Retinal pigment epithelial cells: autocrine and paracrine stimulation of extracellular matrix contraction

BACKGROUND: This study was carried out to examine the biological activity of contraction promoters produced by dedifferentiating retinal pigment epithelial cells (RPE) and to evaluate the importance of autocrine and paracrine effects within a semi-closed environment like the vitreal cavity. METHODS: RPE at different stages of dedifferentiation in culture were examined for their ability (a) to generate tractional forces in vitro, with and without serum stimulation, and (b) to produce and release contraction-stimulating proteins. Autocrine versus paracrine effects of cell-secreted promoters were tested by using RPE or human dermal fibroblasts (HDF) as target cells. The contraction-stimulating activity of the cell-secreted promoters was partially characterized and compared to the activity of defined promoters. RESULTS: Our study confirmed that RPE can synthesize and secrete cell-contraction-promoting factor(s) active in stimulating the development of tractional forces by RPE as well as HDF. The quantity of biological activity secreted per cell decreases with progressive dedifferentiation, yet the responsiveness of the cell to contraction promoters increases. The contraction promoter(s) synthesized by RPE is partially distinct from the promoters in serum, TGF-beta 1 and beta 2, IGF-1, ET-1 and PDGF. The contraction-promoting effects of the RPE product(s) can be completely blocked by staurosporine. CONCLUSION: De-differentiation of RPE is characterized by increasing capacity to generate tractional forces and decreasing synthetic capacity. RPE within a semi-closed system like the vitreal cavity can, theoretically, act both as promoting and active component of traction-related events (tractional retinal detachment).     

Retinal pigment epithelium abnormalities in mice with adenomatous polyposis coli gene disruption

In order to assess the effects of dihydropyridine calcium antagonist on sympathetic nerve activity (SNA) in experimental chronic heart failure (CHF), felodipine was given to rats with CHF induced by coronary artery ligation. Anesthetized CHF (n = 7) and sham-operated (n = 9) rats were injected with a bolus dose of felodipine (20 microg/kg) and then infused with felodipine (30 microg/kg/h) for 3 hours. Control CHF rats (n = 8) were given vehicle in the same way. After felodipine treatment, mean blood pressure (MBP) rapidly decreased to 75-85 mmHg, and there was a reflex tachycardia and reflex activation of renal SNA. The heart rate (HR) had returned to baseline level after 3 hours of continuous felodipine infusion, and the SNA returned to baseline level after 2 hours of infusion. At the end of the experiment, renal SNA was 65.4 +/- 11.5% of the baseline level in CHF rats receiving felodipine (P < 0.05) and 94.1 +/- 22.8% in CHF rats receiving vehicle (P > 0.05), but there was no statistical difference between the two groups. Arterial baroreceptor sensitivity (assessed by phenylephrine infusion), which was impaired in CHF rats (-2.7 +/- 0.2 SNA%/mmHg in all CHF rats together vs. -3.6 +/- 0.4 in sham-operated rats, P < 0.5) did not differ significantly from that in sham-operated rats during felodipine infusion (-3.2 +/- 0.4 in felodipine-treated CHF rats vs. -3.7 +/- 0.6 in sham-operated rats) but deteriorated without felodipine treatment (-2.1 +/- 0.3 in CHF rats receiving vehicle, P < 0.05). The biphasic renal SNA response during felodipine infusion suggests that felodipine does not cause persistent sympathetic activation and relatively improves baroreceptor sensitivity in CHF rats.     

Redox gene therapy protects human IB-3 lung epithelial cells against ionizing radiation-induced apoptosis

Toxicity to nontumor-derived tissue has proven to be a significant obstacle in achieving therapeutic levels of gamma irradiation in the treatment of cancer. The formation of reactive oxygen species (ROS) such as superoxide radicals (O2-) following irradiation is thought to be a major determinant of cellular damage. To this end, we describe the generation of two recombinant adenoviral vectors expressing the radical-scavenging enzymes MnSOD and CuZnSOD to test therapeutic strategies of radioprotection. Using a human lung epithelial cell line (IB-3), we have demonstrated that infections with both Ad.CMVMnSOD or Ad.CMVCuZnSOD significantly increase both the levels of SOD protein and enzymatic activity as compared to control cells. This increase in SOD expression reduced the level of apoptosis at 72 hr post-irradiation by 50% as compared to mock- or Ad.CMVLacZ-infected cells. Such studies provide the foundation for radioprotective gene therapies in the treatment of cancer.     

Retinal pigment epithelium cells cultured on synthetic biodegradable polymers

Glycation of proteins of the vessel wall is thought to play an important role in the pathogenesis of vascular complications in diabetes by affecting structure and function of these proteins. Adhesive proteins in the extracellular matrix (ECM) of endothelial cells (ECs) are essential for attachment of ECs to the subintima. In this study, we investigated the effect of glycation of ECM and purified adhesive proteins on EC adhesion and spreading. ECM was incubated with the reactive sugar glucose-6-phosphate (0-500 mmol/l) for different time periods (0-14 days) at 37 degrees C. Degree of glycation, measured in an enzyme-linked immunosorbent assay using a monoclonal antibody specific for advanced glycation end products, increased in a time- and concentration-dependent manner. Glycation of ECM with 50 mmol/l glucose-6-phosphate resulted in increased coverage by ECs as measured in a cell adhesion assay and was the result of an increase in number of adhered cells, while cell size was unaffected. Glycation of ECM with higher concentrations of glucose-6-phosphate resulted in decreased coverage by ECs caused by both a reduction in number of adhered ECs and impaired spreading. Experiments with purified glycated matrix proteins indicate that the decrease in EC adhesion and spreading on glycated ECM may result from glycation of vitronectin. Impaired EC adhesion and spreading caused by vitronectin glycation may result in impaired endothelial function and contribute to vascular disease.     

Somatic gene therapy in squamous epithelial carcinomas of the head-neck area

Gene therapy is an important new approach to the treatment and prevention of human diseases. Somatic gene therapy involves the introduction of novel genetic material into somatic cells to express therapeutic gene products. Two main strategies in somatic gene therapy of cancer are applied: the genetic correction of the defect, or the elimination of cancer cells by cytotoxic drugs or the immuno system. Gene transfer can be accomplished by physical and chemical methods or nonreplicating viruses. The different transfer systems vary strongly in their efficiency of transfection, plasmid maintenance, and protein expression. The clinical application could be performed in two ways: Firstly, by in vivo application of genome modifying substances injected directly into the tumour; secondly, by ex vivo application of genetically modified tumour cells as a tumour vaccine. The following review will discuss some of the gene therapy strategies that could be effective in managing head and neck cancer.     

Choroidal melanoma-associated retinal and retinal pigment epithelial changes

Many clinical findings associated with choroidal melanoma are the result of secondary changes in the adjacent tissues. There may be alterations in the choriocapillaris, the RPE, the Bruch's membrane, and the sensory retina. Proper identification of these changes is essential for diagnosis and management.     

Glutamate-stimulated proliferation of rat retinal pigment epithelial cells

We investigated the effects of glutamate on cell proliferation and the expression of basic fibroblast growth factor (bFGF) and its receptor (FGF-R1) mRNA in cultured rat retinal pigment epithelial (RPE) cells. The number of primary RPE cells was significantly higher after treatment with 0.2 to 1.0 mM glutamate (maximum at 1.0 mM) for 7 days than in controls. Glutamate-stimulated cell proliferation was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not by 6,7-dinitroquinoxaline-2,3-dione or L(+)-2-amino-3-phosphonopropionic acid. Proliferation was increased to a similar extent by N-methyl-D-aspartate (NMDA), but not by kainate, alpha-amino-3-hydroxy-3-methyl-4-isoxazolepropionic acid or trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. NMDA-receptor-like immunoreactivity was detected in most cells cultured. Treatment of cells with glutamate increased the level of bFGF mRNA and, to a lesser extent, that of FGF-R1 mRNA, which peaked 2 and 4 days, respectively, after glutamate was added. The increase in bFGF mRNA induced by glutamate was inhibited by MK-801. These findings suggest that glutamate might stimulate proliferation of RPE cells through activation of NMDA receptors and expression of bFGF and further suggest that glutamate may be involved in the proliferative changes of RPE cells in retinal wound healing.     

Retinal vascular changes in congenital hypertrophy of the retinal pigment epithelium

The overlying retinal blood vessels were abnormal in five cases of congenital hypertrophy of the retinal pigment epithelium. This illustrated the well-recognized association between outer retinal degeneration and obliteration of the overlying retinal vasculature. The proposed pathophysiological mechanisms, however, seem inadequate to explain completely the morphological changes of the retinal blood vessels in the presence of atrophy of the outer retina.     

Retinoic acid metabolism in cultured retinal pigment epithelial cells

PURPOSE: To examine the stability of retinoic acid administered to cultured bovine retinal pigment epithelial (RPE) cells and to determine if RPE cells metabolize retinoic acid by a cytochrome P-450 mechanism. METHODS: Retinoic acid metabolism was examined in cultured RPE cells and subcellular fractions quantitatively by a thin-layer chromatography procedure and qualitatively by normal and reverse phase high-performance liquid chromatography. RESULTS: Cultured bovine RPE cells were found to have an activity that converts retinoic acid into more polar metabolites rapidly released from the cell. The highest specific activity for this process is found in the post-mitochondrial pellet (100,000g), is induced by retinoic acid, and is inhibited by ketoconazole. The major product of the RPE cell-mediated metabolism of retinoic acid is 4-oxo-retinoic acid, a known P-450 monooxygenase product of retinoic acid. The retinoic acid metabolizing activity is greatest in primary RPE cultures and decreases with aging in culture. CONCLUSIONS: These data suggest that a cytochrome P-450 monooxygenase is involved in the metabolism of retinoic acid in RPE cells, and this is similar to the findings of other investigators using other cells and tissues. The authors' findings suggest that the RPE may be important in the deactivation of this biologically potent retinoid in the retina.     

Harvest and storage of adult human retinal pigment epithelial sheets

PURPOSE: To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RPE) cells. METHODS: Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 50% gelatin containing 300 mM sucrose and stored at 4 degrees C. The viability of the cells, as well as their ability to proliferate in vitro, was studied for 96 hours after harvesting. Light microscopy (LM), transmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclude contamination. RESULTS: LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connections. The initial viability of intact RPE sheets was 86%, with a progressive decline in viability with increased storage time. Cells harvested within 24 hours after death maintained greater viability than those harvested after 24 hours (p < 0.05). Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vitro. CONCLUSION: Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplantation if harvested within 24 hours of death and maintained 82% viability for as long as 48 hours if stored at 4 degrees C.     

PEDF (pigment epithelium-derived factor) promotes increase and maturation of pigment granules in pigment epithelial cells in neonatal albino rat retinal cultures

AIMS: To clarify the association of p53 and CD34 expression with development of malignant solitary fibrous tumour we have studied 10 cases of solitary fibrous tumour arising in the pleura, retroperitoneum and pelvic cavity with clinicopathological features of malignancy. METHODS AND RESULTS: Tumours were localized solid masses with or without necrosis in eight and they nearly totally occupied the pleural cavity in two. Basic histology of the tumours was the proliferation of spindle cells arranged in 'patternless' pattern or in interlacing bundles with nuclear atypia and mitotic activities of various degree. In two, high-grade foci were present within low or intermediate-grade tumours. Recurrent tumours also showed more atypical features than primary tumours in two. Immunohistochemical studies showed CD34 positivity in seven, but three of them showed marked diminution or complete loss of CD34 expression in high-grade foci or a recurrent tumour. Three high-grade cases showed totally negative staining for CD34. p53 was strongly expressed in cases with fatal outcome, clinical recurrence, nuclear atypia, high mitotic activity or local invasion, whereas almost negative in benign tumours. CONCLUSIONS: Malignant solitary fibrous tumours may occur de novo or by transformation within benign or low-grade tumours and may be associated with p53 mutation. Although CD34 is a useful marker in the diagnosis of solitary fibrous tumour, one should bear in mind that its expression can be lost in high-grade tumours.     

Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films

Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm. The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line. Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls. The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2). After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS. The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology. Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions. These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.     

Stimulation of retinal pigment epithelial cell growth by neuropeptides in vitro

PURPOSE: The proliferation of many cell types are regulated by cytokines and neuropeptides by autocrine and paracrine mechanisms. Retinal pigment epithelial (RPE) cells are also regulated by cytokines. But RPE cells are very close to the neural retina which has some neuropeptides. The present study was to investigate the effects of neuropeptides on the growth of RPE cells. METHODS: RPE cells were obtained from the eyes of 11 day old chick embryos and cultured in Dulbecco's modified Eagle's culture medium containing 10% fetal calf serum. The growth of RPE cells was evaluated by [3H]-thymidine uptake. RESULTS: Substance P, beta-endorphin and calcitonin gene-related peptide markedly stimulated the growth of RPE cells. The effects of methionine-enkephalin, somatostatin and vasoactive intestinal peptide were intermediate. The strongest effects of substance P, beta-endorphin and calcitonin gene-related peptide were observed at 10(-6) to 10(-7) M. The stimulation of RPE cells with beta-endorphin was inhibited by naloxone, suggesting that the stimulation with beta-endorphin is mediated by an opioid receptor. beta-endorphin and substance P induced RPE cell growth stimulating activity. Leucine-enkephalin and neuropeptide Y did not affect the growth of RPE cells. CONCLUSIONS: These results suggest that neuropeptides play an important role in the regulation of RPE cell growth.     

Giant pigment epithelial tear and exudative retinal detachment complicating choroidal effusions

PURPOSE: To report a case of a giant pigment epithelial tear and transient exudative retinal detachment occurring in a patient with hypotonic choroidal effusions after trabeculectomy. METHODS: Case report and brief literature review. RESULT: The retinal and choroidal detachments settled, disclosing an extremely large crescentic pigment epithelial defect in the temporal midperiphery. CONCLUSIONS: An exudative retinal detachment secondary to a peripheral pigment epithelial rip may complicate choroidal effusions. Recognition of this association may prevent unnecessary investigation or surgery.     

Inwardly rectifying K+ currents in isolated human retinal pigment epithelial cells

PURPOSE: K+ channels in the retinal pigment epithelium (RPE) play a number of important roles, including the establishment of membrane potential, the transport of K+ between the subretinal space and choroid, and the generation of the c-wave of the electroretinogram. Previous studies on amphibian RPE demonstrated that these functions are likely served by an inwardly rectifying K+ channel. The aim of this study was to characterize inwardly rectifying K+ channels in cultured and freshly isolated adult human RPE (hRPE) cells. METHODS: Single cells were dispersed enzymatically from primary cultures of adult hRPE or from fresh adult hRPE-choroid. Ionic currents were recorded using either the perforated-patch or whole-cell configuration of the patch-clamp technique. RESULTS: In 5 mM external K+, roughly 20% of cultured hRPE cells exhibited a strong inwardly rectifying K+ conductance that passed inward but little outward current. This conductance increased when [K+]o was increased and exhibited a voltage-dependent block by external Na+ at negative potentials. In contrast, all freshly isolated hRPE cells exhibited a mild inwardly rectifying K+ conductance that mediated substantial outward current at physiological voltages. This conductance decreased when [K+]o was increased and showed no voltage-dependent block by external Na+. CONCLUSION: The authors conclude that fresh hRPE cells express a mild inwardly rectifying K+ conductance. The operation of this conductance at physiological voltages makes it a likely candidate for the resting K+ conductances of the apical and basolateral membranes. Cultured hRPE cells express a functionally different channel type that may reflect a change in phenotype.