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Purpose Transgenic mice overexpressing serum retinol-binding protein (RBP4-Tg) develop progressive retinal degeneration, characterized by microglia activation, yet the precise mechanisms underlying retinal degeneration are unclear. Previous studies showed RBP4-Tg mice have normal ocular retinoid levels, suggesting that degeneration is independent of the retinoid visual cycle or light exposure. The present study addresses whether retinal degeneration is light-dependent and RBP4-dependent by testing the effects of dark-rearing and pharmacological lowering of serum RBP4 levels, respectively. Methods RBP4-Tg mice reared on normal mouse chow in normal cyclic light conditions were directly compared to RBP4-Tg mice exposed to chow supplemented with the RBP4-lowering compound A1120 or dark-rearing conditions. Quantitative retinal histological analysis was conducted to assess retinal degeneration, and electroretinography (ERG) and optokinetic tracking (OKT) tests were performed to assess retinal and visual function. Ocular retinoids and bis-retinoid A2E were quantified. Results Dark-rearing RBP4-Tg mice effectively reduced ocular bis-retinoid A2E levels, but had no significant effect on retinal degeneration or dysfunction in RBP4-Tg mice, demonstrating that retinal degeneration is light-independent. A1120 treatment lowered serum RBP4 levels similar to wild-type mice, and prevented structural retinal degeneration. However, A1120 treatment did not prevent retinal dysfunction in RBP4-Tg mice. Moreover, RBP4-Tg mice on A1120 diet had significant worsening of OKT response and loss of cone photoreceptors compared to RBP4-Tg mice on normal chow. This may be related to the very significant reduction in retinyl ester levels in the retina of mice on A1120-supplemented diet. Conclusions Retinal degeneration in RBP4-Tg mice is RBP4-dependent and light-independent.

Serum retinol-binding protein 4 (RBP4) is the sole specific transport protein for retinol in the blood, but it is also an adipokine with retinol-independent, proinflammatory activity associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. Moreover, two separate studies reported that patients with proliferative diabetic retinopathy have increased serum RBP4 levels compared to patients with mild or no retinopathy, yet the effect of increased levels of RBP4 on the retina has not been studied. Here we show that transgenic mice overexpressing RBP4 (RBP4-Tg mice) develop progressive retinal degeneration, characterized by photoreceptor ribbon synapse deficiency and subsequent bipolar cell loss. Ocular retinoid and bisretinoid levels are normal in RBP4-Tg mice, demonstrating that a retinoid-independent mechanism underlies retinal degeneration. Increased expression of pro-interleukin-18 (pro-IL-18) mRNA and activated IL-18 protein and early-onset microglia activation in the retina suggest that retinal degeneration is driven by a proinflammatory mechanism. Neither chronic systemic metabolic disease nor other retinal insults are required for RBP4 elevation to promote retinal neurodegeneration, since RBP4-Tg mice do not have coincident retinal vascular pathology, obesity, dyslipidemia, or hyperglycemia. These findings suggest that elevation of serum RBP4 levels could be a risk factor for retinal damage and vision loss in nondiabetic as well as diabetic patients.

Purpose Diabetic retinopathy is a leading cause of vision loss. Previous studies have shown signaling pathways mediated by Stat3 (signal transducer and activator of transcription 3) play a primary role in diabetic retinopathy progression. This study tested CLT-005, a small molecule inhibitor of Stat3, for its dose-dependent therapeutic effects on vision loss in a rat model of diabetic retinopathy. Methods Brown Norway rats were administered streptozotocin (STZ) to induce diabetes. CLT-005 was administered daily by oral gavage for 16 weeks at concentrations of 125, 250, or 500 mg/kg, respectively, beginning 4 days post streptozotocin administration. Systemic and ocular drug concentration was quantified with mass spectrometry. Visual function was monitored at 2-week intervals from 6 to 16 weeks using optokinetic tracking to measure visual acuity and contrast sensitivity. The presence and severity of cataracts was visually monitored and correlated to visual acuity. The transcription and translation of multiple angiogenic factors and inflammatory cytokines were measured by real-time polymerase chain reaction and Multiplex immunoassay. Results Streptozotocin-diabetic rats sustain progressive vision loss over 16 weeks, and this loss in visual function is rescued in a dose-dependent manner by CLT-005. This positive therapeutic effect correlates to the positive effects of CLT-005 on vascular leakage and the presence of inflammatory cytokines in the retina. Conclusions The present study indicates that Stat3 inhibition has strong therapeutic potential for the treatment of vision loss in diabetic retinopathy.

Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.

13-cis Retinoic acid (13cRA), a stereoisomeric form of retinoic acid, is naturally generated in the body and is also used clinically to treat acute promyelocytic leukaemia, some skin diseases, and cancer. Furthermore, it has been suggested that 13cRA modulates brain neurochemical systems, as increased 13cRA levels are correlated with depression and increased suicidal tendencies. However, the mechanism for the generation of endogenous 13cRA is not well understood. This study identified and characterized a novel enzyme in zebrafish brain, 13-cis isomerohydrolase (13cIMH), that exclusively generated 13-cis retinol, which can be oxidized to 13cRA. 13cIMH shares 74% amino acid sequence identity with human RPE65, an 11-cis isomerohydrolase in the visual cycle, and retains the key residues essential for RPE65’s isomerohydrolase activity. Similar to RPE65, 13cIMH is a membrane-associated protein, requires all-trans retinyl ester as its intrinsic substrate, and its enzymatic activity is dependent on iron. The purified 13cIMH converted atRE exclusively to 13-cis retinol with Km = 2.6 μM and kcat = 4.4 × 10−4s−1. RT-PCR, Western blot analysis and immunohistochemistry detected 13cIMH expression in the brain. These results suggest that 13cIMH may play a key role in the generation of 13cRA and in the modulation of neuronal functions in the brain.

Stargardt disease (STGD) is the major form of inherited juvenile macular degeneration. Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in retinal pigment epithelium (RPE) cells in STGD and contributes to the disease pathogenesis. However, the precise role of A2E in vision loss is unclear. Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key enzyme in the visual cycle. Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase activity in mice. Likewise, A2E also inhibited isomerohydrolase activity in cells coexpressing RPE65, lecithin retinol acyltransferase (LRAT), and cellular retinaldehyde-binding protein. In vitro isomerohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in a concentration-dependent manner, but did not inhibit LRAT activity. The inhibition type for isomerohydrolase was found to be reversible and competitive with K i = 13.6 μM. To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were performed. As shown by fluorimetric titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence. Consistently, the fluorescence of RPE65 decreased upon incubation with A2E. Both of these experiments suggest a direct, specific binding of A2E to RPE65. The binding constant for A2E and purified RPE65 was calculated to be 250 nM. These results demonstrate that A2E inhibits the regeneration of 11- cis retinal, the chromophore of visual pigments, which represents a unique mechanism by which A2E may impair vision in STGD.

Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells) and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs)). Pigment epithelium-derived factor (PEDF) contains an N-terminal 34-amino acid peptide (PEDF-34) that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR) model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS). OIR significantly increased the number of circulating Tie2-GFP+at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10trex-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.

PURPOSE. The final step in the retinoid visual cycle is catalyzed by 11-cis-retinol dehydrogenases (11-cis-RDHs) that oxidize 11cis-retinol (11cROL) to 11-cis-retinaldehyde (11cRAL). Genetic studies in mice indicate that the full repertoire of 11-cis-RDH enzymes remains to be identified. This study was conducted to characterize the 11-cis-RDH activity of RDH10 in vitro and specifically to determine whether RDH10 can functionally and physically interact with visual cycle proteins. METHODS. Human RDH10 was expressed in COS1 cells to measure its 11-cis-RDH activity in the presence or absence of purified recombinant cellular retinaldehyde-binding protein (CRALBP). The RPE visual cycle was reconstituted in HEK-293A cells by co-expressing RDH10, CRALBP, RPE-specific 65-kDa protein (RPE65) and lecithin retinol acyltransferase (LRAT). The cells were subsequently treated with all-trans-retinol (atROL), and retinoid profiles were quantified by HPLC. Immunocytochemical and co-immunoprecipitation analyses were performed to determine whether RDH10 physically interacts with other visual cycle proteins. RESULTS. RDH10 oxidized 11cROL to generate 11cRAL in vitro in the presence of CRALBP. RDH10 can use both NAD and NADP as cofactors for 11-cis-RDH activity, although NAD cofactor confers more robust activity. In a cell culture model co-expressing RDH10 with RPE65, LRAT and CRALBP, the visual chromophore 11cRAL was generated from atROL. Immunohistochemistry showed that RDH10 co-localizes with RPE65 and CRALBP in vivo in primary bovine RPE cells. Immunoprecipitation analysis demonstrated that RDH10 physically interacts with CRALBP and RPE65. CONCLUSIONS. RDH10 may function in the RPE retinoid visual cycle as an 11-cis-RDH, and thereby partially compensate for the loss of RDH5 function in patients with fundus albipunctatus. (Invest Ophthalmol Vis Sci. 2009;50:5089‐5097) DOI:10.1167/iovs.09-3797

It is well known that retinoid (vitamin A derivatives) metabolism is essential not only for normal vision but also for embryonic development and growth. A number of enzymes, retinoid-binding proteins and transporters are involved in modification, conversion and circulation of retinoids [1–3]. Vitamin A (all-trans retinol, atROL) is bound by retinol-binding protein (RBP) and transported from the liver to ocular tissues and non-ocular tissues. 11-cis retinal (11cRAL) is covalently bound to opsin molecule and served as chromophore for rod and cone visual pigments. A single photon isomerizes 11cRAL to all-trans retinal (atRAL), which triggers the activation of the phototransduction cascade and generates vision [4, 5]. Subsequently, 11cRAL is regenerated in the retinal pigment epithelium (RPE) through the retinoid visual cycle which involves a number of enzymes and retinoid binding proteins [6, 7]. An intact visual cycle is essential for normal vision [8]. Retinoid metabolism is also essential for the generation of retinoic acid which plays an important role in regulating development and cell differentiation [1, 2]. In certain tissues, atROL is first oxidized to atRAL which is then oxidized to all-trans retinoic acid (atRA) by a known enzyme, retinaldehyde dehydrogenase (Raldh). AtRA is known to regulate cell differentiation, development and morphogenesis through the retinoic acid receptors or retinoid X receptors [1–3]. Retinol dehydrogenases (RDH) are the enzymes to catalyze oxidation of retinol into retinal or reduction of retinal into retinol in a cofactor dependent manner in both vision and retinoid signaling. The RDH activity is observed in two distinct classes of enzymes, microsomal retinol dehydrogenases (RoDH) in the short-chain dehydrogenase/reductase (SDR) family and cytosolic alcohol dehydrogenases (ADH) in the medium-chain dehydrogenase/reductase (MDR) family [9–15]. More than 20,000 sequences (including species variants) of SDR have been deposited in the database [15]. A number of studies have been carried out to identify the functionally important residues among the highly conserved residues in the family by evaluating the impacts of mutations of the residues using site-directed mutagenesis [9, 16–23] and crystal structure analyses of the SDR family members such as drosophila alcohol dehydrogenase (DADH [24]), human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD-1 [25]) and bacterial 3β/17β-HSD [26]. Most of the mutations of the conserved residues abolish their enzymatic activities [9, 16–23]. These studies elucidated that the highly conserved residues in SDRs are the key residues and suggested that a triad of Ser-Tyr-Lys residues form the catalytic site, in which Tyr functions as the catalytic base, whereas Ser stabilizes the substrate, and Lys interacts with cofactor and lowers the pKa of the Tyr-OH. The conserved Asn residue interacts with the active site Lys via a water molecule to form a tetrad Asn-Ser-Tyr-Lys [10, 17, 27]. Although the SDR family members share low (approximately 15–30%) sequence identities, functionally important residues, such as catalytic sites (Asn-Ser-Tyr-Lys) and cofactor-binding motifs (Gly-X3-Gly-X-Gly), are highly conserved in the SDR family [9–11, 17]. The available 3-D structures of SDRs have revealed relatively similar α/β folding patterns of a Rossmann-fold [9, 10, 24, 25, 27]. Likewise, a number of studies had been carried out to investigate cofactor specificities of the SDR family. The residues between the first β-sheet and the third α-helix of SDRs are potentially important determinants of the specificity of cofactor toward either NADP(H) or NAD(H) [9, 19, 24, 28–34]. Further, RoDHs (membrane-bound SDR) have been shown to possess hydrophobic domains at the N-terminal and/or C-terminal regions, which may anchor the proteins into the membrane [11, 35–39]. RDH10 (retinol dehydrogenase class 10) was first cloned from bovine, mouse and human RPE [40]. The amino acid sequence homologies among RDH10 from different species are exceptionally high, with a 100% identity between bovine and human RDH10 and a 99% identity between mouse and human RDH10 at the amino acid level [40]. The highly conserved sequence of RDH10 across the evolution suggests its functional significance. We also showed that RDH10 is predominantly localized in the microsomal fraction, similar to other membrane-bound RDHs [40]. A recent study showed that RDH10 is highly expressed in many other non-ocular tissues during forelimb and hindlimb differentiation, whereas other RDHs (RDH1, 5, 6, 7 and 11) have significantly lower expression than RDH10 at this stage [41]. Moreover, mice carrying a miss-sense mutation of RDH10 have an embryonic lethal phenotype, indicating an important role of RDH10 in the early embryonic development [42]. Furthermore, the lethal phenotype of this RDH10 mutant mouse can be rescued by RA treatment of the pregnant mother, suggesting that RDH10 is essential for RA generation at early stages of embryonic development [42]. Although its exact function in RA generation is uncertain, RDH10 is likely to function as the enzyme to convert atROL to atRAL which is the substrate for Raldh2 to synthesize RA [2, 11]. This assumption is supported by a recent study showing that RDH10 and Raldh colocalize in tissues during embryogenesis and organ differentiation [43]. To further study the structure and function of this important enzyme, the present study has characterized the conserved key residues in the predicted catalytic center, cofactor-binding site, and membrane association domain in RDH10.

Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation.UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27(Kip1) were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for beta-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells.UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27(Kip1), and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse.UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation.

Neovascular disease in the retina is the leading cause of blindness in all age groups. Thus, there is a great need to develop effective therapeutic agents to inhibit and prevent neovascularization in the retina. Over the past decade, anti-VEGF therapeutic agents have entered the clinic for the treatment of neovascular retinal disease, and these agents have been effective for slowing and preventing the progression of neovascularization. However, the therapeutic benefits of anti-VEGF therapy can be diminished by the need for prolonged treatment regimens of repeated intravitreal injections, which can lead to complications such as endophthalmitis, retinal tears, and retinal detachment. Recent advances in nanoparticle-based drug delivery systems offer the opportunity to improve bioactivity and prolong bioavailability of drugs in the retina to reduce the risks associated with treating neovascular disease. This article reviews recent advances in the development of nanoparticle-based drug delivery systems which could be utilized to improve the treatment of neovascular disease in the retina.