Fiber Diameter Differentially Regulates Function of Retinal Pigment and Corneal Epithelial Cells on Nanofibrous Tissue Scaffolds.

Biomaterials have significant functions as tissue scaffolds to support cells for regeneration. Nanofibrous scaffolds which mimic the architecture of the extracellular matrix are well suited to support epithelial cells for ocular tissue engineering. This study aimed at investigating the role of scaffold architecture, if any, on the response of ocular epithelial cells. Thus, we have cultured two different types of ocular epithelial cells on nanofibrous scaffolds of two different diameters to evaluate their generic and cell-specific properties. Human adult retinal pigment epithelial (ARPE-19) and human corneal epithelial (HCE-T) cells were cultured on poly(ε-caprolactone) (PCL) nanofibers of different diameters, nominally 500 and 1300 nm. Moduli of the fiber mats were marginally different at 7.4 and 11.1 kPa for 500 and 1300 nm diameter, respectively. The molecular changes in the cells in response to the different fibers were analyzed by qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, MTT assay, and SEM to assess properties such as proliferation, apoptosis, membrane potential, epithelial-mesenchymal transition, stem cell population, VEGF-A secretion, differentiation, and metabolic status of the cells. HCE-T cells revealed characteristic morphology along with higher expression of proliferation, differentiation, and lower apoptotic markers when cultured on PCL nanofibers of 500 nm. However, on nanofibers of 1300 nm, the cells showed higher expression of the corneal stem/progenitor as well as pluripotent stem cell markers. ARPE-19 cells exhibited characteristic hexagonal morphology with elevated expression levels of proliferative markers, phagocytic activity, and lower apoptosis levels. However, on 500 nm nanofibers, they expressed higher levels of pluripotent markers and secretion of VEGF-A. These findings demonstrate that the response can differ markedly from scaffold architecture even if derived from the same tissue and originating from the same germ layer. Furthermore, it paves the way for a target specific outcome and, thereby, for personalized translational medicine.

Dysregulated Tear Fluid Nociception-Associated Factors, Corneal Dendritic Cell Density, and Vitamin D Levels in Evaporative Dry Eye.

Purpose: The purpose of this study was to study the status and association among tear-soluble factors, corneal dendritic cell density, vitamin D, and signs and symptoms in dry eye disease (DED). Methods: A total of 33 control subjects and 47 evaporative dry eye patients were included in the study. DED diagnosis and classification was based on the 2017 Report of the Tear Film & Ocular Surface Society International Dry Eye Workshop (TFOS DEWS II). DED workup, including tear film break-up time (TBUT), Schirmer's test I (STI), corneal and conjunctival staining, ocular surface disease index (OSDI) scoring, and in vivo confocal microscopy (to assess corneal dendritic cell density [cDCD] and subbasal nerve plexus [SBNP] features) was performed in the study subjects. Tear fluid using Schirmer's strip and serum were collected from the subjects. Multiplex ELISA or single analyte ELISA was performed to measure 34 tear-soluble factors levels including vitamin D. Results: Significantly higher OSDI discomfort score, lower TBUT, and lower STI were observed in DED patients. cDCD was significantly higher in DED patients. No significant difference was observed in SBNP features. Tear fluid IL-1ß, IL-17A, MMP9, MMP10, MMP9/TIMP ratio, and VEGF-B were significantly higher in DED patients. Significantly lower tear fluid IL-2, IP-10, NPY, VEGF-A, and vitamin D was observed in DED patients. These dysregulated tear factors showed significant associations with DED signs and symptoms. Conclusions: Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with ocular surface discomfort status, TBUT, STI, and cDCD. This implies a functional relationship between the various tear-soluble factors and dry eye pathogenesis, indicating new molecular targets for designing targeted therapies.

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.

Retinal pigment epithelium-secretome: A diabetic retinopathy perspective.

Diabetic retinopathy is a major complication of diabetes mellitus that can lead to retinal vascular abnormalities and visual impairment. While retinal endothelial pathology is well studied, retinal pigment epithelium (RPE) layer modifications and the patho-physiological regulations are not widely understood. The RPE is a highly specialized pigmented layer regulating not only physiological functions such as transport of nutrients, ions, absorption of light, phagocytosis of photoreceptor membranes, but also secretion of a number of cytokines, chemokines, angiogenic and anti-angiogenic factors. The RPE secretome, though crucial in health and disease, remains elusive in diabetic retinopathy. A knowledge of these secreted factors would help explain and correlate the clinical phase of the disease aiding in improved disease management. A comprehensive knowledge of the secreted factors of the RPE is a potential tool for understanding the differential treatment regime of early diabetic retinopathy, diabetic proliferative retinopathy and diabetic macular edema. In this review, we have delineated the importance of factors secreted by the retinal pigment epithelium and its regulation in the pathogenesis of diabetic retinopathy.

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.

Ocular stem cells: a status update!

Stem cells are unspecialized cells that have been a major focus of the field of regenerative medicine, opening new frontiers and regarded as the future of medicine. The ophthalmology branch of the medical sciences was the first to directly benefit from stem cells for regenerative treatment. The success stories of regenerative medicine in ophthalmology can be attributed to its accessibility, ease of follow-up and the eye being an immune-privileged organ. Cell-based therapies using stem cells from the ciliary body, iris and sclera are still in animal experimental stages but show potential for replacing degenerated photoreceptors. Limbal, corneal and conjunctival stem cells are still limited for use only for surface reconstruction, although they might have potential beyond this. Iris pigment epithelial, ciliary body epithelial and choroidal epithelial stem cells in laboratory studies have shown some promise for retinal or neural tissue replacement. Trabecular meshwork, orbital and sclera stem cells have properties identical to cells of mesenchymal origin but their potential has yet to be experimentally determined and validated. Retinal and retinal pigment epithelium stem cells remain the most sought out stem cells for curing retinal degenerative disorders, although treatments using them have resulted in variable outcomes. The functional aspects of the therapeutic application of lenticular stem cells are not known and need further attention. Recently, embryonic stem cell-derived retinal pigment epithelium has been used for treating patients with Stargardts disease and age-related macular degeneration. Overall, the different stem cells residing in different components of the eye have shown some success in clinical and animal studies in the field of regenerative medicine.