severe ROP (stages 3 to 5) is approximately 15% in PTI with birth weight (BW) of less than 1500 g [14, 15]. However, in a multicenter study conducted in the USA and Canada that included 7,483 with BW of less than 1501 g, the incidence of ROP was 43.1% and that of severe ROP was 12.4% [16] (Table 1). Severe ROP occurs mostly among PTI with birth weight less than 1251 g [16]. Globally, of the 14.9 million PTI in 2010, approximately 184,700 developed any stage of ROP, over 30,000 of whom became visually impaired as a consequence of ROP [17]. Sixty-five percent of the visually impaired due to ROP were born in middle-income regions [17].

Table 1 Summary table of epidemiological and genetic aspects in clinical and experimental studies in ROP
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Retinopathy of prematurity is considered a multifactorial disease that involves prenatal and postnatal factors [18]. Oxidative stress (OS) which can result from inflammation [19, 20] has long been implicated in the etiology of ROP [20, 21]. It is recognized that inflammatory processes can interfere with normal retinal vascularization and, more recently, are also considered important factors in the pathogenesis of ROP [20].

Genetic polymorphisms involve one of two or more sequence variants of a specific DNA sequence and occur with a population frequency of at least 1% [23]. Genetic polymorphisms can influence the activity of encoded enzymes and the susceptibility to develop complications induced by reactive oxygen species (ROS) provided by genes involved in the regulation of OS [1, 22]. Studies have shown associations of genetic polymorphisms in genes involved in the pro-oxidant and pro-inflammatory response to premature birth and diseases related to OS in PTI [23].

Although ROP is strongly associated with extreme prematurity [24], environmental factors have also been implicated in the development of ROP, mainly high oxygen supplementation after birth and fluctuations in oxygenation [25, 26], but also nutrition [27], factors related to the causes of preterm birth [28], use of maternal medications [29], maternal smoking [30], altitude [31], length of day during early gestation [32], and assisted conception [33, 34]. These perinatal factors may alter gene expression through DNA acetylation and methylation, supporting the supposition that epigenetic modifications by external factors may affect gene expression and render PTI susceptible to severe ROP or PTI genetically prone to ROP not to develop retinopathy [35].”

In addition to the contribution of environmental factors, a marked genetic predisposition to ROP is suggested from research based on the candidate gene approach, twin studies, experimental, and clinical studies. The observation that ROP in a subset of PTI progresses to a severe stage, while in others with similar clinical characteristics regresses spontaneously is a strong indication of the genetic contribution to the etiology of ROP [36, 37].

Identifying susceptibility factors for ROP and a better comprehension of its pathogenesis is determinant for its proper prevention and treatment. It may also help to clarify the pathophysiology of other pediatric and adult neovascular retinal diseases. This review focuses on current research that involves inflammation and genetic factors in the pathogenesis of ROP.

The role of inflammation as a stress response: mediators of immune and inflammatory response in ROP

Prenatal and postnatal systemic inflammation might predispose to ROP, and this sensitization effect may constitute a pre-stage of the disease [38]. Inflammatory stimuli such as chorioamnionitis [39] and neonatal bacteremia [40] have been suggested in several studies to be risk factors for ROP, possibly due to systemic inflammation [38]. Systemic inflammation in animal models has also been shown to disrupt the development of retinal BV and leads to aberrant retinal vascularization [41].

Cytokines

Cytokines are intercellular signaling polypeptides released by activated immune cells that are produced during inflammatory processes and in which they participate [42]. There is an overlap in molecular signaling between oxidative and inflammatory compounds, in which complex networks of signaling pathways link oxidative agents and pro-inflammatory cytokines [43] (Fig. 2). The vascular damage of the ischemic phase of proliferative retinopathies is followed by an inflammatory response with the production of pro-inflammatory cytokines, which cause an increase in vascular permeability, immune and other cells recruitment, activation and differentiation, apoptosis, and angiogenesis [44].

Fig. 2
figure 2

Role of oxidative stress and inflammation in the pathogenesis of ROP. After preterm birth, premature infants are exposed to an excess of supplemental oxygen, leading to retinal vascular obliteration due to suppression of pro-angiogenic factors regulated by oxygen, oxidant stress, and excessive production of pro-inflammatory factors by damaged tissues. The vascular dropout results in hypoxia and HIF stabilization with subsequent production of growth factors. The microenvironment of retinal ischemia is characterized by microglial activation and release of many pro-inflammatory cytokines and chemokines, which cause pathological vasoproliferation. The major pro-inflammatory cytokines responsible for early responses are IL-1β, IL-6, and TNF-α. Other pro-inflammatory mediators include IL-17, IL-18, IL-23, IL-33, TGF-β, bFGF, and a variety of other cytokines and chemokines. These cytokines upregulate the synthesis of secondary inflammatory mediators and pro-inflammatory cytokines. IL-6 and TGF-β act as either pro-inflammatory or anti-inflammatory cytokines, under various circumstances. Proangiogenic cytokines, such as IL-1, TNF-α, and VEGF, directly or indirectly stimulate endothelial cells proliferation, migration, and tube formation. IL-1Ra, IL-4, IL-10, IL-11, and IL-13 are major anti-inflammatory cytokines. Except for IL-1Ra, anti-inflammatory cytokines also have at least some pro-inflammatory properties. In ischemic areas, the enhanced production of ROS can further increase the level of pro-inflammatory cytokines. ECM degradation by MMPs activated by inflammatory cytokines, growth factors, and ROS, as well as proteolytic enzymes released from MFs allows EC migration and growth factors recruitment to form new capillaries. AA arachidonic acid; ANGs angiopoietins; bFGF basic fibroblast growth factor; ECM extracellular matrix; EPO erythropoietin; ICAM-1 intercellular adhesion molecule-1; IGF-1 insulin-like growth factor 1; IL Interleukin; IL-1Ra Interleukin 1 receptor antagonist; I-TAC Interferon-inducible T-cell alpha chemoattractant; MCP-1 monocyte chemotactic protein 1; MMPs matrix metalloproteinases; PC prostacyclin; PGs prostaglandins; PLA2 phospholipase A2; PLGF placental growth factor; PPARγ proliferator-activated receptor gamma; ROS reactive oxygen species; TGF-β transforming growth factor beta; TA thromboxane; TNF-α Tumor necrosis factor alpha; VEGF vascular endothelial growth factor; VEGFR vascular endothelial growth factor receptor

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Angiogenesis is strongly orchestrated by a variety of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), and interleukin (IL)-1β [45, 46], and anti-angiogenic cytokines [46]. These cytokines contribute to the proliferation and migration of endothelial cells (EC), which is considered the hallmark of angiogenesis [46, 47].

One study reported significant correlations between levels of different cytokines in the first 3 weeks after birth and ROP development [48]. In an OIR model, the investigation of 94 selected genes known to be related to inflammation showed that many of them were upregulated in association with the clinical appearance of OIR [49]. The same authors analyzed the vitreous levels of 27 cytokines in PTI with stage 4 ROP and found higher levels of interleukin (IL) -6, IL-7, IL-10, IL-15, Eotaxin, bFGF, Granulocyte colony-stimulating factor (G-CSF), interferon-gamma-inducible protein (IP) -10, and mainly, VEGF [50].

Molecules of the IL-1 family acting as the first line of defense against invasive pathogenic microorganisms and physical damage play an important role in inflammatory and immune responses. However, many cytokines in the IL-1 family, such as IL-1α, IL-1β, IL-18, IL-33, and IL-37, contribute significantly to angiogenesis [46, 51]. IL-1β, an important mediator of inflammation [46, 47], in ischemic conditions of the retina is markedly increased in recruited neutrophils, EC, and retinal glial cells [52, 53] and has been implicated in the development of vasoproliferative retinopathies [53]. It has been suggested that in the hypoxic neonatal retina, activated microglial cells produce increased amounts of IL-1β and tumor necrosis factor alpha (TNF-α) that can induce retinal ganglion cell death [54]. TNF-α is also known to contribute to the breakdown of the blood–retinal barrier [54, 55].

In an OIR model, it was shown that retinal microglia is induced to produce IL-1β, leading to microvascular injury by the release of semaphorin 3A (Sema3A) from adjacent neurons [53]. Inhibition of the IL-1 β receptor prevented microglial activation and Sema3A expression in the retina, resulting in a significant decrease in vaso-obliteration and in the subsequent pathological pre-retinal neovascularization [53]. In another OIR model, inhibition of the IL-1 β receptor preserved the choroid and prevented external neuroretinal abnormalities, suggesting IL-1 β as a potential therapeutic target in ROP [56].

A mouse model of premature birth, in which chorioamnionitis was induced with an injection of IL-1β in utero, revealed that IL-1β causes sustained eye inflammation accompanied by delayed development of the retinal BV and thinning of the choroid, with all deleterious effects being prevented by antenatal administration of IL-1 receptor antagonist (IL-1Ra) [57]. However, in a study with preterm infants (PTI), the levels of IL-1β in the vitreous were identical and below detectable levels in patients with ROP and in control patients [50].

Cytokines have pro- and anti-inflammatory properties and regulate the human immune response acting in conjunction with specific cytokine inhibitors and soluble cytokine receptors [58]. IL-1Ra, IL-4, IL-10, IL-11, and IL-13 are considered anti-inflammatory cytokines [58].

The IL-1Ra was found at significantly elevated levels in the vitreous and tears of PTI with ROP, along with increased levels of VEGF, complement component proteins, and metalloproteinase 9 [59], possibly as a compensatory mechanism to prevent angiogenic effects of IL-18 and IL-1β [60].

In vitro, the inflammatory response induced in microglial cells was markedly reduced by IL-10 which inhibited the expression of TNF-α, MIP-1α, and regulated on activation, normal T cell expressed and secreted (RANTES) [61]. However, in an OIR mouse model, hypoxia guided the behavior of the macrophage to a pro-angiogenic phenotype via IL-10-activated pathways, implicating IL-10 in promoting pathological angiogenesis [62].

IL-38 is a novel cytokine from the IL-1 family that shares high-sequence homology with IL-1Ra [46, 63] and lower homology with IL-1β and other IL-1 family proteins [46]. A recent study in the OIR mice found that administration of IL-38 may help prevent pathogenic neovascularization and inflammation, suggesting that IL-38 is an anti-angiogenic cytokine and may have therapeutic potential for angiogenesis-related diseases [46].

IL-18 is a pleiotropic pro-inflammatory cytokine with an immunoregulatory activity [48]. Studies have suggested that the association of IL-18 with ROP may be as an immunoregulator and modulator of angiogenesis [48], promoting the regression of pathological neovascularization instead of inhibiting its development [64]. IL-6 is known to be a strong inducer of the acute-phase protein response; however, it has both pro-inflammatory and anti-inflammatory properties [58]. Twenty-four hours after birth, elevated levels of IL-6 and TNF-α were observed in PTI who subsequently needed treatment for ROP [65].

Tetrahydrobiopterin (BH4) is a crucial cofactor in several metabolic processes, with a fundamental role in maintaining inflammatory and neurovascular homeostasis [66]. A deficiency in BH4 can produce the uncoupling of endothelial nitric oxide synthase (eNOS), causing a reduction in nitric oxide bioavailability and increased ROS production [66, 67]. BH4 is involved in retinal vascular damage induced by oxygen due to its reduction caused by hyperoxia, which can result in decreased eNOS activity and increased superoxide [66, 68].

Chemokines

Chemokines are a family of low-molecular weight peptides that induce the activation and migration of specific cells, especially immune cells, such as leukocytes and microglia, and are involved in the inflammatory responses [69, 70]. The participation of chemokines in angiogenesis, growth control, and hematopoiesis has also been demonstrated [69].

IL-8, the first chemokine to be characterized, plays important roles in both eye inflammation and pathological neovascularization [71]. In a study involving PTI with early-onset clinical sepsis, elevated plasma levels of IL-8 in the first days of life were associated with later development of ROP requiring treatment [72]. In another study, high concentrations of IL-8 during the first three weeks after premature birth were associated with an increased risk for pre-threshold ROP [73]. According to these results, in an OIR rat model, an increased level of an IL-8 homolog was observed during the peak of pathological neovascularization [69].

Monocyte chemotactic protein 1 (MCP-1), one of the most produced and transitory chemokines during inflammation [74], is expressed by activated microglia of the neuroretina and simultaneously an attraction factor for various cells of the immune system, including macrophages/microglia [75]. MCP-1 was found to be significantly increased in umbilical cord blood from PTI who developed ROP compared to PTI who did not develop ROP [76].

Low concentrations of the chemokine RANTES in the blood [48, 77] and vitreous [50] have been found in PTI who have developed severe ROP, suggesting that RANTES may play a protective role. In agreement, high concentrations of RANTES have been associated with a lower risk of ROP [73].

The current modulation of well-known angiogenic cytokines such as anti-VEGF therapy demonstrated efficacy in ocular neovascularization [46, 78]. However, some patients are refractory to anti-VEGF agents, suggesting that other angiogenic or anti-angiogenic cytokines that contribute in a coordinated manner to angiogenesis need to be identified [46, 51].

Microglia

Microglia cells, the retinal-resident macrophages that provide neuroprotection against transient pathophysiological insults and play an important role in neuronal homeostasis, under sustained pathological stimuli become overactivated and release exaggerated amounts of inflammatory mediators that may promote tissue damage [79]. IL -1β, IL-6, TNF-α, interferon-gamma (IFN- γ), and TGF-β are produced by a variety of cell types, being macrophages and monocytes the most important sources at inflammatory sites [42].

The macrophage population residing in many tissues is mainly derived from the yolk sac and fetal liver; however, after tissue injury, inflammatory monocytes recruited from the bone marrow complement it [80, 81]. Recent studies have indicated that macrophages play different roles in the process of intraocular neovascularization (Fig. 3) [82]. Macrophages can be divided into at least two main phenotypes with different functions: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages with a major role in resolving inflammation [82, 83]. After tissue hypoxia, it has been proved that cytokines are involved in the recruitment of monocytes and polarization of macrophages, as well as in angiogenesis [82, 84]. The inflammatory microenvironment leads the macrophages to M1 polarization in an initial phase [85]. The change in the microenvironment in the late inflammatory phase drives macrophages toward the M2 polarization [85].

Fig. 3
figure 3

Mechanisms that influence the main macrophage polarization phenotypes and response to its activation. After injury, resident and recruited macrophages experience remarkable phenotypic and functional variations in response to mediators released into the tissue microenvironment. The dominant phenotype regulates inflammation and tissue repair and may play an active role in the development or inhibition of retinal neovascularization in the OIR model. Cytokines are involved in pathogenesis, monocyte recruitment, and macrophage polarization and therefore are also key factors in the regulation of angiogenesis. IGF-1 insulin-like growth factor 1; IL Interleukin; IFN-γ interferon-gamma; iNOS inducible nitric oxide synthase; LPS lipopolysaccharide; MCP-1 monocyte chemotactic protein 1; MMPs matrix metalloproteinases; OIR oxygen-induced retinopathy; PDGF Platelet-derived growth factor; PLGF placental growth factor; TGF-β transforming growth factor beta; TNF-α Tumor necrosis factor alpha; VEGF vascular endothelial growth factor

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The M1 phenotype can be polarized by lipopolysaccharide and IFN-γ, while other cytokines such as IL-4, IL-10, and IL-13 can induce M2 polarization [82, 86]. In the hypoxic microenvironment, it has been suggested that MCP-1 may play a role in the recruitment of monocytes to the vitreous and retina [82].

It is also increasingly clear that epigenetic modifiers can regulate the fate of macrophages [87]. Differentiation toward M1 or M2 polarization and inflammation in situ are regulated by defined microRNAs subsets [85, 88].

M1 macrophages are seen as phagocytic and pro-inflammatory, secreting large amounts of pro-inflammatory cytokines, such as IL-1β, IL-23, and proteases, reactive nitrogen and oxygen intermediates, and little amount of anti-inflammatory IL-10 [85].

M2 macrophages, instead of M1, have been reported to increase angiogenesis in vivo and highly express bFGF, insulin-like growth factor 1 (IGF-1), placental growth factor (PLGF), and MCP-1 [82, 83]. In the OIR model, the M2 phenotype was concentrated around neovascular tufts, promoting the development of retinal neovascularization [89].

Specific molecular targets associated with macrophages can be considered as a potential treatment in the future for retinal neovascularization; however, further studies are needed [82].

Genetic contribution

There is growing evidence that ROP is influenced by genetic predisposition, epigenetic regulation, and environmental factors [35, 90]. The fact that PTI of the same gestational age (GA) and exposed to identical environmental risk factors can develop ROP characterized by different degrees of severity strongly supports a genetic contribution to the etiopathogenesis of ROP [36, 37]. A study concluded that in PTI with extreme phenotypes, the known clinical risk factors were not significantly associated with the development of ROP, suggesting that other clinical, maternal, or genetic factors may predispose or protect from ROP [2].

The evidence of a genetic influence in ROP also comes from two studies with monozygotic and dizygotic twins that obtained an estimated heritability for ROP of 70% and 73%, respectively [91, 92] (Table 1).

In animal models of OIR, studies of different strains of rats observed differences in the avascular area of the retina and the expression of VEGF between the strains, these phenotypic differences also support the influence of a genetic factor [93, 94].

Epidemiological data and the role of β-adrenergic receptor

The influence of a genetic component in the disease was initially based on racial and regional risk profiles resulting from epidemiological studies [95, 96]. The CRYO-ROP study found that although ROP occurred with similar incidence rates in the Caucasian and black populations, severe ROP was less common in black PTI [95, 97]. This result was found in other later studies [2, 98], although one study found an opposite result, a higher incidence of ROP requiring treatment in black PTI than in Caucasian PTI [99]. Studies have found a higher risk of ROP in Asians and Alaskan natives than in Caucasians [100, 101]. One mechanism to explain some of the racial differences observed in ROP is polymorphisms of the β-blocker receptor. A polymorphism of the G protein-coupled 5 kinase receptor desensitizes β-adrenergic receptors causing resistance to noradrenergic stimuli. Retinal EC have β-adrenergic receptors and this theory of polymorphism is reinforced by reports showing an association of cutaneous hemangiomas with ROP, indicating possible common pathogenesis [102]. Cutaneous hemangiomas show a profound reduction with systemic β-blocker treatment [103, 104] and a β-blocker in eye drops, propranolol 0.2%, reduced the progression of ROP in a recent multicenter clinical trial, being promising in the treatment of ROP [105].

Some studies have reported that the incidence of ROP [106] or progression to severe stages [98, 107,108,109] is more frequent in males than in females. However, in other studies, no difference was observed in the incidence of ROP by gender [110, 111].

Wnt pathway

There are several studies on genetic polymorphisms in genes of the canonical Wnt pathway (dependent on beta-catenin) in ROP (Table 2). Variants of genes in the Wnt pathway cause familial exudative vitreoretinopathy (FEVR) or Norrie disease, which are diseases of the retinal development with characteristics similar to ROP although occurring in full-term infants [36, 90, 112]. Both of these diseases are hereditary disorders and have in common dysgenesis of the retinal vessels with a variable breakdown of the blood–retinal barrier, often leading to exudative and tractional retinal detachment [112, 113]. Molecular genetic studies have identified four genes that cause FEVR (NDP, FZD4, LRP5, and TSPAN12), which when mutated cause X-linked, AD, and AR FEVR (also some sporadic cases) [36]. Norrie disease results from mutations in the NDP gene [114].

Table 2 Studies investigating genetic polymorphisms of candidate genes in ROP
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Several studies correlate variants in genes of the Wnt pathway associated with FEVR or Norrie disease with an increased risk of severe ROP, suggesting involvement of genes associated with the Wnt pathway at least in a low portion of patients with severe ROP [36, 112, 115]. An important limitation of these results is the difficulty in distinguishing between PTI with severe ROP and PTI with FEVR [36]. Due to overlapping phenotypes, ROP is generally differentiated from FEVR by premature birth and lack of family history [112]. However, an ambiguous birth history can confuse the diagnosis [112] and some authors proposed the designation of ROPER (ROP vs. FEVR) to more accurately classify these patients [116].

Vascular endothelial growth factor

VEGF-A plays a key role in physiological and pathological angiogenesis [117, 118]. Studies had implicated VEGF and VEGF receptor (VEGFR)-2 in ROP development [119] and the VEGFA-VEGFR system is the main target for anti-angiogenic treatment [120].

In two studies of the VEGFA -634 polymorphism, the G allele had a higher frequency between PTI with ROP [121, 122]. However, another study reported a higher frequency of C allele in severe ROP [123] and other studies have not confirmed an association between this polymorphism and ROP [124, 125].

VEGFA gene − 460C > T and + 13553C > T polymorphisms have also been associated with ROP [123, 126]. Although − 460C > T has been included in other studies, it was not associated with ROP [125, 127].

The frequency of polymorphisms in the VEGFR-1 and -2 genes did not affect the development of ROP in two studies [125, 126].

Nitric oxide and endothelial nitric oxide synthase

The isoform of nitric oxide-producing enzymes fairly specific for EC, eNOS, has been found to play a notable role in angiogenesis and vasculogenesis [128, 129]. Functional polymorphisms of the eNOS gene affect the expression of eNOS [129] and have been reported to be associated with cardiovascular diseases [130] and diabetic retinopathy in type 1 diabetes [129, 131].

ROP was associated with single-nucleotide polymorphisms (SNPs) of the eNOS gene (T-786C [132, 133] and G894T [132]), but these results have not been replicated in other studies [129, 134].

Neurotrophins and serotonine

Neurotrophins (NTs) are members of a family of polypeptide growth factors that control several aspects related to the survival, differentiation, and function of neurons in the central and peripheral nervous systems and with important functions in non-neuronal cells [135, 136]. NTs family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) [137].

NTs bind to tropomyosin kinase (Trk) receptors, NGF with high affinity for TrkA receptor, BDNF for TrkB receptor, and NT-3 for TrkC, resulting in activation of signaling pathways, namely phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK), and phospholipase C gamma (PLCγ)/protein kinase C (PKC) [138]. BDNF and NGF have been described as playing an important role in the process of angiogenesis [137]. In different cell models, both NGF and BDNF promote the proliferation, migration, differentiation, and survival of EC [139, 140]. Moreover, both have been shown to induce VEGF expression, thus also having an indirect angiogenic role [141, 142].

Studies have associated lower-serum BDNF concentrations in the first week of life in PTI with the development of severe ROP [48]. A large-scale study of candidate genes in a cohort of PTI found SNPs (rs7934165 and rs2049046) in intronic regions of the BDNF gene associated with severe ROP [143]. This result supports the line of thought that neurovascular connections play a role in the development of ROP [144].

Different studies have shown a close relationship between BDNF and glial cell line-derived neurotrophic factor (GDNF) with the serotonergic system in brain development and neuroplasticity [145, 146]. The synthesis and release of the neurotransmitter serotonin in the retina, and the existence of several types of serotonin receptors expressed in the retina, support its retinal neuromodulatory role [147]. Several data show that serotonin is implicated in retinal physiology and pathophysiology and photoreceptor survival; however, retinal signaling pathways activated by serotonin receptors have been little investigated to date [147].

Hypoxia-inducible factor

Hypoxia-responsive genes, such as VEGF and erythropoietin (EPO), are regulated mainly through hypoxia-inducible factor (HIF) [148], a heterodimeric transcription factor consisting of two subunits, HIF-1-alpha (or HIF-2-alpha and HIF3-alpha, their analogs) and HIF1-beta (also known as ARNT) [149]. During hypoxia, the HIF-1α level increases and it binds to HIF-1β in the nucleus to trigger the transcription of genes involved in angiogenesis and adaptation of cells to hypoxia [149].

Endothelial PAS Domain Protein 1 (EPAS1), also called HIF-2α, has high homology to HIF-1α and, like HIF-1α, EPAS1 is stabilized during hypoxia and forms a heterodimer with the ARNT translocator and transactivates the VEGF promoter [150]. In addition, this heterodimer complex has also been shown to transactivate Flt-1, which encodes VEGFR-1 [150].

In a study with a hyperoxia/normoxia treatment, using a murine model of ROP, HIF-2α-knockdown mice showed no evidence of retinal neovascularization when compared to wild-type mice [151]. The expression of EPO mRNA was also significantly decreased when compared to control mice [151]. A candidate gene study in PTI found an association between EPAS1 and the development of severe ROP [152]. It is possible that polymorphisms in the EPAS1 gene in PTI predispose to increased expression of angiogenic factors, such as VEGF and EPO [152].

Erythropoietin

EPO is an oxygen-regulated growth factor [44] and also an important angiogenic factor, its production being regulated by HIF [149]. EPO plays an important role in both the first and second phases of ROP [153]. A candidate gene study investigated the influence of an EPO polymorphism in the development of ROP, but no statistical significance was observed [152].

Insulin-like growth factor 1

IGF-1 is a growth factor supplied by the placenta and amniotic fluid that is crucial for fetal development, including healthy retinal angiogenesis [153]. It has been reported that it is also essential for postnatal vascular eye development and that a prolonged period of low levels of IGF-1 can predict the development of ROP and other diseases related to prematurity [154]. In patients with a genetic defect in the production of IGF-1, a reduction in retinal vascularization was observed, which has not been restored after the administration of IGF-1 [155].

Because the level of IGF-1 is determined by the IGF-1 receptor [156], it is possible that the most prevalent IGF-1 receptor polymorphism (c.3174G > A), which exhibits low levels of free plasma IGF-1, has a role in ROP [36]. However, the association of this polymorphism with the risk of advanced ROP has not been proven by studies in different populations [157, 158].

Angiopoietins

Angiopoietins (ANGs) are growth factors that regulate physiological and pathological neovascularization, specifically in association with VEGF [159, 160]. Although ANG-1 and ANG-2 bind to the Tie2 tyrosine kinase receptor, ANG-2 is a functional ANG-1 antagonist [161, 162]. ANG-1 contributes to the maintenance of vascular integrity [163], while ANG-2 stimulates neovascularization [164] and is upregulated by VEGF and hypoxia [165, 166].

The influence of the ANG-2 (− 35G > C) gene polymorphism on ROP was investigated in two studies; however, no significance was reported [167, 168].

Mediators of immune and inflammatory response

Studies involving PTI have demonstrated the presence of multiple and complex associations of polymorphisms that occur in genes involved in the pro-inflammatory and pro-oxidant response with premature birth and the occurrence of SO diseases complicating prematurity [1, 23]

Regarding the association with ROP, one study suggested an increased risk of ROP progression with the presence of SNPs from the IL-10, IL-1β, and TNF-α genes, without changing the risk with an SNP of the Toll-like receptor-4 (TLR-4) gene, although none of these trends reached formal statistical significance [169].

Heme oxygenase 1

Heme oxygenase 1 (HMOX-1) is an enzyme that breaks down heme into iron ions, carbon monoxide, and biliverdin [134]. HMOX-1 products perform important physiological functions in the vascular system, related to the protection of the endothelium through a cytoprotective, promitogenic, and anti-inflammatory action [170].

The effectiveness of this enzyme is affected by repeated polymorphisms in the HMOX-1 gene promoter [170]. Despite this, no significant association was found between HMOX-1 and ROP in a candidate gene study [134].

Renin–angiotensin system

It has been shown that the renin–angiotensin system may influence the early stages of retinal vascularization [171] and retinal neovascularization was prevented by blocking the renin–angiotensin system in a rat model of ROP [172].

Angiotensin-converting enzyme gene polymorphism has been associated with ROP in the population of Kuwait [173], but not in another population [174].

A study of Angiotensin gene polymorphism found no association with ROP [134], while studies on the angiotensin receptor 1 gene found an association with the development of ROP [152] or found no association [134].

Matrix metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that hydrolyze the extracellular matrix (ECM) [175] and play an important role in inflammatory responses and angiogenesis among other various biological processes [176]. “A” disintegrin and metalloproteinase (ADAM) is also a family of enzymes involved in the degradation of components of the ECM [177].

Based on studies in animal models of OIR, some subtypes of the ADAM family have been suggested to be implicated in the ROP pathogenesis [178, 179]. Genetic studies in humans are needed to assess the influence of ECM, metalloproteinases, and the ADAM family on the pathogenesis of ROP.

Other studies

Apart from the studies described involving factors related to the pathogenesis of ROP, a large study mentioned above identified candidate genes with an unknown relationship with ROP, such as the complement factor H gene [152].

From the above, several evidences suggest a genetic contribution to ROP; however, it is not yet clear which genes or genetic polymorphisms are significantly associated with the development and progression of ROP. Many of the ROP candidate gene studies have limitations, essentially small sample size, non-replicable, or conflicting results from several studies, the latter of which may be at least in part, due to differences in neonatal care and clinical characterization in different countries or populations. In some studies, it may also be difficult to separate the contribution of genetic factors associated with ROP from those associated with prematurity itself.

Epigenetic studies on ROP have not been found in the literature, which could be an important contribution to the emergence of new methods for the treatment of this pathology. Future studies involving next-generation sequencing and genome-wide association, integrated with metabolomics and proteomics, may provide a better understanding of the genetic risk factors and pathophysiology of ROP and contribute to finding new solutions in the management and treatment of ROP.

Expected clinical applicability of studies on genetics and inflammatory pathways in ROP

The search for biomarkers to detect PTI with increased risk of developing ROP allowed the identification of potential indicators involved in inflammatory and angiogenic pathways. Circulating and genetic biomarkers can be incorporated into models to predict the risk of developing ROP. A risk analysis system that includes biomarkers and clinical risk factors can help neonatologists and ophthalmologists identify high-risk PTI. This may allow the development of more adequate follow-up strategies depending on the risk level of the PTI and reduce the number of ROP screening tests for those at lower risk.

A better understanding of the genetic contribution to the pathogenesis of ROP may also help to find new targets that lead to the development of therapeutic approaches that are more effective and less harmful than current ones. In addition, knowledge of the influence of genetic polymorphisms on phenotypic biomarkers (biochemical and cellular) can contribute to defining the ROP phase and, thus, choosing the most appropriate therapeutic approach over time.

Furthermore, a deeper knowledge of the molecular and genetic mechanisms involved in ROP may help to better understand and treat other oxidative stress diseases associated with prematurity with which ROP shares etiopathogenic factors.

Many molecules and related signaling pathways suspected to be involved in the pathogenesis of ROP are common to other pediatric and adult ischemic retinopathies. Thus, a deeper comprehension of molecular mechanisms in ROP may provide important insights to other retinal neovascular pathologies.

Conclusion

Several pieces of evidence suggest that the pathogenesis of ROP begins in utero. Perinatal inflammation and genetic factors may contribute to the development and progression of ROP.

Studies have implicated the involvement of factors linked to the inflammatory process, such as leukocytes, monocytes, macrophages, cytokines, chemokines, and growth factors, in angiogenesis and pathological vascular development of ROP. Cytokines are also involved in monocyte recruitment and macrophage polarization. Macrophages may be recruited by long-term pathological neovascularization, but they can also promote pathological neovascularization.

Several studies have found genetic polymorphisms in candidate genes associated with ROP or severe ROP. Many pathways and their signaling molecules have been studied due to their connection with the pathogenesis of ROP. Associations were found between genes involved in the WNT signaling pathway, the VEGFA gene and the eNOS gene, and the development of ROP. A large multicenter study found polymorphisms in the BDNF gene associated with severe ROP.

Although multiple genes have been implicated in several investigations, a genetic component with a major impact on ROP has not yet been discovered. Several of these studies have not replicated findings mainly because of limitations in aspects such as sample size, non-replicable or conflicting results, and differences in neonatal care or inclusion criteria. The knowledge of such a genetic component would possibly allow the identification of possible targets to improve the screening and treatment of ROP.

New technologies involving bioinformatics, genomics, and proteomics may contribute to find genes or pathways associated with ROP and help in the future to find better solutions in the management and treatment of ROP.