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It has long been recognized that many diseases are due to
the two deadly “C’s” of either the clotting or complement
systems (1) and stroke is a prime example. Stroke remains
one of the leading causes of disability and death worldwide (2)
and is predominantly due to thromboembolic occlusion
of a major artery feeding the brain. While the initial
management of stroke has understandably focused on
clotting as manifest in the 1996 FDA approval of tissue
plasminogen activator (tPA), often known as a “clot-
busting” agent, there has been increasing recognition
that other factors including inflammation are important
determinants of outcome. In the new issue of Circulation
Research (3), we learn that the complement system
activated through exosomal signaling may also play a
critical role after ischemic stroke. Age-associated increases
in circulating inammatory mediators have been suggested
as contributors to the development of stroke and other
neurological disorders (4). This notion has been supported
by data showing that exposing young mice to plasma
from aged mice led to cognitive functional decline (5). In
contrast, heterochronic parabiosis experiments, in which
the circulations of mice of different ages are connected,
demonstrate that neurogenesis in aged mice can be
restored by exposure to youthful circulation (6). However,
the mechanism by which age-associated inflammatory
mediators inuence stroke remains elusive.
Zhang et al. (3) in Circulation Research investigated the
role of exosomes in the pathogenesis of stroke. Compared
to young controls (3-month-old), exosomes isolated from
aged rats (21- to 23-month-old) serum had larger diameters
and lower concentrations. Aged rats were subjected to a
simulated stroke with permanent distal middle cerebral
artery occlusion (dMCAO). With the blood-brain-barrier
(BBB) disrupted, intravenous injection of exosomes from
aged rats were found in healthy and ischemic brain regions
(the striatum, hippocampus, and cerebellum) for 14 days.
Using cell lineage markers, the authors found that these
injected exosomes had accumulated within neurons,
microglia, and endothelial cells. These results indicate that
after a stroke, exosomes from aged subjects cross a disrupted
BBB and enter neuronal and non-neuronal populations
within the brain.
How do exosomes from younger and older rodents
influence simulated stroke outcomes during aging? To
determine the role of exosomes in stroke during aging,
aged rats were subjected to dMCAO. Injection of exosomes
isolated from young rat serum reduced infarct volume
and improved sensorimotor decits 72 h after stroke with
effects lasting for as long as 35 days. In contrast, injection of
exosomes isolated from aged rat serum worsened infarction
and sensorimotor decits in aged rats. These data provide
evidence that exosomes inuence post-stroke outcomes in
aged subjects and that exosomes from younger and older
rodents have fundamentally different effects on the aged
brain. Notably, young rats subjected to stroke saw no
change in stroke volume or function after infusion of serum
exosomes from old rats.
The investigators found that the benecial and harmful
Editorial Commentary
Backhanded complement: circulating exosomes in aged animals
add insult to injury after stroke
Haobo Li, Cedric Shefeld Jr, J. Sawalla Guseh, Anthony Rosenzweig
Cardiovascular Research Center, Division of Cardiology, Corrigan Minehan Heart Center, Department of Medicine, Massachusetts General
Hospital, Harvard Medical School, Boston, MA, USA
Correspondence to: Anthony Rosenzweig. Cardiovascular Research Center, Division of Cardiology, Corrigan Minehan Heart Center, Department of
Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Email: rosenzwe@helix.mgh.harvard.edu.
Comment on: Zhang H, Lin S, McElroy CL, et al. Circulating Pro-Inflammatory Exosomes Worsen Stroke Outcomes in Aging. Circ Res
2021;129:e121-40.
Received: 14 October 2021; Accepted: 08 November 2021.
doi: 10.21037/exrna-21-21
View this article at: https://dx.doi.org/10.21037/exrna-21-21
4
ExRNA, 2021
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© ExRNA. All rights reserved. ExRNA 2021 | https://dx.doi.org/10.21037/exrna-21-21
effects of exosomes may have a cellular basis centered not
on the neuron, but the microglia which are known to play
key roles in the primary immune responses within the
central nervous system. First, they found that activated
microglia resided in the penumbra and that aged exosomes
increased the activated population. Second, they found
that the activated microglial population took on a classical
M1 proinflammatory activation pattern (Iba+CD86+) and
not a protective M2 phenotype (Iba+CD206+). Third, aged
exosome exposure led to worsened postinfarction synaptic
activity with a reduction in the total length and number of
dendritic spines in area neurons. Interestingly, exosomes
(from young or old plasma) did not influence resting
neuronal potentials. However, exosomes from young
animals were able to reverse microglial activation. Finally,
a series of elegant experiments reveal that microglial
depletion using a small molecule inhibitor of CSF1R was
able to reduce brain injury and improve motor performance
in older postinfarction rodents. Taken together, the
authors conclude that aged exosomes serve to activate
the microglial population which subsequently injures the
neuronal population through synaptic phagoptosis while
young exosomes appear to beneficially reverse microglial
activation. The authors are to be congratulated on
rigorously examining, for the rst time, the role of exosomes
from young and aged subjects in the pathogenesis of stroke.
This work highlights news areas of study concerning the
therapeutic potential of circulating exosomes in elderly
patients after stroke.
How do exosomes from young and older rodent
populations differ? To further delineate the molecular
mechanisms governing exosomal exacerbation of stroke,
Zhang et al. (3) compared protein profiles in exosomes
from young and aged rats. One hundred and twenty-six
proteins were differentially expressed (71 upregulated and
55 downregulated) and were enriched for gene ontology
categories that broadly involved inammation, phagoptosis,
and complement activation. Among 126 differently
expressed proteins, three complement proteins (CD46, C3a,
and C3b) were further validated by western blot, where
CD46 has higher and C3a and C3b had lower expression
levels in exosomes from young rats compared to aged rats.
In aged rats subjected to dMCAO, compared to injection
of exosomes from young rats, these complement proteins,
C3a/C3b and C3a receptor (C3aR), were increased in active
microglia in the penumbra 48 and 72 h after injection of
exosomes from aged rats. These data suggest an injection of
exosomes from aged rats may exacerbate poststroke brain
injury by inducing C3a/C3b and C3aR in active microglia.
Indeed, in aged rats subjected to dMCAO and injection of
exosomes from aged rats, application of C3aR inhibitor,
SB290157, reduced activated microglia in the penumbra
and decreased phagoptosis as well as reducing poststroke
synaptic damage and infarction, leading to improvement
of sensorimotor performance. Taken together, these data
suggest an essential role of C3aR, and more generally
complement proteins, in the development of stroke in
aged subjects. However, it is still unclear whether these
benecial effects of C3aR inhibitor are through inhibiting
C3aR locally in the brain or C3aR carried by the injected
exosomes.
This interesting work (3) provides new insights into the
contribution of exosomes in the development of stroke in
aged subjects. First, the BBB is disrupted during stroke
in aged subjects, allowing exosomes to cross and deliver
proinammatory mediators that exacerbate stroke outcomes.
Second, through the delivery of complement system
proteins (e.g., C3a/C3b, C3aR) blood exosomes regulate
microglial phagoptosis, stroke-induced brain damage, and
sensorimotor performance. Third, microglial depletion by
inhibition of C3aR is effective in attenuating stroke-induced
brain damage in aged subjects, suggesting that C3aR could
be a therapeutic target for the treatment of stroke. Finally,
exposure of aged rats to young blood exosomes reversed
poststroke synaptic and neurological functional declines,
indicating a therapeutic anti-inammatory potential for the
application of young blood exosomes in protecting the brain
against stroke.
As with all interesting and provocative work, multiple
questions are raised by Zhang et al. (3) that warrant
further investigation. First, since exosomes can be secreted
by multiple organs/tissues and multiple cell types (7),
identifying the source(s) of circulating exosomes and
the changes seen in aging would be of great interest
and might enable complementary therapeutic strategies
blocking exosome release. Second, exosomes carry multiple
bioactive molecules in addition to proteins examined here,
including coding and noncoding RNAs as well as DNA (8).
It seems likely that other exosome molecular cargoes
are also contributing to the age-related effects observed.
Third, although Zhang et al. (3) identified complement
system members as contributing to the adverse effects
of old exosomes, the drivers of the benefits seen with
young exosomes remain largely undefined. Fourth, since
stroke can alter the profile of exosome synthesis and
secretion (9) and the current study focused on injection of
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© ExRNA. All rights reserved. ExRNA 2021 | https://dx.doi.org/10.21037/exrna-21-21
exogenous exosomes, more work will be needed to parse
the roles of endogenous exosomes at different timepoints
in this process. Finally, before clinical translation can be
investigated, demonstration of similar exosome biology in
human stroke as well as an understanding of how exosomes
might be altered by reperfusion (still the mainstay of
ischemic treatment) or patient behaviors and co-morbidities
will be essential.
Despite these issues, Zhang et al. (3) have admirably
provided evidence of the contribution of exosomes in a rat
model of stroke-induced brain injury and have expanded
our understanding of ischemic stroke beyond clotting to
include a key role for the complement system (e.g., C3aR)
in the postinfarction process. These findings provide
valuable insight into the molecular correlates of stroke
during aging with possible implications for therapeutic
intervention among our aging population. Exploring the
potential of exosomal, or C3aR specific interventions, for
the treatment of stroke will be an exciting opportunity for
future investigation.
Acknowledgments
Funding: This work was supported by the National Institutes
of Health (R01AG061034, R35HL155318 to AR), the
American Heart Association (20CDA35310184 to HL;
19AMFDP34990046 to JSG), Sarnoff Cardiovascular Research
Foundation (to CS), and Massachusetts General Hospital
Sanchez-Ferguson Faculty Scholar Program (to JSG).
Footnote
Provenance and Peer Review: This article was commissioned
by the editorial ofce, ExRNA. The article has undergone
external peer review.
Conflicts of Interest: The authors have completed the
ICMJE uniform disclosure form (available at https://
dx.doi.org/10.21037/exrna-21-21). HL was supported
by the American Heart Association (20CDA35310184).
CS was supported by the Sarnoff Cardiovascular
Research Foundation. JSG was supported by the
Robert Wood Johnson Harold Amos Medical Faculty
Development Program, American Heart Association
(19AMFDP34990046) and the Massachusetts General
Hospital Sanchez-Ferguson Faculty Scholar Program.
AR was supported by the National Institutes of Health
(R01AG061034, R35HL155318). AR is a member of the
Leducq Foundation Scientic Advisory Committee. In this
capacity, his primary role is to review grant applications
and existing programs supported by the foundation. AR
is a scientific co-founder of a biotech start-up, LQTT,
focused on developing small molecule therapies for
Long QT Syndrome. Neither the Leducq Foundation
nor LQTT were involved in AR’s contributions to this
manuscript. The authors have no other conicts of interest
to declare.
Ethical Statement: The authors are accountable for all
aspects of the work in ensuring that questions related
to the accuracy or integrity of any part of the work are
appropriately investigated and resolved.
Open Access Statement: This is an Open Access article
distributed in accordance with the Creative Commons
Attribution-NonCommercial-NoDerivs 4.0 International
License (CC BY-NC-ND 4.0), which permits the non-
commercial replication and distribution of the article
with the strict proviso that no changes or edits are made
and the original work is properly cited (including links
to both the formal publication through the relevant
DOI and the license). See: https://creativecommons.org/
licenses/by-nc-nd/4.0/.
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doi: 10.21037/exrna-21-21
Cite this article as: Li H, Shefeld C Jr, Guseh JS, Rosenzweig A.
Backhanded complement: circulating exosomes in aged animals
add insult to injury after stroke. ExRNA 2021.
