Page 16 - Read Online

Page 12 Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08

Tsuneya Ikezu (Boston University, US) told us about EV protein networks from iPSC-derived neural cells
and brain tissue of patients with Alzheimer’s Disease (AD). High-level proteomic analyses of EVs from iPSC
differentiated brain cell types and from brain tissues of patients with AD, mild cognitive impairment, and
healthy controls led to specific markers for cell-type specific EVs and could clearly separate the EV types
by protein composition. The statistical analyses could also distinguish EV protein subsets between healthy
control brain EVs, patients with mild cognitive impairment, and AD patients, including some that might
show progressive changes towards AD. Certain proteome modules resembled pathways found in activated
astrocytes, including inflammatory processes. This information could form a basis for EV proteomics in
liquid biopsy for AD development and monitoring.

Faisal Alibhai (University Health Network, Toronto, Canada) provided insight into a near universal human
phenomenon, aging, and the associated changes in circulating EV cargo and function. Parabiosis of murine
circulation involving young and old mice rejuvenates multiple organ systems in the old mouse, but the young
mouse shows age related defects. Thus, blood circulating factors play roles in aging (and youth), begging the
question of the roles of EVs in this. Faisal isolated EVs from young and old murine populations, noting that
the particles from old individuals were both fewer and smaller than those from young plasma. Curiously,
EV markers (CD63, CD81, TSG101) were more abundant in old plasma, while markers of lipoproteins
were more abundant in young plasma, suggesting that the particle differences noted may be skewed in
this fashion. In functional studies, peritoneal macrophages were treated with EVs from old and young
plasma, and then were stimulated with LPS. Under both circumstances, old EVs decreased IL1B and IL12B
expression (and other cytokines) more than young EVs. Old EVs also reduced VEGF-mediated endothelial
cell tube formation. The miR content of old EVs targeted numerous pathways that could be dysregulated in
aging (inflammation and senescence). In an attempt to reduce aging effects with senolytic therapy (dasatinib
and quercetin in this case) in old mice, the treatments reduced miR levels in plasma EVs, and the EVs no
longer blocked endothelial cell responses to VEGF. Thus, aging cells manifest senescent phenomena via aged
EVs, and some of the effects may be reversed by senolytic therapies (or young EVs).

The last talk of the Pathology session was about the utility of neural stem cell EVs to improve outcomes in
a porcine ischemic stroke model, presented by Steven Stice (University of Georgia, US). As mesenchymal
stem cells (MSCs, and their EVs) are seeing use in many inflammatory-related neurologic diseases. Steve’s
group found that neural stem cell (NSC) EVs better promoted an anti-inflammatory immune environment
than MSC EVs in a murine stroke model. They chose a pig model as a better representative of human brains
than rodent models. In this study, pigs were trained for motor and behavioral function; subjected to ischemic
strokes; treated with NSC EVs; monitored by imaging and functional recovery studies; underwent further
clinical testing and imaging; and followed finally, by an autopsy. Pigs treated with NSC EVs showed stunning
functional recovery compared to surviving untreated pigs. On MRI assessment, the extent of midline shift -
a pathology-induced mass effect leading to brain displacement off the midline - is correlated with survival.
NSC EV-treated pigs showed reduced midline shift and increase in survival rate. On the pig-adapted
Modified Rankin scale (mRS), pigs that received NSC EVs, irrespective of a high or low midline shift,
showed reduced mRS scores, indicating less disability. Consistent with reduced inflammatory responses, pigs
treated with NSC EVs demonstrated attenuated microglia activation both in vitro and in vivo.

The final session of Day 3 was devoted to EVs and viruses, and was hosted by Nihal Altan-Bonnet (NIH,
National Heart, Lung, and Blood Institute, US) and Steve Gould (Johns Hopkins University, US). It started
with James Erickson (George Mason University, US) discussing how to separate EVs from virions in
Coronavirus infections. The lab has used several techniques to separate virus (typically HIV) from EVs
in virally infected cells, including the use of differential ultracentrifugation with a high-resolution density
(iodixanol) gradient ultracentrifugation. They applied this strategy to isolate a betacoronavirus (OC43,
BSL2 compatible) from infected cells (lung cancer cell line). Incubating these virus fractions with Vero cells