Corneal stromal regeneration-keratoconus cell therapy: a review.

BACKGROUND: Keratoconus is a corneal ectatic disease caused by stromal thinning leading to astigmatism and progressive loss of vision. Loss of the keratocytes and excessive degradation of collagen fibres by matrix metalloproteinases are the molecular signatures of the disease. Despite several limitations, corneal collagen cross-linking and keratoplasty are the most widely used treatment options for keratoconus. In the pursuit of alternative treatment modalities, clinician scientists have explored cell therapy paradigms for treating the condition. METHODS: Articles pertaining to keratoconus cell therapy with relevant key words were used to search in PubMed, Researchgate, and Google Scholar. The articles were selected based on their relevance, reliability, publication year, published journal, and accessibility. RESULTS: Various cellular abnormalities have been reported in keratoconus. Diverse cell types such as mesenchymal stromal cells, dental pulp cells, bone marrow stem cells, haematopoietic stem cells, adipose-derived stem cells apart from embryonic and induced pluripotent stem cells can be used for keratoconus cell therapy. The results obtained show that there is a potential for these cells from various sources as a viable treatment option. CONCLUSION: There is a need for consensus with respect to the source of cells, mode of delivery, stage of disease, and duration of follow-up, to establish a standard operating protocol. This would eventually widen the cell therapy options for corneal ectatic diseases beyond keratoconus.

Autophagy in dry eye disease: Therapeutic implications of autophagy modulators on the ocular surface.

Dry eye disease (DED) is a chronic ocular surface disorder, associated with inflammation, which can cause severe morbidity, visual compromise, and loss of quality of life, affecting up to 5-50% of the world population. In DED, ocular surface damage and tear film instability due to abnormal tear secretion lead to ocular surface pain, discomfort, and epithelial barrier disruption. Studies have shown the involvement of autophagy regulation in dry eye disease as a pathogenic mechanism along with the inflammatory response. Autophagy is a self-degradation pathway in mammalian cells that reduces the excessive inflammation driven by the secretion of inflammatory factors in tears. Specific autophagy modulators are already available for the management of DED currently. However, growing studies on autophagy regulation in DED might further encourage the development of autophagy modulating drugs that reduce the pathological response at the ocular surface. In this review, we summarize the role of autophagy in the pathogenesis of dry eye disease and explore its therapeutic application.

Rare eye diseases in India: A concise review of genes and genetics.

Rare eye diseases (REDs) are mostly progressive and are the leading cause of irreversible blindness. The disease onset can vary from early childhood to late adulthood. A high rate of consanguinity contributes to India's predisposition to RED. Most gene variations causing REDs are monogenic and, in some cases, digenic. All three types of Mendelian inheritance have been reported in REDs. Some of the REDs are related to systemic illness with variable phenotypes in affected family members. Approximately, 50% of the children affected by REDs show associated phenotypes at the early stages of the disease. A precise clinical diagnosis becomes challenging due to high clinical and genetic heterogeneity. Technological advances, such as next-generation sequencing (NGS), have improved genetic and genomic testing for REDs, thereby aiding in determining the underlying causative gene variants. It is noteworthy that genetic testing together with genetic counseling facilitates a more personalized approach in the accurate diagnosis and management of the disease. In this review, we discuss REDs identified in the Indian population and their underlying genetic etiology.

Generation of iPSC line (GLNNFi001-A) from peripheral blood mononuclear cells of a patient with macular corneal dystrophy.

Macular corneal dystrophy (MCD) an autosomal recessive disorder leading to severe visual impairment is caused by mutations in the carbohydrate sulfotransferase 6 (CHST6) gene. Here, we describe generation of a iPSC line from peripheral blood mononuclear cells (PBMCs) from a 38 year old MCD patient harboring a homozygous mutation, p.V172L in the CHST6 gene. The iPSC line GLNNFi001-A was generated using an integration-free Sendai virus containing (KLF4, c-MYC, OCT4 and SOX2).

Retinoblastoma genetics screening and clinical management.

BACKGROUND: India accounts for 20% of the global retinoblastoma (RB) burden. However, the existing data on RB1 gene germline mutations and its influence on clinical decisions is minimally explored. METHODS: Fifty children with RB underwent complete clinical examination and appropriate multidisciplinary management. Screening of germline RB1 gene mutations was performed through next-generation sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. The mutation and non-mutation groups were compared for clinical parameters especially severity, progression and recurrence. RESULTS: Twenty-nine patients had bilateral RB (BLRB) and 21 had unilateral RB (ULRB). The genetic analysis revealed 20 RB1 variations in 29 probands, inclusive of 3 novel mutations, known 16 mutations and heterozygous whole gene deletions. The mutation detection rate (MDR) was 86.2% in BLRB and 19% in ULRB. Associations of disease recurrence (p = 0.021), progression (p = 0.000) and higher percentage of optic nerve invasion, subretinal seeds and high-risk pathological factors were observed in the mutation group. Clinical management was influenced by the presence of germline mutations, particularly while deciding on enucleation, frequency of periodic follow up and radiotherapy. CONCLUSIONS: We identified novel RB1 mutations, and our mutation detection rate was on par with the previous global studies. In our study, genetic results influenced clinical management and we suggest that it should be an essential and integral component of RB-care in India and elsewhere.

Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults.

Purpose: Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. Methods: Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O2) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. Results: Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. Conclusions: VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult. These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.

Trehalose augments autophagy to mitigate stress induced inflammation in human corneal cells.

PURPOSE: Cornea acts as a structural barrier and protects the eye from environmental stresses. Inflammation in ocular surface causes discomfort and visual distortion. Defective autophagy has been associated with inflammation and ocular surface diseases. Therefore, we explored the protective role of trehalose on inflammation and desiccation-triggered stress in human corneal cells in vitro and in dry eye patients. METHOD: TNF-α and desiccation stress induced human corneal cells (piHCF and HCE-T) with or without trehalose treatment were analyzed for the expression levels of inflammatory and autophagy related markers by qPCR, western blotting, multiplex ELISA and fluorescence imaging. Dry eye patients (N = 9) were enrolled and administered with trehalose in one eye and carboxymethylcellulose (CMC) in the contralateral eye (B.I.D, for 30 days). Dry eye signs OSDI, TBUT, Schirmer's Test, and tear cytokines were measured in dry eye patient's pre and post treatment. RESULTS: Cells treated with trehalose exhibits increased levels of autophagy markers LC3II and LAMP1 compared to untreated cells. Trehalose reduced the mRNA and secreted cytokines levels of IL-6, IL-8 and MCP-1 in corneal cells under TNF-α and desiccation stress mediated inflammation compared to controls. Further, trehalose reduced stress driven p38 phosphorylation in corneal cells. Additionally, topical administration of trehalose alleviated the clinical symptoms and tears cytokine levels in dry eye patients compared to CMC. CONCLUSION: Trehalose reduces stress induced inflammation through p38MAPK inhibition and autophagy activation. The anti-inflammatory mechanism of trehalose was independent to NFκB pathway. Further, topical administration of trehalose ameliorated dry eye associated symptoms and associated tear cytokines levels.

Autophagy in corneal health and disease: A concise review.

Autophagy is a well-conserved self-eating mechanism of cell survival during periods of nutrient deprivation, stress and injury. Autophagy is implicated in many pathophysiological conditions across all organ systems. The cornea is an avascular transparent tissue that is prone to damage by trauma, injury and infection. Following insult, the cornea undergoes a complex wound healing process, which is regulated by multiple factors including autophagy. The involvement of autophagy in keratoconus and HSV-1 infection has been demonstrated, underlining the importance of this mechanism in corneal disorders. However, the role of autophagy in corneal wound repair, fibrosis and angiogenesis is still unclear. Recently, we characterized the expression of autophagy-related genes in cornea and are studying their role in the modulation of corneal conditions including fibrosis and dystrophies. Preliminary results presented within this review article support further investigation of the dynamic modulation of autophagy-related genes in corneal health and disease. This article provides an overview of how autophagy modulates corneal function.

Protective Role of Decellularized Human Amniotic Membrane from Oxidative Stress-Induced Damage on Retinal Pigment Epithelial Cells.

Oxidative stress is an important cause for several retinal aging diseases. Cell therapy using a decellularized human amniotic membrane (dHAM) as a tissue scaffold for retinal pigment epithelial cells has a potential therapeutic role under such pathological conditions. This is attributed by the anti-inflammatory, antimicrobial, low-immunogenicity aspects of dHAM, apart from harboring a drug reservoir potential. The underlying mechanisms for maintaining the physiological properties of transplanted cells and their survival in a diseased milieu using dHAM has remained unexplored/unanswered. Hence, we investigated the potential role of dHAM in preserving the cellular functions of retinal pigment epithelium in an oxidative stress environment. Adult human retinal pigment epithelial (ARPE-19) cells were cultured on dHAM or tissue culture dishes under hyperoxia. Gene expression, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), and scanning electron microscopy (SEM) were performed to assess the levels of reactive oxygen species, proliferation, apoptosis, epithelial-mesenchymal transition, phagocytosis, and secretion of vascular endothelial factors. These results indicate reduced epithelial-mesenchymal transition, generation of reactive oxygen species (p ≤ 0.0001), and apoptosis (p ≤ 0.05) in cells cultured on dHAM, compared to those on tissue culture dishes under oxidative stress conditions. Concomitantly, the secretion of the vascular endothelial growth factor was significantly reduced (p ≤ 0.01) on dHAM. Phagocytic activity was significantly higher (p ≤ 0.001) in cells cultured on dHAM and were comparable to those cells cultured on tissue culture dishes. SEM images showed a clustered growth pattern on dHAM compared to an elongated morphology when cultured on tissue culture dishes under oxidative stress conditions. These findings demonstrate the utility of dHAM as a scaffold for growing retinal epithelial cells and to maintain their physiological properties in an oxidative stress condition with a potential to develop regenerative medicine strategies to treat degenerative eye diseases.

Chloroquine Protects Human Corneal Epithelial Cells from Desiccation Stress Induced Inflammation without Altering the Autophagy Flux.

Dry eye disease (DED) is a multifactorial ocular surface disorder affecting millions of individuals worldwide. Inflammation has been associated with dry eye and anti-inflammatory drugs are now being targeted as the alternate therapeutic approach for dry eye condition. In this study, we have explored the anti-inflammatory and autophagy modulating effect of chloroquine (CQ) in human corneal epithelial and human corneal fibroblasts cells exposed to desiccation stress, (an in-vitro model for DED). Gene and protein expression profiling of inflammatory and autophagy related molecular factors were analyzed in HCE-T and primary HCF cells exposed to desiccation stress with and without CQ treatment. HCE-T and HCF cells exposed to desiccation stress exhibited increased levels of activated p65, TNF-α, MCP-1, MMP-9, and IL-6. Further, treatment with CQ decreased the levels of active p65, TNF-α, MCP-1, and MMP-9 in cells underdesiccation stress. Increased levels of LC3B and LAMP1 markers in HCE-T cells exposed to desiccation stress suggest activation of autophagy and the addition of CQ did not alter these levels. Changes in the phosphorylation levels of MAPKinase and mTOR pathway proteins were found in HCE-T cells under desiccation stress with or without CQ treatment. Taken together, the data suggests that HCE-T cells under desiccation stress showed NFκB mediated inflammation, which was rescued through the anti-inflammatory effect of CQ without altering the autophagy flux. Therefore, CQ may be used as an alternate therapeutic management for dry eye condition.

Resveratrol reverses the adverse effects of bevacizumab on cultured ARPE-19 cells.

Age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR) are one of the major causes of blindness caused by neo-vascular changes in the retina. Intravitreal anti-VEGF injections are widely used in the treatment of wet-AMD and PDR. A significant percentage of treated patients have complications of repeated injections. Resveratrol (RES) is a polyphenol phytoalexin with anti-oxidative, anti-inflammatory and anti-proliferative properties. Hence, we hypothesized that if RES is used in combination with bevacizumab (BEV, anti-VEGF), it could reverse the adverse effects that precipitate fibrotic changes, drusen formation, tractional retinal detachment and so on. Human retinal pigment epithelial cells were treated with various combinations of BEV and RES. There was partial reduction in secreted VEGF levels compared to untreated controls. Epithelial-mesenchymal transition was lower in BEV + RES treated cultures compared to BEV treated cultures. The proliferation status was similar in BEV + RES as well as BEV treated cultures both groups. Phagocytosis was enhanced in the presence of BEV + RES compared to BEV. Furthermore, we observed that notch signaling was involved in reversing the adverse effects of BEV. This study paves way for a combinatorial strategy to treat as well as prevent adverse effects of therapy in patients with wet AMD and PDR.

SYNGAP1: Mind the Gap.

A cardinal feature of early stages of human brain development centers on the sensory, cognitive, and emotional experiences that shape neuronal-circuit formation and refinement. Consequently, alterations in these processes account for many psychiatric and neurodevelopmental disorders. Neurodevelopment disorders affect 3-4% of the world population. The impact of these disorders presents a major challenge to clinicians, geneticists, and neuroscientists. Mutations that cause neurodevelopmental disorders are commonly found in genes encoding proteins that regulate synaptic function. Investigation of the underlying mechanisms using gain or loss of function approaches has revealed alterations in dendritic spine structure, function, and plasticity, consequently modulating the neuronal circuit formation and thereby raising the possibility of neurodevelopmental disorders resulting from synaptopathies. One such gene, SYNGAP1 (Synaptic Ras-GTPase-activating protein) has been shown to cause Intellectual Disability (ID) with comorbid Autism Spectrum Disorder (ASD) and epilepsy in children. SYNGAP1 is a negative regulator of Ras, Rap and of AMPA receptor trafficking to the postsynaptic membrane, thereby regulating not only synaptic plasticity, but also neuronal homeostasis. Recent studies on the neurophysiology of SYNGAP1, using Syngap1 mouse models, have provided deeper insights into how downstream signaling proteins and synaptic plasticity are regulated by SYNGAP1. This knowledge has led to a better understanding of the function of SYNGAP1 and suggests a potential target during critical period of development when the brain is more susceptible to therapeutic intervention.

Genetic analysis and clinical phenotype of two Indian families with X-linked choroideremia.

PURPOSE: This study aims to describe the phenotype and genotype of two Indian families affected with X-linked choroideremia (CHM). MATERIALS AND METHODS: In these two families, the affected individuals and unaffected family members underwent a comprehensive ophthalmic examination including an optical coherence tomography (OCT) and electroretinogram. Blood samples were collected from the families for genetic analysis. Next generation sequencing (NGS) was done using a panel of 184 genes, which covered previously associated genes with retinal dystrophies. Sequencing data were analyzed for the CHM, RPGR, and RP2 genes that have been implicated in CHM and X-linked retinitis pigmentosa (XLRP), respectively. The identified variants were confirmed by Sanger sequencing in available individuals and unrelated controls. RESULTS: In two unrelated male patients, NGS analysis revealed a previously reported 3'-splice site change c.820-1G>C in the CHM gene in the first family and hemizygous mutation c.653G>C (p.Ser218X) in the second family. The asymptomatic family members were carriers for these mutations. Spectral domain-OCT showed loss of outer retina, preservation of the inner retina, and choroidal thinning in the affected males and retinal pigment epithelial changes in the asymptomatic carriers. The identified mutations were not present in 100 controls of Indian origin. There were no potential mutations found in XLRP-associated (RPGR and RP2) genes. CONCLUSION: This report describes the genotype and phenotype findings in patients with CHM from India. The identified genetic mutation leads to lack of Rab escort protein-1 (REP-1) or affects the production of a REP-1 protein that is likely to cause retinal abnormalities in patients.

Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal cells: A comparative study with implications in transplantation medicine.

PURPOSE: Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, chemical, or radiation burns or by diseases (aniridia and Stevens-Johnson syndrome). Autologous cell transplantation is a widely used therapeutic modality for restoring the corneal surface in such pathological conditions. Ex vivo cultured limbal, conjunctival, and oral biopsies have been widely used to reconstruct the corneal surface with variable outcomes. Culture characterization of the ex vivo cultured cells would provide insight and clues into the underlying signaling mechanisms that would aid in determining the probable transplantation outcome. Comparison of the vital proteins and genes among the three ex vivo cultured tissues has implications in clinical practice. To address this issue, we characterized and compared the proliferative and differentiated properties of ex vivo cultured limbal, conjunctival, and oral biopsies used for cell-based therapy for corneal surface restoration. METHODS: Limbal, conjunctival, and oral biopsies were collected with informed patient consent. Explant cultures were established on the denuded human amniotic membrane with corneal lineage differentiation medium. The day 14 cultures were characterized for epithelial and corneal lineage-specific markers using reverse transcription (RT)-PCR for cytokeratin 3, 4, 12, 13, 15, connexin 43, vimentin, p63α, and ABCG2 markers. mRNA expression was estimated in day 14 cultures with real-time quantitative real time (qRT)-PCR for pluripotency markers (OCT4, SOX2, NANOG), putative corneal stem cell markers (ABCG2 and p63α), proliferation markers (cyclin d1, Ki-67, PCNA, and CDC20), apoptotic markers (BCL2, BAX, caspase 3, and caspase 9), Notch signaling pathway markers (Notch1, Jagged1, Hes1, Hes3, Hes5, and Hey1), and autophagic markers (LC3A, LC3B, ATG7, RAB7, LAMP1, and LAMP2). Fluorescence-activated cell sorter profiling was performed for pluripotent markers and putative corneal stem cell markers ABCG2 and p63α. RESULTS: The protein and mRNA expression levels of the pluripotent markers were lower, whereas those of the putative stem/progenitor markers ABCG2, ΔNp63α, and Notch signaling molecules (Notch1 and Jagged1) were elevated in limbal cultures. The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures. CONCLUSIONS: In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation. This implicates the probable factors that might drive a successful transplantation. Our findings provide the initial steps toward understanding transplantation medicine in an ex vivo model.

Identification of Novel Mutations in ABCA4 Gene: Clinical and Genetic Analysis of Indian Patients with Stargardt Disease.

Stargardt disease (STGD) is the leading cause of juvenile macular degeneration associated with progressive central vision loss, photophobia, and colour vision abnormalities. In this study, we have described the clinical and genetic features of Stargardt patients from an Indian cohort. The next generation sequencing was carried out in five clinically confirmed unrelated patients and their family members using a gene panel comprising 184 retinal specific genes. Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation. Genetic analysis revealed ABCA4 mutations in all of the five unrelated patients. Among these, four patients were found with compound heterozygous mutations and another one had homozygous mutation. All the affected individuals showed signs and symptoms consistent with the disease phenotype. We report two novel ABCA4 mutations in Indian patients with STGD disease, which expands the existing spectrum of disease-causing variants and the understanding of phenotypic and genotypic correlations. Screening for causative mutations in patients with STGD using panel of targeted gene sequencing by NGS would be a cost effective tool, might be helpful in confirming the precise diagnosis, and contributes towards the genetic counselling of asymptomatic carriers and isolated patients.

MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch.

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30mmHg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.

Genetic and genomic perspective to understand the molecular pathogenesis of keratoconus.

Keratoconus (KC; Mendelian Inheritance in Man (OMIM) 14830) is a bilateral, progressive corneal defect affecting all ethnic groups around the world. It is the leading cause of corneal transplantation. The age of onset is at puberty, and the disorder is progressive until the 3 rd -4 th decade of life when it usually arrests. It is one of the major ocular problems with significant social and economic impacts as the disease affects young generation. Although genetic and environmental factors are associated with KC, but the precise etiology is still elusive. Results from complex segregation analysis suggests that genetic abnormalities may play an essential role in the susceptibility to KC. Due to genetic heterogeneity, a recent study revealed 17 different genomic loci identified in KC families by linkage mapping in various populations. The focus of this review is to provide a concise update on the current knowledge of the genetic basis of KC and genomic approaches to understand the disease pathogenesis.