Molecular interaction between fibronectin and heparan sulfate on trabecular meshwork (TM) exosome surfaces.

<p>(A) Serum-free media conditioned by human TM cells for 2.5 hours was split into 7 equal portions and purified heparan sulfate was added at increasing log₁₀ concentrations. Mixtures were incubated for 1 hour on a rocker at room temperature. Exosomes were then isolated from each sample and Fn content was assessed by dot blot. The intensity of Fn staining from 3 biological replicates (cell strains isolated from 3 different human donor eyes) were measured with ImageJ. The bar graph data is the mean±SEM of the 3 replicates. One representative dot blot is shown underneath. *p<0.05, [heparan sulfate] vs no heparan sulfate, Student’s t-test. (B) A dose-response curve was generated from the data shown in A (3 biological replicates). When no exogenous heparan sulfate was added, an endogenous concentration on the exosome of 1pM was used to generate the curve and estimate an EC₅₀ value. <b>C,</b> Conditioned media was split into 3 equal portions. Exosome-depleted FBS (10% final v:v) was added to two portions. Exosomes were prepared, resuspended in PBS with or without a bacterial heparanase II and incubated at 34°C for 1 hour with periodic shaking (approx. 10 minute intervals). Fn content of exosomes was assessed by western blot. Blot shown is representative of 3 biological replicates from cell strains isolated from different donor eyes.</p

Human trabecular meshwork (TM) explants release exosomes.

<p>(A) TM explants from six human donor eyes were cultured separately for 72 hours. Extracellular vesicles (EVs) were prepared from the conditioned media and analyzed for size by nanoparticle tracking analysis (NTA) and protein content by western blot (inset). NTA data is binned in 10 nm increments and represent the mean±SD of six individual biological replicates. Western blots are representative of the results obtained from the six individual biological replicates. AnxA2, Annexin 2; AnxA6, Annexin 6; MFG-E8, lactadherin. (B) A single human TM explant was cultured for 9 days (media collected every 3 days). This media was concentrated by ultrafiltration and mixed into the bottom portion of an Iodixanol (OptiPrep) density gradient and ultracentrifuged overnight. Fractions were collected, their density was determined and the concentration of EVs in each fraction was determined by NTA. Fractions corresponding to the density of exosomes (1.07–1.10 g/ml, gray box) were combined and analyzed for size distribution by NTA (inset).</p

List of TM exosomal proteins bound to heparin beads identified by mass-spec in two biological replicates.

<p>List of TM exosomal proteins bound to heparin beads identified by mass-spec in two biological replicates.</p

Trabecular meshwork (TM) exosomes bind fibronectin on the external surface.

<p>Serum-free medium was conditioned for 2.5 hours with primary TM cell cultures. Conditioned media was split in half and either exosome-depleted FBS (10% final v:v) or purified human plasma fibronectin (Fn, 2.5μg/ml [final]) was added to one portion. Exosomes were prepared from both portions (and from unconditioned media + FBS or purified Fn) and Fn content was assessed by Western blot. The blots shown represent the results from a minimum 3 biological replicates (cell strains isolated from different human donors).</p

Treatment of human trabecular meshwork (TM) explants with dexamethasone (Dex) reduces the amount of exosome bound fibronectin (Fn).

<p>TM explants were dissected from human donor eyes and split in half. Each half was cultured for 72 hours in control (Ctrl) media or media containing 100nM dexamethasone (Dex). Conditioned media from control and Dex treated TM explants was probed for fibronectin (Fn) and myocilin (Myoc) by western blot. Band intensity was normalized to dry TM tissue weight. Dex treatment increased the secretion of (A) Fn and (B) Myoc in the conditioned media (mean ± SD, n = 3). Exosomes were isolated, sized and counted by nanoparticle tracking analysis. (C) Dex treatment does not alter the mean or (D) mode size of exosomes released by human TM explants (control n = 6, Dex n = 6). (E) Dex treatment does not significantly alter the amount of exosomes in the conditioned media (p = 0.1654, Student’s t-test, n = 6). (F) Equal volumes of exosomes were analyzed for Fn content by western blot from six donors (6 control, 6 Dex treated). Fn band intensity was determined using ImageJ and the intensity was normalized to the number of exosomes loaded per lane. The representative bands shown are from a single donor, control = 3.39E+08 particles and Dex = 3.49E+08 particles. Bar graph is the mean±SEM of six control and Dex treated Fn band intensities normalized to the number of particles per lane. *p<0.05, control vs Dex, n = 6, Student’s t-test. Box-and-whisker plots in A, B and C are the max, 3^rd quartile, median, 1^st quartile and min.</p

A Review of Nitric Oxide for the Treatment of Glaucomatous Disease

<p><b>Article full text</b></p> <p><br></p> <p>The full text of this article can be found here<b>.</b> <a href="https://link.springer.com/article/10.1007/s40123-017-0094-6">https://link.springer.com/article/10.1007/s40123-017-0094-6</a></p><p></p> <p><br></p> <p><b>Provide enhanced content for this article</b></p> <p><br></p> <p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p> <p><br></p> <p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p> <p><br></p> <p>Other enhanced features include, but are not limited to:</p> <p><br></p> <p>• Slide decks</p> <p>• Videos and animations</p> <p>• Audio abstracts</p> <p>• Audio slides</p

Treatment of human trabecular meshwork (TM) explants with dexamethasone decreases the amount of exosomal annexins A2/A6 and dipeptidyl peptidase activity.

<p>Exosomes were prepared from media conditioned by TM explants and analyzed for the presence of the heparin/heparan sulfate binding proteins (A) annexin A2 and (B) annexin A6. Intensity of immunoblot staining was measured with ImageJ, normalized to the number of vesicles loaded per lane and shown in bar graphs as mean±SEM. Panel A is representative of 4 biological replicates (equal particle loading per lane, 4 control, 4 Dex treated) and panel B is representative of 6 biological replicates (6 control, 6 Dex treated). *p<0.05 control vs Dex, Student’s t-test; annexin A2 n = 4, annexin A6 n = 6. <b>C,</b> Equal amounts of exosomes from control and Dex treated TM explants were analyzed for DPP4 activity using a peptide cleavage assay. Measured activity was fitted to a standard curve generated with known amounts of recombinant DPP4. Data shown as the mean±SD of 4 biological replicates with technical measurements in triplicate. *p<0.05 control vs Dex, Student’s T-test, n = 4.</p

Time-course of ligand-dependent myocilin recruitment to cell-surface membranes containing GPR-143.

<p>After labeling cell surface proteins with biotin, CHO and MCF7 cells stably expressing MYOC and GPR143 were stimulated with l-DOPA, then proteins were harvested at the times illustrated. (A). Shown are representative western blots of cell lysates subjected to strepavidin chromatography. Proteins in bound (b) and unbound (u) fractions were probed for myocilin (MYOC) and actin abundance. (B). Specific bands from blots were analyzed using ImageJ to quantify band intensity and kinetic results of bound fraction for all three proteins analyzed for an individual experiment is shown. Results are representative of 4 experiments for each cell type tested. The mean values of the bound MYOC at time zero and at 20 min are compared for the experiments in aggregate, and found to be significantly different (* p<0.05).</p

Ligand-dependent co-localization of myocilin and GPR143.

<p>Confocal micrographs of GPR143 linked to GFP and myocilin identified by immunofluorescence. Cells were harvested at 0, 5, 20, 40, 60 after exposure to 1 µM l-DOPA. Overlaid images illustrate co-localization of GPR143 and myocilin starting at 5 minutes and continuing through 20 minutes, with little co-localization at 40 minutes and no co-localization at time 0 and 60 minutes. Insets (shown for 5, 20 and 40 min) are 4x computer magnification of a region denoted with an arrow illustrating co-localization. Bar equals 25 µM.</p

Co-localization of myocilin and arrestin 2.

<p>Confocal micrograph of arrestin 2 linked to GFP and myocilin observed by immunofluorescence at time 0, 10, and 25 minutes after l-DOPA treatment. The images are overlaid to illustrate co-localization at 10 and lack of co-localization at 0 and 25 minutes. Bar equalsµM.</p