Etoposide Upregulates Survival Favoring Sphingosine-1-Phosphate in Etoposide-Resistant Retinoblastoma Cells.

Improved knowledge of retinoblastoma chemotherapy resistance is needed to raise treatment efficiency. The objective of this study was to test whether etoposide alters glucosyl-ceramide, ceramide, sphingosine, and sphingosine-1-phosphate (sphingosine-1-P) levels in parental retinoblastoma cells (WERI Rb1) or their etoposide-resistant subclones (WERI EtoR). WERI Rb1 and WERI EtoR were incubated with 400 ng/ml etoposide for 24 h. Levels of glucosyl-ceramides, ceramides, sphingosine, sphingosine-1-P were detected by Q-TOF mass spectrometry. Statistical analysis was done by ANOVA followed by Tukey post-hoc test (p < 0.05). The mRNA expression of sphingolipid pathways enzymes in WERI Rb1, WERI EtoR and four human retinoblastoma tissue samples was analyzed by quantitative real-time PCR. Pathways enzymes mRNA expression confirmed similarities of human sphingolipid metabolism in both cell lines and tissue samples, but different relative expression. Significant up-regulation of sphingosine was seen in both cell lines (p < 0.001). Only sphingosine-1-P up-regulation was significantly increased in WERI EtoR (p < 0.01), but not in WERI Rb1 (p > 0.2). Both cell lines upregulate pro-apoptotic sphingosine after etoposide incubation, but only WERI EtoR produces additional survival favorable sphingosine-1-P. These data may suggest a role of sphingosine-1-P in retinoblastoma chemotherapy resistance, although this seems not to be the only resistance mechanism.

Alterations in basement membrane immunoreactivity of the diabetic retina in three diabetic mouse models.

BACKGROUND: The mouse retina contains three kinds of basement membrane (BM) structures; the inner limiting membrane (ILM), Bruch's membrane (BrM), and the BM surrounding the capillaries. We aimed to investigate possible variations of individual BM components and to detect effects caused by diabetes in three different diabetic mouse models. METHODS: After 4 and 6 months of diabetes (defined by blood glucose > 250 mg/dl), we analyzed by immunohistochemistry the laminin, collagen IV, and nidogen-1 and nidogen-2 protein composition of the BMs obtained from diabetic and non-diabetic Leptin-receptor deficient (db/db) mice and insulin receptor (IR)/insulin receptor substrate-1 (IRS-1) double heterozygous knockout mice. In addition, C57BL/6 J mice were rendered diabetic by intraperitoneal injections of streptozotocin (STZ). RESULTS: All analyzed BM proteins were detected in all of the three BMs with the exception of collagen IV, which was not detectable in the ILM of db/db mice and IR/IRS-1 mice. We present the first analysis of nidogen expression in diabetic BM. The staining patterns did not differ between the type-1 diabetic model (STZ) or the type-2 diabetic models (db/db and IR/IRS-1) and the wild-type controls, with only one exception: both the db/db mice and the IR/IRS-1 mice but not the STZ mice showed a decreased nidogen-1 immunoreactivity in the BrM after 4 months of diabetes, but not after 6 months. CONCLUSIONS: The BMs in the three mouse strains differ with regard to protein immunoreactivity in the inner limiting membrane. Changes in BM composition may affect both the assembly and the function of the retinal BM. However, there are no marked differences in the BM composition between type-1 and type-2 diabetes. These results provide evidence for BM remodelling during diabetic retinopathy.

A cleaning solution for silicone intraocular lenses: "sticky silicone oil".

AIM: The aim of the study was to compare the efficacy of perfluorobutylpentane (F4H5) and perfluorohexyloctane (F6H8) in dissolving silicone oil from the surface of silicone intraocular lenses (IOL). METHODS: Droplets of stained silicone oil were applied to an object slide either lying flat or tilted by 30 degrees . Mixing with H(2)O, F4H5 or F6H8 was documented by a digital camera. Droplets of silicone oil were applied to silicone lenses and washed off by repeated rinsing with F4H5 or F6H8. The silicone lenses of 11 patients with silicone oil remnants on the posterior IOL surface were rinsed intraoperatively with F4H5 during removal surgery. RESULTS: Only F4H5 was able to mix with silicone oil and to remove it form the surface of a glass object slides. Rinsing with 25 mul F4H5 reduced the amount of silicone oil 1000 mPas or 5000 mPas attached on a silicone lens to 15% and 28%, respectively. A hanging droplet of silicone oil 5000 beneath a silicone lens was completely removed from below by F4H5. In all patients sufficient IOL cleaning was possible using F4H5. There was no significant postoperative inflammation in the vitreous or anterior chamber. CONCLUSION: Polydimethylsiloxanes dissolve effectively in F4H5 due to its lipophilic chemical structure. A much smaller volume of F4H5 than F6H8 is able to remove silicone oil from silicone lenses completely. Intraocular use of F4H5 is safe, and initial clinical data underlines its effectiveness as a cleaning agent after contact of silicone lenses with silicone oil.

Lens epithelial cells express CD95 and CD95 ligand treatment induces cell death and DNA fragmentation in vitro.

PURPOSE: Despite advances in intraocular lens design and material, posterior capsule opacification remains one of the major problems in modern cataract surgery. Therefore, the use of antiproliferative agents has been advocated. CD95 ligand (CD95L, Fas, Apo-1) is a death ligand that triggers apoptosis in susceptible target cells. Apoptosis allows for the safe disposal of cells without damaging the surrounding tissue. The goal of this study was to characterize and evaluate CD95L-induced cell death in cultured lens epithelial cells (LEC). METHODS: Expression of CD95 in untreated porcine LEC was investigated by flow cytometry. Cell death after CD95L or CD95 agonistic antibody treatment was assessed by crystal violet assay and DNA fragmentation was measured by comet assay. RESULTS: The presence of CD95 was observed in LEC. CD95L treatment resulted in a time--and concentration-dependent killing of LEC, which was synergistically enhanced by the addition of cyclohexamide. CD95L treatment induced DNA fragmentation. CONCLUSIONS: The present study confirms the use of apoptosis-inducing CD95L in the inhibition of LEC proliferation. Further studies are needed before clinical application of CD95L to inhibit posterior capsule opacification will be feasible.

Specific features of apoptosis in human lens epithelial cells induced by mitomycin C in vitro.

BACKGROUND: Posterior capsule opacification (PCO) is still one of the major complications following modern cataract surgery. Several attempts have been made to find an appropriate therapeutic concept to significantly lower the rate of PCO. Here, we wanted to focus on the antimetabolic strategy, reducing PCO by using mitomycin C, further characterizing the pathway of apoptosis in human lens epithelial cells (hLECs). METHODS: Human lens epithelial cells were obtained from anterior lens capsules during cataract surgery. The expression of Fas, TRAMP, TRAIL-R1-R4, Apo-3L and TRAIL mRNA was investigated by means of RT-PCR using specific primers. For investigations on bcl-2, bax, p53 and the active form of caspase 3, cell cultures of hLECs were pretreated with mitomycin C and processed for immunocytochemistry thereafter. RESULTS: We detected the expression of the receptors Fas, TRAMP, TRAIL-R2 and TRAIL-R3 in hLECs. We further obtained evidence of the upregulation of the intracellular apoptotic signalling cascade, represented by bcl-2 and bax, the transcription factor p53 and the active form of caspase 3, after pretreatment with mitomycin C. CONCLUSION: We demonstrated the presence of the apoptosis-receptor system in hLECs. Furthermore, we demonstrated the possibility of the induction of key proteins of the apoptotic signalling cascade in these cells by the antimetabolic drug mitomycin C. This could have important implications on the strategies regarding both the prevention and the treatment of PCO after cataract surgery.

Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

Multidrug resistance-associated proteins in glaucoma surgery.

BACKGROUND: Multidrug resistance (MDR) describes the phenomenon of cross-resistance between different cytostatic agents which are structurally and functionally dissimilar. Two recently discovered proteins, lung resistance protein (LRP) and the multidrug resistance-related protein (MRP) have been implicated in the development of MDR. Since resistance to chemotherapeutic agents is a common problem in filtration surgery, especially in cases of complicated glaucoma, we decided to investigate the presence of MRP and LRP in surgically removed Tenon specimens from glaucoma patients. METHODS: The presence of MRP and LRP in surgically removed Tenon tissue (n=15) was analyzed by immunohistochemistry. The expression by cultured Tenon fibroblasts was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorocytometry. RESULTS: LRP expression was detected in 8 of 10 Tenon specimens. Positive staining for MRP was obtained in 5 of 10 specimens. Negative controls with non-immune mouse IgG did not display any specific staining. RT-PCR and fluorocytometry revealed constitutive expression of MRP and LRP, at the RNA and protein level respectively, that was unaltered by pretreatment of the cells with mitomycin C or 5-fluorouracil. CONCLUSION: Our results demonstrate, that besides P-glycoprotein, other components of the MDR-system are present in conjunctival fibroblasts. Future developments in the use of chemotherapeutic agents in association with of filtration surgery need to take account of the presence of these counteracting mechanisms.

Elevated vitreous nitric oxide levels in patients with proliferative diabetic retinopathy.

PURPOSE: The aim of this study was to determine nitric oxide levels in the vitreous of patients with proliferative diabetic retinopathy. METHODS: Using the spectrophotometric method based on Griess reaction, we measured levels of nitrite, the stable product of nitric oxide, in the vitreous of 21 eyes of 21 patients who underwent vitrectomy for the treatment of proliferative diabetic retinopathy with tractional retinal detachment, prospectively. Three samples were excluded from the study because of blood contamination. The control group was made up of vitreous samples from 15 eyes of 15 normal cadavers and five eyes of five patients who were undergoing vitrectomy for macular hole surgery. RESULTS: Nitrite levels were 0. 524 +/- 0.27 microM and 0.383 +/- 0.17 microM in the vitreous of patients with proliferative diabetic retinopathy of diabetes type I and type II, respectively. In 15 cadaver eyes and five vitreous samples from patients who underwent macular hole surgery, nitrite levels were below the detection limit (less than 0.08 microM). There was no significant difference between nitrite levels in patients with type I and type II diabetes (P =.56), whereas there was a significant difference between diabetes groups and controls (P <. 00001). CONCLUSION: Vitreous nitric oxide levels are elevated in patients with proliferative diabetic retinopathy with tractional retinal detachment. Nitric oxide may play a role in the pathogenesis of proliferative diabetic retinopathy.

Age-related macular degeneration. The lipofusion component N-retinyl-N-retinylidene ethanolamine detaches proapoptotic proteins from mitochondria and induces apoptosis in mammalian retinal pigment epithelial cells.

10-20% of individuals over the age of 65 suffer from age-related macular degeneration (AMD), the leading cause of severe visual impairment in humans living in developed countries. The pathogenesis of this complex disease is poorly understood, and no efficient therapy or prevention exists to date. A precondition for AMD appears to be the accumulation of the age pigment lipofuscin in lysosomes of retinal pigment epithelial (RPE) cells. In AMD, these cells seem to die by apoptosis with subsequent death of photoreceptor cells, and light may accelerate the disease process. Intracellular factors leading to cell death are not known. Here we show that the lipophilic cation N-retinyl-N-retinylidene ethanolamine (A2E), a lipofuscin component, induces apoptosis in RPE and other cells at concentrations found in human retina. Apoptosis is accompanied by the appearance of the proapoptotic proteins cytochrome c and apoptosis-inducing factor in the cytoplasm and the nucleus. Biochemical examinations show that A2E specifically targets cytochrome oxidase (COX). With both isolated mitochondria and purified COX, A2E inhibits oxygen consumption synergistically with light. Inhibition is reversed by the addition of cytochrome c or cardiolipin, a negatively charged phospholipid that facilitates the binding of cytochrome c to membranes. Succinate dehydrogenase activity is not altered by A2E. We suggest that A2E can act as a proapoptotic molecule via a mitochondria-related mechanism, possibly through site-specific targeting of this cation to COX. Loss of RPE cell viability through inhibition of mitochondrial function might constitute a pivotal step toward the progressive degeneration of the central retina.

Transplantation of iris pigment epithelium into the choroid slows down the degeneration of photoreceptors in the RCS rat.

BACKGROUND: Trophic factors [e.g. basic fibroblast growth factor (bFGF)] released by transplanted retinal pigment epithelial (RPE) cells are able to slow down the hereditary degeneration of the retina in the Royal College of Surgeons rat in sites distant from the site of transplantation where rod outer segment (ROS) phagocytic activity is not reconstituted by the transplants. METHODS: To investigate whether iris pigmented epithelial (IPE) cells are also able to generate this rescue by trophic factors, we transplanted IPE cells from Long-Evans rats into the choroid and subretinal space of 17 young RCS rats. The eyes were enucleated after 6 months and prepared for light microscopy. Six age-matched RCS rats served as controls. Light microscope sections from the whole choroid, healthy choriocapillaris, transplanted cells and the maximum thickness of the choroid, and outer nuclear layer parameters were analyzed by computer-assisted morphometry. RESULTS: In transplanted animals photoreceptor cells were rescued from degeneration although the majority of the transplanted IPE cells were located in the choroid. In the non-transplanted group photoreceptors were absent. CONCLUSIONS: Transplantation of IPE cells slows down degeneration of the photoreceptors in the RCS rat. This photoreceptor-sparing effect by the IPE cells was observed even when the transplants were predominantly located within the choroid. The beneficial effect observed may be related to trophic factors possibly secreted by the transplanted IPE cells.

Ultimate fate of rod outer segments in the retinal pigment epithelium.

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.

Retinal damage by light in the golden hamster: an ultrastructural study in the retinal pigment epithelium and Bruch's membrane.

The mechanism of the toxicity of light on the retina remains unclear despite a large number of investigations. The purpose of this study is to identify and localize the ultrastructural changes and the site of the earliest damage after intense light exposure. Nine adult Syrian golden hamsters (Mesocricetus auratus) have been maintained under constant illumination with a high-pressure mercury lamp (HQJ R 80 W Deluxe, Osram, Berlin, light intensity 1000 lx) for 12 h, followed by an additional 3 h in the dark. Light damage is assessed by light and electron microscopy. Morphological evaluation reveals focal damage to the retinal pigment epithelial (RPE) cells in close proximity to less-affected RPE cells and normal photoreceptors. Collagen fibers in Bruch's membrane lose their parallel orientation. Occasionally, fusion of cell membranes of neighboring rod outer segments (ROS) is also observed. Continuous, 12 h exposure of hamsters to intense light results in initial focal damage to some RPE cells, such that severely damaged RPE cells are found adjacent to intact RPE cells. Only slight damage to the photoreceptors is evident, suggesting that the sequence of the pathological changes resulting from light begins with damage to the RPE cells and associated Bruch's membrane.

Upregulation of the RAS-GTPase activating protein (GAP)-binding protein (G3BP) in proliferating RPE cells.

Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)-based differential expressed mRNA analysis (DEmRNA-PCR). DEmRNA-PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3'-end of the coding sequence of the human RAS-GTPase activating protein (GAP)-binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA-PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation- and proliferation-dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy.

Rescue effects of IPE transplants in RCS rats: short-term results.

PURPOSE: The aim of this study was to investigate the possible rescue effect of subretinal iris pigment epithelial (IPE) cell transplantation in Royal College of Surgeons (RCS) rats by light and electron microscopic histology. METHODS: IPE cells were harvested from 20- to 26-day-old Long-Evans rats and were directly trans planted transsclerally into the subretinal space of 32 16- to 20-day-old RCS rats using a 32-gauge Hamilton syringe. Specimens of transplanted eyes were embedded for electron microscopy after 8 weeks. Specimens from the iris and retinal pigment epithelium (RPE) of Long-Evans rats and RPE from RCS rats without surgical treatment were also embedded. Sham surgery was also performed in 8 eyes. RESULTS: The IPE cells transplanted into the subretinal space were localized between host RPE and retina, had round cell shapes without polar organization, and contained phagosomes resulting from rod outer segment (ROS) uptake. The underlying host RPE cells were heavily pigmented. RPE cells from RCS rats revealed fragmentation of endoplasmic reticulum, which distinguishes them ultrastructurally from pigment epithelial cells of Long-Evans rats. Ultrastructural alterations were observed in the cytoplasm of transplanted cells. Melanin granules in the IPE cells were found in large vacuoles, which also contained phagosomes originating from ROS uptake. In 13 eyes, 1 to 4 rows and 5 to 8 rows of saved photoreceptors were detected facing transplanted IPE cells in 6 (46%) and 4 (31%) eyes, respectively, 2 months after surgery. However, in 10 (53%) and 7 (37%) of 19 eyes, 1 to 4 rows and 5 to 8 rows, respectively, were also found at sites without IPE cells in the plane of section. ROS directed toward transplanted IPE cells were seen in one case, but these rods were shortened and disorganized. At most sites between transplanted cells and inner segments of photoreceptors, outer segments and cellular debris were absent. In eyes without transplanted cells no photoreceptor cells were alive at the age of 2 months. After sham surgery 6 (75%) eyes had 1 to 4 rows and 2 (25%) 5 to 8 rows of photoreceptors. CONCLUSIONS: Transplanted IPE cells can take up and degrade ROS in vivo in RCS rats. Uptake of ROS alters the morphology of pigment granules in transplanted IPE cells. Pigmentation is an uncertain marker for identifying transplanted pigment cells. IPE transplants are not as good as RPE transplants in rescuing photoreceptors. However, there is a significant difference between transplanted eyes and nontreated eyes. The rescue effect of IPE cells was not significantly different from that of sham surgery.

DNA fingerprint analysis reveals differences in mutational patterns in experimentally induced rat peritoneal tumors, depending on the type of environmental mutagen.

We performed tumor DNA fingerprint analysis using the synthetic minisatellite probe S3315x2 based on the 33.15-repeat unit. The aim of the study was to investigate fingerprinting patterns of peritoneal tumors induced experimentally in Wistar rats by two carcinogens with unknown mechanism of action (crocidolite asbestos and nickel powder) and, as a positive control, benzo[a]pyrene. The carcinogens were administered intraperitoneally into rats. The banding patterns obtained with DNA from 71 peritoneal tumors were compared to the corresponding normal tissues. DNA derived from peritoneal tumors induced by the three carcinogens differed with respect to mutation frequencies and mutation patterns. The mutation frequencies in these tumors, revealed by DNA fingerprinting, were 18.2% for benzo[a]pyrene, 14.8% for crocidolite asbestos, and 40.9% for nickel powder. The alterations detected in the banding pattern of benzo[a]pyrene-induced peritoneal tumors were exclusively additional bands. On the contrary, in the DNA from asbestos-induced peritoneal tumors, only deletions of bands were observed on the autoradiographs. In the DNA from nickel-induced peritoneal tumors, both types of mutations occurred. The different mutation frequencies and mutation patterns appear to discriminate between benzo[a]pyrene, crocidolite asbestos, and nickel powder, and may be related to the mechanisms of action of these compounds.

Melanin granules of retinal pigment epithelium are connected with the lysosomal degradation pathway.

Melanosomes are closely related to lysosomes and lipofuscin granules. This paper indicates the potential involvement of lysosomal degradation processes in retinal pigment epithelial (RPE) cells. RPE cells cultured on Bruch's membrane and choroid were fed with indigestible latex beads. The RPE cells from this preparation were treated with chloroquine to investigate whether membrane swelling, typical for lysosomes under this condition, can be induced in melanosomes. To investigate the fate of indigestible material associated with rod outer segments (ROS), gold-labeled ROS were injected transsclerally into the subretinal space of Long Evans rats using a 32 gauge Hamilton syringe. The degradation of labeled ROS was observed after 5 and 12 days by electron microscopy. The following results were observed. Latex particles fuse with the melanin granules of the RPE. Following chloroquine treatment, the membranes of melanin granules fused, and formed large clusters and vacuoles. Gold granules were detected inside both early stage melanosomes and mature melanin granules of the RPE cells 5 and 12 days following subretinal injection of the labeled ROS. Higher numbers of gold granules were predominantly found in immature melanosomes containing still melanofilaments and in small fused mature melanin granules. In conclusion the effect of chloroquine clearly demonstrates that the melanosomes possess active proton pumps which is typical for lysosomes. In RPE cells stressed by overload with rod outer segments or by ingestion of undegradable material (latex beads, gold particles), fusion of these phagosomes with melanosomes of different maturity is more a general rule than an exception. Therefore, melanosomes are connected to lysosomal pathways in RPE cells.

Cellular transport of subretinal material into choroidal and scleral blood vessels: an electron microscopic study.

BACKGROUND: The fate of indigestible material injected into the subretinal space of rats was investigated. METHODS: The non-toxic dye Monastral Blue (MB), which cannot be digested within the lysosomal compartment, was injected transsclerally into the subretinal space of Long Evans and Wistar rats. After 5 and 12 days respectively the eyes were enucleated and examined by light and electron microscopy. Cryo sections were made of eyes 5 days after MB injection for the application of immunohistochemical techniques using markers for epithelial cells (cytokeratin) and macrophages (ED 1). RESULTS: Retina, choroid and sclera were not altered in their morphology in the circumference of the MB-containing bubble generated by subretinal injection. After both 5 and 12 days no injected material was found extracellularly in the subretinal space. Especially high amounts of MB were found, in particular 5 days after injection, in lysosomes and melanosomes of RPE cells as well as in cells between choroidal melanocytes. Cells containing MB were seen in contact with choroidal and scleral blood vessels. These MB-containing cells in the choroid and in the sclera were positive for macrophage antibodies. CONCLUSION: Subretinal injection was confirmed as a suitable method for placing fluids into the subretinal space without affecting the morphology of the retina. Subretinal injected material was shown to be incorporated into lysosomes and melanosomes of RPE cells. The injected material was subsequently transported through Bruch's membrane to be finally removed from the eye via choroidal and scleral veins, the process involving macrophages.

Detection of mRNA for proteins involved in retinol metabolism in iris pigment epithelium.

BACKGROUND: To investigate in iris pigment epithelium (IPE) the expression of mRNA for proteins involved in retinol metabolism we used a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. METHODS: RNA was prepared from freshly isolated bovine IPE and retinal pigment epithelium (RPE) cells and reverse transcribed. The expression of mRNA for cellular retinaldehyde binding protein (CRALBP), p63 (RPE63), the presumed retinal pigment epithelial membrane receptor for retinoids, and 11-cis-dehydrogenase (11cisRDH) was determined by RT-PCR using specific primers. Semi-quantitative expression data were obtained by using a series of fivefold dilution of each cDNA with a fixed number of PCR cycles. RESULTS: Bovine IPE and RPE cells express mRNA for CRALBP, 11cisRDH, and RPE63. The mRNA expression for CRALBP and 11cisRDH is high and equal in both cell types. However, RPE63 mRNA expression in IPE cells is relatively low compared with the expression in RPE cells. CONCLUSIONS: The presence of mRNA for CRALBP, RPE63, and 11cisRDH suggests that IPE cells may be able to metabolize retinol.

The mRNA expression of cytokines and their receptors in cultured iris pigment epithelial cells: a comparison with retinal pigment epithelial cells.

It has been suggested that human iris pigment epithelial (IPE) cells isolated from iridectomized tissue could be used as autologous cells for transplantation into the subretinal space in diseases with dysfunctional retinal pigment epithelium (RPE). RPE cells synthesize a number of cytokines and their receptors which are important for its proper function. Nearly nothing is known about the capacity of IPE to synthesize cytokines or responding to them. To compare the mRNA expression of 36 cytokines or their receptors in cultured adult IPE cells and RPE cells we used semi-quantitative reverse transcription polymerase chain reactions (RT-PCR). Included in our assay were cytokines with known expression in RPE to get a broad basis for comparing IPE cells: basic fibroblast growth factor (bFGF or FGF-2), and one of its receptor (FGFR-1), epidermal growth factor (EGF), and its receptor EGF-R, transforming growth factor beta(TGFbeta), and its type III receptor TGFbeta-R3, the platelet-derived growth factors and receptors (PDGF A, PDGF B, PDGF-Ralpha, PDGF-Rbeta), tumor necrosis factor alpha(TNFalpha), and two receptors TNF-R1 and TNF-R2, insulin (INS) with receptor INS-R, insulin-like growth factors (IGF1, IGF2), and receptors (IGF1-R, IGF2-R), vascular endothelial growth factor (VEGF), and two receptors (VEGF-R1 or FLT-1 and VEGF-R2 or FLK-1), the receptor for VEGF-C: VEGF-R3 or FLK-4, interleukin 6 (IL6), and its receptor (IL6-R), nerve growth factor (NGF), interleukin 1alpha(IL1alpha), and a receptor (IL1-R). In addition, cytokines or their receptors not known to be expressed in RPE were included to widen our picture of cytokine gene expression in the eye: stem cell factor (SCF), its receptor (SCF-R), low-affinity nerve growth factor receptor p75 (p75(NGF-R), ciliary neutrothropic factor (CNTF), and its receptor (CNTF-R), glycoprotein 130 interleukin 6 transducer (gp130 (IL6-SD), leukemia inhibitory factor (LIF), and its receptor (LIF-R). Semi-quantitative expression data were obtained using series of fivefold dilutions of each cDNA and a fixed number of PCR cycles. The expression of RPE 65, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta2-microglobulin (B2MG) was used as a control for cellular origin, RNA quality and PCR conditions. With the exception of insulin and tumor necrosis factor alphaall other cytokines analysed and their receptors were expressed in both IPE and RPE cells, even though the levels varied. No qualitative or quantitative difference were observed in the mRNA expression level of 34 (94%) of the cytokines or receptors between IPE and RPE. In contrast, the mRNA expression level of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 [VEGF-RS (FLK-1)] was lower in IPE than in RPE cells. As an increased expression of VEGF in the RPE in maculae with age-related macular disease could be involved in its pathogenesis, a decreased expression of angiogenic growth factors in IPE cells could possibly be beneficial for the therapy of age-related maculopathy if indeed other tasks of non-functional RPE cells could be performed by IPE cells. The similarity of the mRNA expression pattern in 94% of the cytokines analyzed supports the assumption that IPE cells potentially can perform functions of RPE cells in the appropriate environment.

Capillaries are present in Bruch's membrane at the ora serrata in the human eye.

PURPOSE: Because earlier studies indicate that the choroid close to the ora serrata may have unique anatomic features such as wandering cells, blood vessels in Bruch's membrane, and accumulated pigment in the retinal pigment epithelium (RPE), the morphology of the normal human eye at the ora serrata region was investigated. METHODS: Specimens from the ora serrata region of two normal human eyes (male donors, 48 and 52 years old) were investigated by light and electron microscope. Specimens from all quadrants were studied in one eye. RESULTS: The elastic layer of Bruch's membrane extended as far as 15 microm into the peripheral choroid; capillaries were included between the elastin layer and the RPE. Nasally, from the anterior end to 2 mm posterior of the ora serrata, the RPE cells contained more melanin than did those in the adjacent posterior region. Melanin granules in the RPE cells close to the ora either formed large clusters or appeared unusually small because of fragmentation. A unique, fine lamellar, membranous material with a fingerprint-like structure was found between the basal folds of the RPE. This material is also found within the extracellular matrix of the choroid and in association with red blood cells. CONCLUSIONS: The morphology of Bruch's membrane is varied near the ora serrata because capillaries and wandering cells are present in its outer collagenous layer. Unique, fine lamellar, fingerprint-like structures are extruded from the RPE and are removed from the eye together with red blood cells. Capillaries within the inner collagenous region of Bruch's membrane at the ora serrata may not necessarily represent a pathologic response but may be a normal characteristic of thick regions of Bruch's membrane.