Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs.

The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9±0.3 pathways vs. temporal: 0.7±0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.

Segmental differences found in aqueous angiographic-determined high - and low-flow regions of human trabecular meshwork.

This work sought to compare aqueous angiographic segmental patterns with bead-based methods which directly visualize segmental trabecular meshwork (TM) tracer trapping. Additionally, segmental protein expression differences between aqueous angiographic-derived low- and high-outflow human TM regions were evaluated. Post-mortem human eyes (One Legacy and San Diego eye banks; n = 15) were perfused with fluorescent tracers (fluorescein [2.5%], indocyanine green [0.4%], and/or fluorescent microspheres). After angiographic imaging (Spectralis HRA+OCT; Heidelberg Engineering), peri-limbal low- and high-angiographic flow regions were marked. Aqueous angiographic segmental outflow patterns were similar to fluorescent microsphere TM trapping segmental patterns. TM was dissected from low- and high-flow areas and processed for immunofluorescence or Western blot and compared. Versican expression was relatively elevated in low-flow regions while MMP3 and collagen VI were relatively elevated in high-flow regions. TGF-ß2, thrombospondin-1, TGF-ß receptor1, and TGF-ß downstream proteins such as α-smooth muscle actin were relatively elevated in low-flow regions. Additionally, fibronectin (FN) levels were unchanged, but the EDA isoform (FN-EDA) that is associated with fibrosis was relatively elevated in low-flow regions. These results show that segmental aqueous angiographic patterns are reflective of underlying TM molecular characteristics and demonstrate increased pro-fibrotic activation in low-flow regions. Thus, we provide evidence that aqueous angiography outflow visualization, the only tracer outflow imaging method available to clinicians, is in part representative of TM biology.

Cellular and cytoskeletal alterations of scleral fibroblasts in response to glucocorticoid steroids.

Steroid-induced ocular hypertension can be seen even after trabecular meshwork (TM) bypass/ablation. Thus, the purpose was to investigate steroid-response in cells distal to the TM by using primary scleral fibroblasts. Primary scleral cell cultures were generated using mid-depth scleral wedges from human donor corneo-scleral rims (n = 5) after corneal transplantation. Cells were treated with dexamethasone (DEX; 100 nM) and compared to media (MED)/vehicle (DMSO) controls. Cell size, shape, and migration were studied using the IncuCyte Live-Cell Analysis System. Cytoskeleton was compared using Alexa Fluor-568 Phalloidin and senescence tested by evaluating beta-galactosidase. Western blot comparison was performed for α-SMA, FKBP-51, fibronectin, phospho-myosin light chain, and myocilin. Scleral fibroblasts upregulated FKBP-51 in response to DEX indicating the existence of steroid-responsive pathways. Compared to controls, DEX-treated cells proliferated slower (~50%; p < 0.01-0.02), grew larger (~1.3-fold; p < 0.001), and migrated less (p = 0.01-0.006). Alexa Fluor 568 Phalloidin actin stress fiber labeling was more diffuse in DEX-treated cells (p = 0.001-0.004). DEX-treated cells showed more senescence compared to controls (~1.7-fold; p = 0.01-0.02). However, DEX-treated cells did not show increased cross-linked actin network formation or elevated myocilin/fibronectin/α-SMA/phospho-myosin light chain protein expression. For all parameters, MED- and DMSO-treated control cells were not significantly different. Primary scleral fibroblasts, grown from tissue collected immediately distal to the TM, demonstrated scleral-response behaviors that were similar to, but not identical with, classic TM steroid-response. Further study is needed to understand how these scleral cellular alterations may contribute to steroid-response IOP elevation after TM bypass/ablation surgery.

Visual Assessment of Aqueous Humor Outflow.

In the past decade, many new pharmacological and surgical treatments have become available to lower intraocular pressure (IOP) for glaucoma. The majority of these options have targeted improving aqueous humor outflow (AHO). At the same time, in addition to new treatments, research advances in AHO assessment have led to the development of new tools to structurally assess AHO pathways and to visualize where aqueous is flowing in the eye. These new imaging modalities have uncovered novel AHO observations that challenge traditional AHO concepts. New behaviors including segmental, pulsatile, and dynamic AHO may have relevance to the disease and the level of therapeutic response for IOP-lowering treatments. By better understanding the regulation of segmental, pulsatile, and dynamic AHO, it may be possible to find new and innovative treatments for glaucoma aiming at these new AHO behaviors.

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.