Corneal Epithelial Regeneration: Old and New Perspectives.

Corneal blindness is the fifth leading cause of blindness worldwide, and therapeutic options are still often limited to corneal transplantation. The corneal epithelium has a strong barrier function, and regeneration is highly dependent on limbal stem cell proliferation and basement membrane remodeling. As a result of the lack of corneal donor tissues, regenerative medicine for corneal diseases affecting the epithelium is an area with quite advanced basic and clinical research. Surgery still plays a prominent role in the treatment of epithelial diseases; indeed, innovative surgical techniques have been developed to transplant corneal and non-corneal stem cells onto diseased corneas for epithelial regeneration applications. The main goal of applying regenerative medicine to clinical practice is to restore function by providing viable cells based on the use of a novel therapeutic approach to generate biological substitutes and improve tissue functions. Interest in corneal epithelium rehabilitation medicine is rapidly growing, given the exposure of the corneal outer layers to external insults. Here, we performed a review of basic, clinical and surgical research reports on regenerative medicine for corneal epithelial disorders, classifying therapeutic approaches according to their macro- or microscopic target, i.e., into cellular or subcellular therapies, respectively.

Bioactive Properties of Carotenoids in Human Health.

Research shows that certain bioactive compounds in our diet have beneficial effects on humanhealth[...].

Eupatilin rescues ciliary transition zone defects to ameliorate ciliopathy-related phenotypes.

Ciliopathies are clinically overlapping genetic disorders involving structural and functional abnormalities of cilia. Currently, there are no small-molecule drugs available to treat ciliary defects in ciliopathies. Our phenotype-based screen identified the flavonoid eupatilin and its analogs as lead compounds for developing ciliopathy medication. CEP290, a gene mutated in several ciliopathies, encodes a protein that forms a complex with NPHP5 to support the function of the ciliary transition zone. Eupatilin relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290. In rd16 mice harboring a blinding Cep290 in-frame deletion, eupatilin treatment improved both opsin transport to the photoreceptor outer segment and electrophysiological responses of the retina to light stimulation. The rescue effect was due to eupatilin-mediated inhibition of calmodulin binding to NPHP5, which promoted NPHP5 recruitment to the ciliary base. Our results suggest that deficiency of a ciliopathy protein could be mitigated by small-molecule compounds that target other ciliary components that interact with the ciliopathy protein.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Transient and localized optogenetic activation of somatostatin-interneurons in mouse visual cortex abolishes long-term cortical plasticity due to vision loss.

Unilateral vision loss through monocular enucleation (ME) results in partial reallocation of visual cortical territory to another sense in adult mice. The functional recovery of the visual cortex occurs through a combination of spared-eye potentiation and cross-modal reactivation driven by whisker-related, somatosensory inputs. Brain region-specific intracortical inhibition was recently recognized as a crucial regulator of the cross-modal component, yet the contribution of specific inhibitory neuron subpopulations remains poorly understood. Somatostatin (SST)-interneurons are ideally located within the cortical circuit to modulate sensory integration. Here we demonstrate that optogenetic stimulation of visual cortex SST-interneurons prior to eye removal decreases ME-induced cross-modal recovery at the stimulation site. Our results suggest that SST-interneurons act as local hubs, which are able to control the influx and extent of cortical cross-modal inputs into the deprived cortex. These insights critically expand our understanding of SST-interneuron-specific regulation of cortical plasticity induced by sensory loss.

Expression of the angiogenic mediator, angiopoietin-like 4, in the eyes of patients with proliferative sickle retinopathy.

The recent success of therapies directly targeting the angiogenic mediator, vascular endothelial growth factor (VEGF), for the treatment of proliferative diabetic retinopathy has encouraged clinicians to extend the use of anti-VEGF therapies for the treatment of another ischemic retinal vascular disease, proliferative sickle cell retinopathy (PSR), the most common cause of irreversible blindness in patients with sickle cell disease. However, results from case reports evaluating anti-VEGF therapies for PSR have been mixed. This highlights the need to identify alternative therapeutic targets for the treatment of retinal neovascularization in sickle cell patients. In this regard, angiopoietin-like 4 (ANGPTL4) is a novel angiogenic factor regulated by the transcription factor, hypoxia-inducible factor 1, the master regulator of angiogenic mediators (including VEGF) in ischemic retinal disease. In an effort to identify alternative targets for the treatment of sickle cell retinopathy, we have explored the expression of ANGPTL4 in the eyes of patients with PSR. To this end, we examined expression and localization of ANGPTL4 by immunohistochemistry in autopsy eyes from patients with known PSR (n = 5 patients). Complementary studies were performed using enzyme-linked immunosorbent assays in aqueous (n = 8; 7 patients, 2 samples from one eye of same patient) and vitreous (n = 3 patients) samples from a second group of patients with active PSR. We detected expression of ANGPTL4 in neovascular tissue and in the ischemic inner retina in PSR, but not control, eyes. We further observed elevated expression of ANGPTL4 in the aqueous and vitreous of PSR patients compared to controls. These results suggest that ANGPTL4 could contribute to the development of retinal neovascularization in sickle cell patients and could therefore be a therapeutic target for the treatment of PSR.

Stem Cell Therapies for Reversing Vision Loss.

Current clinical trials that evaluate human pluripotent stem cell (hPSC)-based therapies predominantly target treating macular degeneration of the eyes because the eye is an isolated tissue that is naturally weakly immunogenic. Here, we discuss current bioengineering approaches and biomaterial usage in combination with stem cell therapy for macular degeneration disease treatment. Retinal pigment epithelium (RPE) differentiated from hPSCs is typically used in most clinical trials for treating patients, whereas bone marrow mononuclear cells (BMNCs) or mesenchymal stem cells (MSCs) are intravitreally transplanted, undifferentiated, into patient eyes. We also discuss reported negative effects of stem cell therapy, such as patients becoming blind following transplantation of adipose-derived stem cells, which are increasingly used by 'stem-cell clinics'.

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.

Epigallocatechin-3-gallate protects retinal vascular endothelial cells from high glucose stress in vitro via the MAPK/ERK-VEGF pathway.

Diabetic retinopathy (DR) is a frequent microvascular complication of diabetes, and one of the most common causes of legal blindness in the world. Epigallocatechin-3-gallate (EGCG) produces an anti-oxidative and anti-inflammatory effect against various human diseases. In this study, we determined the effect of EGCG on a human retinal endothelial cell (HREC) line. The cell viability was determined by a standard MTT assay, while the cell cycle and apoptosis rate were analyzed by flow cytometry. Inflammatory marker expression was detected by enzyme-linked immunosorbent assay. Treatment of HRECs with EGCG (20 and 40 mM) led to a significant decrease in the apoptosis rate (2.35 ± 0.56 and 1.24 ± 0.32%). The culture supernatant of cells treated with high glucose concentrations showed significantly higher levels of TNF-α (598.7 ± 89.7 vs 193.2 ± 38.5 pg/mL; P < 0.001), IL-6 (6.16 ± 0.51 vs 1.61 ± 0.21 ng/mL; P < 0.001), and ICAM-1 (31.6 ± 4.4 vs 14.8 ± 2.9 ng/mL; P < 0.001) compared to the cells in the control group. EGCG decreased the expression level of phosphorylated p38-mitogen activated protein kinase (MAPK) and extracellular regulated kinase (ERK)1/2. Moreover, EGCG was shown to significantly inhibit the expression of vascular endothelial growth factor (VEGF). Therefore, EGCG treatment ameliorated the negative effect of high glucose concentrations on the cell viability and apoptotic rate. The protective effects of EGCG under high glucose conditions may be attributed to the regulation of inflammatory cytokines and inhibition of the MAPK/ERK-VEGF pathway.

Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis.

UNLABELLED: RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved cone-mediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations. SIGNIFICANCE STATEMENT: LCA is a severe early onset retinal dystrophy. Recent clinical trials of gene therapy have implicated the need of an alternative or combination therapy to improve cone survival and function in patients with LCA caused by RPE65 mutations. Using a mouse model carrying the most frequent LCA-associated mutation (R91W), we found that the mutant RPE65 underwent ubiquitination-dependent proteasomal degradation due to misfolding. Treatment of the mice with a chemical chaperone partially corrected stability, enzymatic activity, and subcellular localization of R91W RPE65, which was also accompanied by improvement of cone survival and vision. These findings identify an in vivo molecular pathogenic mechanism for R91W mutation and provide a feasible pharmacological approach that can delay vision loss in patients with RPE65 mutations.

Let There Be Light: Gene and Cell Therapy for Blindness.

Retinal degenerative diseases are a leading cause of irreversible blindness. Retinal cell death is the main cause of vision loss in genetic disorders such as retinitis pigmentosa, Stargardt disease, and Leber congenital amaurosis, as well as in complex age-related diseases such as age-related macular degeneration. For these blinding conditions, gene and cell therapy approaches offer therapeutic intervention at various disease stages. The present review outlines advances in therapies for retinal degenerative disease, focusing on the progress and challenges in the development and clinical translation of gene and cell therapies. A significant body of preclinical evidence and initial clinical results pave the way for further development of these cutting edge treatments for patients with retinal degenerative disorders.

Concise Review: Patient-Specific Stem Cells to Interrogate Inherited Eye Disease.

Whether we are driving to work or spending time with loved ones, we depend on our sense of vision to interact with the world around us. Therefore, it is understandable why blindness for many is feared above death itself. Heritable diseases of the retina, such as glaucoma, age-related macular degeneration, and retinitis pigmentosa, are major causes of blindness worldwide. The recent success of gene augmentation trials for the treatment of RPE65-associated Leber congenital amaurosis has underscored the need for model systems that accurately recapitulate disease. With the advent of patient-specific induced pluripotent stem cells (iPSCs), researchers are now able to obtain disease-specific cell types that would otherwise be unavailable for molecular analysis. In the present review, we discuss how the iPSC technology is being used to confirm the pathogenesis of novel genetic variants, interrogate the pathophysiology of disease, and accelerate the development of patient-centered treatments. Significance: Stem cell technology has created the opportunity to advance treatments for multiple forms of blindness. Researchers are now able to use a person's cells to generate tissues found in the eye. This technology can be used to elucidate the genetic causes of disease and develop treatment strategies. In the present review, how stem cell technology is being used to interrogate the pathophysiology of eye disease and accelerate the development of patient-centered treatments is discussed.

Dental pulp stem cells: a new cellular resource for corneal stromal regeneration.

Corneal blindness afflicts millions of individuals worldwide and is currently treated by grafting with cadaveric tissues; however, there are worldwide donor tissue shortages, and many allogeneic grafts are eventually rejected. Autologous stem cells present a prospect for personalized regenerative medicine and an alternative to cadaveric tissue grafts. Dental pulp contains a population of adult stem cells and, similar to corneal stroma, develops embryonically from the cranial neural crest. We report that adult dental pulp cells (DPCs) isolated from third molars have the capability to differentiate into keratocytes, cells of the corneal stoma. After inducing differentiation in vitro, DPCs expressed molecules characteristic of keratocytes, keratocan, and keratan sulfate proteoglycans at both the gene and the protein levels. DPCs cultured on aligned nanofiber substrates generated tissue-engineered, corneal stromal-like constructs, recapitulating the tightly packed, aligned, parallel fibrillar collagen of native stromal tissue. After injection in vivo into mouse corneal stroma, human DPCs produced corneal stromal extracellular matrix containing human type I collagen and keratocan and did not affect corneal transparency or induce immunological rejection. These findings demonstrate a potential for the clinical application of DPCs in cellular or tissue engineering therapies for corneal stromal blindness.

CRALBP supports the mammalian retinal visual cycle and cone vision.

Mutations in the cellular retinaldehyde-binding protein (CRALBP, encoded by RLBP1) can lead to severe cone photoreceptor-mediated vision loss in patients. It is not known how CRALBP supports cone function or how altered CRALBP leads to cone dysfunction. Here, we determined that deletion of Rlbp1 in mice impairs the retinal visual cycle. Mice lacking CRALBP exhibited M-opsin mislocalization, M-cone loss, and impaired cone-driven visual behavior and light responses. Additionally, M-cone dark adaptation was largely suppressed in CRALBP-deficient animals. While rearing CRALBP-deficient mice in the dark prevented the deterioration of cone function, it did not rescue cone dark adaptation. Adeno-associated virus-mediated restoration of CRALBP expression specifically in Müller cells, but not retinal pigment epithelial (RPE) cells, rescued the retinal visual cycle and M-cone sensitivity in knockout mice. Our results identify Müller cell CRALBP as a key component of the retinal visual cycle and demonstrate that this pathway is important for maintaining normal cone-driven vision and accelerating cone dark adaptation.

Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness.

Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good 'cobblestone-like' morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.

Molecular mechanisms of diabetic retinopathy, general preventive strategies, and novel therapeutic targets.

The growing number of people with diabetes worldwide suggests that diabetic retinopathy (DR) and diabetic macular edema (DME) will continue to be sight threatening factors. The pathogenesis of diabetic retinopathy is a widespread cause of visual impairment in the world and a range of hyperglycemia-linked pathways have been implicated in the initiation and progression of this condition. Despite understanding the polyol pathway flux, activation of protein kinase C (KPC) isoforms, increased hexosamine pathway flux, and increased advanced glycation end-product (AGE) formation, pathogenic mechanisms underlying diabetes induced vision loss are not fully understood. The purpose of this paper is to review molecular mechanisms that regulate cell survival and apoptosis of retinal cells and discuss new and exciting therapeutic targets with comparison to the old and inefficient preventive strategies. This review highlights the recent advancements in understanding hyperglycemia-induced biochemical and molecular alterations, systemic metabolic factors, and aberrant activation of signaling cascades that ultimately lead to activation of a number of transcription factors causing functional and structural damage to retinal cells. It also reviews the established interventions and emerging molecular targets to avert diabetic retinopathy and its associated risk factors.

Restoration of the majority of the visual spectrum by using modified Volvox channelrhodopsin-1.

We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox channelrhodopsin-1 (mVChR1), which is a chimera of Volvox channelrhodopsin-1 and Chlamydomonas channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468-640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.

Glaucomics: a call for systems diagnostics for 21(st) century ophthalmology and personalized visual health.

This article analyzes and theorizes the current knowledge silos at the intersection of omics science, ophthalmology, personalized medicine, and global visual health. Visual disorders represent one of the largest health care expenditures in the United States, costing $139 billion per year. In middle-income and industrialized countries, glaucoma is a World Health Organization priority category eye disease, known for difficulties in its early diagnosis, chronic progressive nature, and large person-to-person differences in drug efficacy and safety. A complex disease, glaucoma is best conceptualized as a syndrome displaying an ostensibly common clinical end-point, but with vastly heterogeneous molecular underpinnings and host-environment interactions. About 12% of all global blindness is attributable to glaucoma. Glaucomics is a term that we coin here so as to introduce omics science and systems diagnostics to ophthalmology, a field that can benefit enormously from personalized medicine, and which has sadly lagged behind in systems diagnostics compared to fields such as oncology. We define glaucomics as the integrated use of multi-omics and systems science approaches towards rational discovery, development, and tandem applications of diagnostics and therapeutics, for glaucoma specifically, and for personalized visual health, more broadly. We propose that glaucoma is one of the neglected lowest hanging fruits and actionable targets for omics and systems diagnostics in 21(st) century ophthalmology for the salient reasons we describe here. Additionally, we offer an analysis on two of the most pertinent neglected tropical diseases (NTDs), trachoma and river blindness, which continue to plague visual health in developing countries. We conclude with a call for research on omics applications in glaucoma and personalized visual health.

Allosteric regulation of pathologic angiogenesis: potential application for angiogenesis-related blindness.

Angiogenesis-related blindness (ARB) includes age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity, all of which are based on pathologic angiogenesis. Current treatment options such as surgery, laser photocoagulation, and steroid have shown limitations because they do not directly resolve the pathologic events in the retina. Furthermore, recently approved and developed therapeutic drugs only focus on direct inhibition of growth factors and suppression of downstream signaling molecules of activated receptor proteins by orthosteric ligands. In this regard, allosteric regulation of receptors and ligands can be a valuable mechanism in the development of novel drugs for ARB. In this review, we briefly address the clinical significance of ARB for further discussion on allosteric regulation of pathologic angiogenesis for ARB. Interestingly, key molecules in the pathogenesis of ARB can be targets for allosteric regulation, sharing characteristics as allosteric proteins. With investigation of allostery by introducing well-established models for allosteric proteins and currently published novel allosteric modulators, we discuss the potential of allosteric regulation for ARB. In conclusion, we hope that allosteric regulation of pathologic angiogenesis in ARB can open new opportunities for drug development.

Hedgehog regulates Norrie disease protein to drive neural progenitor self-renewal.

Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.