Radicicol and geldanamycin prevent neurotoxic effects of anti-cancer drugs on cultured embryonic sensory neurons

Cultured dorsal root ganglion (DRG) neurons from chick embryos were extremely susceptible to the antineoplastic drugs, cisplatin, vincristine and taxol even in the presence of saturating levels of the neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). We previously reported that a low concentration of radicicol enhanced the survival and neurite outgrowth of the embryonic sensory and sympathetic neurons, although the effect was decreased at higher doses. The neurotoxic effects of these anti-cancer drugs were completely prevented by the addition of radicicol (20 nM) to the cultures. Recent studies showed that the major intracellular target of radicicol and geldanamycin is the heat shock protein 90 (HSP90) chaperone, interfering with its function. In this study, geldanamycin at low doses (about 2 nM) also appeared to be neurotrophic on DRG neurons in the presence or absence of neurotrophins, but higher doses of geldanamycin (> 5 nM) had severe cytotoxic effects on neurons. Higher doses of radicicol (500 nM), however, still promoted neurites and prevented apoptosis of the isolated DRG neurons in the absence of neurotrophins. Geldanamycin at low doses was also found to be neuroprotective against anti-cancer drugs as shown with radicicol. Treatment of neurons with optimal doses of geldanamycin and radicicol together was cytotoxic instead of neurotrophic. These two antibiotics may share a common target to provide a trophic effect to the cultured neurons. However, different cellular effects of the two antibiotics are not easily explained. It is presumed that the novel activity might be mediated via suppression of HSP90 function, although the possibility that limited doses of these antibiotics interact with specific target molecule(s) other than HSP90 and suppress apoptosis cannot be ruled out. Present results indicate that radicicol has therapeutic potential for neurodegenerative diseases, especially for anti-cancer drug-induced sensory neuropathy.     

Inhibitors of the HSP90 molecular chaperone: attacking the master regulator in cancer

The heat shock protein 90 (HSP90) chaperones represent some 1-2% of all cellular protein and are key players in protein quality control in cells. They are over expressed in many human cancers and the fact that many oncogenic proteins are clients has prompted much recent research on HSP90 inhibitors as new cancer therapeutics. A brief introduction is followed by a detailed review of the various classes of inhibitors, both natural product-based and synthetic, that have emerged over the last decade. The natural products geldanamycin, radicicol and novobiocin have provided the start points for new drugs in this area and their medicinal chemistry is reviewed, including the exciting recent results emerging from clinical trials using geldanamycin analogues. The detailed understanding of the binding mode of these compounds to HSP90 has been significantly enhanced by X-ray crystallography of HSP90 constructs co-crystallised with various ligands. Efforts to replace the natural product inhibitors with more drug-like synthetic compounds have mushroomed over the last 4 years. The purines and the 3,4-diarylpyrazoles have proven to be the most successful and their medicinal chemistry is reviewed with particular emphasis on structure-based design. Protein/ligand co-crystal structures have shown that conserved water molecules in the active site are a vital part of the hydrogen-bonding network established on binding both natural product and synthetic inhibitors. Medicinal chemists have used this information to develop high affinity lead compounds. Recent research provides the platform for exciting developments in the area of HSP90 inhibition over the next few years.     

The role of oxidative stress in hormesis induced by sodium arsenite in human embryo lung fibroblast (HELF) cellular proliferation model

Hormetic dose-response relationships induced by environmental agents are often characterized by a low-dose stimulation and a high-dose inhibition. The mechanisms underlying hormesis induced by environmental agents still remain an enigma; however, hormetic consequences may have significant implications for health risk assessments. To investigate the role of oxidative stress in hormetic phenomena associated with cell proliferation induced by sodium arsenite, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and heat-shock proteins (HSP) and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in human embryo lung fibroblast (HELF) cells after treatment with sodium arsenite at various concentrations for differing times. Results showed that sodium arsenite induced significant cell proliferation at low concentrations (0.5 microM for 12, 24, and 48 h), but inhibited cell growth at high amounts (5 and 10 microM for 24 and 48 h), reflected as a beta concentration-response curve. Data indicated that the relationship between ROS levels and sodium arsenite exposure concentration displayed a positive correlation. It was found out that sodium arsenite at high concentrations induced LPO damage. The activities of SOD were enhanced at low metal concentrations but inhibited with high amounts in a concentration-dependent manner. Similarly, heat-shock protein 27 (HSP27) levels were increased by sodium arsenite of low concentrations with early exposure time (3, 6, and 12 h), but decreased with high metal concentrations with greater exposure time (24 and 48 h).Sodium arsenite decreased HSP70 expression at lower concentrations, but increased HSP70 expression at higher concentration. The results indicated that this cellular hormetic model of cell proliferation induced by sodium arsenite occurred in HELF cells, which may explain contradictory effects seen with this metal. Sodium arsenite at low concentrations induced enhanced ROS generation without cytotoxicity and a cellular protective effect. In contrast, sodium arsenite at high concentrations produced marked ROS formation, marked oxidative stress, and cellular damage, as evidenced by LPO.     

The Protective Effect of Radicicol Against Renal Ischemia–Reperfusion Injury in Mice

Overexpression of heat shock protein 70 kDa (HSP70) is known to confer cellular protection against ischemia–reperfusion (I/R) injury. Radicicol, a HSP90 inhibitor, has been reported to induce the expression of HSP70 protein. Here we studied whether radicicol attenuated renal I/R injury in vivo. Treatment of mice with radicicol ameliorated renal I/R injury and increased renal HSP70 mRNA and protein. Administration of radicicol with quercetin, an inhibitor of HSP70 induction, eliminated the renoprotective effect of radicicol. Our results suggest that the up-regulation of renal HSP70 protein by radicicol leads to a novel drug therapy against renal I/R injury.     

EPO治疗干预对RPE细胞氧化损伤的保护作用

目的研究EPO治疗干预过氧化氢诱导人RPE细胞氧化损伤的作用机制。方法 ARPE-19细胞株随机分为正常对照组、过氧化氢诱导组(模型组)和EPO治疗干预组。MTT法检测细胞活力变化,免疫组织化学法检测各组细胞Caspase-9蛋白的表达变化。结果 MTT法检测结果显示,过氧化氢可以显著降低细胞的活性,与对照组相比较,t=11.8692,P<0.01。EPO干预组与模型组比较,10IU/mLEPO干预组、20IU/mLEPO干预组、40IU/mLEPO干预组细胞活力逐渐升高,t值分别为5.6569、5.7056、9.4299(P<0.01)。模型组Caspases-9呈强阳性表达,与正常组比较,表达升高,t=147.5805,P<0.01。经不同浓度的EPO干预,Caspase-9的表达不同程度的减弱,与模型组比较,10IU/mLEPO干预组、20IU/mLEPO干预组、40IU/mLEPO干预组表达逐渐下降,t值分别为18.7409、61.8718、54.9153,P<0.01。结论过氧化氢诱导RPE细胞氧化损伤细胞活力下降,EPO可以保护过氧化氢诱导的RPE细胞氧化损伤,其机制可能与抑制凋亡,减弱凋亡诱导因子Caspase-9的表达有关。     

Arsenite-induced apoptosis is prevented by antioxidants in zebrafish liver cell line.

This study evaluated oxidative stress-induced apoptosis as a possible mechanism of arsenite toxicity in zebrafish liver cell line (ZFL cells). The heat shock protein 70 (HSP70), a chaperone protein, appears to provide protection against oxidative stress and apoptosis. Using the MTT assay, we demonstrated that survival of ZFL cells treated with arsenite for 24h decreased in a dose-dependent manner. The possible mechanisms that promote the cytotoxicity of arsenite were addressed. Cell viability assays revealed that arsenite caused a dose-dependent increase in cell death, and pretreatment of the ZFL cells with antioxidants blunted these effects. Antioxidants such as N-acetyl-cysteine (NAC, 5 mM) and dithiothreitol (DTT, 80 microM) significantly prevented ZFL cells from arsenite-induced death. Nuclear staining was performed using 1 microg/ml Hoechst, and cells were analyzed with a fluorescent microscope. Arsenite (30 microM) induced massive apoptosis that was identified by morphology and condensation and fragmentation of the nuclei of the ZFL cells. Pretreatment with NAC or DTT before arsenite insult effectively protected the cells against oxidative stress-induced apoptosis from the arsenite. Using a transfected human hsp 70 promoter-enhanced green fluorescent protein (EGFP) reporter, pHhsp70-EGFP, the induction of HSP70 against oxidative stress-induced apoptosis by arsenite was observed. The induction of HSP70 by arsenite increased in a dose-dependent manner, and pretreatment of transfected ZFL cells with NAC or DTT before arsenite insult reduced EGFP expression. Taken together, our results provide evidence that stimulation of the heat shock response is a sensitive biomarker of arsenic exposure and that arsenite causes oxidative stress-induced apoptosis in ZFL cells.     

线粒体DNA损伤与视网膜色素上皮细胞关系的研究进展

线粒体 DNA （mtDNA） 是线粒体内具有遗传效应的双股闭环 DNA 分子, 对细胞及其功能具有重要作用。视网膜色素上皮 （RPE） 细胞活动亦由大量线粒体参与。因 RPE 细胞代谢活跃, 当发生氧化应激时可引起线粒体及 mtDNA 损伤; 当线粒体及 mtDNA 损伤无法及时修复而使损伤积累, 可引起 RPE 及线粒体功能障碍, 并诱发启动细胞凋亡, 进而引发某些眼病, 如年龄相关性黄斑变性等。现就 mtDNA 与 RPE 细胞的功能关系、 mtDNA 损伤修复及检测方法作一综述。     

Paeoniflorin attenuates atRAL-induced oxidative stress,mitochondrial dysfunction and endoplasmic reticulum stress in retinal pigment epithelial cells via triggering Ca<Superscript>2+</Superscript>/CaMKII-dependent activation of AMPK

Abnormal accumulation of the free-form all-trans-retinal (atRAL), a major intermediate of human visual cycle, is considered to be a key cause of retinal pigment epithelial (RPE) dysfunction in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration (AMD). Paeoniflorin (PF), a monoterpene glucoside isolated from Paeonia lactiflora Pall., has been used in clinical treatment of retinal degenerative diseases in China for several years; however, the underlying mechanism remains unclear. The aim of this study is to investigate the protective effect of PF against atRAL toxicity in human ARPE-19 cells and its molecular mechanism. The results of our study showed that the pre-treatment of PF dose-dependently attenuated atRAL-induced cell injury by the reduction of Nox1/ROS-associated oxidative stress, mitochondrial dysfunction and GRP78-PERK-eIF2α-ATF4-CHOP-regulated endoplasmic reticulum (ER) stress in ARPE-19 cells. Additionally, our data showed that PF mainly exerted its activity via triggering calcium-calmodulin dependent protein kinase II (CaMKII)-mediated activation of AMP-activated protein kinase (AMPK). AMPK inhibition significantly reversed the protective effect of PF against atRAL toxicity in ARPE-19 cells. Overall, our findings provided the novel mechanism of PF protecting human RPE cells, which may prevent the progression of retinal degenerative diseases.     

Estrogenic activity of procymidone in primary cultured rainbow trout hepatocytes (Oncorhynchus mykiss).

It has been shown that procymidone, a dicarboximide fungicide, alters sexual differentiation in vivo and in vitro. The aim of this study was to evaluate the estrogenic activity of this fungicide using the synthesis of vitellogenin (Vtg) in rainbow trout hepatocyte as a biological marker. The cells were treated for 24 h with procymidone 150 microM, using 17beta-estradiol 20 microM as a positive control. The doses were chosen on the basis of cell viability (Neutral Red and MTT tests) and solubility. The results show that procymidone leads to a qualitative and quantitative increase in Vtg synthesis. In Western immonoblots, the 170 and 30 kDa bands, which respectively correspond to the monomeric form of Vtg and posvitine, were brighter in cells treated with procymidone and 17beta-estradiol than those corresponding to the negative controls (cells treated for 24 h with DMSO 0.1% alone); ELISA showed that the cells treated with the fungicide and 17beta-estradiol had a 48 and 76%, respectively, higher Vtg concentration than the negative controls (P<0.01). Western blotting also revealed the induction of HSP27 (27 KDa), which further confirms the estrogenic acitivity of procymidone as it is known that the 3' region of HSP27/28 containing the gene mRNAs is induced by estrogen treatment. Procymidone increased also the production of both HSP70 protein (70 KDa) and free oxygen radicals. This last finding is in agreement with the toxic mechanism of dicarboximide fungicides. It can therefore be presumed that the estrogenic activity of procymidone in primary cultured trout hepatocytes is related to oxidative damage which, as many other studies have shown, can increase the levels of estrogens such as 17beta-estradiol, and thus increase Vtg synthesis     

A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro

Diabetic retinopathy is a common complication of diabetes mellitus (DM). The oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose closely approximates the molecular basis for the loss of vision associated with this disease. We investigate a novel algae-derived polysaccharide compound for its role in protecting ARPE-19 cells from high glucose-induced oxidative damage. ARPE-19 cells were cultured for 4?d with normal concentration of D-glucose, and exposed to either normal or high concentrations of D-glucose in the presence or absence of the polysaccharide compound at variety of concentrations for another 48?h. Taurine was used as a positive control. Activity of super oxide dismutase (SOD) and concentration of glutathione (GSH) were measured as well as cytotoxicity of high glucose and the polysaccharide compound. To analyse cellular damage by high glucose, activation of Annexin V and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) were examined. Our results showed that a significant cellular damage on ARPE-19 cells after 48?h treatment with high glucose, accompanied by a decrease in SOD activity and GSH concentration; high glucose also caused ARPE-19 cell apoptosis and activation of p38MAPK and ERK. As the non-toxic polysaccharide compound protected ARPE-19 cells from high glucose-induced cellular damage, the compound recovered SOD activity and concentration of GSH in the cells. The compound also abrogated the cell apoptosis and activation of p38MAPK and ERK. Therefore, the polysaccharide compound derived from algae extracts could be unique candidate for a new class of anti-DM and anti-oxidative damage.     

Induction of vascular endothelial growth factor by 4-hydroxynonenal and its prevention by glutathione precursors in retinal pigment epithelial cells

Although 4-hydroxynonenal, a highly reactive lipid peroxidation product, is implicated in several age-related disorders such as Alzheimer's and Parkinson's diseases, its role in age-related macular degeneration is not known. The purpose of this study was to determine whether 4-hydroxynonenal increases vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial cells (ARPE-19), a source of VEGF in choroidal neovascularization observed in age-related macular degeneration. In addition, it was the purpose of this study to assess whether glutathione (GSH) and GSH precursors can inhibit the effects of 4-hydroxynonenal. At 1 micro M, 4-hydroxynonenal did not alter cell viability, but elevated VEGF secretion and mRNA expression by 35% (p<0.05) and 1.9-fold (p<0.05), respectively. However, at concentrations 5 microM and above, 4-hydroxynonenal reduced VEGF secretion as well as cell viability. At 1 and 10 microM, 4-hydroxynonenal did not induce apoptosis in ARPE-19 cells. 4-Hydroxynonenal (1 microM) reduced intracellular GSH by 25% (p<0.05) and increased oxidative stress by 50% (p<0.05). GSH precursor pretreatment for 1 h, which increased intracellular GSH levels by 50% (p<0.05), as well as GSH co-treatment, inhibited the VEGF-inductive and cytotoxic effects of 4-hydroxynonenal. Thus, 4-hydroxynonenal (1 microM) induces VEGF expression and secretion in ARPE-19 cells. This effect is likely due to GSH depletion and an associated increase in intracellular oxidative stress, resulting in increased VEGF mRNA levels. 4-Hydroxynonenal-mediated VEGF secretion as well as cytotoxicity can be reversed with GSH precursor pretreatment or GSH co-treatment.     

Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells.

The cytotoxicity of mercuric chloride and the effects of mercuric chloride on glutamate and calcium uptake and the factors regulating glutamate uptake were studied in retinal pigment epithelium (RPE) cell cultures. RPE cells isolated from pig eyes and human RPE cell line (D407) cells were cultured to confluency and further subcultured according to the test protocol in question. The cytotoxicity caused by 15 min of exposure to mercuric chloride (0.01--1000 microM) was evaluated by WST-1 assay based on the activity of mitochondrial dehydrogenases. [(3)H]Glutamate uptake was measured after the cells were exposed to 0.1--100 microM mercuric chloride and the selected regulators of protein kinase C (PKC) pathway: PKC activator SC10, PKC inhibitor chelerythrine chloride, phospholipase A(2)/C inhibitor manoalide, tyrosine kinase inhibitor lavendustin A, competitive NMDA receptor antagonist AP7 and IP(3) receptor antagonist heparin. Intracellular calcium was monitored with Fluo-3 probe starting immediately after the exposure to 1--1000 microM mercuric chloride. Mercuric chloride showed concentration-dependent effects on cell viability, on glutamate uptake and on intracellular calcium concentration. The results give some support to the concept that glutamate uptake is affected by PKC. The PKC inhibitor chelerythrine chloride decreased glutamate uptake by 25%, but the PKC activator SC10 could partly prevent the inhibitory effect of mercuric chloride. Lavendustin A, manoalide and heparin had smaller, but statistically significant, effects. All these substances act on mediators which can regulate the activity of PKC. However, PKC is not likely to be the only regulator of glutamate uptake. The rise observed in [Ca(2+)](i) may initiate various cellular events during mercury intoxication.     

Induction of stress proteins in rat cardiac myocytes by antimony.

The effects of nonlethal concentrations of potassium antimonyl tartrate (PAT) were examined in cultured neonatal rat cardiac myocytes. PAT (5, 10 microM) significantly increased cellular reduced glutathione (GSH) and heme oxygenase activity after 18 h. GSH levels and heme oxygenase activity were increased 2.5- and 5.4-fold, respectively, by 10 microM PAT after 18 h. In addition, total cytochrome P450 levels were decreased by PAT after an 18-h exposure. PAT exposures were associated with the induction of specific stress proteins. Nonlethal concentrations of PAT produced a dose-dependent increase in HO-1, HSP70, and HSP25/27 protein levels but did not increase HSP60 levels. Pretreatment of cardiac myocytes with low concentrations of PAT (0.5-10 microM) protected against a subsequent lethal concentration of PAT (200 microM). This protection was blocked if cells were treated with the protein synthesis inhibitor cycloheximide. Results demonstrate that low concentrations of PAT increase GSH levels and stress protein synthesis, which may be responsible for the protection that low-level PAT exposure offers against the subsequent toxicity of higher concentrations of PAT.     

Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells

Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H2O2, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H2O2-, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H2O2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST), (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD.     

Oxidative stress,polyunsaturated fatty acidsderived oxidation products and bisretinoids as potential inducers of CNS diseases: focus on age-related macular degeneration

Many pathologies of the central nervous system (CNS) originate from excess of reactive free radicals, notably reactive oxygen species (ROS), and oxidative stress. A phenomenon which usually runs in parallel with oxidative stress is unsaturated lipid peroxidation, which, via a chain reaction, contributes to the progression of disbalanced redox homeostasis. Among long-chain (LC) polyunsaturated fatty acids (PUFAs) abundantly occurring in the CNS, docosahexaenoic acid (DHA), a member of ω-3 LC-PUFAs, deserves special attention, as it is avidly retained and uniquely concentrated in the nervous system, particularly in retinal photoreceptors and synaptic membranes; owing to the presence of the six double bonds between carbon atoms in its polyene chain (C=C), DHA is exquisitely sensitive to oxidative damage. In addition to oxidative stress and LC-PUFAs peroxidation, other stress-related mechanisms may also contribute to the development of various CNS malfunctions, and a good example of such mechanisms is the process of lipofuscin formation occurring particularly in the retina, an integral part of the CNS. The retinal lipofuscin is formed and accumulated by the retinal pigment epithelial (RPE) cells as a consequence of both visual process taking place in photoreceptor-RPE functional complex and metabolic insufficiency of RPE lysosomal compartment. Among various retinal lipofuscin constituents, bisretinoids, originating from all-trans retinal substrate – a photometabolite of visual pigment cofactor 11-cis-retinal (responsible for photon capturing), are endowed with cytotoxic and complement-activating potential which increases upon illumination and oxidation. This survey deals with oxidative stress, PUFAs (especially DHA) peroxidation products of carboxyalkylpyrrole type and bisretinoids as potential inducers of the CNS pathology. A focus is put on vision-threatening disease, i.e., age-related macular degeneration (AMD), as an example of the CNS disorder whose pathogenesis has strong background in both oxidative stress and lipid peroxidation products.     

The potential use of cultured hepatocytes in predicting the hepatotoxicity of xenobiotics

1. A protocol is proposed for screening for hepatotoxicity of xenobiotics in vitro in which hepatocytes exposed to the compounds are evaluated for both cytotoxic and metabolic effects. Four established hepatotoxins have been studied. 2. alpha-Amanitin at 1.5 pg/mg cell protein inhibited RNA synthesis by 93% and reduced albumin synthesis to 56% of the control after 13 h treatment. 3. D-Galactosamine at 40 microM inhibited glycogen synthesis by 31%, glucuronidation of p-nitrophenol by 13% and albumin synthesis by 10%, and produced an increase in cytosolic enzyme leakage. 4. Thioacetamide decreased ureogenesis after 24 h of treatment at 230 microM (31% inhibition) and after 48 h at 2.3 microM (25% inhibition). 5. Ultrastructural alterations of hepatocytes were found after 48 h exposure to 1 mM acetaminophen and were preceded by extensive leakage of the enzymes GOT and LDH. Membrane damage was observed after 24 h exposure to 0.1 mM acetaminophen.     

Oxidative damage of retinal pigment epithelial cells and age‐related macular degeneration

Damage to the retinal pigment epithelial (RPE) cells is an early and crucial event in the molecular pathways leading to clinically relevant age‐related macular degeneration (AMD) changes. Oxidative stress, the major environmental risk factor for atrophic AMD, causes RPE injury that results in a chronic inflammatory response, drusen formation, and RPE atrophy. RPE degeneration ultimately leads to a progressive irreversible degeneration of photoreceptors. In vitro studies show that oxidant‐treated RPE cells undergo apoptosis, a possible mechanism by which RPE cells are lost during the early phase of atrophic AMD. The main target of oxidative injury appears to be mitochondria, an organelle known to accumulate genomic damage during aging. Addition of GSH, the most abundant intracellular thiol antioxidant, protects RPE cells from oxidant‐induced apoptosis. Similar protection occurs with dietary enzyme inducers that increase GSH synthesis. In addition, enhancing survival signaling preserves RPE cells under oxidative stress. These results indicate that therapeutic or nutritional intervention to enhance the antioxidant capacity and survival signaling of RPE may provide an effective way to prevent or treat AMD. This review describes major molecular and cellular events leading to RPE death, and presents currently used and new experimental, forthcoming therapeutic strategies. Drug Dev Res 68:213–225, 2007. © 2007 Wiley‐Liss, Inc.     

Low catecholamine concentrations protect adult rat ventricular myocytes against apoptosis through cAMP-dependent extracellular signal-regulated kinase activation

Catecholamines have complex effects on cardiac myocyte growth and survival, including the triggering of apoptosis at high concentration. Here, we examined whether at a lower concentration, catecholamine protected adult rat ventricular myocytes from apoptosis in vitro. Myocytes were exposed to staurosporine (ST, 10 microM) for 18 h, with or without epinephrine (0.1 or 10 microM) or fetal calf serum (10%). Apoptosis was assessed after 48 h of culture in terms of DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method, DNA gel electrophoresis). Epinephrine (0.1 microM) and serum reduced ST-induced myocyte apoptosis by approximately 50% (n = 12 cultures, P <.001), whereas epinephrine and serum alone did not influence the low apoptotic rate in control cultures. In contrast, 10 microM epinephrine induced marked apoptosis in ST-free conditions. The protective effects of 0.1 microM epinephrine and serum were blunted by the tyrosine kinase inhibitor genistein (n = 12 cultures, P <. 001). Extracellular signal-regulated kinase (ERK) activity was stimulated by 0.1 microM epinephrine but not by 10 microM epinephrine. Furthermore, the protective effect of epinephrine was mimicked by isoproterenol (1 microM) and forskolin (1 microM) but not by phenylephrine (10 microM) and was blunted by propranolol (10 microM) but not by prazozin (10 microM). Finally, isoproterenol and forskolin activated ERK, an effect that was blunted by propranolol. In conclusion, low epinephrine concentrations attenuate ST-induced apoptosis of adult cardiac myocytes in vitro, an effect mediated by coupling between the cAMP pathway and ERK activation. This suggests that a minimal adrenergic tone is essential for myocyte survival in conditions of unusual stress.     

The identification of small molecules that stimulate retinal pigment epithelial cells: potential novel therapeutic options for treating retinopathies

Introduction: Combinatory strategies using pharmacology and stem cell therapy have emerged due to their potential in the treatment of retinal pigment epithelium (RPE) cell related diseases, and a variety of different stem cell sources have been evaluated both in animal models and in humans. RPE cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs) are already in clinical trials, holding great promise for the treatment of age-related macular disease (AMD) and hereditary RPE-related retinal dystrophies. Highly efficient protocol for RPE generations have been developed, but they are still time-consuming and laborious.

Areas covered: The authors review RPE related diseases, as well as the known functions of RPE cells in retinal homeostasis. The authors also discuss small molecules that target RPE in vivo as well as in vitro to aid RPE differentiation from pluripotent stem cells clinically. The authors base this review on literature searches performed through PubMed.

Expert opinion: Using high-throughput systems, technology will provide the possibility of identifying and optimizing molecules/drugs that could lead to faster and simpler protocols for RPE differentiation. This could be crucial in moving forward to create safer and more efficient RPE-based personalized therapies.