Technical brief: Pump-probe paradigm in an integrating cavity to study photodecomposition processes.

PURPOSE: Assaying photodecomposition is challenging because light must be used to initiate the photodamage and light must be used to monitor the photodecomposition. The experimental requirements are as follows: 1) During exposure of the actinic beam, continuously monitor the spectral characteristics of the sample, 2) uniformly expose the reactants to the actinic source, 3) obtain informative spectra in the presence of light scatter, and 4) achieve sufficient sensitivity for dilute reactants. Traditional spectrophotometers cannot address these issues due to sample turbidity, the inability to uniformly expose the cuvette contents to the incident beam, the inability to simultaneously perform spectral scans, and inherent low sensitivity. Here, we describe a system that meets these challenges in a practical way. METHODS: Light access to a 8.6 ml quartz integrating sphere containing 10 µM all-trans retinol in PBS was provided by three ports at right angles allowing for the following: 1) actinic light delivery from light-emitting diodes (LEDs) firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec (10,000 µsec scan time) via an OLIS RSM 1000 ultraviolet/visual (UV/Vis) rapid-scanning spectrophotometer (RSM), and 3) light exit to the detector photomultiplier. The RSM spectral intermediate slit was partially covered to allow for a "dark" period of 2,000 µsec when no scanning light was admitted to the cuvette. During that interval, the LED was flashed, and the photomultiplier was temporarily blocked by a perforated spinning shutter disk. The absorbance per centimeter, which is increased due to the internal reflectance of the integrating sphere compared to a standard 1 cm rectangular cuvette, was calculated according to Fry et al. (2010) Applied Optics 49:575. Retinoid photodecomposition was confirmed with high-performance liquid chromatography (HPLC). RESULTS: Using the RSM to trigger the LED flash and photomultiplier shutter closure during the "dark" period allowed actinic flashes to be placed between scans. Exposure of the all-trans retinol to 366 nm flashes resulted in marked reduction in absorbance and a blue shift of the λmax. A white LED, despite its higher photon output, did not support all-trans retinol photolysis. Singular value decomposition (SVD) analysis revealed three spectral intermediates with mechanism, I -> II -> III. HPLC analysis of the reactants at the beginning and the conclusion of the light exposure confirmed the retinol photodecomposition. CONCLUSIONS: The highly reflecting cavity acts as a multipass cuvette that markedly increased the light path length and, thus, sensitivity. Triggering the LED during a dark period within the scan time allowed the actinic flashes to be interleafed between scans in a pump-probe paradigm. Furthermore, the entire sample was exposed to scan beam and actinic flashes, which is not possible in traditional spectrophotometers. Finally, the integrating cavity cuvette allowed use of turbid samples. SVD was useful for resolving spectral intermediates. Although the identity of the intermediates was not determined here, the ability to define molecular intermediates during photodecomposition reactions will allow future studies to isolate and identify the degradation products and determine the mechanism of light-induced retinoid degradation and that of retinoid-binding protein-mediated photoprotection.

BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFß to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFß, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFß-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFß1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFß1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFß showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFß, as well as TGFß receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFß or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFß released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

Optical and spectroscopic properties of human whole blood and plasma with and without Y2O3 and Nd³âº:Y2O3 nanoparticles.

The optical properties of human whole blood and blood plasma with and without Y2O3 and Nd³âº:Y2O3 nanoparticles are characterized in the near infrared region at 808 nm using a double integrating sphere technique. Using experimentally measured quantities of diffuse reflectance and diffuse transmittance, a computational analysis was conducted utilizing the Kubelka-Munk, the Inverse Adding Doubling, and Magic Light Kubelka-Munk and Monte Carlo Methods to determine optical properties of the absorption and scattering coefficients. Room temperature absorption and emission spectra were also acquired of Nd³âº:Y2O3 nanoparticles elucidating their utility as biological markers. The emission spectra of Nd³âº:Y2O3 were taken by exciting the nanoparticles before and after entering the whole blood sample. The emission from the 4F(3/2) → 4I(11/2) manifold transition of Nd³âº:Y2O3 nanoparticles readily propagates through the blood sample at excitation of 808 nm and exhibits a shift in relative intensities of the peaks due to differences in scattering. At 808 nm, in both whole blood and plasma samples, a direct relationship was found with absorption coefficient and Y2O3 nanoparticle concentration. Results for the whole blood indicate a small inverse relationship with Y2O3 nanoparticle concentration and scattering coefficient and in contrast a direct relation for the plasma.

Enhancement of nonlinear optical properties of BaTiO3 nanoparticles by the addition of silver seeds.

Barium titanate (BaTiO3) is a technologically important material because of its nonlinear properties, such as its strong second harmonic generation and high third order susceptibility. While many nonlinear effects have been extensively studied on the bulk scale, there are still questions regarding the strength of nonlinear effects in nanoparticles. The nonlinear properties of BaTiO3 nanoparticles and nanorods have been studied using the closed aperture z-scan technique. Silver was then grown photochemically on the surface of the BaTiO3 nanoparticles, and it was found that the third order susceptibility increases dramatically.

Overexpression of TC-PTP in murine epidermis attenuates skin tumor formation.

T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, has been shown to function as a tumor suppressor during skin carcinogenesis. In the current study, we generated a novel epidermal-specific TC-PTP-overexpressing (K5HA.Ptpn2) mouse model to show that TC-PTP contributes to the attenuation of chemically induced skin carcinogenesis through the synergistic regulation of STAT1, STAT3, STAT5, and PI3K/AKT signaling. We found overexpression of TC-PTP increased epidermal sensitivity to DMBA-induced apoptosis and it decreased TPA-mediated hyperproliferation, coinciding with reduced epidermal thickness. Inhibition of STAT1, STAT3, STAT5, or AKT reversed the effects of TC-PTP overexpression on epidermal survival and proliferation. Mice overexpressing TC-PTP in the epidermis developed significantly reduced numbers of tumors during skin carcinogenesis and presented a prolonged latency of tumor initiation. Examination of human papillomas and squamous cell carcinomas (SCCs) revealed that TC-PTP expression was significantly reduced and TC-PTP expression was inversely correlated with the increased grade of SCCs. Our findings demonstrate that TC-PTP is a potential therapeutic target for the prevention of human skin cancer given that it is a major negative regulator of oncogenic signaling.

Evidence for two retinoid cycles in the cone-dominated chicken eye.

In the classic retinoid cycle, 11-cis retinol is synthesized in the retinal pigment epithelium (RPE) by two enzymes: Isomerase I (RPE65) and lecithin:retinol acyltransferase (LRAT). The purpose of this study is to provide experimental evidence for two active isomerases in the cone-dominated chicken eye: an LRAT-dependent Isomerase I in the RPE and an ARAT (acyl CoA:retinol acyltransferase)-dependent isomerase (Isomerase II) in the retina. First, we show that whole chicken retina in vitro, removed from the RPE/choroid and sclera, produces 11-cis retinoids upon light exposure, indicating the existence of RPE-independent isomerase (Isomerase II) activity in the retina. Reverse transcriptase polymerase chain reaction studies show high levels of RPE65 expression in the RPE, low levels in the retina, and none in primary Muller cell cultures, indicating the presence of Isomerase I in the RPE and a minimal amount in the retina. Activities of the RPE and retina isomerases were then measured by enzyme assays with specific enzyme inhibitors. 2,2'-Bipyridine, a known Isomerase I inhibitor, and N-ethylmaleimide (NEM), a known LRAT inhibitor, significantly reduced Isomerase I activity but not Isomerase II activity. Progesterone, a known ARAT inhibitor, completely blocked Isomerase II activity but not Isomerase I activity. Thus, this study reports novel results for distinguishing the biochemical properties of Isomerase I from those of Isomerase II, as well a difference in their locations in the chicken eye. On the basis of these differences, the cone-dominated chicken eye must contain two retinoid cycles: a classic visual cycle for retinoid exchange between the RPE and the retina supported by Isomerase I in the RPE and an additional visual cycle for retinoid processing in the retina supported by Isomerase II.

Optical absorption and scattering of bovine cornea, lens and retina in the visible region.

Optical properties of bovine ocular tissues were determined at laser wavelengths in the visible region. The inverse adding doubling (IAD), Kubelka-Munk (KM), and inverse Monte Carlo (IMC) methods were applied to the measured values of the total diffuse transmission, total diffuse reflection, and collimated transmission to determine the optical absorption and scattering coefficients of the bovine cornea, lens and retina at 457.9 nm, 488 nm, and 514.5 nm laser lines from an argon ion laser. The optical properties obtained from these three methods were compared, and their validity is discussed.

Epidermal-specific deletion of TC-PTP promotes UVB-induced epidermal cell survival through the regulation of Flk-1/JNK signaling.

UVB exposure can contribute to the development of skin cancer by modulating protein tyrosine kinase (PTK) signaling. It has been suggested that UVB radiation increases the ligand-dependent activation of PTKs and induces PTP inactivation. Our recent studies have shown that T-cell protein tyrosine phosphatase (TC-PTP) attenuates skin carcinogenesis induced by chemical regimens, which indicates its critical role in the prevention of skin cancer. In the current work, we report that TC-PTP increases keratinocyte susceptibility to UVB-induced apoptosis via the downregulation of Flk-1/JNK signaling. We showed that loss of TC-PTP led to resistance to UVB-induced apoptosis in vivo epidermis. We established immortalized primary keratinocytes (IPKs) from epidermal-specific TC-PTP-deficient (K14Cre.Ptpn2fl/fl) mice. Immortalized TC-PTP-deficient keratinocytes (TC-PTP/KO IPKs) showed increased cell survival against UVB-induced apoptosis which was concomitant with a UVB-mediated increase in Flk-1 phosphorylation, especially on tyrosine residue 1173. Inhibition of Flk-1 by either its specific inhibitors or siRNA in TC-PTP/KO IPKs reversed this effect and significantly increased cell death after UVB irradiation in comparison with untreated TC-PTP/KO IPKs. Immunoprecipitation analysis using the TC-PTP substrate-trapping mutant TCPTP-D182A indicated that TC-PTP directly interacts with Flk-1 to dephosphorylate it and their interaction was stimulated by UVB. Following UVB-mediated Flk-1 activation, the level of JNK phosphorylation was also significantly increased in TC-PTP/KO IPKs compared to control IPKs. Similar to our results with Flk-1, treatment of TC-PTP/KO IPKs with the JNK inhibitor SP600125 significantly increased apoptosis after UVB irradiation, confirming that the effect of TC-PTP on UVB-mediated apoptosis is regulated by Flk-1/JNK signaling. Western blot analysis showed that both phosphorylated Flk-1 and phosphorylated JNK were significantly increased in the epidermis of TC-PTP-deficient mice compared to control mice following UVB. Our results suggest that TC-PTP plays a protective role against UVB-induced keratinocyte cell damage by promoting apoptosis via negative regulation of Flk-1/JNK survival signaling.

Chemical Modification of Ginsenoside on Cell Viability and Cytokine Secretion.

BACKGROUND: Rb1 is a ginsenoside steroid glycoside found exclusively in the plant Panax ginseng. In an earlier report, we showed that Rb1 increased cell proliferation and reduced VEGF (vascular endothelial growth factor) secretion by human retinal pigment epithelial (ARPE19) cells. OBJECTIVE: In the present study, we hypothesized that chemical modification of Rb1 changes the level of VEGF secretion by ARPE19 cells. METHOD: Three derivatives of Rb1 were chemically synthesized by hydrogenation (Rb1-H2), acetylation (Rb1-Acyl), and epoxidation (Rb1-Epoxy). Structural modifications were confirmed by 1H Nuclear Magnetic Resonance (NMR) spectra and Mass Spectrometry (MS). To test the biological activity, chemically modified compounds were added to cell culture media and incubated for 72 hours at a concentration of 250 nM at 37°C. Conditioned media were collected and cells were harvested/ counted after treatment. Viable cell numbers were determined by the trypan blue dye exclusion method and VEGF levels by Enzyme-Linked Immunosorbent Assays (ELISA). RESULTS: Consistent with the prior report, results of the present study show Rb1 increased cell proliferation and decreased VEGF secretion. Similar to Rb1's effect on cell proliferation, treatment with Rb1-H2, Rb1-Acyl and Rb1-Epoxy resulted in an increase in cell numbers. In contrast to Rb1- induced decrease in VEGF secretion, treatment with Rb1-H2, Rb-Acyl and Rb1-Epoxy resulted in increased VEGF levels. CONCLUSION: Chemical modifications of the ginsenoside Rb1 significantly affect the biological activity of VEGF secretion by ARPE19 cells. Additional SAR (Structure Activity Relationship) experiments will be conducted to study the detailed mechanisms by which how specific modifications of Rb1 functional groups alter biological activities.

Autocrine and Paracrine Secretion of Vascular Endothelial Growth Factor in the Pre-Hypoxic Diabetic Retina.

Vascular endothelial growth factor (VEGF) is well established as the main agent responsible for vascular leakage and angiogenesis in the diabetic retina. While VEGF can have positive effects on hyperglycemia stressed retinal tissues, it also plays a role in events progressing to the oxygen- stressed, i.e. hypoxic, diabetic retina. Some VEGF makes its way to the retina from systemic sources and some is produced locally within the eye. Hyperglycemia, oxidants, inflammation, and advanced glycation end-products are all stimulants to VEGF production, both in the hypoxic and the pre-hypoxic retina. Endothelial cells, pericytes, Müller cells, microglia, astrocytes, retinal pigment epithelium and neurons have all been known to produce VEGF at some point in retinal development or in disease. Excessive VEGF production in the early diabetic retina can lead to retinal exposure or mechanisms which exacerbate further damage. While Müller cells are likely the most significant producer of VEGF in the pre-hypoxic retina, other VEGF producing cells may also play a role due to their proximity to vessels or neurons. Study of the release of VEGF by retinal cells in hyperglycemia conditions, may help identify targets for early treatment and prevent the serious consequences of diabetic retinopathy.

Oxidative Stress Induces Senescence in Cultured RPE Cells.

The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

Molecular dynamics study of human carbonic anhydrase II in complex with Zn(2+) and acetazolamide on the basis of all-atom force field simulations.

Human carbonic anhydrase II (hCAII) represents an ultimate example of the perfectly efficient metalloenzymes, which is capable of catalyzing the hydration of carbon dioxide with a rate approaching the diffusion controlled limit. Extensive experimental studies of this physiologically important metalloprotein have been done to elucidate the fundamentals of its enzymatic actions: what residues anchor the Zn(2+) (or another divalent cation) at the bottom of the binding pocket; how the relevant residues work concertedly with the divalent cation in the reversible conversions between CO2 and HCO3(-); what are the protonation states of the relevant residues and acetazolamide, an inhibitor complexed with hCAII, etc. In this article, we present a detailed computational study on the basis of the all-atom CHARMM force field where Zn(2+) is represented with a simple model of divalent cation using the transferrable parameters available from the current literature. We compute the hydration free energy of Zn(2+), the characteristics of hCAII-Zn(2+) complexation, and the absolute free energy of binding acetazolamide to the hCAII-Zn(2+) complex. In each of these three problems, our computed results agree with the experimental data within the known margin of error without making any case-by-case adjustments to the parameters. The quantitatively accurate insights we gain in this all-atom molecular dynamics study should be helpful in the search and design of more specific inhibitors of this and other carbonic anhydrases.

Effect of Glucose on Retinal Endothelial Cell Viability and VEGF Secretion.

Previous studies have shown that in diabetic patients, there is an increase of retinal capillaries associated with the development of diabetic retinopathy in the eye. The objective of current study is to investigate the effect of glucose on retinal endothelial cell viability and VEGF secretion. 20,000 cells per well were treated without glucose or with 5.5mM (euglycemic), 18.5mM and 30mM (hyperglycemic) glucose for 24 hours. Viable cells were counted using Trypan blue dye exclusion method. ELISA was used to measure VEGF secretion from cells into the cell medium. The number of viable cells incubated with 5.5mM glucose (physiological control) increased by 53.7% after 24 hours. In comparison, cells treated with 18.5mM glucose decreased by 2.8% while cells treated with 30mM glucose decreased by 20% after 24 hours of incubation. Cells without glucose treatment (0mM control) decreased by 33.3%. In contrast to the decrease of viable cell numbers after treatment with high glucose, there is an increase in VEGF secretion (pg/mL) to the cell medium with increase in glucose concentration from 5.5mM to 0, 18.5, and 30mM. The amount of VEGF secreted per cell also increased with increasing glucose concentrations. Our results show that viability of retinal endothelial cells and VEGF release are highly responsive to changes in glucose concentration. Such glucose-induced changes in retinal endothelial cells may negatively impact the integrity of the microvasculature in the diabetic retina leading to angiogenesis and microaneursyms.

Optical scattering, absorption, and polarization of healthy and neovascularized human retinal tissues.

The optical scattering, absorption, and polarization properties of human retinal tissues are investigated for a number of laser wavelengths in the visible range. The indices of refraction of these tissues are determined by applying Brewster's law. The inverse adding doubling method based on the diffusion approximation and radiative transport theory is applied to the measured values of total diffuse transmission, total diffuse reflection, and index of refraction to determine the optical absorption, scattering, and scattering anisotropy coefficients of the intact retinal tissues from healthy and diseased (neovascularized) human eyes. The polarization studies show that the retinal tissues possess significant intrinsic polarization characteristics, that are more pronounced in diseased tissues than in healthy tissues.

Interphotoreceptor retinoid-binding protein protects retinoids from photodegradation.

Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8842 µW cm(-2)); time periods of 0-60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle.

Optical characterization of bovine retinal tissues.

An in-depth characterization of the optical properties of bovine retinal and retinal pigment epithelium-choroidal tissues has been performed. The indices of refraction of these ocular tissues were determined by applying Brewster's law. The inverse adding doubling method based on the diffusion approximation and radiative transport theory is applied to the measured values of the total diffuse transmission, total diffuse reflection, and collimated transmission to calculate the optical absorption, scattering, and scattering anisotropy coefficients of the bovine retinal and retinal pigment epithelium-choroidal tissues. The values of the optical properties obtained from the inverse adding doubling method are compared with those generated by the Monte Carlo simulation technique. Optical polarization measurements are also performed on bovine retinal tissues. Our studies show that both retina and retinal pigment epithelium-choroid possess strong polarization characteristics.

Vitamin A Homeostasis in the Diabetic Rat.

The concentrations of vitamin A (retinol) and retinyl ester in the plasma and liver of normal and diabetic rats were measured by HPLC (high-performance liquid chromatography). Diabetic rats had severe hyperglycemia, induced by a single streptozotocin injection 5 weeks prior to sampling. In the normal rats, plasma retinyl palmitate was very low, and the level was increased 10-fold by diabetes. Detailed time-course studies showed that rats became hyperglycemic within 48 h of streptozotocin injection, yet the plasma retinyl palmitate level was not elevated until some three weeks later. Severe diabetes did not significantly influence plasma retinol: however. free retinol in the liver was elevated within 10 days of initiation of the disease and continued to increase for the duration of the study. These results show that streptozotocin-induced diabetes significantly alters the concentrations of hepatic retinol and plasma retinyl ester. The biochemical mechanism(s) of this altered vitamin A homeostasis in diabetes and its possible relationship to tissue pathogenesis are not known at present.

Interphotoreceptor retinoid-binding protein (IRBP) promotes retinol uptake and release by rat Müller cells (rMC-1) in vitro: implications for the cone visual cycle.

PURPOSE: Interphotoreceptor retinoid-binding protein's (IRBP) role in facilitating the exchange of retinoids between rod and cone photoreceptors, RPE, and Müller cells in the visual cycle remains a mystery. Interphotoreceptor retinoid-binding protein's ability to bind the pericellular matrix of the cone outer segment and Müller cell villi suggests a function in all-trans and 11-cis retinol targeted trafficking in the cone visual cycle. We hypothesize that IRBP facilitates delivery and uptake of all-trans retinol to and release of 11-cis retinol from rat Müller cells (rMC-1). METHODS: Rat Müller cells were incubated with all-trans retinol and BSA or bovine IRBP (bIRBP). Retinoids in the cell homogenates and conditioned media were analyzed by high performance liquid chromatography (HPLC). RESULTS: Cells incubated with 10 µM retinol and BSA had 2100 pmol of all-trans retinol per milligram homogenate protein compared with 3450 pmol when retinol was delivered by bIRBP; these cells also had 450 pmol all-trans retinyl ester per milligram when retinol was delivered by BSA compared with 270 pmol when retinol was delivered by bIRBP. Conditioned media from cells incubated with retinol delivered by BSA did not contain11-cis retinol. However, cells with retinol delivered by bIRBP released 130 pmol/mL of 11-cis retinol into the cell media. Incubation with 5.0 mM deferoxamine (an iron chelator) reduced IRBP-dependent 11-cis retinol retrieval by 60%. CONCLUSIONS: Promoting Müller cell uptake of all-trans retinol and release of 11-cis retinol is a previously unrecognized function of IRBP that may be critical to cone function and integrity.

Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle.

PURPOSE: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE. METHODS: iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. RESULTS: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media. CONCLUSIONS: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.