Cell Type-Specific Extracellular Vesicles and Their Impact on Health and Disease.

Extracellular vesicles (EVs), a diverse group of cell-derived exocytosed particles, are pivotal in mediating intercellular communication due to their ability to selectively transfer biomolecules to specific cell types. EVs, composed of proteins, nucleic acids, and lipids, are taken up by cells to affect a variety of signaling cascades. Research in the field has primarily focused on stem cell-derived EVs, with a particular focus on mesenchymal stem cells, for their potential therapeutic benefits. Recently, tissue-specific EVs or cell type-specific extracellular vesicles (CTS-EVs), have garnered attention for their unique biogenesis and molecular composition because they enable highly targeted cell-specific communication. Various studies have outlined the roles that CTS-EVs play in the signaling for physiological function and the maintenance of homeostasis, including immune modulation, tissue regeneration, and organ development. These properties are also exploited for disease propagation, such as in cancer, neurological disorders, infectious diseases, autoimmune conditions, and more. The insights gained from analyzing CTS-EVs in different biological roles not only enhance our understanding of intercellular signaling and disease pathogenesis but also open new avenues for innovative diagnostic biomarkers and therapeutic targets for a wide spectrum of medical conditions. This review comprehensively outlines the current understanding of CTS-EV origins, function within normal physiology, and implications in diseased states.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Extracellular-Vesicle-Based Therapeutics in Neuro-Ophthalmic Disorders.

Extracellular vesicles (EVs) have been recognized as promising candidates for developing novel therapeutics for a wide range of pathologies, including ocular disorders, due to their ability to deliver a diverse array of bioactive molecules, including proteins, lipids, and nucleic acids, to recipient cells. Recent studies have shown that EVs derived from various cell types, including mesenchymal stromal cells (MSCs), retinal pigment epithelium cells, and endothelial cells, have therapeutic potential in ocular disorders, such as corneal injury and diabetic retinopathy. EVs exert their effects through various mechanisms, including promoting cell survival, reducing inflammation, and inducing tissue regeneration. Furthermore, EVs have shown promise in promoting nerve regeneration in ocular diseases. In particular, EVs derived from MSCs have been demonstrated to promote axonal regeneration and functional recovery in various animal models of optic nerve injury and glaucoma. EVs contain various neurotrophic factors and cytokines that can enhance neuronal survival and regeneration, promote angiogenesis, and modulate inflammation in the retina and optic nerve. Additionally, in experimental models, the application of EVs as a delivery platform for therapeutic molecules has revealed great promise in the treatment of ocular disorders. However, the clinical translation of EV-based therapies faces several challenges, and further preclinical and clinical studies are needed to fully explore the therapeutic potential of EVs in ocular disorders and to address the challenges for their successful clinical translation. In this review, we will provide an overview of different types of EVs and their cargo, as well as the techniques used for their isolation and characterization. We will then review the preclinical and clinical studies that have explored the role of EVs in the treatment of ocular disorders, highlighting their therapeutic potential and the challenges that need to be addressed for their clinical translation. Finally, we will discuss the future directions of EV-based therapeutics in ocular disorders. Overall, this review aims to provide a comprehensive overview of the current state of the art of EV-based therapeutics in ophthalmic disorders, with a focus on their potential for nerve regeneration in ocular diseases.

Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties.

PURPOSE: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems. METHOD: Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for ß3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites. RESULTS: The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (ß3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 µm vs. 1860.5 µm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05). CONCLUSION: This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

Benzalkonium chloride, a common ophthalmic preservative, compromises rat corneal cold sensitive nerve activity.

PURPOSE: Corneal nerves comprise the densest sensory network in the body. Dysfunction of the corneal cold sensitive neurons (CSN) is implicated in ophthalmic disorders, including Dry Eye Disease, the most common ocular surface disorder. The preservative Benzalkonium chloride (BAK) and the mydriatic agent Phenylephrine hydrochloride (PHE) are considered to be inactive at the level of the CSNs. The purpose of this study is to test the impacts of continuous exposures to BAK or PHE at their clinically used concentrations on corneal nerve structure and function. METHODS: In vivo extracellular electrophysiology of the rat trigeminal ganglion was used to monitor CSN functional response to stimuli mimicking physiological states and stressors of the cornea. Corneal nerve structure was evaluated by immunostaining. RESULTS: Among the tested stimuli, cold probe receptive field stimulation and hyperosmolar stress were the most sensitive methods of detecting activity changes. CSN activity was attenuated after 30 min exposure to either PHE or BAK. After an hour-long washout period, BAK-treated neurons failed to recover activity while PHE-treated neurons showed signs of functional recovery. Intraepithelial nerve density was reduced and nerve fragmentation was increased in BAK-treated corneas, while PHE exposure left corneal nerves structurally intact. CONCLUSIONS: Our study suggests that prolonged ocular instillations of BAK or PHE alter CSN activity through two different processes - irreversible neuronal damage in the case of BAK vs. reversible attenuation in the case of PHE.

VEGF receptor heterodimers and homodimers are differentially expressed in neuronal and endothelial cell types.

PURPOSE: We have previously reported that VEGF-B is more potent than VEGF-A in mediating corneal nerve growth in vitro and in vivo, and this stimulation of nerve growth appears to be different from stimulation of angiogenesis by these same ligands, at least in part due to differences in VEGF receptor activation. VEGF signaling may be modulated by a number of factors including receptor number or the formation of receptor hetero- vs. homodimers. In endothelial cells, VEGF receptor heterodimer (VEGR1/R2) activation after ligand binding and subsequent phosphorylation alters the activation of downstream signaling cascades. However, our understanding of these processes in neuronal cell types remains unclear. The purpose of this study was to identify the presence and distribution of VEGF Receptor-Ligand interactions in neuronal cells as compared to endothelial cells. METHODS: PC12 (rat neuronal cell line), MAEC (mouse aortic endothelial cell line), MVEC (mouse venous endothelial cell line) and HUVEC (human umbilical venous endothelial cell line; control group) were used. Cells were acutely stimulated either with VEGF-A (50 ng/µL) or VEGF-B (50 ng/µL) or "vehicle" (PBS; control group). We also isolated mouse trigeminal ganglion cells from thy1-YFP neurofluorescent mice. After treatment, cells were used as follows: (i) One group was fixed in 4% paraformaldehyde and processed for VEGFR1 and VEGFR2 immunostaining and visualized using confocal fluorescence microscopy and Total Internal Reflection (TIRF) microscopy; (ii) the second group was harvested in cell lysis buffer (containing anti-protease / anti-phosphatase cocktail), lysed and processed for immunoprecipitation (IP; Thermo Fisher IP kit) and immunoblotting (IB; LI-COR® Systems). Immunoprecipitated proteins were probed either with anti-VEGFR1 or anti-VEGFR2 IgG antibodies to evaluate VEGFR1-R2-heterodimerization; (iii) a third group of cells was also processed for Duolink Proximity Ligation Assay (PLA; Sigma) to assess the presence and distribution of VEGF-receptor homo- and heterodimers in neuronal and endothelial cells. RESULTS: TIRF and fluorescence confocal microscopy revealed the presence of VEGFR1 co-localized with VEGFR2 in endothelial and PC12 neuronal cells. Cell lysates immunoprecipitated with anti-VEGFR1 further validated the existence of VEGFR1-R2 heterodimers in PC12 neuronal cells. Neuronal cells showed higher levels of VEGFR1-R2 heterodimers as compared to endothelial cells whereas endothelial cells showed higher VEGFR2-R2 homodimers compared to neuronal cells as demonstrated by Duolink PLA. Levels of VEGFR1-R1 homodimers were very low in neuronal and endothelial cells. CONCLUSIONS: Differences in VEGF Receptor homo- and heterodimer distribution may explain the differential role of VEGF ligands in neuronal versus endothelial cell types. This may in turn influence VEGF activity and regulation of neuronal cell homeostasis.

Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury.

Axon guidance proteins are essential for axonal pathfinding during development. In adulthood, they have been described as pleiotropic proteins with multiple roles in different organs and tissues. While most studies on the roles of these proteins in the cornea have been performed on the Semaphorin family members, with few reports on Netrins or Ephrins, their function in corneal epithelium wound healing and functional nerve regeneration is largely unknown. Here, we studied the expression of ligands belonging to three distinct axon guidance families (Semaphorins, Ephrins, and Netrins) and their most commonly associated receptors in the cornea and trigeminal ganglia (TG) using immunofluorescence staining and RT-qPCR. We also evaluated how their expression recovers after corneal epithelium injury. We found that all ligands studied (Sema3A, Sema3F, EphrinB1, EphrinB2, Netrin-1, and Netrin-4) are abundantly expressed in both the TG and corneal epithelium. Similarly, their receptors (Neuropilin-1, Neuropilin-2, PlexinA1, PlexinA3, EphB2, EphB4, Neogenin, UNC5H1 and DCC) are also expressed in both tissues. Upon corneal epithelium injury, quick recovery of both ligands and receptors was observed at the protein and gene expression levels. While the timing and expression levels vary among these proteins, in general, most of them remained upregulated for several weeks after injury. We propose that the initial protein expression recovery may be related to corneal epithelium recovery since Sema3A, EphrinB2 and Netrin-4 accelerated corneal epithelial cells wound healing. The sustained high expression levels may be functionally related to nerve regeneration and/or patterning. Whilst further studies are required to test this hypothesis, this work contributes to unraveling their function in normal and injured cornea.

Optical coherence elastography for assessing the influence of intraocular pressure on elastic wave dispersion in the cornea.

The cornea is a highly specialized organ that relies on its mechanical stiffness to maintain its aspheric geometry and refractive power, and corneal diseases such as keratoconus have been linked to abnormal tissue stiffness and biomechanics. Dynamic optical coherence elastography (OCE) is a clinically promising non-contact and non-destructive imaging technique that can provide measurements of corneal tissue stiffness directly in vivo. The method relies on the concepts of elastography where shear waves are generated and imaged within a tissue to obtain mechanical properties such as tissue stiffness. The accuracy of OCE-based measurements is ultimately dependent on the mathematical theories used to model wave behavior in the tissue of interest. In the cornea, elastic waves propagate as guided wave modes which are highly dispersive and can be mathematically complex to model. While recent groups have developed detailed theories for estimating corneal tissue properties from guided wave behavior, the effects of intraocular pressure (IOP)-induced prestress have not yet been considered. It is known that prestress alone can strongly influence wave behavior, in addition to the associated non-linear changes in tissue properties. This present study shows that failure to account for the effects of prestress may result in overestimations of the corneal shear moduli, particularly at high IOPs. We first examined the potential effects of IOP and IOP-induced prestress using a combination of approximate mathematical theories describing wave behavior in thin plates with observations made from data published in the OCE literature. Through wave dispersion analysis, we deduce that IOP introduces a tensile hoop stress and may also influence an elastic foundational effect that were observable in the low-frequency components of the dispersion curves. These effects were incorporated into recently developed models of wave behavior in nearly incompressible, transversely isotropic (NITI) materials. Fitting of the modified NITI model with ex vivo porcine corneal data demonstrated that incorporation of the effects of IOP resulted in reduced estimates of corneal shear moduli. We believe this demonstrates that overestimation of corneal stiffness occurs if IOP is not taken into consideration. Our work may be helpful in separating inherent corneal stiffness properties that are independent of IOP; changes in these properties and in IOP are distinct, clinically relevant issues that affect the cornea health.

The Limbal Niche and Regenerative Strategies.

The protective function and transparency provided by the corneal epithelium are dependent on and maintained by the regenerative capacity of limbal epithelial stem cells (LESCs). These LESCs are supported by the limbal niche, a specialized microenvironment consisting of cellular and non-cellular components. Disruption of the limbal niche, primarily from injuries or inflammatory processes, can negatively impact the regenerative ability of LESCs. Limbal stem cell deficiency (LSCD) directly hampers the regenerative ability of the corneal epithelium and allows the conjunctival epithelium to invade the cornea, which results in severe visual impairment. Treatment involves restoring the LESC population and functionality; however, few clinically practiced therapies currently exist. This review outlines the current understanding of the limbal niche, its pathology and the emerging approaches targeted at restoring the limbal niche. Most emerging approaches are in developmental phases but show promise for treating LSCD and accelerating corneal regeneration. Specifically, we examine cell-based therapies, bio-active extracellular matrices and soluble factor therapies in considerable depth.

Hydrogels derived from acellular porcine corneal stroma enhance corneal wound healing.

Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.

Vinaxanthone inhibits Semaphorin3A induced axonal growth cone collapse in embryonic neurons but fails to block its growth promoting effects on adult neurons.

Semaphorin3A is considered a classical repellent molecule for developing neurons and a potent inhibitor of regeneration after nervous system trauma. Vinaxanthone and other Sema3A inhibitors are currently being tested as possible therapeutics to promote nervous system regeneration from injury. Our previous study on Sema3A demonstrated a switch in Sema3A's function toward induction of nerve regeneration in adult murine corneas and in culture of adult peripheral neurons. The aim of the current study is to determine the direct effects of Vinaxanthone on the Sema3A induced adult neuronal growth. We first demonstrate that Vinaxanthone maintains its anti-Sema3A activity in embryonic dorsal root ganglia neurons by inhibiting Sema3A-induced growth cone collapse. However, at concentrations approximating its IC50 Vinaxanthone treatment does not significantly inhibit neurite formation of adult peripheral neurons induced by Sema3A treatment. Furthermore, Vinaxanthone has off target effects when used at concentrations above its IC50, and inhibits neurite growth of adult neurons treated with either Sema3A or NGF. Our results suggest that Vinaxanthone's pro-regenerative effects seen in multiple in vivo models of neuronal injury in adult animals need further investigation due to the pleiotropic effect of Sema3A on various non-neuronal cell types and the possible effect of Vinaxanthone on other neuroregenerative signals.

Silk films with nanotopography and extracellular proteins enhance corneal epithelial wound healing.

Corneal wound healing depends on extracellular matrix (ECM) and topographical cues that modulate migration and proliferation of regenerating cells. In our study, silk films with either flat or nanotopography patterned parallel ridge widths of 2000, 1000, 800 nm surfaces were combined with ECMs which include collagen type I (collagen I), fibronectin, laminin, and Poly-D-Lysine to accelerate corneal wound healing. Silk films with 800 nm ridge width provided better cell spreading and wound recovery than other size topographies. Coating 800 nm patterned silk films with collagen I proves to optimally further increased mouse and rabbit corneal epithelial cells growth and wound recovery. This enhanced cellular response correlated with redistribution and increase in size and total amount of focal adhesion. Transcriptomics and signaling pathway analysis suggested that silk topography regulates cell behaviors via actin nucleation ARP-WASP complex pathway, which regulate filopodia formation. This mechanism was further explored and inhibition of Cdc42, a key protein in this pathway, delayed wound healing and decreased the length, density, and alignment of filopodia. Inhibition of Cdc42 in vivo resulted in delayed re-epithelization of injured corneas. We conclude that silk film nanotopography in combination with collagen I constitutes a better substrate for corneal wound repair than either nanotopography or ECM alone.

The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation.

We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned silk films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned silk films. Cells cultured on nanoscale patterned silk films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/ß-catenin signaling. We conclude that patterned silk film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.

Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development.

The peripheral sensory nerves that innervate the cornea can be easily damaged by trauma, surgery, infection or diabetes. Several growth factors and axon guidance molecules, such as Semaphorin3A (Sema3A) are upregulated upon cornea injury. Nerves can regenerate after injury but do not recover their original density and patterning. Sema3A is a well known axon guidance and growth cone repellent protein during development, however its role in adult cornea nerve regeneration remains undetermined. Here we investigated the neuro-regenerative potential of Sema3A on adult peripheral nervous system neurons such as those that innervate the cornea. First, we examined the gene expression profile of the Semaphorin class 3 family members and found that all are expressed in the cornea. However, upon cornea injury there is a fast increase in Sema3A expression. We then corroborated that Sema3A totally abolished the growth promoting effect of nerve growth factor (NGF) on embryonic neurons and observed signs of growth cone collapse and axonal retraction after 30 min of Sema3A addition. However, in adult isolated trigeminal ganglia or dorsal root ganglia neurons, Sema3A did not inhibited the NGF-induced neuronal growth. Furthermore, adult neurons treated with Sema3A alone produced similar neuronal growth to cells treated with NGF and the length of the neurites and branching was comparable between both treatments. These effects were replicated in vivo, where thy1-YFP neurofluorescent mice subjected to cornea epithelium debridement and receiving intrastromal pellet implantation containing Sema3A showed increased corneal nerve regeneration than those receiving pellets with vehicle. In adult PNS neurons, Sema3A is a potent inducer of neuronal growth in vitro and cornea nerve regeneration in vivo. Our data indicates a functional switch for the role of Sema3A in PNS neurons where the well-described repulsive role during development changes to a growth promoting effect during adulthood. The high expression of Sema3A in the normal and injured adult corneas could be related to its role as a growth factor.

Micro- and Nanoscale Topographies on Silk Regulate Gene Expression of Human Corneal Epithelial Cells.

Purpose: Corneal basement membrane has topographical features that provide biophysical cues to direct cell adherence, migration, and proliferation. In this study, we hypothesize that varying topographic pitch created on silk films can alter epithelial cell morphology, adhesion, and the genetic expression involved in cytoskeletal dynamics-related pathways. Methods: Silicon wafers with parallel ridge widths of 2000, 1000, and 800 nm were produced and used to pattern silk films via soft lithography. Human corneal epithelial cells were cultured onto silk. After 72 hours of incubation, images were taken to study cell morphology and alignment. Cytoskeletal structures were studied by immunofluorescent staining. RNA was collected from cultured cells to perform RNA-Seq transcriptome analysis using the Illumina Hiseq 2500 sequencing system. Differentially expressed genes were identified using DNAstar Qseq then verified using quantitative real-time PCR. These genes were used to perform pathway analyses using Ingenuity Pathways Analysis. Results: Primary human corneal epithelial cell alignment to the surface pattern was the greatest on 1000-nm features. Fluorescent microscopy of f-actin staining showed cell cytoskeleton alignment either in parallel (2000 nm) or perpendicular (1000 and 800 nm) to the long feature axis. Z-stack projection of vinculin staining indicated increased focal adhesion formation localized on the cellular basal surface. RNA-seq analysis revealed differentially expressed genes involved in actin organization, integrin signaling, and focal adhesion kinase signaling (-log (P)>5). Conclusions: Patterned silk film substrates may serve as a scaffold and provide biophysical cues to corneal epithelial cells that change their gene expression, alter cellular adherence, morphology, and may offer a promising customizable material for use in ocular surface repair.

Iris suture fixation: Push-knot needle.

We describe a technique to create a locking knot for iris reconstruction or intraocular lens fixation. A modified needle is prepared by straightening the tip of an ophthalmic viscosurgical device needle or a lacrimal cannula needle. After the suture is passed through 2 sides of an iris defect, the 2 ends are externalized and looped. One strand is then inserted into the needle from the tip out of the ferrule, and the knot is pushed into the anterior chamber by this needle in a controllable manner. Two other locking knots are then made by repeating the process. With this method, only 1 small corneal incision is necessary regardless of the number of knots required and only a small amount of anterior chamber space is needed during the knotting process.

Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation.

Purpose: The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin-derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model. Methods: Solubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies. Results: Results indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls. Conclusions: The addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.

Acute corneal epithelial debridement unmasks the corneal stromal nerve responses to ocular stimulation in rats: implications for abnormal sensations of the eye.

It is widely accepted that the mechanisms for transducing sensory information reside in the nerve terminals. Occasionally, however, studies have appeared demonstrating that similar mechanisms may exist in the axon to which these terminals are connected. We examined this issue in the cornea, where nerve terminals in the epithelial cell layers are easily accessible for debridement, leaving the underlying stromal (axonal) nerves undisturbed. In isoflurane-anesthetized rats, we recorded extracellularly from single trigeminal ganglion neurons innervating the cornea that are excited by ocular dryness and cooling: low-threshold (<2°C cooling) and high-threshold (>2°C) cold-sensitive plus dry-sensitive neurons playing possible roles in tearing and ocular pain. We found that the responses in both types of neurons to dryness, wetness, and menthol stimuli were effectively abolished by the debridement, indicating that their transduction mechanisms lie in the nerve terminals. However, some responses to the cold, heat, and hyperosmolar stimuli in low-threshold cold-sensitive plus dry-sensitive neurons still remained. Surprisingly, the responses to heat in approximately half of the neurons were augmented after the debridement. We were also able to evoke these residual responses and follow the trajectory of the stromal nerves, which we subsequently confirmed histologically. The residual responses always disappeared when the stromal nerves were cut at the limbus, suggesting that the additional transduction mechanisms for these sensory modalities originated most likely in stromal nerves. The functional significance of these residual and enhanced responses from stromal nerves may be related to the abnormal sensations observed in ocular disease.NEW & NOTEWORTHY In addition to the traditional view that the sensory transduction mechanisms exist in the nerve terminals, we report here that the proximal axons (stromal nerves in the cornea from which these nerve terminals originate) may also be capable of transducing sensory information. We arrived at this conclusion by removing the epithelial cell layers of the cornea in which the nerve terminals reside but leaving the underlying stromal nerves undisturbed.

LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis.

The cornea requires constant epithelial renewal to maintain clarity for appropriate vision. A subset of stem cells residing at the limbus is primarily responsible for maintaining corneal epithelium homeostasis. Trauma and disease may lead to stem cell deficiency and therapeutic targeting to replenish the stemness capacity has been stalled by the lack of reliable corneal epithelial stem cell markers. Here we identified the location of Lhx2 in mice (mLhx2) cornea and conjunctival tissue using an Lhx2eGFP reporter model and in human tissues (hLHX2). Lhx2 localized to the basal cells of central cornea, the conjunctiva and the entire limbal epithelium in humans and mice. To ascribe a functional role we generated Lhx2 conditional knockout (cKO) mice and the phenotypic effects in corneas were analyzed by slit lamp microscopy, in cell-based assays and in a model of corneal epithelium debridement. Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure. Lhx2cKO mice produced reduced body hair and spontaneous epithelial defects in the cornea that included neovascularization, perforation with formation of scar tissue and opacification. Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound-healing recovery when compared to wildtype cells. Repeated corneal epithelial wounding resulted in decreased re-epithelialization and multiple cornea lesions in Lhx2cKO mice compared to normal recovery seen in wildtype mice. We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier.

VEGF-B selectively regenerates injured peripheral neurons and restores sensory and trophic functions.

VEGF-B primarily provides neuroprotection and improves survival in CNS-derived neurons. However, its actions on the peripheral nervous system have been less characterized. We examined whether VEGF-B mediates peripheral nerve repair. We found that VEGF-B induced extensive neurite growth and branching in trigeminal ganglia neurons in a manner that required selective activation of transmembrane receptors and was distinct from VEGF-A-induced neuronal growth. VEGF-B-induced neurite elongation required PI3K and Notch signaling. In vivo, VEGF-B is required for normal nerve regeneration: mice lacking VEGF-B showed impaired nerve repair with concomitant impaired trophic function. VEGF-B treatment increased nerve regeneration, sensation recovery, and trophic functions of injured corneal peripheral nerves in VEGF-B-deficient and wild-type animals, without affecting uninjured nerves. These selective effects of VEGF-B on injured nerves and its lack of angiogenic activity makes VEGF-B a suitable therapeutic target to treat nerve injury.