R. Krishnan Kutty

Induction of Heme Oxygenase‐1 mRNA and Protein in Neocortex and Cerebral Vessels in Alzheimer's Disease

Abstract: Previous studies demonstrated the specific association of heme oxygenase (HO)‐1 protein to the neurofibrillary pathology of Alzheimers disease (AD). In this study, we used reverse transcription‐polymerase chain reaction methods to show the increased expression of HO‐1 but not HO‐2 mRNA transcripts in cerebral cortex and cerebral vessels from subjects with AD compared with age‐matched non‐AD controls. Neither the HO‐1 nor the HO‐2 mRNA level was altered in the cerebellum, a brain region usually spared from the pathological alterations of AD. There was no clear evidence that the expression of HO‐1 in these tissues was related to postmortem interval, cause of death, or the age of the subjects studied. Using immunoblotting methods, we further showed that HO‐1 protein content was increased in neocortical and vascular samples from AD subjects compared with controls. Our findings suggest the specific induction of HO‐1 mRNA and protein in the cerebral cortex and cerebral vessels but not HO‐2 mRNA or protein in association with the pathological lesions of the disease.

Advanced Maillard reaction end products, free radicals, and protein oxidation in Alzheimer's disease.

Evidence that free-radical production and oxidative stress are involved in the pathogenesis of Alzheimer disease is increasing. Our laboratory recently found two advanced glycosylation end products associated with the pathological neurofibrillary tangles and senile plaques of Alzheimer’s disease. These glycation-related posttranslational modifications are known to be initiated and potentiated during periods of oxidative stress. Further, we have also demonstrated that heme oxygenase-1, an enzyme induced during oxidative stress and involved in the production of antioxidant molecules, is found in close association with the pathological lesions; being almost completely absent in control brains. We present evidence that the seminal features of Alzheimer’s disease, including amyloid-P deposition, neurofibrillary tangle formation, degeneration of specific neuronal populations, and the persistence of the pathological lesions in vivo and their relative insolubility in vitro may all be related to specific oxidative stress-type mechanisms.

Molecular Characterization and Developmental Expression of a Retinoid- and Fatty Acid-binding Glycoprotein from Drosophila A PUTATIVE LIPOPHORIN

A detailed understanding of the mechanism of lipid transport in insects has been hampered by the inability to identify the proapolipophorin gene that encodes apolipophorins I and II, the principal protein components of lipophorin, the lipid transport vehicle. Here we provide the first molecular description of the Drosophila gene encoding a retinoid- and fatty acid-binding glycoprotein (RFABG) and present evidence that it is a member of the proapolipophorin gene family. The gene, localized to the chromosome 4 (102 F region), encodes a 3351-amino acid protein that could serve as the precursor for the ∼70-kDa and >200-kDa polypeptides associated with RFABG. The N-terminal sequence of the ∼70-kDa polypeptide and that predicted for the >200-kDa polypeptide showed high sequence similarity to blowfly apolipophorin II and apolipophorin I, respectively. The RFABG precursor contains a signal peptide and exhibits a multidomain mosaic protein structure, which is typical of extracellular proteins. It has structural domains similar to lipid-binding proteins, namely vitellogenins and apolipoprotein B. The protein also contains a domain similar to the D domain of von Willebrand factor and mucin. The gene is expressed in the Drosophila embryo during development in cells that make up the amnioserosa and fat bodies. Immunolocalizations using specific antibodies against RFABG reveal that the protein is initially dispersed through the embryonic amnioserosa sac and latter concentrated at skeletal muscle-epidermis apodemeal contact junctions during larval development. This novel gene may play an important role in the transport of lipids, including retinoids and fatty acids, in insects.

Inflammatory Cytokines Regulate MicroRNA-155 Expression in Human Retinal Pigment Epithelial Cells by Activating JAK/STAT Pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ+TNF-α+IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N‐(4‐hydroxyphenyl)retinamide

ARPE‐19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N‐(4‐hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE‐19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide‐induced neuronal differentiation was dose and time dependent, started within 1–2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde‐binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two‐dimensional gel followed by matrix‐assisted laser desorption ionization–time of flight mass spectrometric analysis demonstrated that heat‐shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.

N-(4-hydroxyphenyl)retinamide induces apoptosis in human retinal pigment epithelial cells : Retinoic acid receptors regulate apoptosis, reactive oxygen species generation, and the expression of heme oxygenase-1 and Gadd153

N‐(4‐hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid (RA) derivative and a potential cancer preventive agent, is known to exert its chemotherapeutic effects in cancer cells through induction of apoptosis. Earlier work from our laboratory has shown that relatively low concentrations of 4HPR induce neuronal differentiation of cultured human retinal pigment epithelial (ARPE‐19) cells (Chen et al., 2003, J Neurochem 84:972–981). However, at higher concentrations of 4HPR, these cells showed morphological changes including cell shrinkage and cell death. Here we demonstrate that ARPE‐19 cells treated with 4HPR exhibit a dose‐ and time‐dependent induction of apoptosis as evidenced by morphological changes, mono‐ and oligonucleosome generation, and increased activity of caspases 2 and 3. The 4HPR‐induced apoptosis as well as the activation of caspases 2 and 3 were blocked by both retinoic acid receptors (RAR) pan‐antagonists, AGN193109 and AGN194310, and by an RARα‐specific antagonist AGN194301. 4HPR treatment also increased reactive oxygen species (ROS) generation in ARPE‐19 cells in a time‐dependent manner as determined from the oxidation of 2′,7′‐dichlorofluorescin. In addition, the increase in the expression of heme oxygenase‐1 (HO‐1), a stress response protein, and the growth arrest and DNA damage‐inducible transcription factor 153 (Gadd153) in response to the ROS generation were also blocked by these receptor antagonists. Pyrrolidine dithiocarbamate (PDTC), a free‐radical scavenger, inhibited 4HPR‐induced ROS generation, the expression of its downstream mediator, Gadd153, and apoptosis in the pretreated cells. Therefore, our results, clearly demonstrate that 4HPR induces apoptosis in ARPE‐19 cells and that RARs mediate this process by regulating ROS generation as well as the expression of Gadd153 and HO‐1. J. Cell. Physiol. 209: 854–865, 2006.

Resveratrol Suppresses Expression of VEGF by Human Retinal Pigment Epithelial Cells: Potential Nutraceutical for Age-related Macular Degeneration

Age-related macular degeneration (AMD) is a sight threating retinal eye disease that affects millions of aging individuals world-wide. Choroid-retinal pigment epithelium (RPE)-neuroretina axis in the posterior compartment of the eye is the primary site of AMD pathology. There are compelling evidence to indicate association of vascular endothelial growth factors (VEGF) to AMD. Here, we report the inhibitory actions of resveratrol (RSV) on inflammatory cytokine, TGF-β and hypoxia induced VEGF secretion by human retinal pigment epithelial cells (HRPE). HRPE cultures prepared from aged human donor eyes were used for the studies in this report. HRPE secreted both VEGF-A and VEGF-C in small quantities constitutively. Stimulation with a mixture of inflammatory cytokines (IFN-γ, TNF-α, IL-1β), significantly increased the secretion of both VEGF-A and VEGF-C. RSV, in a dose dependent (10-50 uM) manner, suppressed VEGF-A and VEGF-C secretion induced by inflammatory cytokines significantly. RT-PCR analysis indicated that effects of RSV on VEGF secretion were possibly due to decreased mRNA levels. TGF-β and cobalt chloride (hypoxia mimic) also upregulated HRPE cell production of VEGF-A, and this was inhibited by RSV. In contrast, RSV had no effect on anti-angiogenic molecules, endostatin and pigment epithelial derived factor secretion. Studies using an in vitro scratch assay revealed that wound closure was also inhibited by RSV. These results demonstrate that RSV can suppress VEGF secretion induced by inflammatory cytokines, TGF-β and hypoxia. Under pathological conditions, over expression of VEGF is known to worsen AMD. Therefore, RSV may be useful as nutraceutical in controlling pathological choroidal neovascularization processes in AMD.

Expression of guanylate cyclase-A mRNA in the rat retina: Detection using polymerase chain reaction

A technique based on RNA-PCR was successfully employed for the detection of guanylate cyclase-A (GC-A) mRNA in the rat retina. Three sets of primers designed from the published cDNA sequence of rat brain guanylate cyclase-A (GC-A) produced amplification products of expected sizes from the retina as well as brain. Analysis of retinal PCR products yielded a 970 bp sequence, which showed 100% homology to the cDNA sequence of GC-A (2343-3312 bp region). Northern blot analysis was not very sensitive for the detection of GC-A mRNA in the retina. The results indicate that the mRNA for GC-A (or a closely related form) is probably expressed in the retina, but at a lower level than that found in the brain.

Maitotoxin-induced myocardial cell injury: Calcium accumulation followed by ATP depletion precedes cell death

Maitotoxin, the most potent marine toxin, is known to increase the uptake and the accumulation of Ca2+ into cells, and was used in the present study to investigate the mechanisms of myocardial cell damage induced by Ca2+ overload. In cultured cardiomyocytes, isolated from 2-day-old rats, maitotoxin affected cell viability, as indicated by the leakage of the cytosolic enzyme lactate dehydrogenase (LDH) and of radiolabeled adenine nucleotides into the extracellular medium. Maitotoxin-induced leakage of LDH steadily increased between 30 min and 24 hr, and was preceded by a marked depletion of intracellular ATP. Addition of maitotoxin resulted in a rapid influx of extracellular Ca2+, as detected by preincubating the cells in the presence of 45Ca; this effect evolved in a few minutes, thus preceding the signs of cell death. Cytosolic levels of free Ca2+ ([Ca2+]i) were monitored by loading freshly isolated, suspended cardiomyocytes with the intracellular fluorescent probe fura-2; in these cells, maitotoxin induced a dose-dependent increase in [Ca2+]i, with a lag phase of less than a minute. All these effects of maitotoxin were inhibited by reducing Ca2+ concentration in the culture medium or by incubating the cells with the calcium-channel blocking drug verapamil. It is thus demonstrated that maitotoxin-induced cardiotoxicity is secondary to an inordinate influx of Ca2+ into the cells. It is also suggested that, in those conditions that lead to an inordinate accumulation of Ca2+ into myocardial cells, the unmatched demands of energy and the depletion of ATP play a primary role in the irreversible stage of cell damage.

Maitotoxin-induced liver cell death involving loss of cell ATP following influx of calcium

Maitotoxin, one of the most potent marine toxins known, produced cell death in cultures of rat hepatocytes with a TD50 of 80 pM at 24 hr. The cell death, as indicated by a dose- and time-dependent leakage of lactate dehydrogenase (LDH), was also associated with the leakage of [14C]adenine nucleotides from hepatocytes prelabeled with [14C]-adenine. The toxic effect of maitotoxin was completely abolished by the omission of calcium from the culture medium. The cell death induced by maitotoxin increased with increasing concentrations of calcium in the medium. Treatment of hepatocytes with low concentrations of the toxin (less than 0.5 ng/ml) resulted in increases in 45Ca influx into the cells. At higher concentrations of maitotoxin (greater than 1ng/ml), the initial increase in 45Ca influx was followed by the release of the 45Ca from the cells into the medium. Since the 45Ca release paralleled the LDH leakage, the release of calcium was due to cell death. The 45Ca influx, [14C]adenine nucleotide leakage, and LDH leakage were effectively inhibited by verapamil, a calcium channel blocker. Maitotoxin also induced a time- and dose-dependent loss of ATP from hepatocytes, which preceded the [14C]adenine nucleotide and LDH leakage. Thus, it appears that the cell death resulting from maitotoxin treatment is caused by the elevated intracellular calcium, which in turn inhibits mitochondrial oxidative phosphorylation causing depletion of cell ATP. Loss of cell ATP may be the causative event in the maitotoxin-induced cell death.