Inhibition of monocyte-like cell extravasation protects from neurodegeneration in DBA/2J glaucoma.

BACKGROUND: Glaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J), but their roles, if any, in mediating axon damage remain unclear. METHODS: To understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation). RESULTS: Monocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2 J eyes. CONCLUSIONS: These data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 1

Angiopoietin-1 is required for Schlemm’s canal development in mice and humans

Made available in accordance with publisher's policyPrimary congenital glaucoma (PCG) is a leading cause of blindness in children worldwide and is caused by developmental defects in 2 aqueous humor outflow structures, Schlemm’s canal (SC) and the trabecular meshwork. We previously identified loss-of-function mutations in the angiopoietin (ANGPT) receptor TEK in families with PCG and showed that ANGPT/TEK signaling is essential for SC development. Here, we describe roles for the major ANGPT ligands in the development of the aqueous outflow pathway. We determined that ANGPT1 is essential for SC development, and that Angpt1-knockout mice form a severely hypomorphic canal with elevated intraocular pressure. By contrast, ANGPT2 was dispensable, although mice deficient in both Angpt1 and Angpt2 completely lacked SC, indicating that ANGPT2 compensates for the loss of ANGPT1. In addition, we identified 3 human subjects with rare ANGPT1 variants within an international cohort of 284 PCG patients. Loss of function in 2 of the 3 patient alleles was observed by functional analysis of ANGPT1 variants in a combined in silico, in vitro, and in vivo approach, supporting a causative role for ANGPT1 in disease. By linking ANGPT1 with PCG, these results highlight the importance of ANGPT/TEK signaling in glaucoma pathogenesis and identify a candidate target for therapeutic development

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

Schlemm\u27s Canal Is a Unique Vessel with a Combination of Blood Vascular and Lymphatic Phenotypes that Forms by a Novel Developmental Process.

Schlemm\u27s canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name canalogenesis. Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision. PLoS Biol 2014 Jul 22; 12(7):e1001912

Summary of early SC development.

<p>The images show VECAD (yellow) immunolabeled cells in the IZ in the plane of the future SC at the developmental ages listed. The cartoons represent the corresponding stages. The development of SC starts by sprouting from the LVP and RV. RV and their sprouts have been left out of the cartoons for the sake of simplicity and in no way are meant to suggest lesser importance of RV. (A) As in angiogenesis, sprouts led by tip cells penetrate into the IZ all around the limbus. (B) However, in contrast to angiogenesis, multiple tip cells interact and form tip cell clusters (TCCs). (C) These local clusters of cells connect with each other to form a continuous structure encircling the entire limbus. We call this structure the rSC. (D) The number of cells in the rSC increases and the cellular chain branches. Scale bar, 20 µm.</p

Macrophages are present at sites of tip cell interactions mediated by filopodia.

<p>(A) Tip cells in the IZ interact through their filopodia, and macrophages are present at sites of tip cell interaction. All images are Z-projections of the future SC plane of a P2 eye. The numbers correspond to their location in that eye in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s010" target="_blank">Figure S10</a>. (Left) Endomucin staining of tip cells and their filopodia (arrowheads). (Middle) IBA1 staining marks macrophages. (Right) A merge of the endomucin and IBA1 staining. The interacting filopodia clearly have associated macrophages. (B) Interacting filopodia between two tip cells that are coated with VECAD. The filopodia are intertwined. The numbers label distinct cells. Image was captured using the highly sensitive photon-counting mode on the confocal microscope. The cartoon represents these tip cell–macrophage interactions. RV and their sprouts are not shown for the sake of simplicity and in no way meant to suggest lesser importance of RV. Scale bar, (A) 20 µm and (B) 5 µm.</p

Development from rSC to mature morphology.

<p>(Left, A–E) Z-projections of confocal stacks encompassing the developing SC in <i>Prox1-GFP</i> eyes. (A) At P4.5 VECAD labeling shows multicellular sprouts (*) at the sides of the GFP + tubular rSC. These sprouts have no detectable <i>Prox1</i> expression. (B) By P5, remodeling has formed a central core of PROX1⁺ flattened cells without any discernible internal space or RBCs. Further development continues by sprouting (*, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio.1001912.s013" target="_blank">Figure S13</a>) from the central core. Dotted line delineates developing SC from autofluorescence arising from nearby tissue. (C) At P10, developing SC has expanded considerably in size, and maturation with lumen formation and polarization of PROX1 expression to the inner wall have begun (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001912#pbio-1001912-g015" target="_blank">Figure 15</a>). The developing SC remains connected to the LVP (c) but no connections to the RV are detected after P5. (D) SC looks mature at P17 but continues to grow. (E) Adult SC. (Right) The cartoons distill the essential points of each stage. At P5 PROX1^- sprouts are shown in magenta. At all developmental stages, the SC is connected to the LVP. The adult stage SC cartoon attached to the LVP is seen from the inner wall perspective, where the thin cells are a darker green to depict strong <i>Prox1</i> expression. The SC shown below is from the outer wall perspective, with the paler green large cells having weak or no detectable PROX1 expression. BV, blood vessels; c, collector channels. Scale bar, 50 µm.</p

Cellular proliferation occurs during SC development.

<p>(A–C) Z-projections of the presumptive SC plane at the indicated ages. The top panels at each age show a merged image of endomucin and Ki67 staining. The endomucin staining delineates the developing SC. Ki67 labels dividing cells, labeling both the developing SC and other cells. Bottom panels show endomucin and Ki67 double-labeled cells in the developing SC in blue (voxel overlap of endomucin and Ki67 in a Z-projection). The double-positive proliferating cells closely track the developing SC. Thus, SC growth involves cell proliferation. Scale bar, 50 µm.</p