Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspase-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.     

Neuroactive steroids protect retinal pigment epithelium against oxidative stress

This study was undertaken to assess whether neuroactive steroids, 17beta-estradiol and dehydroepiandrosterone-sulfate, enhance survival and protect DNA of human retinal pigment epithelial cells challenged by oxidative stress, and to investigate the role of sigma1 receptors in the effects of neuroactive steroids. Retinal pigment epithelial cells were treated with various concentrations of neuroactive steroids and then exposed to hydrogen peroxide. Pretreatment with steroids resulted in significant increased viability in a dose-related manner. DNA damage induced by oxidative insult was significantly lower with steroid pretreatment. The effects of 17beta-estradiol and dehydroepiandrosterone-sulfate were antagonized by pretreatment with a sigma1 receptor antagonist. The results suggest that neuroactive steroids protect retinal cells from oxidative stress, and that this effect is mediated by sigma1 receptors.     

Pituitary Adenylate Cyclase-Activating Polypeptide is Protective Against Oxidative Stress in Human Retinal Pigment Epithelial Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent neurotrophic and neuroprotective effects. We have previously shown that PACAP protects against several types of retinal injuries in vivo, including retinal ischemia, glutamate-induced excitotoxicity, UV A-induced lesion, and diabetic retinopathy. We have also shown that PACAP activates antiapoptotic pathways and inhibits proapoptotic signaling in retinal lesions in vivo. PACAP receptors have been identified on the retinal pigment epithelial cells and PACAP has been shown to inhibit interleukin secretion from pigment epithelial cells. It is not known, however, whether PACAP is protective in these cells. Human retinal pigment epithelial cells (ARPE-19 cell line) were exposed to in vitro oxidative stress by hydrogen peroxide. Cell survival was decreased in cells exposed to oxidative stress, which could be significantly and dose-dependently attenuated by 10 pM–1 μM PACAP treatment, as shown by MTT viability test. The protective effect of PACAP could be blocked by the receptor antagonist PACAP6-38. In addition, flow cytometry and JC-1 assay revealed that oxidative stress-induced apoptosis in retinal pigment epithelial cells could be decreased by PACAP treatment. In summary, these results show, for the first time, that PACAP is antiapoptotic in the retinal pigment epithelial cells.     

Sigma receptor ligands protect human retinal cells against oxidative stress

This study was undertaken to investigate the role of sigma receptors during the oxidative damage on human retinal pigment epithelial cells, and to assess whether sigma receptor ligands enhance survival and protect DNA of cells challenged by oxidative stress. Pretreatment with PRE-084, a sigma1 receptor agonist, resulted in significant increased viability in a dose-related manner. DNA damage induced by oxidative insult was significantly lower with PRE-084. The effects of PRE-084 were antagonized by pretreatment with sigma1 receptor antagonists (NE-100 and BD1047), but interestingly were synergized by cotreatment with BD1047 that also presented an affinity for the sigma2 receptor. The results suggest that sigma1 receptors play an important role against retinal damage, even though sigma2 receptor involvement cannot be excluded.     

Effects of PACAP on Intracellular Signaling Pathways in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress

The integrity of retinal pigment epithelial cells is critical for photoreceptor survival and vision. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts retinoprotective effects against several types of injuries in vivo, including optic nerve transection, retinal ischemia, excitotoxic injuries, UVA-induced lesion, and diabetic retinopathy. In a recent study, we have proven that PACAP is also protective in oxidative stress-induced injury in human pigment epithelial cells (ARPE-19 cells). The aim of the present study was to investigate the possible mechanisms of this protection. ARPE cells were exposed to a 24-h hydrogen peroxide treatment. Expressions of kinases and apoptotic markers were studied by complex array kits and Western blot. Oxidative stress induced the activation of several apoptotic markers, including Bad, Bax, HIF-1α, several heat shock proteins, TNF-related apoptosis-inducing ligand, and Fas-associated protein with death domain, while PACAP treatment decreased them. The changes in the expression of MAP kinases showed that PACAP activated the protective ERK1/2 and downstream CREB, and decreased the activation of the pro-apoptotic p38MAPK and c-Jun N-terminal kinase, an effect opposite to that observed with only oxidative stress. Furthermore, PACAP increased the activation of the protective Akt pathway. In addition, the effects of oxidative stress on several other signaling molecules were counteracted by PACAP treatment (Chk2, Yes, Lyn, paxillin, p53, PLC, STAT4, RSK). These play a role in cell death, cell cycle, inflammation, adhesion, differentiation and proliferation. In summary, PACAP, acting at several levels, influences the balance between pro- and anti-apoptotic factors in favor of anti-apoptosis, thereby providing protection in oxidative stress-induced injury of human retinal pigment epithelial cells.     

Lycium barbarum polysaccharides protects retinal ganglion cells against oxidative stress injury

The accumulation of excessive reactive oxygen species can exacerbate any injury of retinal tissue because free radicals can trigger lipid peroxidation,protein damage and DNA fragmentation.Increased oxidative stress is associated with the common pathological process of many eye diseases,such as glaucoma,diabetic retinopathy and ischemic optic neuropathy.Many studies have demonstrated that Lycium barbarum polysaccharides(LBP)protects against oxidative injury in numerous cells and tissues.For the model of hypoxia we used cultured retinal ganglion cells and induced hypoxia by incubating with 200μM cobalt chloride(CoCl2)for 24 hours.To investigate the protective effect of LBP and its mechanism of action against oxidative stress injury,the retinal tissue was pretreated with 0.5 mg/mL LBP for 24 hours.The results of flow cytometric analysis showed LBP could effectively reduce the CoCl2-induced retinal ganglion cell apoptosis,inhibited the generation of reactive oxygen species and the reduction of mitochondrial membrane potential.These findings suggested that LBP could protect retinal ganglion cells from CoCl2-induced apoptosis by reducing mitochondrial membrane potential and reactive oxygen species.     

人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧在高糖状态下的表达

背景：高血糖导致的自由基损伤是糖尿病视网膜病变发病机制的中心环节。 目的：观察高糖对体外培养的人视网膜色素上皮细胞的氧化损伤作用以及高糖对人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧表达的影响。 方法：将培养人视网膜色素上皮细胞,分为对照组、高糖组和甘露醇组,分别用含5.5 mmol/L葡萄糖,33 mmol/L葡萄糖及5.5 mmol/L葡萄糖和27.5 mmol/L甘露醇的DMEM培养液培养。采用相差倒置显微镜观察细胞生长形态,采用免疫荧光染色研究诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达的变化,用氯甲基二氯二氢荧光素二乙酯荧光染色检测视网膜色素上皮细胞中活性氧的产生量。 结果与结论：与对照组相比,应用含33 mmol/L葡萄糖的DMEM培养基处理视网膜色素上皮细胞48 h可见细胞胞体变薄,形态表现多样,不规则细胞增多;高糖培养的视网膜色素上皮细胞诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达增加,活性氧产生明显增多。说明高浓度葡萄糖培养可造成人视网膜色素上皮细胞氧化损伤,使细胞形态发生变化,并导致细胞中3-硝基酪氨酸产生增多。     

Methylprednisolone exerts neuroprotective effects by regulating autophagy and apoptosis

Methylprednisolone markedly reduces autophagy and apoptosis after secondary spinal cord injury. Here, we investigated whether pretreatment of cells with methylprednisolone would protect neuron-like cells from subsequent oxidative damage via suppression of autophagy and apoptosis. Cultured N_2 a cells were pretreated with 10 μM methylprednisolone for 30 minutes, then exposed to 100 μM H_2O_2 for 24 hours. Inverted phase contrast microscope images, MTT assay, flow cytometry and western blot results showed that, compared to cells exposed to 100 μM H_2O_2 alone, cells pretreated with methylprednisolone had a significantly lower percentage of apoptotic cells, maintained a healthy morphology, and showed downregulation of autophagic protein light chain 3B and Beclin-1 protein expression. These findings indicate that methylprednisolone exerted neuroprotective effects against oxidative damage by suppressing autophagy and apoptosis.     

Neuroprotective role of erythropoietin by antiapoptosis in the retina

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light‐induced, oxygen‐dependent, free radical–mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light‐induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide–induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light‐induced oxidative injury. © 2009 Wiley‐Liss, Inc.     

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

Pigment Epithelium-Derived Factor (PEDF), purified from human retinal pigment epithelial (RPE) cell culture medium, is a neurotrophic factor which potentiates the differentiation of human Y-79 retinoblastoma cells and increases the survival of cerebellar granule cells. To investigate the effects of PEDF on non-transformed retinal cells, we used primary cultures of neonatal albino rat retinas, where the three principal cell types of the retinal layers (neuronal, glial and epithelial) were all present and focussed our attention on RPE cells, which are of special relevance for retinal pathophysiology. PEDF had a dramatic effect on these cells. They showed a modified phenotype, with larger dimensions, higher cytoplasmic spreading, presence of phagocytic vacuoles, development of wide intercellular contacts, and increase and maturation of pigment granules. These results suggest that PEDF may have a role in regulating RPE cell differentiation.     

Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

Several studies have investigated the protective functions of brain-derived neurotrophic factor(BDNF) in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293 T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19(ARPE-19) cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293 T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293 T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases.     

Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein synthesized and secreted by retinal pigment epithelial (RPE) cells in early embryogenesis and has been shown to be present in the extracellular matrix between the RPE cells and the neural retina. It induces neuronal differentiation and promotes survival of neurons of the central nervous system from degeneration caused by serum withdrawal or glutamate cytotoxicity. Because the role of PEDF in the retina is still unknown, we examined its ability to protect cultured retinal neurons against hydrogen peroxide (H(2)O(2))-induced cell death. Retinas of 0-2-day-old Sprague-Dawley rats were isolated and dissociated, and the neurons were maintained for 2 weeks in a synthetic serum-free medium. Immunocytochemical labeling showed that 50-60% of the cultured cells were rod photoreceptors. Treatment with H(2)O(2) induced significant death of retinal neurons in a dose- and time-dependent manner. Pretreatment with PEDF prior to insult greatly attenuated H(2)O(2)-induced cytotoxicity, and its effect was shown to be dose dependent. Cytotoxicity was determined by 3,(4,5-dimethylthiazol-2-yl)2, 5-diphenyl-tetrazolium bromide and lactate dehydrogenase assays, and apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay. The present study also showed that H(2)O(2)-induced retinal neuron death was by apoptosis that could be inhibited by PEDF. Combination of PEDF with basic fibroblast growth factor, brain-derived neurotrophic factor, or ciliary neurotrophic factor improves the protection. These data strongly suggest that PEDF is a potential neuroprotective agent in the treatment of retinal degeneration.     

Aberrant optic axons in the retinal pigment epithelium during chick and quail visual pathway development

Examination of a large number of retinal pigment epithelia revealed that, in a small proportion, optic axons in chick and quail eyes aberrantly entered the pigment cell layer between embryonic day (E) 7 to E14. The aberrant retinal axons originated from the main stream of retinal fibers in the optic nerve and invaded the pigment layer from various positions of the optic nerve head or fissure by growing along the basal side of the pigment epithelium. The axon bundles grew several millimeters into the epithelial sheet and arborized at the margin of the eye. As shown by electron microscopy the nerve fibers occurred as bundles of three to several hundred axons. They always were located at the basal side of the epithelium, and were enveloped by processes of epithelial cells. Very large bundles of axons, however, displaced the epithelial cells from the basal matrix. These retinal axons contacted the pigment epithelial basal lamina. The basal extracellular matrix from the retinal pigment epithelium was isolated and used as substratum for in vitro cultures of various types of neural explants. The matrix preparations consisted of a sheet of a 50 nm thick basal lamina with a central lamina densa, two laminae rarae, and a 15 micron thick stroma. Axons from avian retina explants, as well as sensory ganglia, grew on the basal lamina side of the pigment cell matrix with the same growth rate and with the same fiber density as on similarly prepared basal laminae from the neural retina. These experiments show that the matrix from the pigment epithelium of the avian eye does not have negative effects on axonal growth and indicate that a basal lamina from a normally non-innervated tissue can provide a favorable matrix for axonal growth.     

Human retinal pigment epithelial cell implants ameliorate motor deficits in two rat models of Parkinson disease

Intrastriatal transplantation of gelatin microcarrier-attached human retinal pigment epithelial cells (hRPE-GM) may represent an alternative source for cell therapy in Parkinson disease (PD). The use of human retinal pigment epithelial (hRPE) cells in PD relies on the capacity of these cells to produce l-dopa as an intermediate product in the eumelanin synthesis pathway. We investigated the behavioral effects of hRPE-GM implants on forelimb use asymmetries and hindlimb motor deficits in unilateral and bilateral 6-hydroxydopamine (6-OHDA) rat models of PD. We report that intrastriatal unilateral implantation of hRPE-GM in rats with 6-OHDA nigrostriatal lesions produce an amelioration of the contralateral forelimb disuse and the contralateral hindlimb deficits. These results further support the possibility that implantation of cultured hRPE cells may be a promising therapeutic option for patients with PD.     

Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.     

Adenovirus-mediated gene transfer of Bcl-xL impedes neurite regeneration in vitro

Mouse retinal explants were transfected with recombinant adenovirus vector carrying the green fluorescent protein (GFP) gene and the rat bcl-xL gene (Adeno-Bcl-xL) to determine its ability to protect retinal ganglion cells against apoptotic cell death and to promote retinal ganglion cell neurite regeneration. Adeno-Bcl-xL-incubated retinas had reduced apoptosis compared to controls. However, neurite regeneration in adeno-treated retinas was less than that of vector-free retina. These results suggest that the usefulness of adenovirus vectors for gene therapy for retinal ganglion cells may be limited.     

Adenovirus-mediated gene transfer of Bcl-xL impedes neurite regeneration in vitro

Mouse retinal explants were transfected with recombinant adenovirus vector carrying the green fluorescent protein (GFP) gene and the rat bcl-x(L) gene (Adeno-Bcl-xL) to determine its ability to protect retinal ganglion cells against apoptotic cell death and to promote retinal ganglion cell neurite regeneration. Adeno-Bcl-xL-incubated retinas had reduced apoptosis compared with controls. However, neurite regeneration in adeno-treated retinas was less than that of vector-free retina. These results suggest that the usefulness of adenovirus vectors for gene therapy for retinal ganglion cells may be limited.     

Human neuroblastoma (SH-SY5Y) cells are highly sensitive to the lysosomotropic aldehyde 3-aminopropanal

3-Aminopropanal (3-AP), a degradation product of polyamines such as spermine, spermidine and putrescine, is a lysosomotropic small aldehyde that causes apoptosis or necrosis of most cells in culture, apparently by inducing moderate or extensive lysosomal rupture, respectively, and secondary mitochondrial changes. Here, using the human neuroblastoma SH-SY5Y cell line, we found simultaneous occurrence of apoptotic and necrotic cell death when cultures were exposed to 3-AP in concentrations that usually are either nontoxic, or only cause apoptosis. At 30 mM, but not at 10 mM, the lysosomotropic base and proton acceptor NH3 completely blocked the toxic effect of 3-AP, proving that 3-AP is lysosomotropic and suggesting that the lysosomal membrane proton pump of neuroblastoma cells is highly effective, creating a lower than normal lysosomal pH and, thus, extensive intralysosomal accumulation of lysosomotropic drugs. A wave of internal oxidative stress, secondary to changes in mitochondrial membrane potential, followed and gave rise to further lysosomal rupture. The preincubation of cells for 24 h with a chain-breaking free radical-scavenger, alpha-tocopherol, before exposure to 3-AP, significantly delayed both the wave of oxidative stress and the secondary lysosomal rupture, while it did not interfere with the early 3-AP-mediated phase of lysosomal break. Obviously, the reported oxidative stress and apoptosis/necrosis are consequences of lysosomal rupture with ensuing release of lysosomal enzymes resulting in direct/indirect effects on mitochondrial permeability, membrane potential, and electron transport. The induced oxidative stress seems to act as an amplifying loop causing further lysosomal break that can be partially prevented by alpha-tocopherol. Perhaps secondary brain damage during a critical post injury period can be prevented by the use of drugs that temporarily raise lysosomal pH, inactivate intralysosomal 3-AP, or stabilize lysosomal membranes against oxidative stress.