Different-sized extracellular vesicles derived from stored red blood cells package diverse cargoes and cause distinct cellular effects.

BACKGROUND: The formation of extracellular vesicles (EVs) occurs during cold storage of RBCs. Transfusion of EVs may contribute to adverse responses in recipients receiving RBCs. However, EVs are poorly characterized with limited data on whether distinct vesicles are formed, their composition, and potential biological effects. STUDY DESIGN AND METHODS: Stored RBC-derived EVs were purified using protocols that separate larger microvesicle-like EVs (LEVs) from smaller exosome-like vesicles (SEVs). Vesicles were analyzed by electron microscopy, content of hemoglobin, heme, and proteins (by mass spectrometry), and the potential to mediate lipid peroxidation and endothelial cell permeability in vitro. RESULTS: SEVs were characterized by having an electron-dense double membrane whereas LEVs had more uniform electron density across the particles. No differences in hemoglobin nor heme levels per particle were observed, however, due to smaller volumes, SEVs had higher concentrations of oxyHb and heme. Both particles contained antioxidant proteins peroxiredoxin-2 and copper/zinc superoxide dismutase, these were present in higher molecular weight fractions in SEVs suggesting either oxidized proteins are preferentially packaged into smaller vesicles and/or that the environment associated with SEVs is more pro-oxidative. Furthermore, total glutathione (GSH + GSSG) levels were lower in SEVs. Both EVs mediated oxidation of liposomes that were prevented by hemopexin, identifying heme as the pro-oxidant effector. Addition of SEVs, but not LEVs, induced endothelial permeability in a process also prevented by hemopexin. CONCLUSION: These data show that distinct EVs are formed during cold storage of RBCs with smaller particles being more likely to mediate pro-oxidant and inflammatory effects associated with heme.

A Novel Tree Shrew (Tupaia belangeri) Model of Glaucoma.

Purpose: Primates and rodents are used widely as animal models of glaucoma, but each has significant limitations. Researchers need additional animal models that closely resemble the relevant anatomy and pathologic features of the human disease to more quickly advance research. We validate a novel glaucoma animal model in tree shrews (Tupaia belangeri). Methods: Experimental glaucoma was induced in adult tree shrews (n = 8) by injecting 50 µL of a 25 mg/mL ferromagnetic bead solution into the anterior chamber. Beads were directed into the iridocorneal angle with a magnet to impede aqueous outflow. Animals were followed for 3 months with weekly IOP measurements and biweekly spectral domain optical coherence tomography (SD-OCT) images of the optic nerve head. Histopathology of the optic nerve and optic nerve axon counts were completed at the end of the study. Results: The 12-week average mean IOP was 22.7 ± 3.6 and 8.6 ± 2.9 mm Hg in the treated and control eyes, respectively. Longitudinal analysis showed significant retinal nerve fiber layer (RNFL) thinning throughout the study. Axon counts were significantly reduced (59.7%) in treated versus control eyes. SD-OCT imaging showed cupping and posterior displacement of the lamina cribrosa in glaucomatous eyes. RNFL thickness and optic nerve axon counts were reduced consistent with IOP elevation. Optic nerves demonstrated histopathology consistent with glaucomatous optic neuropathy. Conclusions: Tree shrews with experimental glaucoma show key pathologic characteristics of the human disease. The tree shrew model of glaucoma has the potential to help researchers accelerate our understanding of glaucoma pathophysiology.

Effects of particulates and lipids on the hydraulic conductivity of Matrigel.

The hydraulic conductivity of a connective tissue is determined both by the fine ultrastructure of the extracellular matrix and the effects of larger particles in the interstitial space. In this study, we explored this relationship by examining the effects of 30- or 90-nm-diameter latex nanospheres or low-density lipoproteins (LDL) on the hydraulic conductivity of Matrigel, a basement membrane matrix. The hydraulic conductivity of Matrigel with latex nanospheres or LDL particles added at 4.8% weight fraction was measured and compared with the hydraulic conductivity of Matrigel alone. The LDL-derived lipids in the gel were visualized by transmission electron microscopy and were seen to have aggregated into particles up to 500 nm in size. The addition of these materials to the medium markedly decreased its hydraulic conductivity, with the LDL-derived lipids having a much larger effect than did the latex nanospheres. Debye-Brinkman theory was used to predict the effect of addition of particles to the hydraulic conductivity of the medium. The theoretical predictions matched well with the results from adding latex nanospheres to the medium. However, LDL decreased hydraulic conductivity much more than was predicted by the theory. The validation of the theoretical model for rigid particles embedded in extracellular matrix suggests that it could be used to make predictions about the influence of particulates (e.g., collagen, elastin, cells) on the hydraulic conductivity of the fine filamentous matrix (the proteoglycans) in connective tissues. In addition, the larger-than-predicted effects of lipidlike particles on hydraulic conductivity may magnify the pathology associated with lipid accumulation, such as in Bruch's membrane of the retina during macular degeneration and the blood vessel wall in atherosclerosis.

Reprogramming progeny cells of embryonic RPE to produce photoreceptors: development of advanced photoreceptor traits under the induction of neuroD.

PURPOSE: In examining the prospect of producing functional photoreceptors by reprogramming the differentiation of RPE progeny cells, this study was conducted to investigate whether reprogrammed cells can develop highly specialized ultrastructural and physiological traits that characterize retinal photoreceptors. METHODS: Cultured chick RPE cells were reprogrammed to differentiate along the photoreceptor pathway by ectopic expression of neuroD. Cellular ultrastructure was examined with electron microscopy. Cellular physiology was studied by monitoring cellular free calcium (Ca(2+)) levels in dark-adapted cells in response to light and in light-bleached cells in response to 9-cis-retinal. RESULTS: Reprogrammed cells were found to localize red opsin protein appropriately to the apex. These cells developed inner segments rich in mitochondria, and while in culture, some formed rudimentary outer segments, analogous to those of developing photoreceptors in the retina. In response to light, reprogrammed cells reduced their Ca(2+) levels, as observed with developing retinal photoreceptors in culture. Further, on exposure to 9-cis-retinal, the light-bleached, reprogrammed cells increased their Ca(2+) levels, reminiscent of visual cycle recovery. CONCLUSIONS: These results indicate the potential of reprogrammed cells to develop advanced ultrastructural and physiological traits of photoreceptors and point to reprogramming progeny cells of embryonic RPE as a possible alternative in producing developing photoreceptors.

Prevalence and morphology of druse types in the macula and periphery of eyes with age-related maculopathy.

PURPOSE: Macular drusen are hallmarks of age-related maculopathy (ARM), but these focal extracellular lesions also appear with age in the peripheral retina. The present study was conducted to determine regional differences in morphology that contribute to the higher vulnerability of the macula to advanced disease. METHODS: Drusen from the macula (n = 133) and periphery (n = 282) were isolated and concentrated from nine ARM-affected eyes. A semiquantitative light microscopic evaluation of 1-mum-thick sections included 12 parameters. RESULTS: Significant differences were found between the macula and periphery in ease of isolation, distribution of druse type, composition qualities, and substructures. On harvesting, macular drusen were friable, with liquefied or crystallized contents. Peripheral drusen were resilient and never crystallized. On examination, soft drusen appeared in the macula only, had homogeneous content without significant substructures, and had abundant basal laminar deposits (BlamD). Several substructures, previously postulated as signatures of druse biogenesis, were found primarily in hard drusen. Specific to hard drusen, which appeared everywhere, were central subregions and reduced RPE coverage. Macular hard drusen with a rich substructure profile differed from primarily homogeneous peripheral hard drusen. Compound drusen, found in the periphery only, exhibited a composition profile that was not intermediate between hard and soft. CONCLUSIONS: The data confirm regional differences in druse morphology, composition, and physical properties, most likely based on different formative mechanisms that may contribute to macular susceptibility for ARM progression. Two other reasons that only the macula is at high risk despite having relatively few drusen are the exclusive presence of soft drusen and the abundant BlamD in this region.

Comparison of morphology of human macular and peripheral Bruch's membrane in older eyes.

Deposits in macular human Bruch's membrane (BrM) increase with age and have been postulated to be associated with age-related maculopathy. We used two ultrastructural methods to compare these deposits by electron microscopy in macular and peripheral BrM of eight eyes from donors 63-86 years of age. Quick-freeze/deep-etch (QFDE) was used to prepare replicas that showed the ultrastructure of deposits, and osmium-tannic acid-paraphenylenediamine (OTAP) was used to preserve small extracellular lipid particles. We found that an accumulation of lipoprotein-like particles (LLPs) occurred in the peripheral BrM just as it does in the macular region, but with perhaps a somewhat slower time course. The "lipid wall," reported in macular BrM, was also found occasionally in the peripheral regions. The same processes that lead to age-related accumulation of LLPs in macular BrM appear to also occur in the peripheral regions.

Age-related changes in human macular Bruch's membrane as seen by quick-freeze/deep-etch.

Lipid-containing inclusions have been observed in human Bruch's membrane (BrM) and are postulated to be associated with age-related maculopathy (ARM), a major cause of legal blindness in developed countries. The dehydration associated with specimen preparation for thin-section transmission electron microscopy causes loss of these inclusions. Better preservation of the ultrastructure of the inclusions and tissue is achieved by using a quick-freeze/deep-etch preparation. We use this technique to examine normal human macular BrM in order to characterize the deposition of the lipid-rich inclusions and their age-related accumulation within different layers of the tissue. We find that various inclusions mentioned in other studies can be formed by combinations of three basic structures: lipoprotein-like particles (LLPs), small granules (SGs) and membrane-like structures. These inclusions are associated with collagen and elastic fibrils by fine filaments. In younger eyes, these inclusions are found mostly in the elastic (EL) and outer collageneous layer (OCL) and occupy a small fraction of the interfibrillar spacing. As age increases, LLPs and SGs gradually fill the interfibrillar spacing of the EL and inner collageneous layer (ICL) of the tissue, and later form a new sublayer, the lipid wall, within the boundary region between the basal lamina of retinal pigment epithelium (RPE) and ICL. Because the formation of the lipid wall only occurs after these inclusions fill the ICL, and it seems unlikely that the LLPs can pass through the packed layer, this result suggests a possible RPE origin of the LLPs that make up the lipid wall.

Apolipoprotein localization in isolated drusen and retinal apolipoprotein gene expression.

PURPOSE: To evaluate apolipoprotein (Apo) gene expression in native human retinal pigment epithelium (RPE) and neurosensory retina and to detect apolipoproteins within age-related, extramacular drusen. METHOD: Drusen were isolated manually from 10 eyes of 10 donors (age range, 58-93 years) with grossly normal maculas that were preserved in 4% paraformaldehyde within 6 hours of death. In cryosections of druse-enriched pellets (6-57 drusen per eye), the Apos A-I, A-II, B, C-I, C-II, C-III, E, and J were detected by indirect immunofluorescence. Two graders assessed the prevalence and pattern of immunoreactivity. mRNA transcripts were detected by reverse-transcription polymerase chain reaction (RT-PCR), with human hepatoma HepG2 cells as the positive control. RESULTS: Extramacular drusen were classified in two groups on gross appearance: transparent with a reflective shell and cloudy. The proportion of the latter increased significantly with age. All Apos examined were detectable, in descending order of prevalence: ApoE (99.5%), J (99.5%), C-I (93.1%), B (80.4%), A-I (61.0%), A-II (59.2%), C-II (57.7%), and C-III (16.6%). Immunoreactivity was either diffusely distributed throughout the drusen (56.7%), confined to the druse rim (16.0%), or both (21.2%). Six percent displayed evidence of organized substructure reminiscent of active remodeling. The proportion of diffusely labeled drusen decreased significantly with age for ApoE (P=0.034) and ApoE/C-I combined (P=0.027). RT-PCR products for Apos C-I, C-II, E, and J were found in retina and RPE; for ApoA-II in the retina only. The ApoC-III message was undetectable. CONCLUSIONS: To an emerging model of an RPE-secreted large lipoprotein particle implied by previous work, this study adds ApoC-I and ApoC-II, major modulators of lipoprotein lipase activity, and confirms previously demonstrated Apos A-I, B-100, and E. It is possible that a neutral lipid-rich druse shell containing Apos will be visible in the living fundus.

Synaptic input to the on-off directionally selective ganglion cell in the rabbit retina.

A physiologically identified on-off directionally selective (DS) ganglion cell with its preferred-null axis defined was stained with horseradish peroxidase (HRP) and prepared for electron microscopy. A continuous series of thin sections were used to examine the cell's synaptology. Although the DS cell dendrite received the majority of its synaptic input from a heterogeneous population of amacrine cell processes, a frequently observed synaptic profile consisted of a DS cell dendrite receiving synapses from a cluster of several amacrine cell processes. These clusters of processes were assumed to be from a fascicle of amacrine cells, most of which probably belonged to several different cholinergic starburst amacrine cells. The most frequently observed presynaptic profile within the clusters consisted of a synaptic couplet in which two processes synapsed with each other before one of them finally synapsed with the DS ganglion cell dendrite; occasionally, a chain of three serial synapses was seen. In addition, a specific microcircuit that has the potential to exert lateral feedforward inhibition was also observed. This microcircuit consisted of two cone bipolar cell terminal dyad synapses where one dyad contained an amacrine cell process making a reciprocal synapse and a DS ganglion cell dendrite receiving direct excitation; the other dyad synapse, found lateral to the first dyad, contained two amacrine cell processes that both made reciprocal synapses, but one fed forward to make a putative inhibitory synapse with the DS cell dendrite.