The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.

Indiana University-Purdue University Indianapolis (IUPUI) / Dry age-related macular degeneration (AMD) is a progressive neurodegenerative disorder characterized by geographical atrophy of the retinal pigment epithelium (RPE), causing irreversible central vision loss. Systemically-delivered bone marrow-derived cells (BMDCs), programmed to RPE-like cells via expression of human RPE65, regenerate damaged RPE and preserve vision in murine models of retinal degeneration. RPE65 rapidly activates adenylate cyclase (AC), which then activates endogenous Rpe65 and RPE-associated marker Cralbp. Previous studies expressed RPE65 from an integrating lentiviral vector (ILV), which is an unnecessary safety risk due to the potential for insertional mutagenesis, as long- term expression of RPE65 is not required for BMDC programming. Here, we developed a 3rd generation integrase-defective lentiviral vector (IDLV) for programming both murine and human BMDCs to RPE-like cells, reducing insertional mutagenesis risk and expanding the protocol to include human cells. We enhanced IDLV3-RPE65 infection of murine and human BMDCs by preloading concentrated vector on RetroNectin at MOI 50, and infecting with low-speed centrifugation, increasing RPE65 mRNA levels from ~12-fold to ~25-fold (p<0.05). IDLV3-RPE65 infection initiates expression of endogenous Rpe65 mRNA expression in murine BMDC and Cralbp/CRALBP mRNA in both murine and human BMDCs, indicating programming to RPE-like cells. Inhibiting AC in RPE65infected BMDCs abrogated expression of the endogenous genes, confirming the role of AC activation in programming. Critically, IDLV3-RPE65-infected murine BMDCs are recruited to and incorporate into to the RPE layer, and preserve vision in murine models of retinal degeneration. We conclude that BMDCs programmed with IDLV3-RPE65 successfully prevent retinal degeneration progression and are appropriate for testing in human cells, with a view to move into human clinical trial for the treatment of dry AMD. This approach significantly increases the safety of the therapy and is, to the best of our knowledge, the first application of a single IDLV in the generation of therapeutic cells from adult stem cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21685号 / 医博第4491号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 高橋 淳, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Purpose. These experiments were designed to characterize tight junction formation by retinal pigment epithelial (RPE) cells in vitro and to compare the effects on this process of hormonally defined medium (HDM) and serum- containing medium. Methods. Formation of RPE tight junctions was analyzed in freshly isolated rat RPE cells maintained either in HDM or serum-containing medium. Junctions were evaluated functionally by measuring transepithelial electrical resistance (TER) and permeability and structurally by immunolocalization of the junction-associated actin microfilaments. Calcium dependency of the junction was determined by reducing media calcium concentration. Results. RPE cells cultured in serum-free HDM developed calcium-dependent tight junctions, which exhibited TER levels > 150 Ω · cm 2 and low paracellular permeability. Serum-containing media inhibited tight junction formation as indicated by significant reductions in TER and increases in permeability. Junction-associated actin microfilaments and cell density were unchanged. Conclusions. Tight junction formation by RPE cells is inhibited by serum. This activity may play an important role in responses of the RPE layer to injury, contributing to the pathologic progression of blood- retinal barrier dysfunction.

Purpose. In the genetically defective Royal College of Surgeons (RCS) rat model for retinal degeneration, a breakdown occurs in the retinal pigment epithelial (RPE) cell tight junctions just as the photoreceptors begin to degenerate. These experiments sought to determine the impact of the RPE genetic defect on this alteration in the RPE cell tight junctions. Methods. Retinal pigment epithelial cell cultures prepared from RCS and control rats were treated with hormonally defined medium (HDM), base medium conditioned by RCS or control retinas, or unconditioned base medium. The tight junctions formed by these cultures were assayed functionally by measuring transepithelial electrical resistance and permeability. Junction structure was evaluated by immunolocalization of the tight junction protein zonula occludens I and of the junction-associated actin microfilaments. Results. Retinal pigment epithelial cultures from dystrophic rats formed structurally and functionally normal tight junctions when maintained in hormonally defined medium. The junctions remained stable when the medium bathing the apical surface was switched to base medium preconditioned by normal retinas. In contrast, cultures treated with medium preconditioned by degenerating dystrophic retinas or with unconditioned medium exhibited a breakdown in their tight junctions. Conclusions. Retinal pigment epithelial cells isolated from dystrophic RCS rats can form tight junctions normally in vitro. Normal, but not dystrophic, retinas release factors that support RPE tight junctions. Therefore, the junctional abnormality seen in dystrophic rat RPE cells in vivo is probably caused by the loss of trophic factors normally provided by the healthy neural retina rather than by a direct effect of the genetic defect on the tight junctions.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23813号 / 医博第4859号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中本 裕士, 教授 花川 隆, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In patients with age-related macular degeneration, a monolayer of cells in the eyes called retinal pigment epithelium differ from healthy ones in morphology. It is therefore important to quantify the morphological changes, which will help us better understand the physiology, disease progression and classification. Classification of the RPE morphometry has been accomplished with whole tissue data. In this work, we focused on the spatial aspect of RPE morphometric analysis. We used the second-order spatial analysis to reveal the distinct patterns of cell clustering between normal and diseased eyes for both simulated and experimental human RPE data. We classified the mouse genotype and age by the k-Nearest Neighbors algorithm. Radially aligned regions showed different classification power for several cell shape variables. Our proposed methods provide a useful addition to classification and prognosis of eye disease noninvasively.

A high frequency of retinitis pigmentosa (RP) is found in Northern Sweden. In an inventory of autosomal recessive RP patients in Västerbotten County, a great number of cases with a unique phenotype was noticed, denoted Bothnia Dystrophy (BD). The aim of the study was to describe the phenotype, to determine the chromosomal location, and to identify the gene. Patients typically show night blindness from early childhood. Symptoms of defect macular function with a decrease of visual acuity can appear in early adulthood. The retinal fundus shows irregular white spots in a central, and parafoveal pattern along the arcades. Centrally areolar maculopathy develops and round circular atrophies are observed in the periphery. The disease was shown to be associated with a missense mutation in the RLBP1 gene resulting in an amino acid substitution (R234W) in the cellular retinaldehyde-binding protein (CRALBP). The R234W mutation was found in a homozygous state in 61 patients affected with BD. Ten patients were heterozygous for the R234W mutation, and presented a similar phenotype. No additional mutations in the coding sequence or exon-intron junctions were found. CRALBP is localised in retinal pigment epithelium (RPE), and Müller cells of the retina. In the RPE, CRALBP functions as a carrier protein for endogenous retinoids. Dark adaptometry and electrophysiologic testing showed an initial loss of rod function followed by a progressive reduction of the cone responses in older ages. A compromised rod function, dysfunction of the Müller cells, and indications of a disturbed function of the inner retina were found. With prolonged dark adaptation, a gradual increase in retinal sensitivity to light and an improvement of the ERG components occurred. The findings indicate a prolonged synthesis of photopigments, retardation of the visual process in the retinal pigment epithelium and a loss of retinal cells probably starting at a relative early age in BD. To evaluate the subjective visual function in BD patients, a battery of objective tests of visual function and composite score of the 25-item NEI-Visual Function Questionnaire (VFQ-25) were analyzed. We found that weighted distance logMAR visual acuity (WVA), had the strongest association with subjective visual function, and that there was a considerable loss of subjective and objective visual function with increasing age in BD patients. The prevalence of BD is as high as 1:3600 in Västerbotten County. The possibility that recycling of retinoids localized in the RPE might be impaired in BD might give future therapeutic possibilities. Due to the large and clinically well-characterized set of patients with this disease, they constitute a suitable study group.

Age related macular degeneration (AMD) is a public health concern in an aging society. The retinal pigment epithelium (RPE) layer of the eye is a principal site of pathogenesis for AMD. Morphological characteristics of the cells in the RPE layer can be used to discriminate age and disease status of individuals. In this thesis three genotypes of mice of various ages are used to study the predictive abilities of these characteristics. The disease state is represented by two mutant genotypes and the healthy state by the wild-type. Classification analysis is applied to the RPE morphology from the different spatial regions of the RPE layer. Variable reduction is accomplished by principal component analysis (PCA) and classification analysis by the k-nearest neighbor (k-NN) algorithm. In this way the differential ability of the spatial regions to predict age and disease status by cellular variables is explored.