Efficient ultrasound-mediated drug delivery to orthotopic liver tumors - Direct comparison of doxorubicin-loaded nanobubbles and microbubbles.

Liver metastasis is a major obstacle in treating aggressive cancers, and current therapeutic options often prove insufficient. To overcome these challenges, there has been growing interest in ultrasound-mediated drug delivery using lipid-shelled microbubbles (MBs) and nanobubbles (NBs) as promising strategies for enhancing drug delivery to tumors. Our previous work demonstrated the potential of Doxorubicin-loaded C3F8 NBs (hDox-NB, 280 ± 123 nm) in improving cancer treatment in vitro using low-frequency unfocused therapeutic ultrasound (TUS). In this study, we investigated the pharmacokinetics and biodistribution of sonicated hDox-NBs in orthotopic rat liver tumors. We compared their delivery and therapeutic efficiency with size-isolated MBs (hDox-MB, 1104 ± 373 nm) made from identical shell material and core gas. Results showed a similar accumulation of hDox in tumors treated with hDox-MBs and unfocused therapeutic ultrasound (hDox-MB + TUS) and hDox-NB + TUS. However, significantly increased apoptotic cell death in the tumor and fewer off-target apoptotic cells in the normal liver were found upon the treatment with hDox-NB + TUS. The tumor-to-liver apoptotic ratio was elevated 9.4-fold following treatment with hDox-NB + TUS compared to hDox-MB + TUS, suggesting that the therapeutic efficacy and specificity are significantly increased when using hDox-NB + TUS. These findings highlight the potential of this approach as a viable treatment modality for liver tumors. By elucidating the behavior of drug-loaded bubbles in vivo, we aim to contribute to developing more effective liver cancer treatments that could ultimately improve patient outcomes and decrease off-target side effects.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Efficient ultrasound-mediated drug delivery to orthotopic liver tumors - Direct comparison of doxorubicin-loaded nanobubbles and microbubbles.

Liver metastasis is a major obstacle in treating aggressive cancers, and current therapeutic options often prove insufficient. To overcome these challenges, there has been growing interest in ultrasound-mediated drug delivery using lipid-shelled microbubbles (MBs) and nanobubbles (NBs) as promising strategies for enhancing drug delivery to tumors. Our previous work demonstrated the potential of Doxorubicin-loaded C3F8 NBs (hDox-NB, 280 ± 123 nm) in improving cancer treatment in vitro using low-frequency ultrasound. In this study, we investigated the pharmacokinetics and biodistribution of sonicated hDox-NBs in orthotopic rat liver tumors. We compared their delivery and therapeutic efficiency with size-isolated MBs (hDox-MB, 1104 ± 373 nm). Results showed a similar accumulation of hDox in tumors treated with hDox-MBs and unfocused therapeutic ultrasound (hDox-MB+TUS) and hDox-NB+TUS. However, significantly increased apoptotic cell death in the tumor and fewer off-target apoptotic cells in the normal liver were found upon the treatment with hDox-NB+TUS. The tumor-to-liver apoptotic ratio was elevated 9.4-fold following treatment with hDox-NB+TUS compared to hDox-MB+TUS, suggesting that the therapeutic efficacy and specificity are significantly increased when using hDox-NB+TUS. These findings highlight the potential of this approach as a viable treatment modality for liver tumors. By elucidating the behavior of drug-loaded bubbles in vivo, we aim to contribute to developing more effective liver cancer treatments that could ultimately improve patient outcomes and decrease off-target side effects.

Increasing Energetic Demands on Photoreceptors in Diabetes Corrects Retinal Lipid Dysmetabolism and Reduces Subsequent Microvascular Damage.

Mechanisms responsible for the pathogenesis of diabetic retinal disease remain incompletely understood, but they likely involve multiple cellular targets, including photoreceptors. Evidence suggests that dysregulated de novo lipogenesis in photoreceptors is a critical early target of diabetes. Following on this observation, the present study aimed to determine whether two interventions shown to improve diabetic retinopathy in mice-pharmacologic visual cycle inhibition and prolonged dark adaptation-reduce photoreceptor anabolic lipid metabolism. Elevated retinal lipid biosynthetic signaling was observed in two mouse models of diabetes, with both models showing reduced retinal AMP-activated kinase (AMPK) signaling, elevated acetyl CoA carboxylase (ACC) signaling, and increased activity of fatty acid synthase, which promotes lipotoxicity in photoreceptors. Although retinal AMPK-ACC axis signaling was dependent on systemic glucose fluctuations in healthy animals, mice with diabetes lacked such regulation. Visual cycle inhibition and prolonged dark adaptation reversed abnormal retinal AMPK-ACC signaling in mice with diabetes. Although visual cycle inhibition reduced the severity of diabetic retinopathy in control mice, as assessed by retinal capillary atrophy, this intervention was ineffective in fatty acid synthase gain-of-function mice. These results suggest that early diabetic retinopathy is characterized by glucose-driven elevations in retinal lipid biosynthetic activity, and that two interventions known to increase photoreceptor glucose demands alleviate disease by reversing these signals.

Dietary Vitamin A Affects the Function of Incretin-Producing Enteroendocrine Cells in Male Mice Fed a High-Fat Diet.

BACKGROUND: Retinol-binding protein 2 (RBP2) is an intracellular carrier for vitamin A in the absorptive enterocytes. Mice lacking RBP2 (Rbp2-/-) display an unexpected phenotype of obesity, glucose intolerance, and elevated glucose-dependent insulinotropic polypeptide (GIP) levels. GIP and glucagon-like peptide 1 (GLP-1) are incretin hormones secreted by enteroendocrine cells (EECs). We recently demonstrated the presence of RBP2 and other retinoid-related proteins in EECs. OBJECTIVES: Given RBP2's role in intracellular retinoid trafficking, we aimed to evaluate whether dietary vitamin A affects incretin-secreting cell function and gene expression. METHODS: Male Rbp2-/- mice and sex- and age-matched controls (n = 6-9) were fed a high-fat diet (HFD) for 18 wk containing normal (VAN, 4000 IU/kg of diet) or low (VAL, 25% of normal) vitamin A concentrations. Body weight was recorded biweekly. Plasma GIP and GLP-1 levels were obtained fasting and 30 min after an oral fat gavage at week 16. Glucose tolerance tests were also performed. Mice were killed at week 18, and blood and tissue samples were obtained. RESULTS: Rbp2-/- mice displayed greater weight gain on the VAN compared with the VAL diet from week 7 of the intervention (P ≤ 0.01). Stimulated GIP levels were elevated in Rbp2-/- mice compared with their controls fed the VAN diet (P = 0.02), whereas their GIP response was lower when fed the VAL diet (P = 0.03). Although no differences in GLP-1 levels were observed in the VAN diet group, a lower GLP-1 response was seen in Rbp2-/- mice fed the VAL diet (P = 0.02). Changes in incretin gene expression and that of other genes associated with EEC lineage and function were consistent with these observations. Circulating and hepatic retinoid levels revealed no systemic vitamin A deficiency across dietary groups. CONCLUSIONS: Our data support a role for RBP2 and dietary vitamin A in incretin secretion and gene expression in mice fed a HFD.

Genetic deletion of Bco2 and Isx establishes a golden mouse model for carotenoid research.

OBJECTIVE: Low plasma levels of carotenoids are associated with mortality and chronic disease states. Genetic studies in animals revealed that the tissue accumulation of these dietary pigments is associated with the genes encoding ß-carotene oxygenase 2 (BCO2) and the scavenger receptor class B type 1 (SR-B1). Here we examined in mice how BCO2 and SR-B1 affect the metabolism of the model carotenoid zeaxanthin that serves as a macular pigment in the human retina. METHODS: We used mice with a lacZ reporter gene knock-in to determine Bco2 expression patterns in the small intestine. By genetic dissection, we studied the contribution of BCO2 and SR-B1 to zeaxanthin uptake homeostasis and tissue accumulation under different supply conditions (50 mg/kg and 250 mg/kg). We determined the metabolic profiles of zeaxanthin and its metabolites in different tissues by LC-MS using standard and chiral columns. An albino Isx-/-/Bco2-/- mouse homozygous for Tyrc-2J was generated to study the effect of light on ocular zeaxanthin metabolites. RESULTS: We demonstrate that BCO2 is highly expressed in enterocytes of the small intestine. Genetic deletion of Bco2 led to enhanced accumulation of zeaxanthin, indicating that the enzyme serves as a gatekeeper of zeaxanthin bioavailability. Relaxing the regulation of SR-B1 expression in enterocytes by genetic deletion of the transcription factor ISX further enhanced zeaxanthin accumulation in tissues. We observed that the absorption of zeaxanthin was dose-dependent and identified the jejunum as the major zeaxanthin-absorbing intestinal region. We further showed that zeaxanthin underwent oxidation to ε,ε-3,3'-carotene-dione in mouse tissues. We detected all three enantiomers of the zeaxanthin oxidation product whereas the parent zeaxanthin only existed as (3R, 3'R)-enantiomer in the diet. The ratio of oxidized to parent zeaxanthin varied between tissues and was dependent on the supplementation dose. We further showed in an albino Isx-/-/Bco2-/- mouse that supra-physiological supplementation doses (250 mg/kg) with zeaxanthin rapidly induced hypercarotenemia with a golden skin phenotype and that light stress increased the concentration of oxidized zeaxanthin in the eyes. CONCLUSIONS: We established the biochemical basis of zeaxanthin metabolism in mice and showed that tissue factors and abiotic stress affect the metabolism and homeostasis of this dietary lipid.

Toward structural-omics of the bovine retinal pigment epithelium.

The use of an integrated systems biology approach to investigate tissues and organs has been thought to be impracticable in the field of structural biology, where the techniques mainly focus on determining the structure of a particular biomacromolecule of interest. Here, we report the use of cryoelectron microscopy (cryo-EM) to define the composition of a raw bovine retinal pigment epithelium (RPE) lysate. From this sample, we simultaneously identify and solve cryo-EM structures of seven different RPE enzymes whose functions affect neurotransmitter recycling, iron metabolism, gluconeogenesis, glycolysis, axonal development, and energy homeostasis. Interestingly, dysfunction of these important proteins has been directly linked to several neurodegenerative disorders, including Huntington's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, and schizophrenia. Our work underscores the importance of cryo-EM in facilitating tissue and organ proteomics at the atomic level.

Expression and biochemical analyses of proteins involved in the transport of carotenoids and retinoids.

Animals acquire carotenoids from the diet and convert them to retinoids. These lipids must be distributed in the body to support retinoid signaling in peripheral tissues and photoreceptor function in the eyes. However, the hydrophobicity of carotenoids and retinoids limit their diffusion in the aqueous environment of the body. Therefore, membrane proteins and cellular binding proteins transport these lipids between extra- and intracellular compartments and facilitate their metabolism. Mutations in genes encoding these transport proteins are associated with a wide spectrum of blinding disorders. Here, we describe approaches used by our laboratories that have proven successful in expressing these proteins and examining their biochemical properties in the test tube and in cell-based assays. These assays can be utilized for screening of small molecule modulators of their activities to correct pathologies associated with retinoid metabolism.

Acyl-CoA:wax alcohol acyltransferase 2 modulates the cone visual cycle in mouse retina.

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2-/- mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.

Intestinal Enteroendocrine Cell Signaling: Retinol-binding Protein 2 and Retinoid Actions.

Retinol-binding protein 2-deficient (Rbp2-/-) mice are more prone to obesity, glucose intolerance, and hepatic steatosis than matched controls. Glucose-dependent insulinotropic polypeptide (GIP) blood levels are dysregulated in these mice. The present studies provide new insights into these observations. Single cell transcriptomic and immunohistochemical studies establish that RBP2 is highly expressed in enteroendocrine cells (EECs) that produce incretins, either GIP or glucagon-like peptide-1. EECs also express an enzyme needed for all-trans-retinoic acid (ATRA) synthesis, aldehyde dehydrogenase 1 family member A1, and retinoic acid receptor-alpha, which mediates ATRA-dependent transcription. Total and GIP-positive EECs are significantly lower in Rbp2-/- mice. The plasma transport protein for retinol, retinol-binding protein 4 (RBP4) is also expressed in EECs and is cosecreted with GIP upon stimulation. Collectively, our data support direct roles for RBP2 and ATRA in cellular processes that give rise to GIP-producing EECs and roles for RBP2 and RBP4 within EECs that facilitate hormone storage and secretion.

Retinol-binding protein 2 (RBP2): More than just dietary retinoid uptake.

Retinol-binding protein 2 (RBP2, also known as cellular retinol-binding protein 2 (CRBP2)) is a member of the fatty acid-binding protein family and has been extensively studied for its role in facilitating dietary vitamin A (retinol) uptake and metabolism within enterocytes of the small intestine. RBP2 is present in highest concentrations in the proximal small intestine where it constitutes approximately 0.1-0.5% of soluble protein. Recent reports have established that RBP2 binds monoacylglycerols (MAGs) with high affinity, including the canonical endocannabinoid 2-arachidonoylglycerol (2-AG). Crystallographic studies reveal that retinol, 2-AG, or other long-chain MAGs alternatively can bind in the retinol-binding pocket of RBP2. It also has been demonstrated recently that Rbp2-deficient mice are more susceptible to developing obesity and associated metabolic phenotypes when exposed to a high fat diet, or as they age when fed a conventional chow diet. When subjected to an oral fat challenge, the Rbp2-deficient mice release into the circulation significantly more, compared to littermate controls, of the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP). These new findings regarding RBP2 structure and actions within the intestine are the focus of this review.

Mouse models in studies on the etiology of evaporative dry eye disease.

Evaporative dry eye disease (DED) is a common ocular condition impacting the quality of life of millions of patients worldwide. The etiology of evaporative DED is related to dysfunction of meibomian glands (MGs), resulting in suboptimal yield or lipid composition of secreted meibum. The clinical manifestation of evaporative DED involves mechanical obstruction of the MG orifice and decreased tear film stability that leads to chronic eye irritation, inflammation, and progressive damage to the cornea and surrounding tissue. Despite its high prevalence, evaporative DED remains an unmet medical need. The main obstacle in the development of effective therapeutic strategies against this disease is inadequate knowledge about the complex arrays of lipogenic reactions (meibogenesis) in the MGs and a lack of suitable animal models of the human condition. In this review, we discuss the recent advances in the creation of genetically modified mouse models that recapitulate the phenotype of evaporative DED as well as their impact on our understanding of lipid biosynthesis in MGs and therapeutic strategies targeting meibogenesis.

Structure of putative tumor suppressor ALDH1L1.

Putative tumor suppressor ALDH1L1, the product of natural fusion of three unrelated genes, regulates folate metabolism by catalyzing NADP+-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Cryo-EM structures of tetrameric rat ALDH1L1 revealed the architecture and functional domain interactions of this complex enzyme. Highly mobile N-terminal domains, which remove formyl from 10-formyltetrahydrofolate, undergo multiple transient inter-domain interactions. The C-terminal aldehyde dehydrogenase domains, which convert formyl to CO2, form unusually large interfaces with the intermediate domains, homologs of acyl/peptidyl carrier proteins (A/PCPs), which transfer the formyl group between the catalytic domains. The 4'-phosphopantetheine arm of the intermediate domain is fully extended and reaches deep into the catalytic pocket of the C-terminal domain. Remarkably, the tetrameric state of ALDH1L1 is indispensable for catalysis because the intermediate domain transfers formyl between the catalytic domains of different protomers. These findings emphasize the versatility of A/PCPs in complex, highly dynamic enzymatic systems.

Peptide Derivatives of Retinylamine Prevent Retinal Degeneration with Minimal Side Effects on Vision in Mice.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands.

Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format. By examining chemical libraries of bioactive lipids, we provided evidence for the selective interaction of CRBP2 with a subset of nonretinoid ligands with the highest affinity for sn-1 and sn-2 MAGs that contain polyunsaturated C18-C20 acyl chains. We also elucidated the structure-affinity relationship for nonretinoid ligands of this protein. We further dissect the molecular basis for this ligand's specificity by analyzing high-resolution crystal structures of CRBP2 in complex with selected derivatives of MAGs. Finally, we identify T51 and V62 as key amino acids that enable the broadening of ligand selectivity to MAGs in CRBP2 as compared with retinoid-specific CRBP1. Thus, our study provides the molecular framework for understanding the lipid selectivity and diverse functions of CRBPs in controlling lipid homeostasis.

LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis from ß-carotene.

There is increasing recognition that dietary lipids can affect the expression of genes encoding their metabolizing enzymes, transporters, and binding proteins. This mechanism plays a pivotal role in controlling tissue homeostasis of these compounds and avoiding diseases. The regulation of retinoid biosynthesis from ß-carotene (BC) is a classic example for such an interaction. The intestine-specific homeodomain transcription factor (ISX) controls the activity of the vitamin A-forming enzyme ß-carotene oxygenase-1 in intestinal enterocytes in response to increasing concentration of the vitamin A metabolite retinoic acid. However, it is unclear how cells control the concentration of the signaling molecule in this negative-feedback loop. We demonstrate in mice that the sequestration of retinyl esters by the enzyme lecithin:retinol acyltransferase (LRAT) is central for this process. Using genetic and pharmacological approaches in mice, we observed that in LRAT deficiency, the transcription factor ISX became hypersensitive to dietary vitamin A and suppressed retinoid biosynthesis. The dysregulation of the pathway resulted in BC accumulation and vitamin A deficiency of extrahepatic tissues. Pharmacological inhibition of retinoid signaling and genetic depletion of the Isx gene restored retinoid biosynthesis in enterocytes. We provide evidence that the catalytic activity of LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis and maintains optimal retinoid levels in the body.

The Structural and Biochemical Basis of Apocarotenoid Processing by ß-Carotene Oxygenase-2.

In mammals, carotenoids are converted by two carotenoid cleavage oxygenases into apocarotenoids, including vitamin A. Although knowledge about ß-carotene oxygenase-1 (BCO1) and vitamin A metabolism has tremendously increased, the function of ß-carotene oxygenase-2 (BCO2) remains less well-defined. We here studied the role of BCO2 in the metabolism of long chain ß-apocarotenoids, which recently emerged as putative regulatory molecules in mammalian biology. We showed that recombinant murine BCO2 converted the alcohol, aldehyde, and carboxylic acid of a ß-apocarotenoid substrate by oxidative cleavage at position C9,C10 into a ß-ionone and a diapocarotenoid product. Chain length variation (C20 to C40) and ionone ring site modifications of the apocarotenoid substrate did not impede catalytic activity or alter the regioselectivity of the double bond cleavage by BCO2. Isotope labeling experiments revealed that the double bond cleavage of an apocarotenoid followed a dioxygenase reaction mechanism. Structural modeling and site directed mutagenesis identified amino acid residues in the substrate tunnel of BCO2 that are critical for apocarotenoid binding and catalytic processing. Mice deficient for BCO2 accumulated apocarotenoids in their livers, indicating that the enzyme engages in apocarotenoid metabolism. Together, our study provides novel structural and functional insights into BCO2 catalysis and establishes the enzyme as a key component of apocarotenoid homeostasis in mice.

Protective Effects of Flavonoids in Acute Models of Light-Induced Retinal Degeneration.

Degeneration of photoreceptors caused by excessive illumination, inherited mutations, or aging is the principal pathology of blinding diseases. Pharmacological compounds that stabilize the visual receptor rhodopsin and modulate the cellular pathways triggering death of photoreceptors could avert this pathology. Interestingly, flavonoids can modulate the cellular processes, such as oxidative stress, inflammatory responses, and apoptosis, that are activated during retinal degeneration. As we found previously, flavonoids also bind directly to unliganded rod opsin, enhancing its folding, stability, and regeneration. In addition, flavonoids stimulate rhodopsin gene expression. Thus, we evaluated the effect of two main dietary flavonoids, quercetin and myricetin, in ATP-binding cassette subfamily A member 4 -/- /retinol dehydrogenase 8 -/- and wild-type BALB/c mice susceptible to light-induced photoreceptor degeneration. Using in vivo imaging, such as optical coherence tomography, scanning laser ophthalmoscopy, and histologic assessment of retinal morphology, we found that treatment with these flavonoids prior to light insult remarkably protected retina from deterioration and preserved its function. Using high-performance liquid chromatography-mass spectrometry analysis, we detected these flavonoids in the eye upon their intraperitoneal administration. The molecular events associated with the protective effect of quercetin and myricetin were related to the elevated expression of photoreceptor-specific proteins, rhodopsin and cone opsins, decreased expression of the specific inflammatory markers, and the shift of the equilibrium between cell death regulators BCL2-associated X protein (BAX) and B-cell lymphoma 2 toward an antiapoptotic profile. These results were confirmed in photoreceptor-derived 661W cells treated with either H2O2 or all-trans-retinal stressors implicated in the mechanism of retinal degeneration. Altogether, flavonoids could have significant prophylactic value for retinal degenerative diseases. SIGNIFICANCE STATEMENT: Flavonoids commonly present in food exhibit advantageous effects in blinding diseases. They bind to and stabilize unliganded rod opsin, which in excess accelerates degenerative processes in the retina. Additionally, flavonoids enhance the expression of the visual receptors, rod and cone opsins; inhibit the inflammatory reactions; and induce the expression of antiapoptotic markers in the retina, preventing the degeneration in vivo. Thus, flavonoids could have a prophylactic value for retinal degenerative diseases.

The human mitochondrial enzyme BCO2 exhibits catalytic activity toward carotenoids and apocarotenoids.

The enzyme ß-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Deficiency in Acyl-CoA:Wax Alcohol Acyltransferase 2 causes evaporative dry eye disease by abolishing biosynthesis of wax esters.

Lipids secreted by the meibomian glands (MGs) of the eyelids are essential to the protection of the eye's surface. An altered meibum composition represents the primary cause of evaporative dry eye disease (DED). Despite the critical importance of the meibum, its biosynthetic pathways and the roles of individual lipid components remain understudied. Here, we report that the genetic deletion of Acyl-CoA:wax alcohol acyltransferase 2 (AWAT2) causes the obstruction of MGs and symptoms of evaporative DED in mice. The lipid composition of the meibum isolated from Awat2-/- mice revealed the absence of wax esters, which was accompanied by a compensatory overproduction of cholesteryl esters. The resulting increased viscosity of meibum led to the dilation of the meibomian ducts, and the progressive degeneration of the MGs. Overall, we provide evidence for the main physiological role of AWAT2 and establish Awat2-/- mice as a model for DED syndrome that can be used in studies on tear film-oriented therapies.