Role of macular xanthophylls in prevention of common neovascular retinopathies: Retinopathy of prematurity and diabetic retinopathy

Archives of Biochemistry and Biophysics 572 (2015) 40–48

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Role of macular xanthophylls in prevention of common neovascular retinopathies: Retinopathy of prematurity and diabetic retinopathy Xiaoming Gong, Lewis P. Rubin ⇑ Department of Pediatrics, Texas Tech University Health Science Center, Paul L. Foster School of Medicine, El Paso, TX 79905, USA

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Article history: Received 30 November 2014 and in revised form 3 February 2015 Available online 18 February 2015 Keywords: Lutein Zeaxanthin Xanthophylls Retina Diabetes Prematurity Retinopathy Oxidative stress Prevention

a b s t r a c t Retinopathy of prematurity (ROP) and diabetic retinopathy (DR) are important causes of blindness among children and working-age adults, respectively. The development of both diseases involves retinal microvascular degeneration, vessel loss and consequent hypoxic and inflammatory pathologic retinal neovascularization. Mechanistic studies have shown that oxidative stress and subsequent derangement of cell signaling are important factors in disease progression. In eye and vision research, role of the dietary xanthophyll carotenoids, lutein and zeaxanthin, has been more extensively studied in adult onset macular degeneration than these other retinopathies. These carotenoids also may decrease severity of ROP in preterm infants and of DR in working-age adults. A randomized controlled clinical trial of carotenoid supplementation in preterm infants indicated that lutein has functional effects in the neonatal eye and is anti-inflammatory. Three multicenter clinical trials all showed a trend of decreased ROP severity in the lutein supplemented group. Prospective studies on patients with non-proliferative DR indicate serum levels of lutein and zeaxanthin are significantly lower in these patients compared to normal subjects. The present review describes recent advances in lutein and zeaxanthin modulation of oxidative stress and inflammation related to ROP and DR and discusses potential roles of lutein/zeaxanthin in preventing or lessening the risks of disease initiation or progression. Ó 2015 Elsevier Inc. All rights reserved.

Introduction Retinal neovascularization (NV)1 from ischemia-induced retinopathy are common causes of blindness in children (retinopathy of prematurity, ROP) and working-age adults (diabetic retinopathy, DR) in the developed world. Ischemic retinopathies are characterized by microvascular degeneration and retinal ischemia, which can lead to secondary aberrant neovascularization, hemorrhages and blindness [1–3]. This pathologic angiogenesis could be prevented either by direct inhibition of the pathologic NV or by reducing retinal vessel loss, thus, decreasing the hypoxic stimulus that drives the NV. Current therapeutic strategies for ischemic retinopathies have focused on the former approach of inhibiting later stages of pathologic NV. There is great potential in the alternative ⇑ Corresponding author at: Department of Pediatrics, Texas Tech University Health Science Center, Paul L. Foster School of Medicine, 4800 Alberta Avenue, El Paso, TX 79905, USA. Fax: +1 915 545 6785. E-mail address: [email protected] (L.P. Rubin). 1 Abbreviations used: NV, retinal neovascularization; DR, diabetic retinopathy; ROP, retinopathy of prematurity; AMD, age-related macular disease; MPOD, macular pigment optical density; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; IGF-1, insulin-like growth factor-1; BCO2, b-carotene 90 ,100 -dioxygenase; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase-1 http://dx.doi.org/10.1016/j.abb.2015.02.004 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.

strategy of reducing vessel loss or promoting normal vascular repair, thereby reducing the hypoxic stimulus that drives NV [4–6]. Such a strategy could result in reducing retinal vascular degeneration, increasing retinal revascularization, or the combination of both. It is, therefore, of great benefit to learn more about the factors and mechanisms that govern the extent of vessel loss and normal vascular regrowth in ischemic retinopathies. In human retina, two vascular systems (retinal and choroidal) provide blood supply to the highly oxygen-consuming retinal layers. The retinal vascular system provides oxygen and nutrients to the inner retina, whereas the choroidal vasculature supplies the outer retina [3]. In ROP, a delay in physiologic retinal vascular development in preterm infants, suppression of growth factors due to hyperoxia and increase in metabolic demand is associated with hypoxic retinal injury [7]. The hypoxic retina stimulates expression of oxygen-regulated proangiogenic factors, which stimulates retinal NV with sprouting of abnormal vessels from the retina into the vitreous [8,9]. In diabetes, elevated blood glucose and decrease in blood flow result in hyperglycemia and hypoxia in the retina [10,11]. The retina is highly susceptible to oxidative damage by reactive oxygen species (ROS). During pathological conditions, such as retinal ischemia, the imbalance between the production of ROS

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and the ability to scavenge these ROS by endogenous antioxidant systems is exaggerated. ROS triggers several signaling pathways, affects DNA and lipids inside the cell, and subsequently leads to cell death. Antioxidants that can inhibit or prevent the oxidative processes can protect retinal cells from oxidative damage. Antioxidants inhibit microvascular degeneration in animal models of DR [12,13] and oxygen-induced retinopathy (OIR) [14,15]. Recent clinical trials of lutein supplementation in term [16] and preterm infants point to lutein functional effects in the neonatal eye [17]. The carotenoids, lipophilic pigments, are important antioxidants, anti-inflammatory agents and regulators of development, reproduction, cellular differentiation and vision protection. They are characterized by an extended conjugated p-electron system that can only be synthesized by plants and microorganisms. Of the >700 described natural carotenoids, approximately 50–60 are typically consumed in the human diet, but only 15–20 are usually detected in human serum and tissues [18], including a-carotene, bcarotene, b-cryptoxanthin, lycopene, lutein, and zeaxanthin. Of these, the xanthophylls (lutein and zeaxanthin) account for 20– 30% of total carotenoids in human serum but 80–90% of the total carotenoids in the human retina [19]. In early primate development, lutein is the predominant retinal carotenoid. Over time, zeaxanthin levels rise, partly due to conversion of lutein to zeaxanthin. Meso-zeaxanthin, a specific lutein metabolite localized in the retina, has not been detected elsewhere in the body [20]. These xanthophylls are most dense at the center of the fovea in the yellowish pigmented area called the macula lutea and are referred to as macular pigment [21,22]. The macular pigment is tissue protective, acting via antioxidant, anti-inflammatory and light-screening properties [23,24]. Low systemic and retinal levels of lutein and zeaxanthin are adversely associated with the risk of age-related macular disease (AMD), ROP and DR [25–29]. Nevertheless, the molecular mechanisms underlying xanthophyll actions in the retina still remain elusive and whether dietary lutein/zeaxanthin can prevent or lessen severity of ROP and DR in practice remains somewhat inconclusive. To date, the majority of the evidence on protective effects of lutein and zeaxanthin in visual health has addressed AMD and cataract. In this review, we describe various aspects of ROP and DR pathogenesis and discuss the potential role of lutein and zeaxanthin in these common neovascular retinopathies of younger individuals.

Retinal vascular development and pathogenesis of retinopathy of prematurity Retinal vascular development comprises two phases: vasculogenesis and angiogenesis. Vasculogenesis is characterized by de novo formation of blood vessels from endothelial precursor cells within the central retina. Angiogenesis is characterized by the development of new vessels that sprout from preexisting vessels [30] and is responsible for increasing the vascular density and peripheral vascularization in the inner retina. During fetal development, the relative hypoxic intrauterine environment promotes production of vascular endothelial growth factor (VEGF), stimulating retinal vascular development. The development of the human retinal vasculature starts at approximately the 16th week of gestation in a central-to-peripheral wave at a rate of about 0.1 mm/day and continues throughout gestation; it reaches an adult pattern at term (i.e., the 40th week of gestation) [31]. Hence, when the infant is born prematurely, her/his retinal blood supply is incomplete. This immaturity in vascular development predisposes the retina to complications. In addition, the developing retina is highly susceptible to oxidative stress [32]. Prematurity further complicates the infant’s ability to deal with oxidative stress via imbalanced antioxidant to oxidant ratio, resulting in increased ROS.

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ROP is a retinal ischemic and vasoproliferative disease associated with premature birth [33]. It is a major cause of blindness in children in the developed and developing worlds, despite current disease treatment. ROP is characterized initially by a delay in physiologic retinal vascular development, and subsequently by aberrant angiogenesis in the form of intravitreal neovascularization [34]. The more profound the immaturity at birth and persistence of developmental arrest due to exposure of the retina to harmful factors, coupled with deficiencies of factors normally provided in utero, the more aggressive the later pathological response. The development of ROP progresses through two postnatal phases [35], possibly preceded by a prophase of antenatal sensitization via inflammation [36,37]. The first phase begins with the interruption of normal retinal vascular development at the time of preterm birth, accompanied by a sudden reduction in insulin-like growth factor-1 (IGF-1) and VEGF [38]. Premature infants are exposed to higher oxygen tension after birth compared to that in utero, which leads to downregulation of the major hypoxia-triggered VEGF and subsequent regression of developed retinal vessels. This relatively avascular preterm retina responds to increasing hypoxia and metabolic demand by triggering an abnormal proliferation of vessels leading to neovascularization, the second phase of ROP progression (Fig. 1) [39]. In this phase, overproduction of hormones and growth factors to ensure adequate perfusion to the now hypoxic retina occurs; in particular, VEGF, but also IGF-1 are produced. These factors influence production of extracellular matrix proteins (vitronectin, fibronectin and fibrinogen), deposit adhesive fibrins and induce growth, differentiation and migration of endothelial cells [40]. Preterm birth also is associated with reduction of enzymatic and nonenzymatic antioxidants which are produced or accumulated later in gestation, such as superoxide dismutase (SOD), catalase, vitamin C, vitamin E and lutein/zeaxanthin [41]. Moreover, preterm newborns poorly auto-regulate oxygen delivery to tissues during oxygen administration in intensive care [39]. One example of the presence of oxidative stress is levels of 8-hydroxy-20 -deoxyguanosine (8-OHdG), an indicator of oxidative stress, are elevated in leukocytes and urine from preterm infants with ROP [42]. During the development of ROP in the rat OIR model, the retina is subjected to fluctuating oxygen tensions, resulting in retinal hypoxia that triggers overproduction of ROS, which activates nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [4]. Intravitreal NV is promoted by resulting activation of signaling pathways such as Janus kinase and signaling transducer and activator of transcription 3 (JAK/ STAT3) [43]. Inhibiting ROS with the NADPH oxidase inhibitor apocynin reduces the avascular retina by interfering with apoptosis [4]. Increased levels of NOS, which contribute to the nitric oxide (NO) production, is observed in neonatal retina exposed to hypoxia [44]. In the animal model of ROP, increased formation of retinal peroxynitrite and apoptosis of endothelial cells were seen in association with increased tyrosine nitration of PI3K, cleaved caspase3, activation of p38 MAPK signaling pathways, and decreased protein kinase B (Akt) phosphorylation. Blocking tyrosine nitration of PI3K with epicatechin or N-acetylcysteine (NAC) reversed the nitro-oxidation-induced pathogenesis [45]. In addition, application of a NOS inhibitor or genetic ablation of endothelial NOS effectively attenuates the severity of ROP in mice, demonstrating the importance of nitro-oxidative stress in ROP [46]. Repeated oxygen fluctuations in the rat OIR model also increase VEGF production, a major signaling molecule involved in hypoxiainduced NV and in ROP. In the retina, VEGF is primarily secreted by Müller cells and astrocytes. In the second phase of ROP, relative hypoxia induces VEGF production and, hence, promotes pathological vessel proliferation [47]. Levels of IGF-1, another key factor in retinal development, are inversely related to ROP. IGF-1

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trations of neurotrophin-4 and IL-17 were found on postnatal days 0–3; whereas on postnatal days 7–21, there were higher levels of IL-18 [57] and tumor necrosis factor a (TNF-a) [58]. Simultaneous analyses of vitreous levels of 27 cytokines in eyes with ROP showed higher levels of several cytokines in eyes with ROP, including IL-6, IL-7, IL-10, IL-15, eotaxin, basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), macrophage granulocyte colony-stimulating factor (GM-CSF), interferon-c-inducible protein 10 (IP-10) and much higher VEGF [59]. In a mouse OIR model, increasing intake of omega-3-polyunsatuated fatty acids (PUFAs) has been shown to attenuate production of excessive cytotoxic concentrations of TNF-a, and progression from early ROP phases to threshold ROP [60].

Diabetes mellitus and pathogenesis of diabetic retinopathy

Fig. 1. Schematic showing the relationship between oxidative stress and retinopathy of prematurity. Premature birth and hyperoxia can inhibit VEGF, IGF-1, and erythropoietin (EPO) production, resulting in hypoxic and oxidative retinal injury which, in turn, stimulates expression of oxygen-regulated factors such as EPO, VEGF, and production of cytokines, consequently leading to retinal neovascularization or ROP.

deficient mice exhibit poor retinal vascular growth, which suggests low IGF-1 might contribute to suppressing vascular growth in ROP [48]. Interestingly, in preterm infants, low serum IGF-1 directly correlates with severity of ROP [49]. IGF-1 modulates vessel survival via controlling VEGF-induced activation of Akt and ERK1/2 MAPK, essential for endothelial cell proliferation [50]. In this regard, IGF-I appears to act as a permissive factor for VEGF-dependent vascular endothelial cell growth and survival in the first phase of ROP [48]. In preterm infants after birth, lower IGF-1 levels reduce Akt activation and endothelial cell survival, which facilitates avascular retinal hypoxia and leads to VEGF accumulation in the vitreous [48]. IGF-1 levels rise with higher VEGF and may trigger NV through activation of MAPK and Akt signaling pathways in the second phase of ROP [5,6]. Inhibiting IGF-1 and neutralizing VEGF bioactivity can decrease NV and vessel tortuosity. Although exposure of the immature retina to excessive oxygen is an important factor in ROP pathogenesis, accumulating evidence shows perinatal infection and inflammation are associated with increased risk for ROP [37]. Epidemiological studies have shown that the incidence of ROP is higher in infants with early or late onset neonatal sepsis [51–53]. Higher rates of ROP have been demonstrated in infants born to mothers with histological and clinical chorioamnionitis relative to mothers without chorioamnionitis [54,55]. Chorioamnionitis and the accompanying fetal inflammatory response syndrome may increase the risk of ROP by directly sensitizing the developing retina to oxygen-induced changes in VEGF availability and subsequent vascular development and/or by causing systemic hypotension resulting in retinal hypoperfusion/ischemia [37,56]. These clinical data indicate a need for experimental studies to elucidate the pathophysiological mechanisms underlying the increased risk of ROP in infants exposed to chorioamnionitis [56]. Although the role of inflammation in ROP has been poorly investigated, some evidence in humans points to its contribution in ROP. In ROP infants, higher systemic levels of interleukin 6 (IL-6) and C-reaction protein (CRP) and lower concen-

Diabetes mellitus is a heterogeneous metabolic disease characterized by hyperglycemia resulting from defective insulin secretion (type 1), resistance to insulin action (type 2), or both. DR is the most common cause of acquired blindness in individuals between the ages of 20 and 65 years. All patients with diabetes mellitus are at risk. The longer an individual has diabetes, the greater the chance of developing DR. The total number of people with diabetes, estimated to be 285 million worldwide in 2010 [61], is rapidly increasing as a consequence of rising obesity rates, increasing life span and improved disease detection. Despite evidence that tighter control of blood glucose and blood pressure reduces the risk of microvascular diabetes complications, as well as tremendous advances in the clinical management of diabetic eye disease, rates of DR in the US have increased by 89% over the last decade. Significant visual impairment associated with diabetes remains high, and recent estimates show that nearly 5% of U.S. adults with diabetes have sight-threatening diabetic retinopathy (STR); DR rates are higher among African, Latino and Native American populations [62]. Moreover, global estimates of DR and STR based on pooled analysis of population-based studies show 93 million cases of DR and 28 million cases of STR. That suggests that nearly 2.8 million Americans may have sight-threatening diabetic retinopathy [63]. Pathological features of DR are basement membrane thickening, loss of vascular pericytes, increased VEGF synthesis, microaneurysms, blood–retinal barrier breakdown, and NV [64]. Microvascular disease is a manifestation of DR which leads to retinal ischemia/hypoxia and exacerbates the condition [65]. Diabetes causes a number of metabolic abnormalities, including hyperglycemia, dyslipidemia, hypertension and oxidative stress. These factors interact to increase ROS production by mitochondria, especially within the retina and other insulin-independent tissues (small caliber neurons, renal glomeruli and aorta) [66]. Mitochondrial ROS activates specific and well-established biochemical pathways within retinal cells that directly lead to vascular and retinal ganglion cell damage [64,67]. Release of inflammatory mediators, such as interleukin-1b (IL-1b), TNF-a, intracellular adhesion molecule-1 (ICAM-1) and angiotensin II, activation of microglial cells and apoptosis of capillary endothelial cells and retinal ganglion cells may lead to breakdown of the blood–retinal barrier, resulting in vascular leakage, hypoxia and retinal NV – the hallmarks of DR. Elevated blood glucose plays a central role in DR, worsening dyslipidemia and hypertension, and driving oxidative stress that damages the retina. Prolonged elevation of blood glucose level causes oxidative stress via several pathways. As shown in Fig. 2, four major pathways are involved in development of pathologies in DR: (1) increased polyol pathway flux; (2) increased formation of advanced glycation end-products (AGEs), (3) activation of protein kinase C (PKC) isoforms, and (4) increased hexosamine pathway flux [68]. In hyperglycemia, aldose reductase, the first and rate-limiting

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Fig. 2. Schematic representation of the main factors leading to diabetic retinopathy. Diabetes can cause high blood pressure, high and abnormal cholesterol and hyperglycemia. Hyperglycemia induces several biochemical processes with important pathogenic implications. Hyperglycemia-induced activation of abnormal signaling pathways (polyol, AGE/RAGE, PKC and hexosamine) leads to oxidative stress and production of inflammatory cytokines and mediators, and consequently, DR. Several diabetes-induced abnormalities in the retina are influenced by oxidative stress, and are considered to be interrelated.

enzyme in the polyol pathway, reduces glucose to sorbitol, consuming NADPH for generating the intracellular antioxidant GSH. Sorbitol is then oxidized to fructose by the enzyme sorbitol dehydrogenase. The reduction in the availability of NADPH exacerbates intracellular oxidative stress [68,69]. AGEs and its receptor, RAGE, also promote oxidative stress. AGEs induce oxidative damage by overproduction of superoxide through the activation of NADPH oxidase in a PKC-d-dependent manner [70]. Increased intracellular formation of AGE and RAGE activates a cascade of signaling proteins and attributes to intracellular redox imbalance, which leads to overproduction of ROS [71]. In addition, hyperglycemia itself leads to intracellular accumulation of glyceraldehyde 3-phosphate (G3P) which further induces the production of ROS and leads to subsequent accumulation of poly(ADP-ribose) polymerase-1 (PARP), triggering activation of PKC and increased AGE formation [72,73]. Indeed, activation of PKC signaling pathways and overproduction of ROS by the mitochondria electron transport chain are the key factors in the pathogenesis of DR [64,69]. Oxidative stress causes mitochondrial dysfunction by damaging the inner membrane, which leads to imbalance in the electron transport chain and consequently leads to further overproduction of superoxide, ONOO and hydroxyl radicals [74]. ROS also impairs mitochondrial function and transport machinery by damaging the mitochondrial DNA and increases apoptosis of retinal capillary cells during DR [75–77]. Increased ROS levels trigger the release of cytochrome c which, in turn, disrupts mitochondrial membrane potential and initiates apoptosis via activation of caspase-9 and caspase-3 [78]. Several signaling pathways are involved in the pathogenesis of DR. MAPK has been shown to play a role in hyperglycemia-induced cell death in DR [79]. In an in vitro model of hyperglycemia and hypoxia, phosphorylation of c-Jun N-terminal kinases (JNK) and p38 MAPK is induced, leading to overproduction of ROS and disruption of tight junctions in ARPE-19 cells [80]. In streptozotocin-induced diabetic mice, activation of cannabinoid-1 receptor contributes to DR by increasing MAPK activation, oxidative stress, and inflammatory signaling [81]. In addition, PARP and nuclear factor-jB (NF-jB) are also downstream effectors of oxidative stress. PARP is a DNA nick-sensor enzyme activated by

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DNA single-strand breaks. It is involved in DNA repair, gene transcription, cell death, and apoptosis and acts as a co-activator in NF-jB signaling pathways [82,83]. Increased levels of oxidative and nitrosative stress induce DNA single-strand breaks and activate PARP. This, in turn, inhibits the activity of GAPDH by poly(ADP-ribosylation) and, consequently, leads to endothelial dysfunction in diabetic conditions [72]. Therefore, PARP inhibition could decrease the activation of PKC isoforms, hexosamine pathway flux and AGE formation induced by hyperglycemia [72]. As oxidative stress activates PARP which, in turn, initiates the activation NF-jB, application of PARP inhibitor could successfully inhibit hyperglycemia-activated NF-jB signaling pathway in endothelia cell culture [84]. PARP or NF-jB inhibition also reduces hyperglycemia-induced cell death in retinal endothelial cells [83]. NFjB also triggers the Notch-1/Akt and PI3K/Akt signaling pathways. Increased activation of PARP, cleaved caspase-3 and reduced expression of Notch1 and Akt phosphorylation lead to apoptosis in the hyperglycemic retina [85]. Inflammation, shown in Fig. 2, also factors in DR development and progression. Systemic inflammation is an intrinsic response to overfeeding, obesity and diabetes [86]. Diabetes increases the release of retinal inflammatory mediators (ICAM-1, VCAM-1, IL1b and TNF-a) [87] and activates microglial cells in early retinopathy [88]. Clinical studies have correlated elevated serum concentrations of IL-1b, TNF-a, and VEGF with DR presence and severity [89]. VEGF is an important proinflammatory factor in the pathogenesis of DR; its serum levels are associated with the development of DR [90]. Levels of MCP-1, IL-1b, and IL-6, IL-8, TNF-a and VEGF are all elevated in proliferative DR [91]. Leukostasis occurs in diabetic mice and rats, and deletion of the genes for the adhesion protein ICAM or its leukocyte binding partner, CD18, ameliorates leukostasis and permeability [92]. Vascular permeability, leukostasis, CD18 and ICAM expression, and NF-jB activation are normalized by treatment with high-dose aspirin, a cyclooxygenase-2 (COX-2) inhibitor, or a soluble TNF-a receptor–Fc hybrid [93], suggesting that TNF-a and cyclooxygenase-2 contribute to DR. Furthermore, a newly discovered inhibitor of PKC-d prevents TNF-a-induced retinal vascular permeability [94] and VEGF-induced permeability. IL-1b and TNF-a levels increase in the vitreous of patients with proliferative diabetic retinopathy [95,96]. Progressive retinal injury may impair the blood–retina barrier and lead to macrophage migration into the neurosensory retina or increased adherence to the vasculature, as well as accumulation of inflammatory and angiogenic mediators in the vitreous cavity.

Macular xanthophyll lutein/zeaxanthin and their metabolism in the retina Lutein and zeaxanthin are xanthophylls, oxygenated carotenoids that consist of 40-carbon compounds with nine conjugated double bonds in the polyene chain. Their structures are characterized by the presence of two hydroxyl groups in the ionone rings of the basic C40H56 carotene structure. Lutein and zeaxanthin differ in the ionone ring. Lutein has a b-ionone ring and a e-ionone ring, whereas zeaxanthin contains two b-ionone rings. In effect, zeaxanthin is a lutein isomer, differing only in the location of one double bond in one of the hydroxyl groups (Fig. 3). The hydroxyl groups may provide unique biological functions [85]. Relative to hydrocarbon carotenoids, lutein and zeaxanthin are more hydrophilic and polar in blood and tissues. The hydrophilic properties facilitate reaction with singlet oxygen generated in water phase. The relatively higher polarity partly determines the distinctive properties in metabolism, light absorption, capture and stabilization in tissues, and potential orientations in a bilayer membrane [97]. Lutein and zeaxanthin possess absorption bands near the blue to

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Fig. 3. Chemical structures of macular xanthophylls: lutein, zeaxanthin and mesozeaxanthin.

violet end of the visible spectrum, making them ideal filters of blue light, as confirmed in fluorescence emission studies [98]. Dietary xanthophyll absorption generally follows the intestinal pathway for fat and is affected by the same factors [99]: passive diffusion [100] and scavenger receptor-mediated uptake [101]. Xanthophyll-containing chylomicrons enter the hepatic circulation where the bulk is repackaged as plasma lipoproteins for release into the systemic circulation [102]. Unlike non-polar carotenoids such as b-carotene or lycopene, which tend to be primarily localized to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), lutein and zeaxanthin are evenly distributed between high density lipoproteins (HDL) and LDL in fasting blood [103]. Chylomicron levels of xanthophylls peak early, 2 h after ingestion, while peak blood concentrations are observed at 16 h post-ingestion [104]. Blood xanthophyll level can vary considerably across individuals and populations. To very different extents, lutein and zeaxanthin are accumulated in tissues depending on the expression of membrane receptors, such as SR-BI, CD36, LDLR and other unidentified cell surface proteins, and specific binding proteins. These are the only carotenoids normally present in the macula and lens of the human eye [22]. The estimated concentration approaches 1 mM in the fovea of the retina [105], three orders of magnitude greater than serum concentrations, indicating uptake, stabilization and storage of these xanthophylls in the macula is extraordinarily specific and efficient [106]. Studies of tissue-specific distribution of macular xanthophylls show zeaxanthin is preferentially accumulated in the fovea, whereas lutein becomes the dominant carotenoid with increasing radial distance along the retina [107]. Several xanthophyll-binding proteins have been identified and characterized. A Pi isoform of glutathione S-transferase (GSTP1) [108], was identified as a specific zeaxanthin binding protein; it was isolated from the region of the fovea and shows high affinity and specificity for both forms of macular zeaxanthin. The protein StARD3 (steroidogenic acute regulatory domain protein 3), also known as MNL64, has been identified as a specific lutein-binding protein in the macula of human eye [109]. These xanthophylls:binding protein complexes may facilitate the transport of lutein and zeaxanthin and preferential retinal accumulation via protecting the degradation of these xanthophylls from carotenoid cleavage enzymes. Intracellular carotenoid metabolism is catalyzed by a family of carotenoid cleavage oxygenases yielding a structurally diverse class of compounds known as retinoids and apo-carotenoids. The major central cleavage pathway, via b-carotene 15,150 -monooxygenase or BCO1, generates retinoids from provitamin A carotenoids

[110–113], such as a- and b-carotene and b-cryptoxanthin. An alternative eccentric cleavage pathway is catalyzed by b-carotene 90 ,100 -dioxygenase or BCO2 [114–116]. BCO2 is expressed in several tissues not known to be sensitive to vitamin A deficiency and/or that lack BCO1 expression [117]. These observations imply that BCO2 may be involved in biological processes other than vitamin A synthesis. Biochemical studies also show that BCO2 exhibits different substrate choice than BCO1 and can cleave the 90 ,100 double bonds of several carotenoids [118,119], including apo-carotenoids in vitro and in vivo [111,119], which could undergo further a-oxidative catabolism [120]. BCO1 and BCO2 are expressed in human retina [117]. BCO1 may serve as local regulator for vitamin A production from b-carotene, especially at times of dietary insufficiency. The physiological importance of eccentric cleavage and the metabolic function of BCO2 in the retina remain unclear. BCO2 null mice show hepatic mitochondrial dysfunction and increased hepatic cell susceptibilities to oxidative stress, implying BCO2 may be a stress responsive gene [119,121]. A recent report indicates that human BCO2 is structurally inactive and contributes to the specific accumulation of xanthophylls in the retina [122]. Alternatively, BCO2 might cleave the protein-free carotenoids, leaving protein-bound xanthophylls to preferentially accumulate in the retina.

Macular xanthophyll lutein/zeaxanthin and retinopathy of prematurity The roles of lutein and zeaxanthin in protection against certain eye diseases such as cataracts and AMD are under intensive investigations [27,123–125]. In the instances of NV retinopathies, retinal lutein and zeaxanthin influence the development of the visual system by altering input during a critical/sensitive period of visual development. They influence maturation and protect the retina when it is particularly vulnerable to actinic and oxidant stress. During fetal, neonatal and infant development, lutein is the dominant retinal carotenoid, different from the adult central retina, in which zeaxanthin: lutein ratios across the retina are reversed [22]. Studies in non-human primates raised on a standard diet or xanthophyllfree diets [126] demonstrate that, in addition to simply lacking macular pigment, xanthophyll-free monkeys have more drusenlike bodies (indicative of retinal degeneration) within their retinal pigment epithelium (RPE), increased macular hyper-fluorescence, and retinal abnormalities [127]. Some of these findings could be partially attributable to other dietary differences between the groups [128]. A follow-up study on macaques that were more specifically depleted lutein and zeaxanthin (and omega-3 fatty acids in some subjects) localized the pathological effects of lutein and zeaxanthin depletion to the RPE [129]. The RPE lies between the underlying choroidal vasculature and the photoreceptor cell layer, which it nourishes. Some of the retinal changes produced by xanthophyll-depleted diets in monkeys are reversed if xanthophylls are supplemented later in life [129]. The rapid maturation and increased metabolic activity of the developing retina increase tissue susceptibility to hypoxic/oxidant and photo-stress. In animal models [2], poor auto-regulation of choroidal blood flow, similar to the human condition of neonatal ROP, leads to hyper-oxygenation and excess lipid peroxidation. Restricted blood flow induces retinal cellular degeneration. The developing retina in premature infants is particularly susceptible to oxidative damage because of its high proportion of long chain polyunsaturated fatty acid (PUFA) [130,131]. High-energy short-wavelength visible light, high oxygen fluctuation and high metabolic activity in the immature retina also promote the production of ROS which are highly reactive and readily react with lipid, protein and nuclear acids in the retina, thereby resulting in

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irreversible damage to various cell structures. Cumulative oxidative damage is believed in part to be responsible for the pathogenesis of ROP. The components of macular pigment, lutein and zeaxanthin, quench singlet oxygen, scavenge free radicals, and inhibit peroxidation of membrane phospholipids. Detection of non-dietary oxidative metabolites of lutein and zeaxanthin in the retina confirm antioxidant activity of these carotenoids in the eye [18,132]. Xanthophylls are found in significant quantities within the rod outer segment membranes of retina and epithelium/outer cortex of the lens, the sites most susceptible to oxidative damage [133,134]. Xanthophylls can reside perpendicular to the plane of the membrane with hydroxyl groups protruding from the lipid cell membrane into the intra- and extra-cellular plasma, and thus can interact with ROS outside the membrane. This property makes them more effective antioxidants, in contrast with nonpolar carotenoids such as lycopene and b-carotene [135,136]. In addition, xanthophylls, which effectively preserve membrane structure and decrease oxygen diffusion–concentration products, control the rate of chemical reactions with oxygen and help to protect fatty acids from lipid peroxidation [98,137]. In animal models, lutein can be neuroprotective in retinal ischemic retinopathy injury. Lutein prevents the increase of nitrotyrosine and PAR and, hence, apoptosis and cell loss in inner retinal neuron in an animal model of retinal ischemic retinopathy [138]. Lutein can directly protect retinal ganglion cells from H2O2-induced oxidative stress and cobalt chloride-induced hypoxia in vitro [139]. Lutein is present in umbilical cord blood at birth, indicating placental transfer to the fetus, and lutein concentrations peak at the beginning of third trimester [140]. Cord blood concentrations are highly correlated to the corresponding maternal serum concentration [141]. The antioxidant properties of lutein have been also verified in newborn infants in a pilot study [142]. A randomized controlled trial (RCT) conducted among 150 newborns [16] demonstrated that neonatal supplementation of lutein in the first hours of life increases biological antioxidant potential and reduces level of total hydroperoxide. These findings suggest interventions using a dietary antioxidant, such as lutein, may delay the extent of oxidative damage to retinal tissues and, therefore, may impact early retinal development and, perhaps, oxidative stress-induced ischemic retinopathy. One recent area of interest is the potential role of lutein in preventing or lessening severity of neonatal and congenital retinopathies. To date, four RCTs have investigated the relationship between xanthophylls and ROP [17,143–145]. All studies included preterm infants <33 weeks gestational age. Two multicenter placebo-controlled RCTs supplemented a 0.5 ml daily dosage of 0.14 mg lutein and 0.0006 mg zeaxanthin with the objective of ROP prevention [143,144]. Both provided the xanthophyll supplementation via oral feeds of maternal milk, donor human milk, or preterm formula. The supplemented group showed a trend to reduced incidence of ROP versus the control group (6.2% vs 10.3% and 19% vs 27%, respectively). Of note, the progression rate from early ROP phases to threshold and higher stage 3–5 (sight-limiting) ROP in supplemental group was decreased by 50%, although, again, not reaching statistical significance. Lutein and zeaxanthin supplementation with weight-based dosages was performed in a single center, placebo-controlled RCT [145]. Despite increasing supplementation with weight-based dosages, in this small study there were no differences in ROP incidence. Another multicenter RCT [17] compared plasma carotenoid levels in preterm infants (n = 203, gestational age < 33 weeks) fed formula with and without added lutein, lycopene, and b-carotene with carotenoid levels in human-milk-fed term infants. A secondary aim was to evaluate the effectiveness of carotenoid supplementation in reducing visual complications. The infants were

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randomized to receive formula with or without added carotenoids. A human milk reference group received P80% of total nutritional intake as human milk. Preterm formula supplementation (per liter) included 211 lg lutein, 219 lg b-carotene and 143 lg lycopene. At time of discharge, the infants were transitioned to a corresponding post-discharge formula containing 68.7 lg lutein, 85.3 lg b-carotene and 49.5 lg lycopene per liter. Although total ROP incidence was similar between the groups, the supplemented group had less progression to severe ROP versus control group (8% vs 28%). Supplemented infants also had significantly lower C-reactive protein (CRP) levels (P < .001) and higher plasma lutein levels compared to the control infants (P < .01). Furthermore, they had similar plasma lutein levels to human milk fed infants 7.68 vs 5.88 lg/dl, respectively. This study demonstrated a significant correlation between plasma lutein levels by 50 weeks of age with saturated response amplitude in rod photoreceptors (P < .05) and rod photoreceptor sensitivity (P = .05) [17]. These findings indicate lutein promoted photoreceptor maturation and visual acuity across the retinal surface. To date, no clinical trials have been adequately designed specifically to test the hypothesis that lutein affects ROP outcomes. Only one study [143] performed a power analysis to test a lutein effect on ROP, but the study ended early for other reasons. That investigation also assumed a lutein effect might be maximal for less severe ROP, whereas experimentally and in human trials lutein mitigates ROP severity. The three multicenter trials all showed a trend to decreased ROP severity (severe ROP = Stages 3–5 or Plus disease). A meta-analysis of the four RCTs shows an overall 26% in severe ROP cases with lutein supplementation (unpublished data). These findings and experimental data provide a rationale for a clinical trial of lutein supplementation in extremely preterm infants targeting multiple eye and vision outcomes.

Macular xanthophyll lutein/zeaxanthin and diabetic retinopathy Increased oxidative stress and inflammatory mediators are implicated in the development of diabetic retinopathy. As mentioned above, oxidative stress leads to the activation of PKC, the formation of AGEs, sorbitol accumulation and NF-jB activation. Experimental evidence indicates that lutein and zeaxanthin can block these activation pathways via quenching oxygen radicals. In fact, lutein has been shown to reduce the biochemical, histological and functional changes in several mouse models of diabetes without affecting blood glucose levels [146–148]. In the diabetic mouse retina, lutein supplementation reduces the production of ROS, inactivates the NF-jB signaling pathway, and decreases the levels of oxidative markers, thereby preserving retinal function [149]. Lutein also prevents ERK activation, synaptophysin reduction, depletion of brain-derived neurotrophic factor (BDNF), and, consequently, neuronal loss in the diabetic retina [147,149]. Diabetic mice supplemented with lutein/zeaxanthin-rich wolfberry demonstrate normalized retinal thickness, RPE integrity and number of retinal ganglion cells via initiation of adenosine monophosphate-activated protein kinase (AMPK) and reduction of endoplasmic reticulum stress. Supplementation with zeaxanthin in diabetic rats also prevents increase in retinal oxidative stress and proinflammatory cytokines, VEGF and ICAM-1 [150]. Lutein supplementation in rats with STZ-induced diabetes shows not only reduction in diabetic abnormalities in the cerebral cortex [151], but also development of DR, and maintenances of normal retinal function, mitochondrial homeostasis and decreased inflammatory mediators [152]. Although a large number of studies have addressed the role of carotenoids in the development of diabetes, only a few have

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examined their role in development of DR [153]. Type 2 diabetes patients having a higher ratio of serum non-pro-vitamin A carotenoids (lutein, zeaxanthin, lycopene) to pro-vitamin A carotenoids (a-carotene, b-carotene and b-cryptoxanthin) have a 66% reduction in risk for DR after adjustment for confounding variables [153]. Moreover, macular pigment optical density (MPOD) has been reported to be lower in patients with type 2 diabetes than in age-matched controls, and lower still in patients with type 2 diabetes and retinopathy. MPOD is inversely correlated with glycosylated hemoglobin [154]. Supplementation of lutein (6 mg per day) and zeaxanthin (0.5 mg per day) over 3 months has increased MPOD, improved visual acuity, contrast sensitivity and foveal thickness in subjects with non-proliferative DR compared to controls [155]. To date, no intervention studies have been reported testing lutein and zeaxanthin in the prevention or treatment of DR. Animal models present challenges since rodents only display early retinopathy stages but not the later changes seen in humans with DR. Nevertheless, lutein and zeaxanthin supplementation to diabetic rats is a significant step forward toward clinical trials in DR. Conclusions ROP and DR are blinding disorders that affect numerous individuals and cause enormous burden to society. As oxidative stress plays a pivotal role in both ROP and DR, treatments that decrease the production of ROS or increase ROS scavenging ability can be beneficial. The positive outcomes in administration of lutein/zeaxanthin in experimental models of DR as well as in term and preterm infants point to potential efficacy of lutein/zeaxanthin in preventing ischemic retinopathy and hypoxia-induced oxidative stress. The specific distribution patterns of lutein/zeaxanthin in the macula and lens are thought to be associated with unique functions in these two vital ocular tissues. Several, but not all, epidemiological studies and clinical trials have shown higher levels of lutein/zeaxanthin in diet and serum are associated with lower risk of eye diseases. Animal studies also implicate an important role for these carotenoids in protecting the neural retina from photooxidative damage and development of common visually disabling disorders. The sum of evidence suggests lutein/zeaxanthin help to delay or mitigate a series of events in the retina that lead to ROP and DR. Although recent evidence to support the possibility that lutein/ zeaxanthin have an important role in reducing risk of ROP and DR is generally consistent, statistical power has been limited by small sample size. Additional research is needed to establish efficacy of the preventive and therapeutic aspects of these carotenoids in both ROP and/or DR. Large-scale long-term prospective interventional trials should resolve some of these issues in humans, and later identify effective daily dosages of lutein/zeaxanthin. Acknowledgments This work was supported by the Muma Family Endowment and the Laura W. Bush Institute for Women’s Health at Texas Tech University. References [1] P. Lee, C.C. Wang, A.P. Adamis, Surv. Ophthalmol. 43 (1998) 245–269. [2] P. Hardy, I. Dumont, M. Bhattacharya, X. Hou, P. Lachapelle, D.R. Varma, S. Chemtob, Cardiovasc. Res. 47 (2000) 489–509. [3] P.A. Campochiaro, J. Cell. Physiol. 184 (2000) 301–310. [4] M.E. Hartnett, Adv. Ophthalmol. 120 (2010) 25–39. [5] E. Kermorvant-Duchemin, P. Sapieha, M. Sirinyan, M. Beauchamp, D. Checchin, P. Hardy, F. Sennlaub, P. Lachapelle, S. Chemtob, Adv. Ophthalmol. 120 (2010) 51–60.

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