Reappraisal of serum retinol-binding protein as a surrogate marker for retinol and discovery of a novel retinol estimation formula.

BACKGROUND & AIMS: Serum retinol (ROH) is commonly used for population level assessment of vitamin A status. High-performance liquid chromatography (HPLC) is considered most accurate method for measuring ROH. However, with the technical difficulty of using HPLC for routine assays, serum retinol-binding protein (RBP) measured by immunological assays is expected to be a surrogate marker for ROH, with reports of a close correlation between serum RBP and ROH. Nevertheless, RBP is not commonly tested to assess vitamin A status with concerns over RBP alterations under various physiopathological conditions. Thus, we reappraised the extent to which RBP could be used as a surrogate marker in representative disorders that alter serum RBP levels. As a related marker, diagnostic utility of transthyretin (TTR) was also evaluated. METHODS: To evaluate the reliability of ROH and RBP assays, specimen stability was assessed in terms of (1) storage at 25, 4, -20, and -80 °C for 1-28 days, (2) five-cycle freeze-thawing, and (3) fluorescent light exposure for 1-14 days. Sources of variation (sex, age, body mass index [BMI], and drinking habits) and reference intervals for ROH, RBP, and TTR were determined in 617 well-defined healthy individuals. To investigate the influence of disorders that affect serum RBP, patients with five diagnostic groups were enrolled: 26 with chronic kidney disease (CKD); 13 with various malignancies in advanced stages (AdM), 12 with acute bacterial infections (ABI), 6 with liver cirrhosis (LC), and 26 with simple obesity (BMI ≥ 27 kg/m2). RESULTS: The stability of RBP and ROH in serum was confirmed under all conditions. In healthy individuals, serum ROH, RBP, and TTR were appreciably high in males with a slight increase in proportion to age and BMI. The major-axis regression line between RBP (x) and ROH (y) in healthy individuals was y = x, with a correlation coefficient of 0.986. In the LC, AdM, and ABI groups, similar strong correlations were observed; however, the regression lines were shifted slightly rightward from the healthy group line, indicating a positive bias in estimating ROH. Interestingly, the same analyses between TTR and ROH revealed similar strong linear relationships in all groups; however, the regression line of each group showed a leftward (opposite) shift from the healthy group line. Based on these observations, we developed a novel regression model composed of RBP and TTR, which gave much improved accuracy in estimating ROH, even under these pathological conditions. CONCLUSIONS: The perfect RBP-ROH correlation in healthy individuals indicates the utility of RPB as a surrogate marker for ROH. Nevertheless, under RBP-altered conditions, a slight overestimation of ROH is inevitable. However, when the TTR was tested together, the bias can be corrected almost perfectly using the novel ROH estimation formula comprising RBP and TTR.

CLINICAL CHARACTERIZATION OF AQUEOUS AND VITREOUS RETINOL-BINDING PROTEIN 3 CONCENTRATIONS IN RELATION TO DIABETIC RETINOPATHY SEVERITY, RETINAL STRUCTURES, AND SYSTEMIC COMPLICATIONS.

PURPOSE: To evaluate Retinol-Binding Protein 3 (RBP3) from photoreceptors in aqueous and its association with vitreous concentrations, diabetic retinopathy (DR) severity, retinal layer thickness, and clinical characteristics in people with diabetes. METHODS: RBP3 concentration was measured by custom-developed enzyme-linked immunosorbent assay in aqueous and correlated with vitreous concentrations in patients from the 50-Year Medalist study and Beetham Eye Institute at Joslin Diabetes Center. RESULTS: Aqueous RBP3 concentration (N = 131) was elevated in eyes with no to mild DR (mean ± SD 0.7 nM ± 0.2) and decreased in eyes with moderate to severe DR (0.65 nM ± 0.3) and proliferative DR (0.5 nM ± 0.2, P < 0.001) compared to eyes without diabetes. Aqueous and vitreous RBP3 concentrations correlated with each other (r = 0.34, P = 0.001) and between fellow eyes (P < 0.0001). History of retinal surgery did not affect aqueous RBP3 concentrations, but cataract surgery affected both vitreous and aqueous levels. Elevated aqueous RBP3 concentration associated with increased thickness of the outer nuclear layer (P = 0.004) and correlated with hemoglobin A1c, whereas vitreous RBP3 concentrations correlated with diabetic systemic complications. CONCLUSION: These findings suggest that aqueous RBP3 concentration may be an important endogenous clinical retinal protective factor, a biomarker for DR severity, and a promising VEGF-independent clinical intervention target in DR.

Vitamin A biomarkers were associated with α(1)-acid glycoprotein and C-reactive protein over the course of a human norovirus challenge infection.

Retinol binding protein (RBP) is used as a proxy for retinol in population-based assessments of vitamin A deficiency (VAD) for cost-effectiveness and feasibility. When the cut-off of < 0·7 µmol/l for retinol is applied to RBP to define VAD, an equivalence of the two biomarkers is assumed. Evidence suggests that the relationship between retinol and RBP is not 1:1, particularly in populations with a high burden of infection or inflammation. The goal of this analysis was to longitudinally evaluate the retinol:RBP ratio over 1 month of follow-up among fifty-two individuals exposed to norovirus (n 26 infected, n 26 uninfected), test whether inflammation (measured as α-1-acid glycoprotein (AGP) and C-reactive protein (CRP)) affects retinol, RBP and the ratio between the two and assess whether adjusting vitamin A biomarkers for AGP or CRP improves the equivalence of retinol and RBP. We found that the median molar ratio between retinol and RBP was the same among infected (0·68) and uninfected (0·68) individuals. AGP was associated with the ratio and RBP individually, controlling for CRP, and CRP was associated with both retinol and RBP individually, controlling for AGP over 1 month of follow-up. Adjusting for inflammation led to a slight increase in the ratio among infected individuals (0·71) but remained significantly different from the expected value of one. These findings highlight the need for updated recommendations from the WHO on a cut-off value for RBP and an appropriate method for measuring and adjusting for inflammation when using RBP in population assessments of VAD.

Comparative molecular dynamics study on the features of binding and non-binding modes of retinoic acid in cellular retinol-binding protein (I).

Retinoids play crucial roles in various biological processes by interacting with their carrier proteins such as cellular retinol-binding protein (CRBP). Understanding the molecular interactions between retinoids and CRBP enables their pharmacological and biomedical applications. Experimentally, CRBP(I) does not bind to retinoic acid, but when arginine is introduced into 108th residue instead of glutamine (Q108R), it binds to retinoic acid. Here, molecular dynamics simulations were performed to understand the differences in the microscopic and dynamic behaviors of the non-binding wild-type CRBP(I)-retinoic acid and binding Q108R variant-retinoic acid complexes. The ligand RMSD and RMSF, the binding poses of binding motif amino acids, and the number of hydrogen bonds and salt-bridges revealed the relative instability of the non-binding complex. In particular, the ligand's terminal group showed very different dynamics and interactions. So far, most studies have focused on the binding characteristics of retinoids, but the features of their non-binding modes have not been studied well. This study provides some structural insights into the non-binding modes of a retinoid in CRBP, which may be applicable in retinoid-based drug discovery and protein engineering through computational modeling.

Retinol-binding protein-hijacking nanopolyplex delivering siRNA to cytoplasm of hepatic stellate cell for liver fibrosis alleviation.

Activated hepatic stellate cell (aHSC) is mainly responsible for deposition of extracellular collagen matrix that causes liver fibrosis. Although several siRNAs adequately inhibited HSC activation in vitro, they were demonstrated poor RNAi efficiency in vivo. Developing HSC-targeting and cytoplasmic delivery nanocarrier is highly essential to acquire a desirable siRNA therapeutic index for anti-liver fibrosis. Here, we developed a unique crosslinking nanopolyplex (called T-C-siRNA) modified by vitamin A (VA) with the well-designed natures, including the negative charge, retinol-binding protein (RBP) hijacking, and cytoplasmic siRNA release in response to ROS and cis diol molecules. The nanopolyplex was given a yolk-shell-like shape, camouflage ability in blood, and HSC-targeting capability by hijacking the endogenous ligand RBP via surface VA. PDGFR-ß siRNA (siPDGFR-ß) supplied via T-C-siPDGFR-ß nanopolyplex dramatically reduced HSC activation and its production of pro-fibrogenic proteins in vitro and in vivo. Furthermore, T-C-siPDGFR-ß nanopolyplex effectively alleviated CCl4-induced liver injury, decreased hepatic collagen sediment, and recovered liver function in mice. This study provides a sophisticated method for HSC-targeting cytoplasmic RNA delivery using endogenous ligand hijacking and dual sensitivity of ROS and cis diol compounds.

A high fat diet fosters elevated bisretinoids.

High dietary fat intake is associated with metabolic dysregulation, but little is known regarding the effects of a high fat diet (HFD) on photoreceptor cell functioning. We explored the intersection of an HFD and the visual cycle adducts that form in photoreceptor cells by nonenzymatic reactions. In black C57BL/6J mice and albino C57BL/6Jc2j mice raised on an HFD until age 3, 6, or 12 months, chromatographically quantified bisretinoids were increased relative to mice on a standard diet. In vivo measurement of fundus autofluorescence, the source of which is bisretinoid, also revealed a significant increase in the HFD mice. Additionally, mice provided with a diet high in fat presented with elevated retinol-binding protein 4, the protein responsible for transporting retinol in plasma. Vitamin A was elevated in plasma although not in ocular tissue. Bisretinoids form in photoreceptor cell outer segments by random reactions of retinaldehyde with phosphatidylethanolamine. We found that the latter phospholipid was significantly increased in mice fed an HFD versus mice on a control diet. In leptin-deficient ob/ob mice, a genetic model of obesity, plasma levels of retinol-binding protein 4 were higher but bisretinoids in retina were not elevated. Photoreceptor cell viability measured as outer nuclear layer thickness was reduced in the ob/ob mice relative to WT. The accelerated formation of bisretinoid we observed in diet-induced obese mice is related to the high fat intake and to increased delivery of vitamin A to the visual cycle.

Retinol binding protein 4 promotes the phenotypic transformation of vascular smooth muscle cells under high glucose condition via modulating RhoA/ROCK1 pathway.

Phenotypic switch of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of atherosclerosis (AS). High level of retinol binding protein 4 (RBP4) is regarded as a risk factor in cardiac-cerebral vascular disease. This study is performed to clarify the biological function of RBP4 in modulating the phenotypic switch of VSMCs induced via RhoA/ROCK1 signaling pathway. In vivo experiment, all the rats were divided into control group (NC), diabetic group (DM) and diabetic atherosclerosis group (DAS). The expressions of biochemical indicators, RhoA and Rho associated coiled-coil containing protein kinase 1 (ROCK1) were detected. In vitro experiment, VSMCs were cultured under high glucose condition, and ectogenic RBP4, HA-1100, rapamycin, or 3-methyladenine (3-MA) were supplemented to treat the VSMCs, respectively. The proliferation and migration of VSMCs were evaluated. The regulatory relationship between RBP4 and ROCK1 was predicted by bioinformatics analysis, and validated by qRT-PCR and Western blot. The regulatory effects of RBP4 on contractile phenotypic markers such as calponin, MYH11, α-SMA and autophagy markers including LC3II, LC3I, and Beclin-1 as well as mTOR were also detected. Moreover, VSMCs were cultured exposed to ROCK1 overexpressed plasmid or short hairpin RNA (shRNA), the proliferation and migration of VSMCs were evaluated and the regulatory effects of RhoA/ROCK1 signaling pathway on contractile phenotypic markers and autophagy markers were also detected. In vivo, RhoA, ROCK1, and mTOR were highly expressed in the rats intraperitoneally injected with RBP4. In vitro, the expressions of calponin, MYH11, α-SMA, LC3II, LC3I, and Beclin-1 were decreased in VSMCs treated with ROCK1-OA under high glucose condition, conversely, the expressions were increased in VSMCs exposed to ROCK1-shRNA. After incubated with rapamycin additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were up-regulated and the expression of p-mTOR was decreaed in VSMCs of HG+ROCK1-OA. Conversely, after incubated with 3-MA additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were down-regulated and the expression of p-mTOR was elevated in VSMCs of HG+ROCK1-shRNA. Ectogenic RBP4 facilitated high glucose-induced proliferation and migration of VSMCs, and it repressed the expression of calponin, MYH11, α-SMA, LC3II/I, and Beclin-1 in VSMCs. As expected, ROCK1 inhibit or counteracted the biological effects of RBP4 on VSMCs. In addition, the expressions of contractile phenotypic markers, LC3II/I, and Beclin-1 were promoted and mTOR were decreased after the VSMCs treated with autophagy agonist, whereas no significant difference was observed in the expressions of ROCK1, RhoA. RBP4 is an injurious factor in the pathogenesis of diabetic AS, and it promotes the phenotypic switch of VSMCs via activating RhoA/ROCK1 pathway and inhibiting autophagy.

Unraveling the role of resistin, retinol-binding protein 4 and adiponectin produced by epicardial adipose tissue in cardiac structure and function: evidence of a paracrine effect.

PURPOSE: Adipokines produced by adipose tissue have been found to be involved in the pathophysiology of metabolic and cardiovascular diseases. We aimed to investigate the relationships of resistin, retinol-binding protein 4 (RBP4) and adiponectin produced by epicardial adipose tissue with coronary artery disease (CAD) and cardiac structure and function. METHODS: Forty-one non-diabetic males scheduled for cardiothoracic surgery were examined. Anthropometric measurements, echocardiography, coronary angiography, and blood analysis were performed preoperatively. We measured the serum levels of resistin, RBP4, and adiponectin and their mRNA expression in thoracic subcutaneous adipose tissue and two epicardial adipose tissue samples, one close to left anterior descending artery (LAD) (resistin-LAD, RBP4-LAD, adiponectin-LAD), and another close to the right coronary artery (RCA) (resistin-RCA, RBP4-RCA, adiponectin-RCA). RESULTS: Left ventricular (LV) ejection fraction correlated negatively with adiponectin-LAD (rho = - 0.390, p = 0.025). The ratio of early to late diastolic transmitral flow velocity, as an index of LV diastolic function, correlated negatively with resistin-LAD (rho = - 0.529, p = 0.024) and RBP4-LAD (rho = - 0.458, p = 0.049). There was no difference in epicardial adipose tissue mRNA expression of resistin, RBP4, and adiponectin between individuals with CAD and those without CAD. When we compared the individuals with CAD in the LAD with those without CAD in the LAD, there was no difference in resistin-LAD, RBP4-LAD, and adiponectin-LAD. There was no difference in resistin-RCA, RBP4-RCA, and adiponectin-RCA between the individuals with CAD in the RCA and those without CAD in the RCA. CONCLUSION: Elevation of epicardial adipose tissue mRNA expression of adiponectin was associated with LV systolic dysfunction, while that of both resistin and RBP4 was linked to LV diastolic dysfunction.

Free energy simulations to study mutational effect of a conserved residue, Trp24, on stability of human serum retinol-binding protein.

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a ß-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP.Communicated by Ramaswamy H. Sarma.

Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton, and Promotes Nematode Parasitism and Pathogenicity.

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes.

Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice.

Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69 kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150 kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

Factors that affect placental retinol transfer in preterm infants and mothers with retinol deficiency.

BACKGROUND: The retinol level and retinol delivery to the placenta may vary depending on various factors involving the mother and newborn. The present study evaluates the factors affecting retinol levels in newborns and the transplacental retinol passage in preterm newborns. METHODS: In this prospective cohort study, the retinol and retinol binding protein (RBP) in the umbilical cord blood of 44 preterm infants with a gestation age of <30 weeks were studied. Serum retinol and RBP levels were determined using an enzyme-linked immunosorbent assay, and the rate of transplacental retinol passage was calculated. The demographic data of mothers and newborns, the use of vitamins by the mother, the application of antenatal corticosteroids, and any diseases diagnosed during pregnancy were recorded. An evaluation was made of the retinol, RBP, and other factors of the mother and newborn affecting transplacental retinol passage. RESULTS: A retinol deficiency was identified in 68.2% of the study population. Retinol and RBP levels in umbilical cord blood (273.7 ± 150.03 ng/mL, 7.88 ± 5.6 ng/mL, respectively) were significantly higher than the corresponding levels in the mother (206.4 ± 86.26 ng/ mL, 1.04 ± 0.97 ng/mL, respectively). Umbilical cord blood retinol deficiency was more common in the male participants, while the transplacental retinol passage rate was higher in females. The umbilical cord blood RBP was found to be lower in those administered antenatal corticosteroids than in those who did not receive antenatal corticosteroids, and median maternal RBP levels were lower in patients with anemia and pregnancy-induced hypertension than in those with no disease. DISCUSSION: Placental adaptation and contributing factors may vary in populations with severe retinol deficiency. The finding of significantly increased cord blood retinol levels when compared to maternal retinol levels in the present study suggests that some compensatory mechanisms, such as increased placental RBP levels, support the presentation of retinol to the fetus, even if the mother has a retinol deficiency.

Integrative study of gene expression datasets in retinal samples of Diabetic Retinopathy.

Diabetic Retinopathy is prevalent among patients with uncontrolled hyperglycemia resulting in vision loss. Despite numerous challenges to create a link among these conditions, the characterization of pathological neovascularization causing retinal damage due to the prognosis of early non-proliferative diabetic retinopathy to late proliferative diabetic retinopathy needs deep understanding. In this study, meta-analysis-based integration of gene expression datasets for the fibrovascular membrane of PDR and neural retina of NPDR were compared, to investigate the differentially expressed genes involved in retinal angiogenesis. Human samples with gene expression profiling of the same experiment type and platform with sufficient information for analysis were included in the study. The studies from cell lines and non-human studies, human samples that include serum, cornea, lens, and/or other ocular tissues or fluids, and studies that lack basic information for analysis were excluded. The microarray datasets available in the Gene Expression Omnibus database of the early and late stages in DR were screened to find common gene expression profiles. Using the INMEX bioinformatics tool, significantly upregulated and downregulated genes in the neural retina of Non-Proliferative Diabetic Retinopathy and fibrovascular membrane of Proliferative Diabetic Retinopathy were compared and studied by the combine effect size method. Using the STRING database PPI network, 50 upregulated and 50 downregulated genes were used to find the key candidate genes involved in retinal disease/degeneration in eye/retinal tissues. In the extensive gene expression meta-analysis performed using INMEX bioinformatics tool, overall, 7935 differentially expressed genes were identified and the respective heatmap was created by using the visualization tools of INVEX. STRING database PPI network identified Retinol Binding Protein 3, Neural Retina Leucine Zipper, S-Antigen Visual Arrestin, Peripherin 2, and Aryl Hydrocarbon Receptor Interacting Protein Like-1 to be the most highly ranked hub genes. The newly discovered potential genes related to retinal angiogenesis causing FVM formation in DR may provide insight into the cellular pathogenesis of NPDR to PDR.

Imaging retinaldehyde-protein binding in plants using a merocyanine reporter.

Retinoid-binding proteins (RBPs) are a diverse category of proteins that have been most extensively characterized for their role in vertebrate development. Recent work has uncovered new functions of RBPs in invertebrates and plants. Here, we present a methodology for applying a fluorescent chemical probe to characterize RBP binding in plants. This reporter, called merocyanine aldehyde (MCA), fluoresces upon binding to RBPs and therefore enables in vivo investigations into their functions with high spatio-temporal resolution. MCA treatment is simple, fast, non-destructive, and does not require prior knowledge of the RBP encoding genes. Therefore, a major advantage of this methodology is that it can be performed in species that are not genetically tractable. Furthermore, many of the methods presented here apply to diverse species within and beyond the plant kingdom.

Elevated Retinol Binding Protein 3 Concentrations Are Associated With Decreased Vitreous Inflammatory Cytokines, VEGF, and Progression of Diabetic Retinopathy.

OBJECTIVE: To correlate inflammatory cytokines and vascular endothelial growth factor (VEGF) in vitreous and plasma with vitreous retinol binding protein 3 (RBP3), diabetic retinopathy (DR) severity, and DR worsening in a population with type 1 and type 2 diabetes. RESEARCH DESIGN AND METHODS: RBP3, VEGF, and inflammatory cytokines were measured in plasma and vitreous samples (n = 205) from subjects of the Joslin Medalist Study and Beetham Eye Institute. RESULTS: Higher vitreous RBP3 concentrations were associated with less severe DR (P < 0.0001) and a reduced risk of developing proliferative DR (PDR) (P < 0.0001). Higher RBP3 correlated with increased photoreceptor segment thickness and lower vitreous interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and TNF-ß (P < 0.05). PDR was associated with lower vitreous interferon-γ and IL-10 and higher VEGF, IL-6, and IL-15 (P < 0.05), but was not associated with their plasma concentrations. CONCLUSIONS: Higher vitreous RBP3 concentrations are associated with less severe DR and slower rates of progression to PDR, supporting its potential as a biomarker and therapeutic agent for preventing DR worsening, possibly by lowering retinal VEGF and inflammatory cytokines.

Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function.

The systemic transport of dietary vitamin A/all-trans retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant rbpr2 zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body Rbpr2 knockout mouse (Rbpr2-/-) model. These mice were viable on both vitamin A-sufficient and -deficient diets. Rbpr2-/- mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when Rbpr2-/- mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

The FAR protein family of parasitic nematodes.

Fatty acid-and retinol-binding proteins (FARs) belong to a unique family of excreted/secreted proteins (ESPs) found exclusively in nematodes. Much of our understanding of these proteins, however, is limited to their in vitro binding characteristics toward various fatty acids and retinol and has provided little insight into their in vivo functions or mechanisms. Recent research, however, has shown that FARs elicit an immunomodulatory role in plant and animal model systems, likely by sequestering lipids involved in immune signaling. This alludes to the intricate relationship between parasitic nematode effectors and their hosts.

Genetic dissection in mice reveals a dynamic crosstalk between the delivery pathways of vitamin A.

Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. Here, we used a genetic dissection approach in mice to examine the contributions of these proteins in select tissues. We observed that ISX deficiency increased utilization of both preformed and provitamin A. We found that increased storage of retinoids in peripheral tissues of ISX-deficient mice was dependent on STRA6 and induced by retinoid signaling. In addition, double-mutant mice exhibited a partial rescue of the Stra6 mutant ocular phenotype. This rescue came at the expense of a massive accumulation of vitamin A in other tissues, demonstrating that vitamin A is randomly distributed when present in excessive amounts. Remarkably, provitamin A supplementation of mutant mice induced the expression of the RBP receptor 2 in the liver and was accompanied by increased hepatic retinyl ester stores. Taken together, these findings indicate dynamic crosstalk between the delivery pathways for this essential nutrient and suggest that hepatic reuptake of vitamin A takes place when excessive amounts circulate in the blood.

RBP4 regulates androgen receptor expression and steroid synthesis in Sertoli cells from Bactrian camels.

Retinol-binding protein (RBP4) plays an important role in the transport and metabolism of retinol. In addition, RBP4 contributes to testicular homeostasis, including maintenance of spermatogenesis and synthesis of androgens that mediate their physiological functions through the androgen receptor. RBP4 in Sertoli cells regulates testosterone and dihydrotestosterone synthesis and secretion, although the mechanisms have yet to be revealed. In this study, we examined the expression and function of RBP4 in Sertoli cells isolated from Bactrian camels. qRT-PCR analysis of various Bactrian camel tissues revealed high expression of RBP4 in the testis and epididymis. To examine RBP4 function, Sertoli cells isolated from testes were transfected with an RBP4 overexpression plasmid or RBP4-targeting siRNA. RBP4 overexpression resulted in significant inhibition of transcription and translation of the steroidogenic enzymes 3ßHSD and SRD5A1 concomitant with a significant decrease in androgen receptor expression and dihydrotestosterone secretion. Conversely, RBP4 knockdown significantly increased the expression of 3ßHSD, SRD5A1 and androgen receptor and enhanced the secretion of dihydrotestosterone and testosterone. These data reveal a novel role for RBP4 in regulating steroid synthesis in Sertoli cells from Bactrian camels.