Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future

As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.     

The pigmented epithelium sustains cell growth and tissue differentiation of chicken retinal explants in vitro

Embryonic retinae from 5-6-day-old chicks (E5-E6) were cut into stripes either in close contact with (RPE stripes) or in absence of the neighboring retinal pigmented epithelium (R stripes). The stripes were explanted and cultivated in vitro for up to 6 days, during which time they show the following differences in their characteristics of growth and differentiation. Compared with R stripes, RPE stripes morphologically showed a significant increase in size during the first 2 days in culture. Using E5 tissue, this is also demonstrated by a higher rate of cell proliferation (as measured by uptake of radioactive thymidine as well as by DNA contents). In contrast, R stripes after two days in culture show a much stronger neurite growth. After longer periods of culturing (5-6 days) we can show by cholinesterase histochemistry (AChE and BChE) and by PNA-lectin binding that the RPE stripes have started to form all major layers of the in vivo retina, whereas R stripes remain unstratified and start to degenerate earlier. We conclude that the pigment epithelium might exert a specific stimulus on growth and tissue differentiation of the neural retina not only during in vitro, but possibly also during in vivo development. The in vitro methods introduced here could become useful model systems to further investigate the significance of the RPE for developmental, regenerative and even adult processes of the neural retina. Their future applicability in ophthalmologic research is briefly discussed.     

Differential expression of N-ras in metaplastic retinal pigmented epithelium

Changes in expression of proto-oncogenes have been suggested as a key element in controlling the proliferative and differentiated status of a cell. We studied the expression of several of these genes in proliferating normal and metaplastic RPE cells. Using cDNAs to the c-myc, N-ras and n-myc genes we performed Northern blot analysis on the cytoplasmic RNA in normal and metaplastic RPE cells. We found that proliferating normal and metaplastic RPE cells exhibit similar patterns of expression of the c-myc gene. In contrast, when compared to normal cells, proliferating metaplastic RPE cells exhibited early and abnormally high levels of a 4 kb N-ras RNA. These changes in N-ras expression precede the vitreous-induced phenotypic change from an epithelial to a fibroblastic morphology. Understanding what role, if any, the N-ras gene plays in this metaplasia may lead to a better understanding of proliferative disorders such as proliferative vitreoretinopathy.     

Inducible expression of cre recombinase in the retinal pigmented epithelium

PURPOSE: The retinal pigmented epithelium (RPE) expresses many genes that play important roles in the support and maintenance of photoreceptors. The present study was conducted to develop a system amenable to the dissection of the temporal function of these genes, specifically within RPE cells. Transgenic mice were generated and characterized in which the expression of Cre recombinase could be specifically induced within the RPE. METHODS: Transgenic mice carrying the human vitelliform macular dystrophy-2 (VMD2) promoter (P(VMD2))-directed reverse tetracycline-dependent transactivator (rtTA) and the tetracycline-responsive element (TRE)-directed cre were generated. Inducible Cre expression was achieved by feeding doxycycline to these mice and was characterized by using a Cre-activatable lacZ reporter mouse strain (R26R). RESULTS: A beta-galactosidase assay of rtTA/Cre-R26R mice demonstrated that the basal level of Cre expression without doxycycline induction was negligible. Addition of doxycycline led to induction of RPE-specific Cre expression/function at least from embryonic day 9 to postnatal day 60. The highest induction occurred at approximately postnatal day 4. As measured by ERG and histology, retinal function and morphology were normal in 10-month-old rtTA/Cre mice that were treated with doxycycline at weaning age. CONCLUSIONS: Transgenic mice were generated that express Cre recombinase in the RPE in an inducible fashion. These mice will be useful for studies of the RPE-specific role of genes that are expressed in the RPE as well as other cells, particularly for avoiding embryonic lethality and dissecting the function of genes that play dual roles in development and adulthood.     

Vectorial accumulation of cathepsin D in retinal pigmented epithelium: effects of age

The extracellular accumulation of n-acetyl-beta-glucosaminidase (NAG) and a cathepsin D-like enzymatic activity were studied in human retinal pigmented epithelium cultured on nitrocellulose filter inserts. These enzymes accumulated in both the apical and basal extracellular fluid. For NAG, the total extracellular activity accumulated in 24 hours was 7.3 +/- 1.75% of the total cell-associated activity, while the cathepsin D-like activity was 10 +/- 0.71% of the total cell-associated activity. The distribution of the extracellular activity was 60/40%, basal/apical, for NAG and was constant regardless of the age of the donor. In contrast, the distribution for the cathepsin D-like activity ranged from 70/30 to 10/90 and showed a marked age dependence in RPE obtained from donors aged 9-93 years. These levels of activity were stable for at least 2 months in culture. The cathepsin D-like activity differed from the NAG in that the presence of mannose-6-phosphate in the medium did not result in increased extracellular accumulation of cathepsin D activity.     

Effects of hypoxia on retinal pigmented epithelium cells: protection by antioxidants

Age-related macular degeneration and other eye diseases, such as diabetic retinopathy, are probably linked to the effects of oxygen radicals derived from light or metabolic reactions. We have investigated the effects of hypoxia on bovine retinal pigmented epithelial cells (RPE) and the response of these cells to two antioxidants that have previously shown a beneficial action against free radical-linked senescent involution. The main results of the study were as follows: (i) Hypoxia induced apoptotic damage on RPE cells, with LDH leakage and ATP reduction; (ii) both vitamin C (VC) and N-acetyl-cysteine (NAC) treatment protected against hypoxia-induced apoptosis, with less DNA fragmentation. In our opinion, these findings justify further experimental and clinical work to investigate the role of hypoxia in the mechanisms of age-related RPE injury and death as well as the potential of antioxidant administration to prevent or delay retinal degenerative processes caused by oxygen-dependent pathophysiological conditions.     

Studies on the differentiation of retinal pigmented epithelium cells in culture.

Sephadex G-25 fractions of chick embryo extract affect the differentiation of cloned embryonic pigmented epithelial cells (PE) in culture. Both tyrosinase activity and amounts of melanin are markedly affected by the extract fractions. 5-Hydroxyindole decreased melanin accumulation in PE cells while alpha-methyl-p-tyrosine had no apparent effect on the process. Melanin disposal as well as biosynthesis may be important in controlling the accumulation of pigment in PE cells under culture conditions. Triiodothyronine greatly increased visible pigmentation and affected the morphology of cultured PE cells.     

Pigment dispersion syndrome and pigmented pattern dystrophy of retinal pigment epithelium.

The 2 rare entities, pigmentary dispersion syndrome and pigmented pattern dystrophy of the retinal pigment epithelium, were found in a young male patient. Visual function was undisturbed.     

Carbonic anhydrase activity is increased in retinal pigmented epithelium and choriocapillaris of RCS rats

Background: In Royal College of Surgeons (RCS) rats the retinal pigmented epithelium (RPE) exhibits defective phagocytosis of rod outer segments, causing degeneration of the photoreceptor layer. It is not known whether another function of the RPE, ion and fluid transport, is also affected by the disease. One enzyme involved in modulation of RPE transport activities is carbonic anhydrase (CA). To clarify whether changes in CA activity are correlated with the process of retinal degeneration, the localization of CA activity in RCS rat eyes was investigated. Methods: Eyes of 12 RCS rats and 12 age-matched congenic controls of different ages were studied, using a modified histochemical method of Hansson for light and electron microscopy. Results: Control eyes showed CA staining in corneal endothelium, both layers of ciliary epithelium, Müller cells, inner segments of photoreceptors, and RPE cells. In RPE the apical membranes were most intensely stained. In RCS rats, changes in CA staining were seen only in the posterior segment of rats 6 and 7 months of age. Most of the RPE cells were more intensely stained than those of age-matched controls, especially due to increased CA activity in the basolateral membrane infoldings. Adjacent endothelial cells of the choriocapillaris and of retinal capillaries developed staining for CA activity. Conclusion: Changes in CA activity in the RPE and adjacent capillary endothelium, together with previously described changes in RPE morphology in RCS rats, indicate changes in ion and fluid transport across the RPE. Since the retina was already impaired when the increase in CA activity occurred, we hypothesize that the causative factor is not the genetic defect per se, but the destruction of the retina.     

Hydroxychloroquine and ritonavir for COVID-19 infection: a possible synergic toxicity for retinal pigmented epithelium

Graefe's Archive for Clinical and Experimental Ophthalmology -     

Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid

The discoveries of gene variants associated with macular diseases have provided valuable insight into their molecular mechanisms, but they have not clarified why the macula is particularly vulnerable to degenerative disease. Its predisposition may be attributable to specialized structural features and/or functional properties of the underlying macular RPE/choroid. To examine the molecular basis for the macula's disease susceptibility, we compared the gene expression profile of the human RPE/choroid in the macula with the profile in the extramacular region using DNA microarrays. Seventy-five candidate genes with differences in macular:extramacular expression levels were identified by microarray analysis, of which 29 were selected for further analysis. Quantitative PCR confirmed that 21 showed statistically significant differences in expression. Five genes were expressed at higher levels in the macula. Two showed significant changes in the macular:extramacular expression ratio; another two exhibited changes in absolute expression level, as a function of age or AMD. Several of the differentially expressed genes have potential relevance to AMD pathobiology. One is an RPE cell growth factor (TFPI2), five are extracellular matrix components (DCN, MYOC, OGN, SMOC2, TFPI2), and six are related to inflammation (CCL19, CCL26, CXCL14, SLIT2) and/or angiogenesis (CXCL14, SLIT2, TFPI2, WFDC1). The identification of regional differences in gene expression in the RPE/choroid is a first step in clarifying the macula's propensity for degeneration. These findings lay the groundwork for further studies into the roles of the corresponding gene products in the normal, aged, and diseased macula.     

Methionine sulfoxide reductase B2 is highly expressed in the retina and protects retinal pigmented epithelium cells from oxidative damage

Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial enzyme that converts methionine sulfoxide (R) enantiomer back to methionine. This enzyme is suspected of functioning to protect mitochondrial proteins from oxidative damage. In this study we report that the retina is one of the human tissues with highest levels of MSRB2 mRNA expression. Other tissues with high expression were heart, kidney and skeletal muscle. Overexpression of an MSRB2-GFP fusion protein increased the MSR enzymatic activity three-fold in stably transfected cultured RPE cells. This overexpression augmented the resistance of these cells to the toxicity induced by 7-ketocholesterol, tert-butyl hydroperoxide and all-trans retinoic acid. By contrast, knockdown of MSRB2 by a miRNA in stably transfected cells did not convey increased sensitivity to the oxidative stress. In the monkey retina MSRB2 localized to the ganglion cell layer (GLC), the outer plexiform layer (OPL) and the retinal pigment epithelium (RPE). MSRB2 expression is most pronounced in the OPL of the macula and foveal regions suggesting an association with the cone synaptic mitochondria. Our data suggests that MSRB2 plays an important function in protecting cones from multiple type of oxidative stress and may be critical in preserving central vision.     

Mercapturic acid pathway enzymes in bovine ocular lens, cornea, retina and retinal pigmented epithelium

Analogous to the liver, ocular tissues contain large concentrations of glutathione and are exposed to potentially damaging chemical compounds. Since glutathione has been shown to have a detoxification function, via mercapturic acid production in the liver, we investigated whether glutathione has a similar function in ocular tissues. We have demonstrated the presence of all of the enzymes involved in the mercapturic acid pathway i.e. glutathione S-transferase, gamma-glutamyl transpeptidase, cysteinylglycinase, and N-acetyl transferase, in the ocular tissues of bovine lens, cornea, retina, and retinal pigmented epithelium. Therefore glutathione may have another function in ocular tissues, that of the detoxification of xenobiotics.