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

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

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 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

Ultrastructure of cerebral cortex and cerebellum of a 22L scrapie-infected tg44+/+ mouse.

<p>Sections were from same mouse as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000800#ppat-1000800-g005" target="_blank">Figure 5</a>, and were stained with uranyl acetate/lead citrate. (A) Area of neuropil with severe vacuolation where most vacuoles originate within processes and are separated from each other by intact membranes. (B) Several distended astrocytic processes (asterisks) of the perivascular glial limitans are present around a blood vessel Lu: lumen. (C) On higher magnification of boxed area from (B), the endothelial basement is shown to be filled with irregularly orientated amyloid fibrils (arrows). (D) The earliest stage of vascular amyloid is shown. Here the endothelial basement membrane (black arrowheads) is intact, but the pericyte basement membrane (black arrows) is thickened and heavily infiltrated with short amyloid fibrils (white arrowheads). (E) Severe neuritic dystrophy in which several processes show an excessive accumulation of organelles and abnormal electron dense bodies. (F) White matter of the cerebellum showing an empty myelin sheath (seen as vacuoles by light microscopy) and also a dark degenerate axon (asterisk) within an intact myelin sheath. Similar findings were observed in RML scrapie-infected tg44+/+ mice. Scale bars: A, 2 µm; B, E, F, 1 µm; C, 500 nm; D, 500 nm.</p

Survival curves for scrapie-infected mice.

<p>Transgenic mice (tg44+/+ and tg23+/+) expressing only anchorless PrP, C57BL/10 (Prnp+/+) and Prnp+/− mice, expressing normal anchored mouse PrP were compared. Mice were inoculated intracerebrally with 22L scrapie (panel A) and RML scrapie (panel B) and observed weekly for development of disease (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000800#ppat-1000800-t001" target="_blank">Table 1</a>). Mice were euthanized when clinical signs were severe as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000800#ppat-1000800-t001" target="_blank">Table 1</a>. N values for each group are as follows: Panel A (22L): Prnp+/+, 11; Prnp+/−, 17; tg44+/+, 23; tg23+/+, 10. Panel B (RML): Prnp+/+, 8; tg44+/+, 21; tg23+/+, 9.</p

Immunological detection of PrPres in brain at both light and electron microscopic levels.

<p>The 22L scrapie-infected anchorless PrP tg44+/+ mouse shown was clinically positive at 377 dpi. (A–B) Light microscopy of 1 µm thick plastic-embedded tissue labelled with monoclonal antibody 1A8. (A) shows intravascular and perivascular PrPres. In (B), the marked vascular amyloid infiltration is associated with occlusion of the vascular lumen (boxes indicate occluded lumens of two vessels). When these vessels were visualised in the electron microscope the smooth muscle of the vascular media was totally replaced by amyloid and an amorphous electron dense material filled the lumen (not shown). (C–F) Electron microscopy. (C) Low power view of large PrPres amyloid plaque adjacent to a small artery. Vessel lumen is in upper left corner and an endothelial cell with a prominent nucleus is to the right of the lumen. Silver enhanced gold-labeled PrPres is seen within the basement membrane (BM) and within the heavy amyloid accumulation which partially replaces the smooth muscle media layer. Amyloid bundles radiate away from the vessel and through the neuropil at the bottom right. (D) A high magnification illustration of (C) showing PrPres labelling on small bundles of amyloid fibrils at the periphery of the plaque. (E) Marked PrPres accumulation at the endothelial and pericyte basement membranes (arrows) and extending into narrow extracellular spces between nearby neurites and perivascular glial processes. Asterix (*) shows area of astrocytic cytoplasmic swelling. Lu; lumen. (F) Neuropil of cerebrum showing immunogold label for PrPres present over the extracellular spaces between neurites bounded by pairs of adjacent plasmalemmae. No visible amyloid fibrils were visible and the spaces between cellular processes were regular and even. This non-fibrillar PrPres labelling which dissects between neurite and glial cell profiles may extend over large area of neuropil as shown in (G). Asterix (*) on left side shows enlarged glial process with loss of normal cytoplasmic organelles. Similar findings were observed in tg44+/+, tg44+/− and tg23+/− mice. Tg23+/+ mice were not examined by electron microscopy. Scale bars: A and B, 20 µm; C and D, 1 µm; E and G, 2 µm; F, 500 nm.</p

Detection of PrPres by immunoblot using monoclonal antibody D13.

<p>Comparison of PrPres in brain of 22L scrapie-infected tg44+/+ and tg44+/− mice at the time of clinical disease. All lanes were loaded with 0.25 mg brain tissue equivalents. A clinical Prnp+/− mouse is shown for comparison. Lane 1: Prnp+/− (251 dpi), PrPres bands are seen at 21, 28, and 31 kD; lanes 2–5: tg44+/− mice (567, 589, 594, 594 dpi) and lanes 6–9 tg44+/+ (348, 365, 384, 408 dpi). PrPres bands are at 18 and 23 kD. The sizes are lower in these mice due to lack most carbohydrates and lack of GPI <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000800#ppat.1000800-Chesebro1" target="_blank">[19]</a>. PrPres levels in tg44+/− mice were approximately 50% lower than in tg44+/+ mice.</p