Effect of position-specific single-point mutations and biophysical characterization of amyloidogenic peptide fragments identified from lattice corneal dystrophy patients.

Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor ß-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble protein deposits. Using mass spectrometry, we identified increased abundance of a 32 amino acid-long peptide in the 4th fasciclin-like domain-1 (FAS-1) domain of transforming growth factor ß-induced protein (amino acid 611-642) in the amyloid deposits of the patients with lattice corneal dystrophies (LCD). In vitro studies demonstrated that the peptide readily formed amyloid fibrils under physiological conditions. Clinically relevant substitution (M619K, N622K, N622H, G623R and H626R) of the truncated peptide resulted in profound changes in the kinetics of amyloid formation, thermal stability of the amyloid fibrils and cytotoxicity of fibrillar aggregates, depending on the position and the type of the amino acid substitution. The results suggest that reduction in the overall net charge, nature and position of cationic residue substitution determines the amyloid aggregation propensity and thermal stability of amyloid fibrils.

Corneal lenticule storage before reimplantation.

Purpose: To explore the optimal lenticule storage conditions that maintain lenticule integrity and clarity. Methods: A total of 99 lenticules obtained from myopic patients undergoing small incision lenticule extraction (SMILE) were divided into four combinations for short-term storage conditions: PBS, Dulbecco's Modified Eagle's Medium (DMEM), Optisol GS, or anhydrous glycerol. Two thirds of the lenticules were further stored for 4 weeks under eight different conditions. Clarity evaluation with transmittance measurements, cell-death assays with terminal deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL), collagen fibril spacing and necrotic response assessed with transmission electron microscopy (TEM), and immunohistochemistry analysis for human leukocyte antigens (HLAs) and CD45 for immunogenicity, and matrix metalloproteinase (MMP)-2 for keratocyte response, were undertaken at baseline, 48 h (short term), and 4 weeks (long term). Results: The TUNEL and immunogenicity results were comparable among the groups. The mean percentage of TUNEL-positive cells across all groups was 24.3% ± 11.8% and 62.9% ± 20.7% at the 48 h and 4 week time points, respectively. HLA-ABC+, HLA-DR+, and CD45+ cells were extremely rare, and MMP-2 expression ranged from non-detectable to minimal, under all conditions at all time points. Transmittance at 4 weeks was significantly different among groups with the greatest maintenance of clarity seen in the lenticules stored initially in DMEM at 4 °C for 48 h followed by cryopreservation in serum-free medium or glycerol at 4 °C followed by storage at room temperature. At TEM analysis at 4 weeks, the lenticules cryopreserved in liquid nitrogen, regardless of storage solutions, had significantly narrower inter-fibrillar distance than controls, while glycerol-preserved lenticules, at either room temperature or -80 °C, maintained the inter-fibrillar distance. Conclusions: Clarity, structural integrity, and low immunogenicity under various conditions, at 4 °C or room temperature for short-term storage, offer encouragement for lenticule storage. It can be undertaken without access to s specialized and potentially expensive laboratory setup at least within the first 48 h before transportation to larger facilities for long-term storage.

Dental stem cells: a future asset of ocular cell therapy.

Regenerative medicine using patient's own stem cells (SCs) to repair dysfunctional tissues is an attractive approach to complement surgical and pharmacological treatments for aging and degenerative disorders. Recently, dental SCs have drawn much attention owing to their accessibility, plasticity and applicability for regenerative use not only for dental, but also other body tissues. In ophthalmology, there has been increasing interest to differentiate dental pulp SC and periodontal ligament SC (PDLSC) towards ocular lineage. Both can commit to retinal fate expressing eye field transcription factors and generate rhodopsin-positive photoreceptor-like cells. This proposes a novel therapeutic alternative for retinal degeneration diseases. Moreover, as PDLSC shares similar cranial neural crest origin and proteoglycan secretion with corneal stromal keratoctyes and corneal endothelial cells, this offers the possibility of differentiating PDLSC to these corneal cell types. The advance could lead to a shift in the medical management of corneal opacities and endothelial disorders from highly invasive corneal transplantation using limited donor tissue to cell therapy utilizing autologous cells. This article provides an overview of dental SC research and the perspective of utilizing dental SCs for ocular regenerative medicine.

Release of frustration drives corneal amyloid disaggregation by brain chaperone.

TGFBI-related corneal dystrophy (CD) is characterized by the accumulation of insoluble protein deposits in the corneal tissues, eventually leading to progressive corneal opacity. Here we show that ATP-independent amyloid-ß chaperone L-PGDS can effectively disaggregate corneal amyloids in surgically excised human cornea of TGFBI-CD patients and release trapped amyloid hallmark proteins. Since the mechanism of amyloid disassembly by ATP-independent chaperones is unknown, we reconstructed atomic models of the amyloids self-assembled from TGFBIp-derived peptides and their complex with L-PGDS using cryo-EM and NMR. We show that L-PGDS specifically recognizes structurally frustrated regions in the amyloids and releases those frustrations. The released free energy increases the chaperone's binding affinity to amyloids, resulting in local restructuring and breakage of amyloids to protofibrils. Our mechanistic model provides insights into the alternative source of energy utilized by ATP-independent disaggregases and highlights the possibility of using these chaperones as treatment strategies for different types of amyloid-related diseases.

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp.

Protein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer's, Parkinson's and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitro TGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611-633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611-633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.

Descemet Membrane Endothelial Keratoplasty With a Pull-Through Insertion Device: Surgical Technique, Endothelial Cell Loss, and Early Clinical Results.

PURPOSE: To describe a surgical technique for Descemet membrane endothelial keratoplasty (DMEK) using a pull-through, endothelium-in insertion device, the DMEK EndoGlide. We evaluated the endothelial cell loss (ECL) associated with the EndoGlide-DMEK (E-DMEK) technique in both ex vivo and prospective clinical studies. METHODS: The ex vivo study involved calcein acetoxymethyl staining and preparation of DMEK grafts, which were trifolded endothelium-in, loaded into the EndoGlide, pulled through, and unfolded in imaging dishes. Inverted fluorescent microscopy was performed, and ECL was quantified using trainable segmentation software. The prospective clinical series describes the outcomes of consecutive surgeries using the E-DMEK technique. Grafts were pulled through the EndoGlide with forceps and unfolded in the anterior chamber endothelium-down. Our main outcome measure was ECL in both studies. RESULTS: In the ex vivo study with 9 human donor corneas, mean ECL was 15.2% ± 5.4% (n = 9). In our clinical series of 69 eyes, leading indications for surgery were pseudophakic/aphakic bullous keratopathy (47.8%), previous failed grafts (23.2%), and Fuchs endothelial dystrophy (18.8%). Rebubbling and primary graft failure rates related to E-DMEK were 11.6% and 1.5%, respectively. Among eyes with at least 6 months of follow-up, mean preoperative endothelial cell density was 2772 (range 2457-3448) cells/mm, and postoperative endothelial cell density was 1830 (range 541-2545) cells/mm. Mean ECL was 33.6% (range 7.5-80.4; n = 32) at the 7.1 (range 6-11) months follow-up. CONCLUSIONS: The ex vivo and pilot clinical studies suggest that E-DMEK shows acceptable rates of ECL, with safe and promising early clinical outcomes.

Lamellar Dissection Technique for Descemet Membrane Endothelial Keratoplasty Graft Preparation.

PURPOSE: To describe a novel lamellar dissection technique for Descemet membrane endothelial keratoplasty (DMEK) graft preparation, and to evaluate the rate of endothelial cell loss (ECL) and graft preparation failure associated with this technique. METHODS: We conducted an ex vivo laboratory-based study comparing ECL between the lamellar dissection and peeling techniques. Eight pairs of human donor corneas underwent calcein acetoxymethyl staining-all right eyes underwent the peeling technique and all left eyes underwent the lamellar dissection technique. ECL was quantified by image analysis with trainable segmentation software and compared between groups. We also conducted a retrospective analysis of 161 consecutive DMEK graft preparations by a single surgeon using the lamellar dissection technique from 2010 to 2018. Data on donor characteristics and graft preparation failures were obtained. RESULTS: Baseline donor characteristics were comparable in both arms of the laboratory-based study. Mean (SD) ECL with the lamellar dissection and peeling techniques was 13.8% (4.2%) and 11.2% (6.1%), respectively. There was no significant difference between the two (P = 0.327). In the clinical series, there were 2 graft preparation failures in 161 cases (1.2%). Among cases performed on diabetic donor tissue, the rate of graft preparation failure was 4.7%. CONCLUSIONS: The lamellar dissection technique has a similar rate of ECL compared with the peeling technique for DMEK graft preparation. This technique also has a low rate of graft preparation failure and may be a useful technique for diabetic donor tissue.

Nanofibrous substrates support colony formation and maintain stemness of human embryonic stem cells.

Inadequate cell numbers in culture is one of the hurdles currently delaying the application of human embryonic stem cells (hESCs) for transplantation therapy. Nanofibrous scaffolds have been effectively used to expand and differentiate non-colony forming multipotent mesenchymal stem cells (MSC) for the repair of tissues or organs. In the present study, we evaluated the influence of nanofibrous scaffolds for hESC proliferation, increase in colony formation, self-renewal properties, undifferentiation and retention of 'stemness'. Polycaprolactone/collagen (PCL/collagen) and PCL/gelatin nanofibrous scaffolds were fabricated using electrospinning technology. The hESCs were seeded on the nanofibrous scaffolds in the presence or absence of mitomycin-C treated mouse embryonic fibroblasts (MEFs). The hESCs grown on both scaffolds in the presence of the MEFs produced an increase in cell growth of 47.58% (P

Advances in corneal cell therapy.

Corneal integrity is essential for visual function. Transplantation remains the most common treatment option for advanced corneal diseases. A global donor material shortage requires a search for alternative treatments. Different stem cell populations have been induced to express corneal cell characteristics in vitro and in animal models. Yet before their application to humans, scientific and ethical issues need to be solved. The in vitro propagation and implantation of primary corneal cells has been rapidly evolving with clinical practices of limbal epithelium transplantation and a clinical trial for endothelial cells in progress, implying cultivated ocular cells as a promising option for the future. This review reports on the latest developments in primary ocular cell and stem cell research for corneal therapy.

Endothelial approach ultrathin corneal grafts prepared by femtosecond laser for descemet stripping endothelial keratoplasty.

PURPOSE: To investigate the quality of the ultrathin corneal grafts prepared by femtosecond laser from the endothelial side for Descemet stripping endothelial keratoplasty. METHODS: Thirty human corneoscleral buttons were cut from the endothelial side by laser Doppler velocimetry (LDV) with or without viscoelastic materials coating. Two cutting depths were selected: 70 and 90 µm. The postcut endothelial count was determined by specular microscopy, and the graft thickness was evaluated by anterior segment optical coherence tomography. The endothelial viability was determined using Trypan blue/Alizarin red staining, calcein-AM/EthD-1 live/dead cell assay, and scanning electron microscope (SEM). The graft interface smoothness was evaluated by SEM. Another 18 corneoscleral buttons were used as controls for the comparisons. RESULTS: The overall targeted cutting depth and achieved cutting depth were significantly highly correlated (r = 0.84). The central to peripheral corneal thickness ratio was 0.976 and 0.998 for the 70- and 90-µm grafts. The percentage of the damaged endothelial cells assessed by vital staining and SEM showed the 70-µm grafts had noticeably more endothelial damage compared with the 90-µm grafts. But the damage was significantly reduced, to the control corneas level, after coating the endothelium with Viscoat. The 90-µm grafts had a slightly rougher graft interface than the 70-µm grafts, but all the grafts dissected by a Chansue dissector exhibited a generally smooth interface. CONCLUSIONS: The corneal endothelial grafts prepared by LDV femtosecond laser with endothelial approach produced consistently ultrathin grafts in uniform shape with high accuracy and good endothelial and stromal interface quality.

Identification of cell surface markers glypican-4 and CD200 that differentiate human corneal endothelium from stromal fibroblasts.

PURPOSE: There is a lack of definitive cell surface markers to differentiate cultured human corneal endothelial cells (HCECs) from stromal fibroblasts, which could contaminate HCEC cultures. The aim of our study is to discover cell surface antigens on HCECs that can be used to identify and purify HCECs from stromal fibroblasts. METHODS: RNA sequencing (RNA-seq) was used to find differentially overexpressed genes in HCECs and commercial antibodies against these overexpressed antigens were screened by immunofluorescence assay. Similarly, 242 commercial antibodies against cell-surface antigens also were screened. Selected antibodies were used to sort HCECs from stromal fibroblasts by fluorescence-activated cell sorting (FACS). RESULTS: Two monoclonal antibodies, anti-GPC4 and anti-CD200, were identified to stain HCECs specifically. FACS was used successfully to sort HCECs away from stromal fibroblasts. Recovery efficiency of HCECs after sorting using anti-GPC4 antibody was higher compared to anti-CD200 antibody, but purity of HCECs culture using either antibody was comparable. CONCLUSIONS: Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts.