The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm’s canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.

No abstract

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and a-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc À/À mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice. (Am J Pathol 2017, 187: 713e723; http://dx.doi.org/10.1016/j.ajpath.2016 Glucocorticoids (GCs) are one of the most commonly prescribed medications worldwide for the treatment of a plethora of diseases and conditions. Because of their broadspectrum anti-inflammatory and immunosuppressive properties, the worldwide market for GC use is estimated to be >$10 billion per year.1 Approximately 1.2% of US and 0.85% of UK populations are prescribed therapeutic GCs every year.2,3 GCs also remain the mainstay of treatment for a variety of ocular inflammatory diseases involving almost all tissues of the eye, such as eyelids, conjunctiva, cornea, sclera, uvea, retina, and optic nerve. 4 The routes of GC administration in treatment of these disorders can be topical ocular, oral, systemic, intravitreal injections and implants, and periocular injections (including subconjunctival, subtenon, retrobulbar, and peribulbar).5 However, prolonged GC therapy is associated with serious ocular adverse effects, including development of posterior subcapsular cataracts, and the development of GC-induced ocular hypertension (GC-OHT) and iatrogenic open-angle glaucoma.The clinical presentation of GC-induced glaucoma is similar to primary open-angle glaucoma (POAG), and for >50 years, reports have suggested a link between glaucoma and GCs. Development of GC-induced OHT depends on GC dose and duration of treatment, method of administration, potency of GC, and individual susceptibility to GCs. 6e8 There are varying degrees of steroid responsiveness (ie, development of GC-OHT) among individ...

PurposeIncreased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation.MethodsCultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test.ResultsTGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption.ConclusionsTGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.