Pilocarpine mediated excessive calcium accumulation leads to ciliary muscle cell senescence and apoptosis.

The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca2+ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca2+ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca2+ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions.

The ocular immunological alterations in the process of high-risk corneal transplantation rejection.

PURPOSE: This study aims to reveal the immunopathogenesis of the high-risk corneal transplantation using a comparative proteomic approach. METHODS: The immunological properties of ocular tissues (including corneal grafts, aqueous humour, and iris-ciliary body) were analysed using a high-risk rabbit corneal transplantation model employing a comparative proteomic approach. RESULTS: The corneal grafts revealed a dramatic increase in the immune response both at the early (postoperative day 7) and rejection stages, along with the appearance of transplantation stress-induced cellular senescence in the early stage. The aqueous humour (AH) displayed persistent pathological alterations, indicated by the significant enrichment of complement and coagulation cascades pathway in the early stage and interleukin (IL)-17 signalling pathway in the rejection stage. More surprisingly, the pronounced elevation of immune response was also observed in the iris-ciliary body (I-CB) tissues at the early and rejection stages. The enriched immune-related pathways were associated with antigen processing and presentation, complement and coagulation cascades, and IL-17 signalling pathway. Furthermore, proteomic analysis revealed that the implantation of Cyclosporine A drug delivery system (CsA-DDS) into the anterior chamber obviously mitigated corneal transplantation rejection by inhibiting immunoreaction both in the corneal grafts and I-CB tissues. CONCLUSION: The results highlighted the involvement of intraocular immunity both in the grafts and I-CB tissues during corneal transplantation rejection, further suggesting the anterior chamber as an optimal drug-delivery site for its treatment.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

CRISPR-Cas9-mediated deletion of carbonic anhydrase 2 in the ciliary body to treat glaucoma.

The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma.

Aqueous humor as eye lymph: A crossroad between venous and lymphatic system.

Aqueous humor (AQH) is a transparent fluid with characteristics similar to those of the interstitial fluid, which fills the eyeball posterior and anterior chambers and circulates in them from the sites of production to those of drainage. The AQH volume and pressure homeostasis is essential for the trophism of the ocular avascular tissues and their normal structure and function. Different AQH outflow pathways exist, including a main pathway, quite well defined anatomically and referred to as the conventional pathway, and some accessory pathways, more recently described and still not fully morphofunctionally understood, generically referred to as unconventional pathways. The conventional pathway is based on the existence of a series of conduits starting with the trabecular meshwork and Schlemm's Canal and continuing with a system of intrascleral and episcleral venules, which are tributaries to veins of the anterior segment of the eyeball. The unconventional pathways are mainly represented by the uveoscleral pathway, in which AQH flows through clefts, interstitial conduits located in the ciliary body and sclera, and then merges into the aforementioned intrascleral and episcleral venules. A further unconventional pathway, the lymphatic pathway, has been supported by the demonstration of lymphatic microvessels in the limbal sclera and, possibly, in the uvea (ciliary body, choroid) as well as by the ocular glymphatic channels, present in the neural retina and optic nerve. It follows that AQH may be drained from the eyeball through blood vessels (TM-SC pathway, US pathway) or lymphatic vessels (lymphatic pathway), and the different pathways may integrate or compensate for each other, optimizing the AQH drainage. The present review aims to define the state-of-the-art concerning the structural organization and the functional anatomy of all the AQH outflow pathways. Particular attention is paid to examining the regulatory mechanisms active in each of them. The new data on the anatomy and physiology of AQH outflow pathways is the key to understanding the pathophysiology of AQH outflow disorders and could open the way for novel approaches to their treatment.

Characterization of lncRNA and mRNA profiles in ciliary body in experimental myopia.

Currently, researchers have mainly focused on the role of the tissues of the posterior segment of the eyes in the development of myopia. However, the ciliary body, an anterior ocular tissue that contracts to initiate the process of accommodation, may also play an important role in the progression of myopia due to the increased demand for near work. In the present study, we established a lens-induced myopia (LIM) animal model in guinea pigs and investigated the molecular changes in the ciliary body associated with the development of myopia based on RNA sequencing. As a result, 871 differentially expressed (DE) mRNAs and 19 DE lncRNAs were identified in the ciliary body between the LIM group and the normal control group. In addition, the lncRNA-mRNA co-expression analysis was performed to explore the target genes of lncRNAs, which were mainly enriched in the Rap1 signaling pathway, cytokine-cytokine receptor interaction, and complement and coagulation cascades pathways based on the functional enrichment analysis. Among the target genes of lncRNAs, three hub genes, including Ctnnb1, Pik3r1, and Itgb1, were found to be involved in the Rap1 signaling pathway. Interestingly, two crucial genes, Grk1 and Pde6a, which are mainly expressed in retinal photoreceptors, were enriched in visual perception in the ciliary body in functional analysis and were verified to be expressed in the ciliary body. These findings indicate the molecular pathogenetic role of the ciliary body in myopia and provide new insights into the underlying mechanism of myopia development. Further studies are needed to explore the specific contributions of these identified lncRNAs and mRNAs to the development of myopia.

Rat Model of Type 2 Diabetes Mellitus Recapitulates Human Disease in the Anterior Segment of the Eye.

Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.

Morroniside Ameliorates Endotoxin-Induced Uveitis by Regulating the M1/M2 Polarization Balance of Macrophages.

Background: Inflammation is closely associated with the pathogenesis of various ocular diseases. Uveitis is a condition characterized by the inflammation of the uvea and ocular tissues that causes extreme pain, decreases visual acuity, and may eventually lead to blindness. The pharmacological functions of morroniside, isolated from Cornus officinalis, are multifarious. Morroniside exerts various therapeutic effects, e.g., it ameliorates inflammation. However, the specific anti-inflammatory effect of morroniside on lipopolysaccharide-induced uveitis has not been reported widely. In this study, we investigated the anti-inflammatory effect of morroniside on uveitis in mice. Methods: An endotoxin-induced uveitis (EIU) mouse model was constructed and treated with morroniside. The inflammatory response was observed using slit lamp microscopy, and histopathological changes were observed by hematoxylin-eosin staining. The cell count in the aqueous humor was measured using a hemocytometer. The concentrations of TNF-α, IL-6, and IL-1ß in the ciliary body and retina were measured using ELISA kits. The expression of iNOS and Arg-1 in the ciliary body and retina was measured by immunofluorescence costaining, and western blotting was performed to measure the protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 in the ciliary body and retina. Results: Morroniside effectively ameliorated the inflammatory response in EIU mice. Furthermore, morroniside significantly reduced the concentrations of IL-1ß, IL-6, and TNF-α in the ciliary body and retina. Morroniside treatment significantly reduced the expression of iNOS in the ciliary body and retinal tissues. It also significantly inhibited p-JAK2 and p-STAT3 expression and promoted Arg-1 expression. In addition, morroniside boosted the effect of JAK inhibitors on the above indices. Conclusions: Collectively, these findings suggest that morroniside may protect against LPS-induced inflammation in uveitis by promoting M2 polarization through the inhibition of the JAK/STAT pathway.

Regulation of Aqueous Humor Secretion by Melatonin in Porcine Ciliary Epithelium.

Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50-60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT3 receptor was found to be >10-fold higher than that of MT1 and MT2 in porcine ciliary epithelium. Aqueous pre-treatment with MT1/MT2 antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT3 antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl- and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT3 receptors.

Bestrophin-2 and glutamine synthetase form a complex for glutamate release.

Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology1-4. Here we uncover a directional permeability of BEST2 to glutamate that heavily favours glutamate exit, identify glutamine synthetase (GS) as a binding partner of BEST2 in the ciliary body of the eye, and solve the structure of the BEST2-GS complex. BEST2 reduces cytosolic GS activity by tethering GS to the cell membrane. GS extends the ion conducting pathway of BEST2 through its central cavity and inhibits BEST2 channel function in the absence of intracellular glutamate, but sensitizes BEST2 to intracellular glutamate, which promotes the opening of BEST2 and thus relieves the inhibitory effect of GS. We demonstrate the physiological role of BEST2 in conducting chloride and glutamate and the influence of GS in non-pigmented ciliary epithelial cells. Together, our results reveal a novel mechanism of glutamate release through BEST2-GS.

A single-cell transcriptomic atlas of the human ciliary body.

The ciliary body critically contributes to the ocular physiology with multiple responsibilities in the production of aqueous humor, vision accommodation and intraocular immunity. Comparatively little work, however, has revealed the single-cell molecular taxonomy of the human ciliary body required for studying these functionalities. In this study, we report a comprehensive atlas of the cellular and molecular components of human ciliary body as well as their interactions using single-cell RNA sequencing (scRNAseq). Cluster analysis of the transcriptome of 14,563 individual ciliary cells from the eyes of 3 human donors identified 14 distinct cell types, including the ciliary epithelium, smooth muscle, vascular endothelial cell, immune cell and other stromal cell populations. Cell-type discriminative gene markers were also revealed. Unique gene expression patterns essential for ciliary epithelium-mediated aqueous humor inflow and ciliary smooth muscle contractility were identified. Importantly, we discovered the transitional states that probably contribute to the transition of ciliary macrophage into retina microglia and verified no lymphatics in the ciliary body. Moreover, the utilization of CellPhoneDB allowed us to systemically infer cell-cell interactions among diverse ciliary cells including those that potentially participate in the pathogenesis of glaucoma and uveitis. Altogether, these new findings provide insights into the regulation of intraocular pressure, accommodation reflex and immune homeostasis under physiological and pathological conditions.

Evidence of lymphatics in the rat eye retina.

BACKGROUND: The lymphatic structure of the eye is still under debate. It is mainly assumed that the retina is primarily drained by prelymphatics and not by lymphatics per se. We aimed to identify lymphatics in the rat retina. METHODS: Eyes from ten Wistar rats were paraffin-embedded and the lymphatic marker podoplanin (D2-40) was investigated. RESULTS: We identified in the rat retina a blunt-end network of lymphatic endothelial vessels. It consisted of circumferential vessels within the outer and, respectively, inner plexiform layers, connected by radial dichotomous vessels. Moreover, D2-40 expression was found within the choroid, ciliary body, and extraocular muscles. CONCLUSIONS: This in situ evidence is strongly supported by the recent in vitro demonstration of the expression of lymphatic markers in retinal endothelial cells. Further studies of comparative histology should use specific lymphatic markers to test whether other species besides rats have proper retinal lymphatics.

A Mathematical Model of Aqueous Humor Production and Composition.

Purpose: We develop a mathematical model that predicts aqueous humor (AH) production rate by the ciliary processes and aqueous composition in the posterior chamber (PC), with the aim of estimating how the aqueous production rate depends on the controlling parameters and how it can be manipulated. Methods: We propose a compartmental mathematical model that considers the stromal region, ciliary epithelium, and PC. All domains contain an aqueous solution with different chemical species. We impose the concentration of all species on the stromal side and exploit the various ion channels present on the cell membrane to compute the water flux produced by osmosis, the solute concentrations in the AH and the transepithelial potential difference. Results: With a feasible set of parameters, the model predictions of water flux from the stroma to the PC and of the solute concentrations in the AH are in good agreement with measurements. Key parameters which impact the aqueous production rate are identified. A relevant role is predicted to be played by cell membrane permeability to \(\text{K}^+\) and \(\text{Cl}^-\), by the level of transport due to the Na+-H+ exchanger and to the co-transporter of Na+/K+/2Cl-; and by carbonic anhydrase. Conclusions: The mathematical model predicts the formation and composition of AH, based on the structure of the ciliary epithelium. The model provides insight into the physical processes underlying the functioning of drugs that are adopted to regulate the aqueous production. It also suggests ion channels and cell membrane properties that may be targeted to manipulate the aqueous production rate.

Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types.

The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.

The ciliary zonules provide a pathway for immune cells to populate the avascular lens during eye development.

The eye is an immune-privileged site, with both vasculature and lymphatics absent from the central light path. Unique adaptations have made it possible for immune cells to be recruited to this region of the eye in response to ocular injuries and pathogenic insults. The induction of such immune responses is typically activated by tissue resident immune cells, considered the sentinels of the immune system. We discovered that, despite the absence of an embedded vasculature, the embryonic lens becomes populated by resident immune cells. The paths by which they travel to the lens during development were not known. However, our previous studies show that in response to corneal wounding immune cells travel to the lens from the vascular-rich ciliary body across the zonules that link these two tissues. We now examined whether the zonule fibers provide a path for immune cells to the embryonic lens, and the zonule-associated matrix molecules that could promote immune cell migration. The vitreous also was examined as a potential source of lens resident immune cells. This matrix-rich site in the posterior of the eye harbors hyalocytes, an immune cell type with macrophage-like properties. We found that both the zonules and the vitreous of the embryonic eye contained fibrillin-2-based networks and that migration-promoting matrix proteins like fibronectin and tenascin-C were linked to these fibrils. Immune cells were seen emerging from the ciliary body, migrating along the ciliary zonules to the lens, and invading through the lens capsule at its equator. This is just adjacent to where immune cells take up residence in the embryonic lens. In contrast, the immune cells of the vitreous were not detected in the region of the lens. These results strongly suggest that the ciliary zonules are a primary path of immune cell delivery to the developing lens.

A transcriptome atlas of the mouse iris at single-cell resolution defines cell types and the genomic response to pupil dilation.

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Effects of selective EP2 receptor agonist, omidenepag, on trabecular meshwork cells, Schlemm's canal endothelial cells and ciliary muscle contraction.

This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm's canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-ß2 (TGF-ß2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and ß-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-ß2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-ß2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.

Piezo2 downregulation via the Cre-lox system affects aqueous humor dynamics in mice.

Purpose: Proper aqueous humor (AH) dynamics is crucial for maintaining the intraocular pressure (IOP) in the eye. This study aims to investigate the function of Piezo2, a newly discovered mechanosensitive ion channel, in regulating AH dynamics. Methods: Immunohistochemistry (IHC) analysis and western blotting were performed to detect Piezo2 expression. The Cre-lox system was applied to create a conditional knockout model of Piezo2. IOP and aqueous humor outflow facility in live animals were recorded with a Tonometer and a syringe-pump system for up to 2 weeks. Results: We first detected Piezo2 with robust expression in the human trabecular meshwork (TM), Schlemm's canal (SC), the ciliary body's epithelium, and ciliary muscle. In addition, we found Piezo2 in human retinal ganglion cells (RGCs) and astrocytes in the optic nerve head (ONH). Through the Cre-lox system, Piezo2 can be successfully downregulated in mouse iridocorneal angle tissues. However, Piezo2 downregulation cannot significantly influence the IOP and outflow facility through the conventional pathway. Instead, we observed an effect of downregulated Piezo2 on decreasing the intercept in the flow rate versus pressure plot. According to the Goldmann equation, Piezo2 may function in regulating unconventional outflow, AH production, and episcleral venous pressure. Conclusions: These findings, for the first time, demonstrate that Piezo2 acts as an essential mechanosensor in maintaining the proper aqueous humor dynamics in the eye.

Let-7, Lin28 and Hmga2 Expression in Ciliary Epithelium and Retinal Progenitor Cells.

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues. Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2. Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2. Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Overlapping Immunohistochemical Features of Adenocarcinoma of the Nonpigmented Ciliary Body Epithelium and Renal Cell Carcinoma.

PURPOSE: To find immunohistochemical markers that distinguish adenocarcinoma of the nonpigmented ciliary epithelium (NPCE) from metastatic carcinoma, especially metastatic renal cell carcinoma. DESIGN: Retrospective case series. METHODS: Three cases of adenocarcinoma of the NPCE were examined histologically with hematoxylin-eosin stain and immunohistochemical stains including vimentin, AE1/AE3, Cam 5.2, CK7, PAX2, PAX8, AMACR, and CAIX. We also reviewed previously reported cases of this tumor. RESULTS: We found that the immunohistochemical profile of adenocarcinoma of the NPCE can overlap with renal cell carcinoma. Both tumors can express vimentin, cytokeratin AE1/AE3, Cam 5.2, PAX2, PAX8, and AMACR. One of the adenocarcinomas of the NPCE in our series also expressed CD10 and the renal cell carcinoma marker (RCC Ma). Carbonic anhydrase IX (CAIX) was not detected in any of the 3 tumors. CONCLUSIONS: Adenocarcinomas arising in phthisic eyes can be diagnostically challenging. We have found it particularly difficult to distinguish adenocarcinoma of the NPCE from metastatic carcinoma, especially metastatic clear cell renal cell carcinoma and papillary renal cell carcinoma. Because of the immunophenotypic overlap, most patients will require systemic workup including imaging of the kidneys to be certain of the diagnosis.