Biomedical subjects

P Elizabeth Rakoczy

Publications and source records attributed to P Elizabeth Rakoczy.

At least 19 recordsLinked to original sources

PubMed2006

Contrasting actions of prolonged mitogen-activated protein kinase activation on cell survival.

Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.

Adenosine Triphosphate↗

PubMed2005

Long-term effect of therapeutic laser photocoagulation on gene expression in the eye.

Microarray-based gene expression analysis demonstrated that laser photocoagulation (LPC) of mouse eyes had a long-term effect on the expression of genes functionally related to tissue repair, cell migration, proliferation, ion, protein and nucleic acid metabolism, cell signaling, and angiogenesis. Six structural genes, including five crystallins (Cryaa, Cryba1, Crybb2, Crygc, Crygs) and keratin 1-12 (Krt1-12), the anti-angiogenic factor thrombospondin 1 (Tsp1), the retina- and brain-specific putative transcription factor tubby-like protein 1 (Tulp1), and transketolase (Tkt), a key enzyme in the pentose-phosphate pathway, were all shown to be up-regulated by real-time PCR and/or Western blotting. Immunohistochemistry localized five of these proteins to the laser lesions and surrounding tissue within the retina and pigmented epithelium. This is the first study demonstrating long-term changes in the expression of these genes associated with LPC. Therefore, it suggests that modulated gene expression might contribute to the long-term inhibitory effect of LPC. In addition, these genes present novel targets for gene-based therapies aimed at treating microangiopathies, especially diabetic retinopathy, a disease currently only treatable with LPC.

Animals↗

PubMed2005

Mouse models of age-related macular degeneration.

Recent advances in genetic technologies have greatly accelerated our ability to find disease-related genes and to generate animal models. The availability of ocular tissues with known genetic diseases are greatly contributing to our understanding of retinal disease processes including age-related macular degeneration (AMD), and panretinal and cone degenerations. While the macula is a highly specialised area of the retina not present in many mammals, the use of animal models such as mouse strains will give basic physiology and visual processing genetics relevant to human AMD. This review aims to provide a framework for better understanding some of the existing animal models and the knowledge that has been derived from their evaluations.

Animals↗

PubMed2005

VEGF differentially regulates transcription and translation of ZO-1alpha+ and ZO-1alpha- and mediates trans-epithelial resistance in cultured endothelial and epithelial cells.

Tight junctions (TJ) between retinal pigmented epithelial (RPE) and retinal endothelial cells maintain the outer and inner blood-retinal barrier, and the breakdown of these barriers is associated with retinal diseases. Vascular endothelial growth factor (VEGF) increases vascular permeability and is thought to be involved in age-related maculopathy. However, to date, little is known about the effect of VEGF on RPE cell junctions. We have investigated the effect of VEGF on TJ formation by examining two essential proteins, ZO-1alpha(+) and ZO-1alpha(-). Cultured vascular endothelial cells in the presence of 5 ng/ml VEGF significantly down-regulate ZO-1alpha(+) and ZO-1alpha(-) transcripts and proteins with significant loss of their trans-epithelial resistance (TER). Immunoconfocal analysis with an anti-ZO-1 antibody has confirmed the relocation of ZO-1 protein from membrane to cytoplasm. By contrast, in the presence of 5 ng/ml VEGF, cultured RPE cells (ARPE19 and RPE51) significantly up-regulate ZO-1alpha(+) and ZO-1alpha(-) transcripts and proteins resulting in a significant increase in their TER. Subsequent immunoconfocal analysis has demonstrated increased ZO-1 membrane assembly in VEGF-treated RPE cells. Thus, VEGF has a dual capability with respect to the regulation of the expression of some TJ proteins at the transcriptional and post-translational levels depending on cell type.

Cells, Cultured↗

PubMed2005

Are stem cell characteristics altered by disease state?

Autologous stem cell transplantation combined with gene therapy can potentially be used to treat genetically inherited diseases. However, characterization of multipotential cells from a disease state remains extremely limited. We have characterized adult bone marrow stromal cells (MSCs) derived from three retinal degenerative mouse models and compared them to marrow stromal cells derived from their normal strain counterparts. Despite similar profiles soon after harvest, at 30 days postisolation, marrow stromal cells derived from a disease origin were shown to contain a large pool (approximately 89-99%) of undifferentiated marrow stromal cells (CD90(+)/STRO-1(+)) as compared to their normal counterparts (approximately 19-43%). Fetal bovine serum appeared essential for marrow stromal cell proliferation and was not found to induce differentiation, although it could be substituted with other additives including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and leukemia inhibitory factor (LIF). We also showed that resulting CD90(+)/STRO(+) cells derived from both states could be directed into desired lineages expressing at the same rate and that they could be transduced with the same efficiency using different viral vehicles. This investigation has shown the existence of a large pool of undifferentiated stem cells derived from the disease state that have the potential to form the desired cell types when appropriately cued.

Adipocytes↗

PubMed2005

Treatments for choroidal and retinal neovascularization: a focus on oligonucleotide therapy and delivery for the regulation of gene function.

Blinding eye diseases caused by neovascularization of the retinal tissue are the leading cause of blindness in Western societies. Current treatments, such as laser photocoagulation, are limited in their effectiveness at halting the progression of angiogenesis and are unable to reduce the number of vessels once they have developed. In addition, although complete blindness is often avoided, vision is often permanently impaired by the treatment itself. Several less invasive treatments are being developed and one of these is oligonucleotide gene therapy in which short stretches of nucleotides are being used as inhibitors of key, metabolic processes involved in angiogenesis. Combined with this is the development of new and improved nucleotide chemistries aimed at overcoming many of the problems associated with oligonucleotide gene therapy, such as poor longevity because of endonuclease activity. In addition, advancements in delivery systems have further enhanced the efficacy of oligonucleotide gene therapy by increasing cellular penetration and localizing delivery to specific cell types and organs.

Choroidal Neovascularization↗

PubMed2004

Discovery of a novel control element within the 5'-untranslated region of the vascular endothelial growth factor. Regulation of expression using sense oligonucleotides.

The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5'- and 3'-untranslated regions, many of which are yet to be described. In this study we examined the 5'-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S(1) and S(2)) were designed based on its sequence, and a third oligonucleotide (S(3)) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S(1) and S(2) resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S(3) had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S(1). Further in vivo studies showed that S(1) and S(2) mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct.

5' Untranslated Regions↗

PubMed2004

Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue.

BACKGROUND: Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10-15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function. METHODS: rAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection. RESULTS: rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health. CONCLUSION: This work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina.

Journal Article↗

PubMed2004

Concurrent enhancement of transcriptional activity and specificity of a retinal pigment epithelial cell-preferential promoter.

PURPOSE: To develop a transgene expression system in retinal pigment epithelial cells with the aim of enhancing the transcriptional activity of a weak RPE-specific/preferential promoter. METHODS: The transgene expression system was established by introducing a chimeric transcriptional activator (GAL4-VP16) and its DNA binding sequence and using truncated human and mouse RPE65 promoters in combination with a luciferase reporter gene. Two groups of expression plasmids were constructed for transfection. The group for co-transfection contained two DNA constructs where the reporter and GAL4-VP16 were separately expressed in pLuc and pGV series. The other group, pLuc-GV series, was prepared as single DNA constructs expressing both the reporter and GAL4-VP16. The transcriptional activities of the DNA constructs were assayed by transfection of human RPE cells (RPE51 and D407) and other cell lines (HEK293, COS-1, Hela, HepG2, and F2000). RESULTS: We found that the transcriptional activity of the human RPE65 promoter was dramatically enhanced 10-13 fold in RPE cells co-transfected with DNA constructs phR65luc and phR65GV when compared to the human RPE65 promoter alone. A comparatively lower, 4-5 fold, increase was observed following transfection with the single DNA construct phR65luc-GV. In RPE cells, when the transcriptional responses to GAL4-VP16 expression were compared between the RPE65 promoter of phR65luc and the minimal promoter of pLuc, the increase in transcriptional activity was about 10 fold higher in phR65luc constructs. Low or non-significant enhancement of promoter activity was observed with these constructs following transfection of the non-RPE cell lines. CONCLUSIONS: Our results indicate that the current transgene expression system dramatically amplifies transcriptional activity of weak and cell-specific/preferential promoters (e.g., the hRPE65 promoter) whilst retaining relative cell specificity.

Animals↗

PubMed2004

Predilection of the macular region to high incidence of choroidal neovascularization after intense laser photocoagulation in the monkey.

OBJECTIVE: To determine the key factors for creating a high incidence model of choroidal neovascularization (CNV) in the monkey. METHODS: Intense laser photocoagulation was performed in 8 eyes of 4 monkeys using krypton red and green-yellow and Alcon frequency-doubled diode ophthalmic lasers. Eight to 13 lesions were delivered to an area between the temporal vascular arcades in each eye. Development of CNV was monitored by fluorescein angiography at 2 and 4 weeks after laser treatment, and the results were correlated with histological analysis. RESULTS: A much higher incidence of CNV occurred in the macular region, which refers to an anatomic area equivalent to a mean +/- SD 2.5 +/- 0.4 times the horizontal diameter of the optic disc in the fundus. Regardless of the type of ophthalmic laser used, 72% of lesions developed fluorescein leakage within the macula, compared with 12% outside the macula (P<.001). By histological analysis, 89% of lesions developed microscopic CNV within the macula vs 22% outside the macula (P<.001). CONCLUSION: The macular region is predisposed to creation of laser-induced CNV in the monkey.Clinical Relevance The predilection of the macular region to a high incidence of laser-induced CNV may account for the high recurrence rate of subfoveal CNV after laser treatment in humans.

Animals↗

PubMed2004

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide.

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (x=81.51%) was significantly better (P=0.0036) than those that possessed 4 positive charges (x=56.8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P<0.0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases.

Animals↗

PubMed2004

Impurity of recombinant adeno-associated virus type 2 affects the transduction characteristics following subretinal injection in the rat.

We recently reported that different purification methods of recombinant adeno-associated virus type 2 (rAAV2) affect the transduction characteristics following subretinal injection. In this study, we examined the roles of contaminant proteins from the HEK-293 cells and helper adenovirus, inactivation of helper adenovirus and cell stress induced by DNA-damaging agents in rAAV-mediated retinal transduction. Our results showed that contaminating factors/proteins resulting from the helper E1 deleted adenovirus are possibly responsible for efficient RPE transduction. Future studies of these factors will undoubtedly lead to development of new therapeutic approaches to PR- and RPE-specific retinal diseases.

Adenovirus E1 Proteins↗

PubMed2003

Differentiation of marrow stromal cells into photoreceptors in the rat eye.

Retinal degenerations and dystrophies are the major causes of genetically inherited blindness that are characterized by the apoptotic death of the photoreceptor cell layer of the retina. To date, no treatment exists for these diseases and only recently have they been considered as candidates for gene and stem cell therapies. Here we report the ability of adult CD90+ marrow stromal cells (MSCs) to be induced by activin A, taurine, and EGF into cells (20-32%) expressing photoreceptor-specific markers rhodopsin, opsin, and recoverin in vitro. CD90+ cells were either transduced with recombinant adeno-associated virus expressing green fluorescent protein (GFP) or bromodeoxyuridine (BrdU) labeled and then injected into the subretinal space of adult Royal College of Surgeons rats. Fundus photography and angiography showed no adverse effects of CD90+ MSC transplantation. GFP-expressing cells or BrdU-positive cells covered approximately 30% of the entire retinal area. By 2 weeks after injection, CD90+ MSCs integrated into the host retina, forming structures similar to the photoreceptor layer and expressed a photoreceptor-specific marker. No teratoma formation was observed in the recipient retina. The subretinally delivered CD90+ MSCs did not stain for proliferating cell nuclear antigen, indicating that they primarily undergo differentiation rather than proliferation. In addition, we established that transplanted cells can attract synaptic vesicles and hence are potentially capable of signal transduction. This study demonstrates for the first time the partial differentiation of adult CD90+ MSCs into photoreceptors in vitro and in vivo. Our results establish a proof of concept for CD90+ MSC differentiation with autologous transplantation, which may provide a promising therapeutic strategy for the treatment of some forms of genetically inherited retinal degenerations.

Animals↗

PubMed2003

Practical considerations of recombinant adeno-associated virus-mediated gene transfer for treatment of retinal degenerations.

BACKGROUND: Photoreceptor (PR) and retinal pigment epithelium (RPE) are the principal cell targets in retinal gene therapy. Recombinant adeno-associated virus (rAAV) has emerged as a very promising vector for gene therapy in hereditary retinal diseases. Gene transfer at different stages of the disease is a practical consideration for future clinical application. METHODS: A rAAV carrying the enhanced green fluorescent protein gene driven by a cytomegalovirus promoter was produced by either co-infecting the 293 cell line with E1-defective adenovirus and purified by CsCl(2) density gradient (CsCl(2)-rAAV), or by transfecting with an adenoviral helper plasmid and purified by iodixanol density gradient followed by heparin column chromatography (heparin-rAAV). The impact of different virus preparations on the patterns of transgene expression was investigated after subretinal injection. Furthermore, rAAV-mediated gene transfer was evaluated at both early and advanced stages of retinal degeneration in four disease models including the RCS rat, rd, RPE(65) (-)/(-) and cathepsin D mutant mice that are associated with PR- or RPE-related gene defects. RESULTS: CsCl(2)-rAAV predominantly transduced RPE and with less efficiency in PR. In contrast, heparin-rAAV predominantly transduced PR but with much less efficiency in RPE. Subretinal injection of either rAAV preparation induced no changes to retinal morphology and retinal-choroidal vasculature. The product of transgene, however, could be observed in multiple tracts in the brain. In the four disease models, target cells were efficiently transduced not only at the early stage, but also at the late stage of disease as long as the target cells were present. CONCLUSIONS: Different preparations of rAAV have an impact on the patterns of transgene expression after subretinal injection. Patients at advanced stages of retinal degeneration may still benefit from rAAV-mediated gene therapy. The possible side effects of transgenic products on the central nervous system should be carefully monitored once therapeutic genes are employed.

Animals↗

PubMed2003