Ram Kannan

A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.

αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.

AlphaB crystallin regulation of angiogenesis by modulation of VEGF

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.

GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC.

The purpose of the present study was to identify and localize glutathione (GSH) transport in an in vitro tissue culture model of blood-brain barrier (BBB). The localization of Na+-dependent GSH transport in an immortalized cell line of human cerebrovascular endothelial cells (HCEC) and asymmetry of transport in Transwell studies were investigated. Initial studies with cultured HCEC established a significant (45%) Na+-dependency for GSH uptake in cultured HCEC pretreated with acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT). Transendothelial electrical resistance (TEER) and uptake of [35S]GSH from luminal and abluminal fluids of HCEC were measured in Na+-containing and Na+-free (choline chloride) buffers using cells grown on gelatin-coated membrane filters. TEER of HCEC monolayers in regular medium was 40.1 +/- 8.0 ohms cm2. Human astrocyte-conditioned medium (ACM) caused no change in TEER, but increased GGT activity approximately threefold when measured in cell lysates. Luminal and abluminal GSH uptake increased in a time-dependent fashion and were not affected by inhibition of GGT activity with acivicin. Sodium dependency was only observed for luminal uptake (Na+-containing 2.41 +/- 0.15 vs. Na+-free 0.96 +/- 0.03 pmol/30 min/million cells, p < 0.001) but not for abluminal uptake (1.02 +/- 0.13 vs. 1.11 +/- 09, p > 0.05). Apparent efflux via the luminal membrane was lower in the presence of sodium as compared to that without sodium, further suggesting that a Na+-dependent uptake process for GSH is operative at this membrane. GSH uptake and efflux were also demonstrated in neonatal rat and fetal human astrocytes, both exhibiting partial Na+-dependency of uptake. In conclusion, our results show for the first time, that HCEC and astrocytes take up GSH by both Na+-dependent and -independent mechanisms. The Na+-dependent GSH transport process in HCEC appears to be localized to luminal plasma membranes of HCEC.

Novel roles for α-crystallins in retinal function and disease

α-Crystallins are key members of the superfamily of small heat shock proteins that have been studied in detail in the ocular lens. Recently, novel functions for α-crystallins have been identified in the retina and in the retinal pigmented epithelium (RPE). αB-Crystallin has been localized to multiple compartments and organelles including mitochondria, golgi apparatus, endoplasmic reticulum and nucleus. α-Crystallins are regulated by oxidative and endoplasmic reticulum stress, and inhibit apoptosis-induced cell death. α-Crystallins interact with a large number of proteins that include other crystallins, and apoptotic, cytoskeletal, inflammatory, signaling, angiogenic, and growth factor molecules. Studies with RPE from αB-crystallin deficient mice have shown that αB-crystallin supports retinal and choroidal angiogenesis through its interaction with vascular endothelial growth factor. αB-Crystallin has also been shown to have novel functions in the extracellular space. In RPE, αB-crystallin is released from the apical surface in exosomes where it accumulates in the interphotoreceptor matrix and may function to protect neighboring cells. In other systems administration of exogenous recombinant αB-crystallin has been shown to be anti-inflammatory. Another newly described function of αB-crystallin is its ability to inhibit β-amyloid fibril formation. α-Crystallin minichaperone peptides have been identified that elicit anti-apoptotic function in addition to being efficient chaperones. Generation of liposomal particles and other modes of nanoencapsulation of these minipeptides could offer great therapeutic advantage in ocular delivery for a wide variety of retinal degenerative, inflammatory and vascular diseases including age-related macular degeneration and diabetic retinopathy.

GSH transport in immortalized mouse brain endothelial cells: evidence for apical localization of a sodium-dependent GSH transporter.

Abstract: We have previously shown GSH transport across the blood‐brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC‐4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+‐dependent and Na+‐independent GSH transport and have localized the Na+‐dependent transporter using domain‐enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC‐4 cells in which γ‐glutamyltranspeptidase and γ‐glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+‐dependent uptake in MBEC‐4 cells was confirmed with parallel uptake studies with Cos‐7 cells that did not show this activity. Molecular form of uptake was varified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (∼40‐70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos‐1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+‐dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+‐dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P‐glycoprotein 170 by western blot analysis. Localization of Na+‐dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.

Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia.

This study evaluated the role of crystallins in retinal degeneration induced by chemical hypoxia. Wild-type, alphaA-crystallin (-/-), and alphaB-crystallin (-/-) mice received intravitreal injection of 12 nmol (low dose), 33 nmol (intermediate dose) or 60 nmol (high dose) cobalt chloride (CoCl(2)). Hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) stains were performed after 24 h, 96 h, and 1 week post-injection, while immunofluorescent stains for alphaA- and alphaB-crystallin were performed 1 week post-injection. The in vitro effects of CoCl(2) on alphaB-crystallin expression in ARPE-19 cells were determined by real time RT-PCR, Western blot, and confocal microscopy and studies evaluating subcellular distribution of alphaB-crystallin in the mitochondria and cytosol were also performed. Histologic studies revealed progressive retinal degeneration with CoCl(2) injection in wild-type mice. Retinas of CoCl(2) injected mice showed transient increased expression of HIF-1alpha which was maximal 24h after injection. Intermediate-dose CoCl(2) injection was associated with increased retinal immunofluorescence for both alphaA- and alphaB-crystallin; however, after high-dose injection, increased retinal degeneration was associated with decreased levels of crystallin expression. Injection of CoCl(2) at either intermediate or high dose in alphaA-crystallin (-/-) and alphaB-crystallin (-/-) mice resulted in much more severe retinal degeneration compared to wild-type eyes. A decrease in ARPE-19 total and cytosolic alphaB-crystallin expression with increasing CoCl(2) treatment and an increase in mitochondrial alphaB-crystallin were found. We conclude that lack of alpha-crystallins accentuates retinal degeneration in chemically induced hypoxia in vivo.

Deficiency of αB crystallin augments ER stress-induced apoptosis by enhancing mitochondrial dysfunction

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

PURPOSE The chaperone proteins, α-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer α-crystallin-derived mini-chaperone peptides (α-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer αA- or αB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, α-crystallin-derived mini-peptides and recombinant full-length αB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for α-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated αB-crystallin mini-chaperone peptides from H2O2-induced cell death was studied. RESULTS Primary hfRPE cells exposed to oxidative stress and either αA- or αB-crystallin mini-chaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length αB-crystallin was minimal while a time-dependent uptake of αB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing αB-crystallin mini-chaperone were also taken up and protected hfRPE from H2O2-induced cell death at significantly lower concentrations than free αB-crystallin mini-chaperone peptide. CONCLUSIONS αA- and αB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of αB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.

Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.