Engineering a Biomimetic In Vitro Model of Bruch's Membrane Using Hagfish Slime Intermediate Filament Proteins.

Bruch's membrane resides in the subretinal tissue and regulates the flow of nutrients and waste between the retinal pigment epithelial (RPE) and vascular layers of the eye. With age, Bruch's membrane becomes thicker, stiffer, and less permeable, which impedes its function as a boundary layer in the subretina. These changes contribute to pathologies such as age-related macular degeneration (AMD). To better understand how aging in Bruch's membrane affects surrounding tissues and to determine the relationship between aging and disease, an in vitro model of Bruch's membrane is needed. An accurate model of Bruch's membrane must be a proteinaceous, semipermeable, and nonporous biomaterial with similar mechanical properties to in vivo conditions. Additionally, this model must support RPE cell growth. While models of subretinal tissue exist, they typically differ from in vivo Bruch's membrane in one or more of these properties. This study evaluates the capability of membranes created from recombinant hagfish intermediate filament (rHIF) proteins to accurately replicate Bruch's membrane in an in vitro model of the subretinal tissue. The physical characteristics of these rHIF membranes were evaluated using mechanical testing, permeability assays, brightfield microscopy, and scanning electron microscopy. The capacity of the membranes to support RPE cell culture was determined using brightfield and fluorescent microscopy, as well as immunocytochemical staining. This study demonstrates that rHIF protein membranes are an appropriate biomaterial to accurately mimic both healthy and aged Bruch's membrane for in vitro modeling of the subretinal tissue.

Regulation of Aqueous Humor Secretion by Melatonin in Porcine Ciliary Epithelium.

Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50-60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT3 receptor was found to be >10-fold higher than that of MT1 and MT2 in porcine ciliary epithelium. Aqueous pre-treatment with MT1/MT2 antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT3 antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl- and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT3 receptors.

Mechanobiological implications of age-related remodelling in the outer retina.

The outer retina consists of the light-sensitive photoreceptors, the pigmented epithelium, and the choroid, which interact in a complex manner to sustain homeostasis. The organisation and function of these cellular layers are mediated by the extracellular matrix compartment named Bruch's membrane, situated between the retinal epithelium and the choroid. Like many tissues, the retina experiences age-related structural and metabolic changes, which are relevant for understanding major blinding diseases of the elderly, such as age-related macular degeneration. Compared with other tissues, the retina mainly comprises postmitotic cells, making it less able to maintain its mechanical homeostasis over the years functionally. Aspects of retinal ageing, like the structural and morphometric changes of the pigment epithelium and the heterogenous remodelling of the Bruch's membrane, imply changes in tissue mechanics and may affect functional integrity. In recent years, findings in the field of mechanobiology and bioengineering highlighted the importance of mechanical changes in tissues for understanding physiological and pathological processes. Here, we review the current knowledge of age-related changes in the outer retina from a mechanobiological perspective, aiming to generate food for thought for future mechanobiology studies in the outer retina.

Single-cell RNA-sequencing analysis of the ciliary epithelium and contiguous tissues in the mouse eye.

The ciliary epithelium plays a central role in ocular homeostasis but cells of the pigmented and non-pigmented layers are difficult to isolate physically and study. Here we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptional signatures of cells harvested from the ciliary body and contiguous tissues. Microdissected tissue was dissociated by collagenase digestion and the transcriptomes of individual cells were obtained using a droplet-based scRNA-seq approach. In situ hybridization was used to verify the expression patterns of selected differentially-expressed genes. High quality transcriptomes were obtained from 10,024 cells and unsupervised clustering distinguished 22 cell types. Although efforts were made to specifically isolate the ciliary body, approximately half of the sequenced cells were derived from the adjacent retina. Cluster identities were assigned using expression of canonical markers or cluster-specific genes. The transcriptional signature of cells in the PCE and NPCE were distinct from each other and from cells in contiguous tissues. PCE cell transcriptomes were characterized by genes involved in melanin synthesis and transport proteins such as Slc4a4. Among the most differentially expressed genes in NPCE cells were those encoding members of the Zic family of transcription factors (Zic1, 2, 4), collagen XVIII (Col18a1), and corticotrophin-releasing hormone-binding protein (Crhbp). The ocular melanocyte population was distinguished by expression of the gap junction genes Gjb2 and Gjb6. Two fibroblast signatures were detected in the ciliary body preparation and shown by in situ hybridization to correspond to uveal and scleral populations. This cell atlas for the ciliary body and contiguous layers represents a useful resource that may facilitate studies into the development of the ciliary epithelium, the production of the aqueous and vitreous humors, and the synthesis of the ciliary zonule.

Let-7, Lin28 and Hmga2 Expression in Ciliary Epithelium and Retinal Progenitor Cells.

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues. Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2. Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2. Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Overlapping Immunohistochemical Features of Adenocarcinoma of the Nonpigmented Ciliary Body Epithelium and Renal Cell Carcinoma.

PURPOSE: To find immunohistochemical markers that distinguish adenocarcinoma of the nonpigmented ciliary epithelium (NPCE) from metastatic carcinoma, especially metastatic renal cell carcinoma. DESIGN: Retrospective case series. METHODS: Three cases of adenocarcinoma of the NPCE were examined histologically with hematoxylin-eosin stain and immunohistochemical stains including vimentin, AE1/AE3, Cam 5.2, CK7, PAX2, PAX8, AMACR, and CAIX. We also reviewed previously reported cases of this tumor. RESULTS: We found that the immunohistochemical profile of adenocarcinoma of the NPCE can overlap with renal cell carcinoma. Both tumors can express vimentin, cytokeratin AE1/AE3, Cam 5.2, PAX2, PAX8, and AMACR. One of the adenocarcinomas of the NPCE in our series also expressed CD10 and the renal cell carcinoma marker (RCC Ma). Carbonic anhydrase IX (CAIX) was not detected in any of the 3 tumors. CONCLUSIONS: Adenocarcinomas arising in phthisic eyes can be diagnostically challenging. We have found it particularly difficult to distinguish adenocarcinoma of the NPCE from metastatic carcinoma, especially metastatic clear cell renal cell carcinoma and papillary renal cell carcinoma. Because of the immunophenotypic overlap, most patients will require systemic workup including imaging of the kidneys to be certain of the diagnosis.

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

Progressive RPE atrophy and photoreceptor death in KIZ-associated autosomal recessive retinitis pigmentosa.

Background: To evaluate the long-term progression of autosomal recessive retinitis pigmentosa (RP) due to mutations in KIZ using multimodal imaging and a quantitative analytical approach.Methods: Whole exome sequencing (WES) and targeted capture sequencing were used to identify mutation. Fundus photography, short-wavelength autofluorescence (SW-AF), spectral-domain optical coherence tomography (SD-OCT) imaging, and electroretinography (ERG) were analyzed. Serial measurements of peripheral retinal pigment epithelium (RPE) atrophy area with SW-AF, as well as the ellipsoid zone (EZ) width using SD-OCT were performed.Results: Two homozygous variants in KIZ-a c.226C>T mutation as well as a previously unreported c.119_122delAACT mutation-were identified in four unrelated patients. Fundus examination and ERG revealed classic rod-cone dysfunction, and SD-OCT demonstrated outer retinal atrophy with centrally preserved EZ line. SW-AF imaging revealed hyperautofluorescent rings with surrounding parafoveal, mid-peripheral and widespread loss of autofluorescence. The RPE atrophy area increased annually by 4.9%. Mean annual exponential rates of decline for KIZ patients were 8.5% for visual acuity and 15.9% for 30 Hz Flicker amplitude. The average annual reduction distance of the EZ distance was 66.5 µm per year.Conclusions: RPE atrophy progresses along with a loss of photoreceptors, and parafoveal RPE hypoautofluorescence is commonly seen in KIZ-associated RP patients. KIZ-associated RP is an early-onset severe rod-cone dystrophy.

Evaluation of the iris thickness changes for the Chinese families with GPR143 gene mutations.

PURPOSE: Pathogenic variants of the G-protein coupled receptor 143 (GPR143) gene may result in Ocular albinism type I (OA1). In this study, we describe the clinical features and investigate the GPR143 gene mutations in six Chinese families with OA1 and evaluate the thickness changes of iris for the affected males and female carriers. METHODS: Families were ascertained, and patients underwent complete ophthalmologic examinations, including the best corrected visual acuity (BCVA), anterior segment of the eyes, vitreous and fundus changes. Spectral domain optical coherence tomography (SD-OCT) was used to measure the full iris thickness, the stroma/anterior border (SAB) layer, and the posterior epithelial layer (PEL) at the pupillary and ciliary regions. DNA was extracted from the peripheral blood vessels after confirmed consent information. GPR143 gene was directly sequenced by the Sanger method. RESULTS: The affected males had variable reduced visual acuity, nystagmus and macular hypoplasia. Four novel frameshift mutations and two previously reported missense/nonsense mutations in the GPR143 gene were detected in these families. The thickness of the iris was significantly reduced at the ciliary region in the affected males, compared with that in the normal controls and the female carriers. CONCLUSIONS: Pathogenic variants in the GPR143 gene may disturb the normal melanogenesis in the pigmented tissues of the eye, result in macular hypoplasia, and alter the thickness of the iris.

Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.

Danon disease presenting with early onset of hypertrophic cardiomyopathy and peripheral pigmentary retinal dystrophy in a female with a de novo novel mosaic mutation in the LAMP2 gene.

Purpose: To describe the phenotype and genotype in a young woman with Danon disease. Methods: The patient underwent an ophthalmic examination including best corrected visual acuity (BCVA), fundus photography and fundus autofluorescence (FAF), full-field electroretinography (full-field ERG), multifocal ERG, optical coherence tomography (OCT) and SAP-Humphrey 30-2 at the ages of 20 and 25. Electrooculography, fluorescein angiography (FA), indocyanine angiography and OCT angiography were performed only once. Genetic testing using a Next-Generation Sequencing panel and immunohistochemical analysis of LAMP2 protein expression were performed in the patient's explanted heart, and the patient's cardiologic and ophthalmologic records were retrospectively reviewed. Results: A de novo, novel, mosaic mutation, c.135dupA; p.(Trp46Metfs*10) was identified in exon 2 of the LAMP2 gene. Immunohistochemical investigation of the myocardium in the explanted heart revealed pronounced deficiency of LAMP2 protein in cardiomyocytes. The color photographs, FAF images and FA revealed more extensive peripheral pigmentary retinal dystrophy (PPRD) at the 5-year follow-up examination. No changes were observed in BCVA, OCT, SAP-Humphrey 30-2 or multifocal ERG findings at follow-up. Full-field ERG showed an asymmetric interocular reduction in ERG response at follow-up: the b-wave amplitude of the rod response had decreased by 29% in the right eye, but by only 6 % in the left eye. The a-wave amplitude of single-flash response had decreased by 9 % in the left eye, while it had increased by 3% in the right eye. Conclusions: Although PPRD progressed slowly, it was an important clue in the diagnosis of the life-threatening condition of Danon disease.

Calf melanin immunomodulates RPE cell attachment to extracellular matrix protein.

PURPOSE: It is widely accepted that RPE melanin has a protective effect against oxidative damage in RPE cells. It is possible that an additional protective characteristic of melanin is the ability to modulate RPE cell immune response. In this study, in vitro modeling was used to probe the relationship between RPE pigmentation and immune response by monitoring IL-6 expression and secretion in calf melanin pigmented ARPE-19 cells seeded onto glycated extracellular matrix as a stressor. METHODS: ARPE-19 cells were left unpigmented or were pigmented with either calf melanin or latex beads, and were then seeded onto RPE-derived extracellular matrix (ECM) or tissue culture-treated plates (no ECM). ECMs were modified by glycation. IL-6 expression was measured using qPCR and IL-6 secretion was determined using an ELISA, both at 30 min and 24 h after seeding. MTT assay was used to quantify cell attachment to glycated matrices 30 min after seeding. In unpigmented ARPE-19 cells, rate of cell attachment to substrate was monitored for 60 min after seeding using a hemacytometer to count unattached cells. Additionally, cell viability was evaluated using the Neutral Red assay 24 h after seeding. RESULTS: A significant increase in IL-6 expression was observed in calf melanin pigmented cells versus latex bead and unpigmented controls (p < 0.0001) 30 min after seeding onto ECM. Twenty-four hours after seeding, a significant decrease in IL-6 expression was observed in calf melanin pigmented cells (p < 0.0001) versus controls, implicating down-regulation of the cytokine. Additionally, calf melanin pigmented cell populations showed significant increase in attachment compared to unpigmented controls on either no ECM or unmodified ECM. CONCLUSIONS: Pigmentation of RPE cells with calf melanin resulted in significant changes in IL-6 expression regardless of ECM modification, in vitro. These findings suggest that melanin in the RPE may participate in immune response modulation in the retina with particular regard to cell attachment to protein substrates. The results of this study further implicate the role of chemical changes to melanin in regulating inflammation in retinal disease.

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium.

Purpose: Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. Methods: Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer. Results: Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%. Conclusions: Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

Immune Privilege and Eye-Derived T-Regulatory Cells.

Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs), which are generated by the anterior chamber-associated immune deviation (ACAID), and ocular resident cells including corneal endothelial (CE) cells, ocular pigment epithelial (PE) cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF-ß), cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2α), and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

Expression and regulation of alarmin cytokine IL-1α in human retinal pigment epithelial cells.

Human retinal pigment epithelial (hRPE) cells play important immune-regulatory roles in a variety of retinal pathologic processes, including the production of inflammatory cytokines that are essential mediators of the innate immune response within the ocular microenvironment. The pro-inflammatory "alarmin" cytokine IL-1α has been implicated in both infectious and non-infectious retinal diseases, but its regulation in the retina is poorly understood. The purpose of this study was to elucidate the expression and regulation of IL-1α within hRPE cells. To do this, IL-1α mRNA and protein in hRPE cells was assessed by RT-PCR, qPCR, ELISA, Western blot, and immunofluorescence following treatment with a variety of stimuli and inhibitors. ER stress, LPS, IL-1ß, and TLR2 activation all significantly increased intracellular IL-1α protein. Increasing intracellular calcium synergized both LPS- and Pam3CSK4-induced IL-1α protein production. Accordingly, blocking calcium signaling and calpain activity strongly suppressed IL-1α protein expression. Significant but more moderate inhibition occurred following blockage of TLR4, caspase-4, or caspase-1. Neutralizing antibodies to IL-1ß and TLR2 partially eliminated LPS- and TLR2 ligand Pam3CSK4-stimulated IL-1α protein production. IFN-ß induced caspase-4 expression and activation, and also potentiated LPS-induced IL-1α expression, but IFN-ß alone had no effect on IL-1α protein production. Interestingly, all inhibitors targeting the PI3K/Akt pathway, with the exception of Ly294002, strongly increased IL-1α protein expression. This study improves understanding of the complex mechanisms regulating IL-1α protein expression in hRPE cells by demonstrating that TLR4 and TLR2 stimulation and exposure to IL-1ß, ER stress and intracellular calcium all induce hRPE cells to produce intracellular IL-1α, which is negatively regulated by the PI3K/Akt pathway. Additionally, the non-canonical inflammasome pathway was shown to be involved in LPS-induced hRPE IL-1α expression through caspase-4 signaling.

Effect of berberine on lipopolysaccharide-induced monocyte chemotactic protein-1 and interleukin-8 expression in a human retinal pigment epithelial cell line.

PURPOSE: In this study, we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in a human retinal pigment epithelial cell line (ARPE-19) caused by lipopolysaccharide (LPS) stimulation. METHODS: ARPE-19 cells were cultured to confluence. Berberine and LPS were added to the medium. MCP-1 and IL-8 mRNA were measured by real-time polymerase chain reaction. MCP-1 and IL-8 protein concentrations in the media were measured using enzyme-linked immunosorbent assay. RESULTS: After stimulation with LPS, MCP-1 and IL-8 mRNA in ARPE-19 cells reached maximum levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached maximum levels at 24 h. Berberine dose-dependently inhibited MCP-1 and IL-8 mRNA expression of the cells and protein levels in the media stimulated with LPS. CONCLUSIONS: These findings indicate that berberine inhibited the expression of MCP-1 and IL-8 induced by LPS.

ApoA-I Mimetic Peptide 4F Reduces Age-Related Lipid Deposition in Murine Bruch's Membrane and Causes Its Structural Remodeling.

PURPOSE: Accumulation of lipoprotein-derived lipids including esterified and unesterified cholesterol in Bruch's membrane of human eyes is a major age-related change involved in initiating and sustaining soft drusen in age-related macular degeneration (AMD). The apolipoprotein (apo) A-I mimetic peptide 4F is a small anti-inflammatory and anti-atherogenic agent, and potent modifier of plasma membranes. We evaluated the effect of intravitreally-injected 4F on murine Bruch's membrane. METHODS: We tested single intravitreal injections of 4F doses (0.6 µg, 1.2 µg, 2.4 µg, and placebo scrambled peptide) in ApoEnull mice ≥10 months of age. After 30 days, mice were euthanized. Eyes were processed for either direct immunofluorescence detection of esterified cholesterol (EC) in Bruch's membrane whole mounts via a perfringolysin O-based marker linked to green fluorescent protein or by transmission electron microscopic visualization of Bruch's membrane integrity. Fluorescein isothiocyanate-conjugated 4F was traced after injection. RESULTS: All injected eyes showed a dose-dependent reduction of Bruch's membrane EC with a concomitant ultrastructural improvement compared to placebo treated eyes. At a 2.4 µg dose of 4F, EC was reduced on average by ~60% and Bruch's membrane returned to a regular pentalaminar structure and thickness. Tracer studies confirmed that injected 4F reached intraocular targets. CONCLUSION: We demonstrated a highly effective pharmacological reduction of EC and restoration of Bruch's membrane ultrastructure. The apoA-I mimetic peptide 4F is a novel way to treat a critical AMD disease process and thus represents a new candidate for treating the underlying cause of AMD.

Phenotypic heterogeneity of human retinal pigment epithelial cells in passaged cell populations.

Human retinal pigment epithelial (RPE) cells at different population doublings (PDs) were cultured for 28 days to examine their phenotypic heterogeneity in a confluent state. In an early population (PD = 2.8), cells showed a cobblestone-like appearance (type I), which gradually became small and tight, and eventually exhibited dark pigmentation. Some cells showed a dome-like structure (type II), which detached from the culture surface during culture. With increasing PD, the cells showed active migration that caused a shift in phenotype from a single layer of large, flattened cells (type III) to a multiple cell layers (stratified) with flattened, irregularly shaped cells (type IV). Immunostaining of specific RPE markers, ZO-1 and Na+/K+-ATPase revealed that cells have markedly decreased expressions in a late population (PD = 10.1). RPE phenotypes were classified into four types by measuring the nuclear size and local density. The frequencies of type I cells decreased with increasing PD value, while the frequencies of type III and IV cells increased along with the decrease in type I. The frequencies of type IV cells at PD = 10.1 had increased by 10.3-fold compared with PD = 2.8. From these results, the nuclear size and local density were proposed as indicators for understanding phenotypic heterogeneity of RPE cells in the passaged cell population during cell expansion. It is concluded that the population doubling level is an important factor to affect the transition of RPE phenotype and thereby to modulate the quality of cultured cells.

Human limbal neurospheres prevent photoreceptor cell death in a rat model of retinal degeneration.

BACKGROUND: The culture of retinal progenitors from an accessible adult stem cell source such as the limbus could provide a useful autologous source of retinal cell therapies. The human corneoscleral limbus contains multipotent stem cells that can be cultured as floating neurospheres. Previous work in rodents has demonstrated neuronal and photoreceptor differentiation from limbal neurosphere cultures. Here, this study has examined undifferentiated cultured adult human limbal neurospheres as donor cells for retinal cell therapies by transplantation into a rat model of retinal degeneration. METHODS: Gene expression in limbal neurospheres was examined by immunostaining and western blot. Human limbal neurospheres were transplanted into the subretinal space of Royal College of Surgeon's rats. Rats were monitored by optical coherence tomography for 6 weeks then processed for retinal histology. RESULTS: Human limbal neurospheres expressed the neural lineage markers, Nestin, sex determining region box-2 and N-cadherin, and the retinal transcription factors microphthalmia-associated transcription factor, sex determining region box-2 and orthodentical homeobox-2. Human limbal neurospheres could be cultured to express NeuN, neurofilament and rhodopsin. Rats receiving saline or no injection underwent complete degeneration of the retinal outer nuclear layer after 3 weeks. In contrast, rats injected with human limbal neurospheres or retinal pigment epithelial cells maintained the outer nuclear layer for up to 6 weeks. Gene expression in transplanted limbal neurospheres was inconsistent with the production of mature retinal pigment epithelial or photoreceptor cells. CONCLUSIONS: Human limbal neurospheres represent an accessible source of autologous donor cells for the treatment of retinal diseases.

Deletion of Efemp1 Is Protective Against the Development of Sub-RPE Deposits in Mouse Eyes.

Purpose: EFEMP1 (fibulin-3) is mutated in Malattia Leventinese/Doyne's honeycomb retinal dystrophy (ML/DHRD), an inherited macular dystrophy similar to AMD. Both ML/DHRD and AMD are characterized by the presence of sub-RPE deposits. Efemp1 knockout mice do not develop sub-RPE deposits. This study was to test whether sub-RPE deposits can be induced in Efemp1 knockout mice by experimentally applied stress conditions that cause wild-type mice to develop sub-RPE deposits. Methods: Efemp1 knockout and control mice at 6, 18, or 24 months old were fed with a synthetic high-fat diet (HFD). Beginning 1 month after starting the HFD, one group of mice was exposed to cigarette smoke daily for 1 month, and another group of mice was subjected to photochemical injury every other day for 2 weeks from a 488-nm argon laser. After the treatments, histologic analysis was performed to assess whether sub-RPE deposits were induced. Results: Basal laminar deposits (BLamDs), a form of sub-RPE deposits, were observed in the 18- and 24-month-old wild-type mice but not in Efemp1 knockout mice in any age groups after exposure to HFD and cigarette smoke or laser injury. Conclusions: Mice lacking fibulin-3 do not develop sub-RPE deposits. Environmental oxidative stressors (HFD/cigarette smoke or HFD/laser) known to cause BLamD formation in wild-type mice failed to induce BLamD formation in Efemp1 knockout mice. These results suggest that fibulin-3 is a central player in the development of BLamD, and deletion of fibulin-3 is protective against the development of BLamD.