Effect of topical bovine colostrum in wound healing of corneal surface after acute ocular alkali burn in mice.

The purpose of this study was to evaluate the effect of bovine colostrum (BC) in the regeneration of corneal epithelial cells on an ocular alkali burn model. Twenty-four C57BL/6 mice were categorized into two gender/age-matched groups for treatment. Two days after inducing a corneal alkali burn in all left eyes with 4 µl of sodium hydroxide 0.15 mol/l, both eyes of group 1 were treated with BC 4 times per day, and both eyes of group 2 were treated with isotonic saline solution (SS). The epithelial defect was photographed and measured by fluorescein staining on days two, four, seven, and ten. Ocular burn damage was assessed with a pre-established classification in clock hours from the limbus. After 10 days both eyes were processed, half of the group's corneas were assessed histopathologically, and the other half was used for pro/anti-inflammatory cytokine quantification using ELISA. BC treated (Group 1) corneas revealed significantly improved fluorescein staining score for limbal involvement when compared to SS treated (Group 2) corneas at days 4 (p = 0.013), 7 (p < 0.001), and 10 (p < 0.001), respectively. No differences were noted in limbal involvement at day 2 between the two groups (p > 0.99). The overall change (difference in slope) in fluorescein staining for limbal involvement between days 2 and 10 was -0.1669 (p = 0.006). Histologic examinations and cytokine measurements of group 2 demonstrated a strong inflammatory component compared to group 1. Our data indicates that topical application of BC facilitates corneal re-epithelialization and wound healing by suppressing the inflammatory process in an ocular alkali burn model.

The impact of lipids, lipid oxidation, and inflammation on AMD, and the potential role of miRNAs on lipid metabolism in the RPE.

The accumulation of lipids within drusen, the epidemiologic link of a high fat diet, and the identification of polymorphisms in genes involved in lipid metabolism that are associated with disease risk, have prompted interest in the role of lipid abnormalities in AMD. Despite intensive investigation, our understanding of how lipid abnormalities contribute to AMD development remains unclear. Lipid metabolism is tightly regulated, and its dysregulation can trigger excess lipid accumulation within the RPE and Bruch's membrane. The high oxidative stress environment of the macula can promote lipid oxidation, impairing their original function as well as producing oxidation-specific epitopes (OSE), which unless neutralized, can induce unwanted inflammation that additionally contributes to AMD progression. Considering the multiple layers of lipid metabolism and inflammation, and the ability to simultaneously target multiple pathways, microRNA (miRNAs) have emerged as important regulators of many age-related diseases including atherosclerosis and Alzheimer's disease. These diseases have similar etiologic characteristics such as lipid-rich deposits, oxidative stress, and inflammation with AMD, which suggests that miRNAs might influence lipid metabolism in AMD. In this review, we discuss the contribution of lipids to AMD pathobiology and introduce how miRNAs might affect lipid metabolism during lesion development. Establishing how miRNAs contribute to lipid accumulation in AMD will help to define the role of lipids in AMD, and open new treatment avenues for this enigmatic disease.

Lipids, oxidized lipids, oxidation-specific epitopes, and Age-related Macular Degeneration.

Age-related Macular Degeneration (AMD) is the leading cause of blindness among the elderly in western societies. While antioxidant micronutrient treatment is available for intermediate non-neovascular disease, and effective anti-vascular endothelial growth factor treatment is available for neovascular disease, treatment for early AMD is lacking due to an incomplete understanding of the early molecular events. The role of lipids, which accumulate in the macula, and their oxidation, has emerged as an important factor in disease development. These oxidized lipids can either directly contribute to tissue injury or react with amine on proteins to form oxidation-specific epitopes, which can induce an innate immune response. If inadequately neutralized, the inflammatory response from these epitopes can incite tissue injury during disease development. This review explores how the accumulation of lipids, their oxidation, and the ensuing inflammatory response might contribute to the pathogenesis of AMD. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder .

Synthesis of Positional Isomeric Phenylphenalenones.

A series of isomeric phenylphenalenones in which the phenyl ring is located at all possible peripheral positions of the phenalenone nuclei was synthesized. The structural characteristics of the series, in which topological variation is permitted with minimal electronic disturbance, could, in principle, allow for easy pharmacophore recognition when the compounds are aligned in steroidomimetic conformations.

Pentraxin 3 recruits complement factor H to protect against oxidative stress-induced complement and inflammasome overactivation.

The discovery that genetic abnormalities in complement factor H (FH) are associated with an increased risk for age-related macular degeneration (AMD), the most common cause of blindness among the elderly, raised hope of new treatments for this vision-threatening disease. Nonetheless, over a decade after the identification of this important association, how innate immunity contributes to AMD remains unresolved. Pentraxin 3 (PTX3), an essential component of the innate immunity system that plays a non-redundant role in controlling inflammation, regulates complement by interacting with complement components. Here, we show that PTX3 is induced by oxidative stress, a known cause of AMD, in the retinal pigmented epithelium (RPE). PTX3 deficiency in vitro and in vivo magnified complement activation induced by oxidative stress, leading to increased C3a, FB, and C3d, but not C5b-9 complex formation. Increased C3a levels, resulting from PTX3 deficiency, raised the levels of Il1b mRNA and secretion of activated interleukin (IL)-1ß by interacting with C3aR. Importantly, PTX3 deficiency augmented NLRP3 inflammasome activation, resulting in enhanced IL-1ß, but not IL-18, production by the RPE. Thus, in the presence of PTX3 deficiency, the complement and inflammasome pathways worked in concert to produce IL-1ß in sufficient abundance to, importantly, result in macrophages accumulating in the choroid. These results demonstrate that PTX3 acts as an essential brake for complement and inflammasome activation by regulating the abundance of FH in the RPE, and provide critical insights into the complex interplay between oxidative stress and innate immunity in the early stages of AMD development. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Complement factor H binds malondialdehyde epitopes and protects from oxidative stress.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

Synthesis of 8-Phenylphenalenones: 2-Hydroxy-8-(4-hydroxyphenyl)-1H-phenalen-1-one from Eichhornia crassipes.

2-Hydroxy-8-(4-hydroxyphenyl)-1H-phenalen-1-one (1), the first reported 8-phenylphenalenone from the roots of Eichhornia crassipes (water hyacinth), was synthesized starting from 2-methoxynaphthalene in 11 steps and with an overall yield of 2%. A cascade Friedel-Crafts/Michael annulation reaction between acryloyl chloride and 2-methoxynaphthalene afforded 9-methoxyperinaphthanone that, after transformation to 9-methoxy-2-(4-methoxyphenyl)-1H-phenalen-1-one by means of standard Suzuki-Miyaura methodology, was subjected to a reductive carbonyl transposition to afford 8-(4-methoxyphenyl)perinaphthanone. Dehydrogenation, epoxidation, and demethylation of the latter afforded 1.

Biology of p62/sequestosome-1 in Age-Related Macular Degeneration (AMD).

p62/sequestosome-1 is a multidimensional protein that interacts with many signaling factors, and regulates a variety of cellular functions including inflammation, apoptosis, and autophagy. Our previous work has revealed in the retinal pigment epithelium (RPE) that p62 promotes autophagy and simultaneously enhances an Nrf2-mediated antioxidant response to protect against acute oxidative stress. Several recent studies demonstrated that p62 contributes to NFkB mediated inflammation and inflammasome activation under certain circumstances, raising the question of whether p62 protects against or contributes to tissue injury. Herein, we will review the general characteristics of p62, focusing on its pro- and anti-cell survival roles within different physiological/pathological contexts, and discuss the potential of p62 as a therapeutic target for AMD.

p62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium.

As a signaling hub, p62/sequestosome plays important roles in cell signaling and degradation of misfolded proteins. p62 has been implicated as an adaptor protein to mediate autophagic clearance of insoluble protein aggregates in age-related diseases, including age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE). Our previous studies have shown that cigarette smoke (CS) induces oxidative stress and inhibits the proteasome pathway in cultured human RPE cells, suggesting that p62-mediated autophagy may become the major route to remove impaired proteins under such circumstances. In the present studies, we found that all p62 mRNA variants are abundantly expressed and upregulated by CS induced stress in cultured human RPE cells, yet isoform1 is the major translated form. We also show that p62 silencing exacerbated the CS induced accumulation of damaged proteins, both by suppressing autophagy and by inhibiting the Nrf2 antioxidant response, which in turn, increased protein oxidation. These effects of CS and p62 reduction were further confirmed in mice exposed to CS. We found that over-expression of p62 isoform1, but not its S403A mutant, which lacks affinity for ubiquitinated proteins, reduced misfolded proteins, yet simultaneously promoted an Nrf2-mediated antioxidant response. Thus, p62 provides dual, reciprocal enhancing protection to RPE cells from environmental stress induced protein misfolding and aggregation, by facilitating autophagy and the Nrf2 mediated antioxidant response, which might be a potential therapeutic target against AMD.

Nrf2 signaling is impaired in the aging RPE given an oxidative insult.

Age-related macular degeneration (AMD) represents the leading cause of blindness in the elderly, yet no definitive therapy exists for early, dry disease. Several lines of evidence have implicated oxidative stress-induced damage to the retinal pigment epithelium (RPE) in the pathogenesis of AMD, suggesting that the aging RPE may exhibit increased susceptibility to cell damage induced by exogenous stressors. The transcription factor Nrf2 serves as the master regulator of a highly coordinated antioxidant response in virtually all cell types. We compared Nrf2 signaling in the RPE of young (2 months) and old (15 months) mice under unstressed and stressed (sodium iodate) conditions. The aging RPE expressed higher levels of the Nrf2 target genes NQO1, GCLM, and HO1 compared with the RPE of younger mice under unstressed conditions, suggesting an age-related increase in basal oxidative stress. Moreover, the RPE of older mice demonstrated impaired induction of the protective Nrf2 pathway following oxidative stress induced with sodium iodate. The RPE of old mice exposed to sodium iodate also exhibited higher levels of superoxide anion and malondialdehyde than young mice, suggesting inadequate protection against oxidative damage. Induction of Nrf2 signaling in response to sodium iodate was partially restored in the RPE of aging mice with genetic rescue, using conditional knockdown of the Nrf2 negative regulator Keap1 (Tam-Cre; Keap1loxP) compared to Keap1loxP mice. These data indicate that the aging RPE is vulnerable to oxidative damage due to impaired Nrf2 signaling, and that Nrf2 signaling is a promising target for novel pharmacologic or genetic therapeutic strategies.

Decreased membrane complement regulators in the retinal pigmented epithelium contributes to age-related macular degeneration.

Dysregulated complement is thought to play a central role in age-related macular degeneration (AMD) pathogenesis, but the specific mechanisms have yet to be determined. In maculae of AMD specimens, we found that the complement regulatory protein, CD59, was increased in regions of uninvolved retinal pigmented epithelium (RPE) of early AMD, but decreased in the RPE overlying drusen and in geographic atrophy, an advanced form of AMD. While CD46 immunostaining was basolaterally distributed in the RPE of unaffected controls, it was decreased in diseased areas of early AMD samples. Since oxidized low-density lipoproteins (oxLDL) collect in drusen of AMD and are a known complement trigger, we treated ARPE-19 cells with oxLDL and found that cellular CD46 and CD59 proteins were decreased by 2.9- and nine-fold (p < 0.01), respectively. OxLDLs increased complement factor B mRNA and Bb protein, but not factor D, I or H. OxLDLs increased C3b, but not C3a, C5 or C5b-9. C5b-9 was increased by 27% (p < 0.01) when the medium was supplemented with human serum, which was sufficient to induce poly(ADP-ribose) polymerase cleavage, a marker of apoptosis. The decreased levels of CD46 and CD59 were in part explained by their release in exosomal and apoptotic membranous particles. In addition, CD59 was partially degraded through activation of IRE1α. Collectively, these results suggest that a combination of impaired complement regulators results in inadequately controlled complement by the RPE in AMD that induces RPE damage.

Oxidized low-density-lipoprotein-induced injury in retinal pigment epithelium alters expression of the membrane complement regulatory factors CD46 and CD59 through exosomal and apoptotic bleb release.

Genetic and immunohistochemical studies have identified the alternative complement pathway as an important component of age-related macular degeneration (AMD). The objective of this chapter is to review the impact of complement regulators on complement activation in the macula as it relates to AMD. Our laboratory and other investigators have identified CD46 and CD59 as important retinal pigment epithelium (RPE) cell membrane complement regulators, which are decreased in AMD. Using oxidized low-density lipoproteins (oxLDLs), which are found in Bruch's membrane in AMD, we found that CD46 and CD59 were decreased in RPE cells in part, by their release in exosomes and apoptotic particles. The release of complement regulators could potentially impair complement regulation on RPE cells and contribute to lesion formation in the outer retina and Bruch's membrane during the development of AMD.

Mitophagy initiates retrograde mitochondrial-nuclear signaling to guide retinal pigment cell heterogeneity.

Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment.Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O2-: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.

Advanced glycation endproduct changes to Bruch's membrane promotes lipoprotein retention by lipoprotein lipase.

Lipoprotein particles accumulate in Bruch's membrane before the development of basal deposits and drusen, two histopathologic lesions that define age-related macular degeneration (AMD). We therefore, sought to determine which molecules could participate in lipoprotein retention. Wild-type or lipoprotein lipase-deficient mice were injected with low-dose D-galactose or PBS subcutaneously for 8 weeks to induce advanced glycation endproduct (AGE) formation. Some mice were also injected with the AGE breaker phenacylphiazolium bromide and D-galactose. Rhodamine-labeled low-density lipoproteins were injected into mice, and the fluorescence was measured up to 72 hours later. AGEs, proteoglycans, and other lipid-retaining molecules were evaluated by IHC. Lipoprotein lipase distribution was assessed in AMD samples by IHC. D-galactose-treated mice retained lipoproteins in the retinal pigment epithelial and Bruch's membrane to a greater extent than either PBS- or phenacylphiazolium bromide/D-galactose-treated mice at 24 and 72 hours after injection (P ≤ 0.04). Immunolabeling for carboxymethyllysine, biglycan, and lipoprotein lipase was found in D-galactose-treated mice only. Mice deficient for lipoprotein lipase treated with D-galactose did not retain lipoproteins to any measureable extent. Human AMD samples had lipoprotein lipase labeling within drusen, basal deposits, and the choroid. Mice treated with D-galactose to induce AGE formation in Bruch's membrane retain intravenously injected lipoproteins. Our results suggest that lipoprotein retention in Bruch's membrane is mediated by lipoprotein lipase.

ßA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for ßA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express ßA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant ßA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that ßA3/A1-crystallin is essential for normal astrocyte function in the retina.

Bactericidal Efficacy of High Irradiance Ultraviolet A Photoactivation of Riboflavin Versus Standard Corneal Cross-Linking Protocol In Vitro.

PURPOSE: The purpose of this study was to compare the efficacy of high ultraviolet A (UVA) irradiance photoactivation of riboflavin (vitamin B2) versus the standard corneal cross-linking protocol on bacterial viability. METHODS: Methicillin-sensitive Staphylococcus aureus (MSSA) Newman strain and methicillin-resistant multidrug-resistant S. aureus (MDR-MRSA) USA300, CA409, CA127, GA656, and NY315 strains were exposed to a UVA energy dose of 5.4 to 6 J/cm 2 by 2 high irradiance regimens: A) 30 mW/cm 2 for 3 minutes and B) 10 mW/cm 2 for 10 minutes with B2 0.1%. Control groups included B2/UVA alone, CA409 exposed to standard B2 0.1% + UVA (3 mW/cm 2 for 30 minutes), and an untreated sample. Cell viability was assessed. Triplicate values were obtained. The Mann-Whitney test and Student t test were used for statistical analysis. RESULTS: There was no difference comparing the median bacterial load (log CFU/mL) of the untreated samples versus regimen A: Newman P = 0.7, CA409 P = 0.3, USA300 P = 0.5, CA127 P = 0.6, GA656 P = 0.1, and NY315 P = 0.2 ( P ≥ 0.1); and B: Newman P = 0.1, CA409 P = 0.3, USA300 P = 0.4, CA127 P = 0.6, GA656 P = 0.1, and NY315 P = 0.3 ( P ≥ 0.1). Standard regimen killed 100% of CA409. CONCLUSIONS: Photoactivation of B2 by high UVA irradiance does not seem to be effective for bacterial eradication in this study.

Cell Death in AMD: The Rationale for Targeting Fas.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the developed world. While great advances have been made in the treatment of the neovascular ("wet") form of the disease, there is still a significant need for therapies that prevent the vision loss associated with the advanced forms of dry, atrophic AMD. In this atrophic form, retinal pigment epithelial (RPE) and photoreceptor cell death is the ultimate cause of vision loss. In this review, we summarize the cell death pathways and their relation to RPE and retinal cell death in AMD. We review the data that support targeting programmed cell death through inhibition of the Fas receptor as a novel approach to preserve these structures and that this effect results from inhibiting both canonical death pathway activation and reducing the associated inflammatory response. These data lay the groundwork for current clinical strategies targeting the Fas pathway in this devastating disease.

Nrf2 deficiency decreases NADPH from impaired IDH shuttle and pentose phosphate pathway in retinal pigmented epithelial cells to magnify oxidative stress-induced mitochondrial dysfunction.

The nuclear factor-erythroid 2-related factor-2 (Nrf2), a major antioxidant transcription factor, is decreased in several age-related diseases including age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western society. Since Nrf2's mito-protective response is understudied, we investigated its antioxidant response on mitochondria. Control and Nrf2-deficient retinal pigmented epithelial (RPE) cells were compared after treating with cigarette smoke extract (CSE). Mitochondrial antioxidant abundance and reactive oxygen species (ROS) were quantified. Mitochondrial function was assessed by TMRM assay, NADPH, electron transport chain activity, and Seahorse. Results were corroborated in Nrf2-/- mice and relevance to AMD was provided by immunohistochemistry of human globes. CSE induced mitochondrial ROS to impair mitochondrial function. H2 O2 increase in particular, was magnified by Nrf2 deficiency, and corresponded with exaggerated mitochondrial dysfunction. While Nrf2 did not affect mitochondrial antioxidant abundance, oxidized PRX3 was magnified by Nrf2 deficiency due to decreased NADPH from decreased expression of IDH2 and pentose phosphate pathway (PPP) genes. With severe CSE stress, intrinsic apoptosis was activated to increase cell death. PPP component TALDO1 immunolabeling was decreased in dysmorphic RPE of human AMD globes. Despite limited regulation of mitochondrial antioxidant expression, Nrf2 influences PPP and IDH shuttle activity that indirectly supplies NADPH for the TRX2 system. These results provide insight into how Nrf2 deficiency impacts the mitochondrial antioxidant response, and its role in AMD pathobiology.

Apolipoprotein B100 secretion by cultured ARPE-19 cells is modulated by alteration of cholesterol levels.

Cholesteryl ester rich apolipoprotein B100 (apoB100) lipoproteins accumulate in Bruch's membrane before the development of age-related macular degeneration. It is not known if these lipoproteins come from the circulation or local ocular tissue. Emerging, but incomplete evidence suggests that the retinal pigmented epithelium (RPE) can secrete lipoproteins. The purpose of this investigation was to determine (i) whether human RPE cells synthesize and secrete apoB100, and (ii) whether this secretion is driven by cellular cholesterol, and if so, (iii) whether statins inhibit this response. The established, human derived ARPE-19 cells challenged with 0-0.8 mM oleic acid accumulated cellular cholesterol, but not triglycerides. Oleic acid increased the amount of apoB100 protein recovered from the medium by both western blot analysis and (35) S-radiolabeled immunoprecipitation while negative stain electron microscopy showed lipoprotein-like particles. Of nine statins evaluated, lipophilic statins induced HMG-CoA reductase mRNA expression the most. The lipophilic Cerivastatin (5 µM) reduced cellular cholesterol by 39% and abrogated apoB100 secretion by 3-fold. In contrast, the hydrophilic statin Pravastatin had minimal effect on apoB100 secretion. These data suggest that ARPE-19 cells synthesize and secrete apoB100 lipoproteins, that this secretion is driven by cellular cholesterol, and that statins can inhibit apoB100 secretion by reducing cellular cholesterol.

Periocular triamcinolone enhances intraocular gene expression after delivery by adenovirus.

PURPOSE: A noninvasive imaging technique was used for serial assessment of gene expression after intraocular gene transfer. Bioluminescence after intravitreous administration of an adenovirus vector containing the firefly luciferase gene was measured serially and noninvasively. The optical signal was then used as a bioassay to determine whether periocular immune modulation affects intraocular transgene expression. METHODS: Sixty-two, 8-week-old, male BALB/c mice were used. The correlation of optical signal intensity was determined by tissue luciferase level after injecting 30 mice with one of three intravitreous doses of Ad-Luc-GFP (10(8), 5 x 10(8), or 10(9) particles in 1 microL). Ocular bioluminescence was measured at days 2, 5, 8, and 14. The bioluminescence was then directly compared with measured tissue luciferase levels. The remaining 32 mice were divided into two groups. One group (n = 16), was injected with periocular corticosteroid (400 mug in 10 microL). Two days later, Ad-Luc-GFP was administered by intravitreous injection (10(9) particles in 1 microL). The remaining mice (n = 16) were injected with the same dose of intravitreous Ad-Luc-GFP without corticosteroid pretreatment. Ocular bioluminescence was then assessed longitudinally on days 2, 4, 6, 8, 11, 14, 22, and 30 after intravitreous injection in n = 10 mice per group. The optical signal intensity in each group was compared over the study period. The remaining 12 mice (n = 6, each group) were used to assess histologic differences between the two groups. RESULTS: In vivo measurement of ocular bioluminescence was well correlated with tissue luciferase levels (Spearman's correlation, r = 0.969, P < 0.001). Periocular TA injection markedly decreased the acute inflammatory reaction associated with intravitreous Ad-Luc-GFP and was associated with a significant increase in the duration of peak luciferase expression as well as the total period of luciferase expression. CONCLUSIONS: A significant enhancement of intraocular transgene expression is associated with periocular pretreatment with corticosteroid. Histologic evidence of immune cell reduction in ocular tissues in corticosteroid-treated eyes implies a local immune response. Periocular treatment with corticosteroids may enhance adenovirus-mediated gene expression in the eye.