Exosomes TER in recipient cells when stressed donor

Exosomes are 30–150 nm vesicles that
are secreted by almost every cell type in the human body, which occurs via
fusion of multivesicular bodies (MVBs) and the plasma membrane and subsequent
release by their parent cells (Tong
et al., 2016). Exosomes were initially considered
waste disposal material but recent evidence has progressively changed this
view, and exosomes are currently considered as a broad intercellular
communication system that can internalize, transport and transfer all types of
biomolecules, from nucleic acids to peptides and proteins (Soria
et al., 2017). Thus exosomes play a fundamental
biological role in the regulation of normal physiological as well as aberrant
pathological processes. In this manuscript, we are elucidating the role of
exosomes derived from oxidatively stressed RPE in the development of age
related macular degeneration (AMD). Our hypothesis is that the dry AMD
pathology occurs in patches, such that damage occurs in multiple locations in
the area of the macula, that slowly coalesce. Do these small areas of damage
occur randomly, or does this phenomenon involve long-distance communication
between a damaged and a healthy part of the RPE? And if so, what would mediate
this long-distance communication-Exosomes? The main results of this study were
as follows- (a) The concentration of Exosomes released apically were
significantly increased in stressed ARPE-19 cells as compared to control; (b)
Exosomes were released from the donor cells and uptaken by the recipient cells
as seen by live imaging. The exosomes released from oxidatively stressed cells
significantly reduces transepithelial resistance (TER) which is the measure of
tight junction integrity in the recipient cells; (c) This reduction in TER is
rapid and partially dependent on VEGF pathway; (d) Mass spec analysis
identified 1248 unique protein sequences present in exosomes. We utilized
pathway analysis tools to identify proteins involved in tight junction
function. One of the proteins increased in exosomes released from H2O2
stressed cells was Histone deacetylase 6 (HDAC6). HDAC6 is known to be
involved in endothelial cell barrier function (Chistiakov
et al., 2015, Yu et al,
2016). HDAC6 was further evaluated in this
study and was found to be involved in reduction of TER in recipient cells when
stressed donor exosomes (with increased HDAC6 expression) were transferred to
healthy recipient cells. Taken together, our data suggest that exosomes from
oxidatively stressed donor cells were taken up much faster by recipient cells
as compared to control exosomes and communicate stress message to the healthy
cells making neighboring healthy cells sick.

It is currently
believed that exosomes mediate information exchange between cells through four
ways: (1) Exosomes are used as a signal complex directly stimulating recipient
cells through binding to a cell surface ligand; (2) Exosomes transfer receptors
between cells; (3) Exosomes deliver functional proteins or infectious particles
to recipient cells; and (4) Exosomes transfer genetic information to recipient
cells through mRNAs, microRNAs, or transcription factors (Camussi et al., 2010). Once
exosomes are absorbed by the recipient cell, stored lipids, proteins, mRNAs,
miRNAs, and other molecules can then affect the function and cellular phenotype
of the recipient cell by regulating signal cascade pathways, key enzyme
reactions, cellular homeostasis, or other mechanisms. However, the
physiological and pathological status of the source cell and the type of source
and recipient cells determine the mechanism used and its effect.

A major
breakthrough in exosome research was the finding of their nucleic acid contents
such as messenger RNAs (mRNAs), small non-coding microRNAs (miRNAs) and
mitochondrial DNA (mtDNA) which can be transported to other cells (Valadi et
al., 2007) (Guescini et
al., 2010). Since then, exosomes became more and more
interesting in many research fields indicating a novel role as regulators in
cell–cell communications during diverse biological processes (Thery et al.,
2002a) (Simons and
Raposo, 2009) (Gupta et al.,
2010) (Cocucci et
al., 2009) (Simpson et
al., 2008) (Bang and
Thum, 2012).

Recent studies have shown that exosomes
released from donor cells have influence on the neighboring cells. It has been
demonstrated that stressed RPE cells release high quantity of exosome which
packaged VEGF receptors in their membrane, and VEGFR?1 and ?2 mRNA within the exosome and thus promote vasculogenesis/angiogenesis
when they interact with neighboring endothelial cells (Atienzar-Aroca
et al., 2016). Hajrasouliha et. al. reported that exosomes
released from retinal astroglial cells (RACs) suppress retinal vessel leakage
and inhibit CNV but not RPE exosomes (Hajrasouliha
et al., 2013). Wang et al proposed that exosomes released
from aged RPE contribute to the formation of drusen (Wang et al.,
2009). Apart from RPE, influence of exosomes on
neighboring cells have been shown in many other cell types. In mammalian heart,
it is well recognized that endothelial cells play a critical role in
cardiomyocyte survival and myocardial contraction (Record et
al., 2014). Aliwadi et. al. have observed that exosomes
derived from cardiomyocytes harbor a variety of mRNAs, miRNAs and proteins,
which may be transferred to the adjacent endothelial cells and modulate their
function (Kharaziha et
al., 2012) (Kalani et
al., 2014) (Ailawadi et
al., 2015) (Kowal et al.,
2014) (Akers et al.,
2013). In this study we have shown that when
exosomes from normal RPE cells were transferred to healthy RPE monolayer, there
was no significant effect on the recipient cells but when exosomes from stressed
(H2O2 or Rotenone) RPE cells were transferred to heathy
recipient cells, the integrity of tight junctions were affected as seen by
reduced transepithelial resistance (TER) (Fig 2). This indicates that these
exosomes package stress message in their cargo and transfer it to the healthy
cells, thus making recipient cells sick and communicating disease to the
healthy cells. It has been reported that Microglia spread tau via exosome
secretion and depletion of microglia or inhibition of exosome synthesis
significantly reduced tau propagation in vitro and in vivo (Hsu et al.,
2010; Xiao et al., 2017).

We have shown by live imaging that exosomes
were taken up by the recipient RPE cells. The oxidatively stressed exosomes
were taken up rapidly in as less as 10 to 20 mins which was 5-10 times faster
than the control exosomes (Fig 5). The uptake of exosomes was believed to be
mediated by endocytosis as uptake of exosomes
was interfered when cells were treated with dynasore (Fig 4B), a GTPase inhibitor
that targets dynamin and completely blocks endocytosis in hippocampal neuron (Newton et al., 2006). Similarly, exosomes from RPE cells lacking an
essential ligand, annexin II for exosome generation by donor cells were found
to interfere with uptake mechanism (Fig 4A). This endocytosis process was further
confirmed by transepithelial resistance which was not reduced by exosomes
derived from annexin II knockdown cells or in recipient cells treated with
dynasore as exosomes were not endocytosed or uptaken by the recipient cells to
induce its effect (Fig 4).  Both direct and indirect evidence exists to
suggest that exosomes are internalized into the recipient cells. Exosomes have
been shown to transfer functional cargo into the recipient cells {Gutierrez-Vazquez,
2013 #68}{Pulliam, 2015 #69}{Villarroya-Beltri, 2014 #70}. A number of research
groups have provided evidence to suggests that exosomes are usually taken up
into endosomal compartments via endocytosis (Morelli et
al., 2004) (Mulcahy et al., 2014).

Mass Spec analysis identified 1248 unique protein sequences
present in exosomes. To reduce the number of proteins and pathways to analyze,
we utilized pathway analysis tools to identify proteins involved in tight
junction function. One of the proteins increased in apical H2O2
was Histone deacetylase 6 (HDAC6). HDAC6 is known to be involved in endothelial
cell barrier function  (Chistiakov et
al., 2015) and HDAC6 inhibitor, TSA was reported to
improve tight junction stability in various endothelial (Edelman et
al., 2005) (Stewart,
2014) and epithelial tissues (Ablonczy et
al., 2011; Desjardins et al., 2016a). Bordin et al. showed that histone
deacetylase inhibitors up-regulate the expression of tight Junction proteins
including cingulin, ZO-1, and ZO-2 in Rat-1 fibroblasts (Bordin et
al., 2004). Our data show increased activity of HDAC6
in stressed exosomes released from the apical side (Fig. 7). When recipient
cells were treated with HDAC6 inhibitors TSA or Tub A, the transfer of stressed
exosomes (H2O2 or Rotenone)  to the recipient cells didn’t induce any
damage to the tight junction integrity of these cells. This is due to the fact
that the effect of increased level of HDAC6 from these stressed exosomes was
blocked by HDAC6 inhibitors in the recipient cells. This is further confirmed
by binding assay where H2O2 apical exosomes were
incubated with TSA to block effect of HDAC6 in these exosomes and when this
complex (with no HDAC6 activity) was used for transfer or cell-communication
assay, there is no reduction in TER in the recipient cells (Fig. 6) which
confirms that the reduction of transepithelial resisitance (TER) or damage to
tight junction is due to the effect of HDAC6 from stressed exosomes in the
recipient cells. ?-tubulin is a endogenous substrate of
HDAC6 {Yu, 2016 #72}{Zhang, 2003 #71}). Acetylation of the ?-tubulin at Lys40 stabilizes microtubule structure {Asthana,
2013 #73}{Yu, 2016 #72} and overexpression of HDAC6
specifically reduces the acetylation levels of ?-tubulin {Hubbert, 2002 #75}. Our data confirms that H2O2 stressed
exosomes which have packaged increased amount of HDAC6 deactylates ?-tubulin when it communicates with the recipient cells
(Fig. 8).

We have shown earlier that all stressors are not created equally
and RPE cells respond differently to specific cellular stressors {Kunchithapautham, 2011 #52}. Smoke and FAC exosomes didn’t reduce TER
significantly as compared to control exosomes (Fig. 9). As seen in earlier
experiments, increase in HDAC6 is linked to decrease in TER. So, we checked
HDAC6 activity in these exosomes and as expected, these exosomes didn’t
packaged increased level of HDAC6 as compared to control exosomes. This
explains the inability of exosomes released from smoke and FAC stressed cells
to damage tight junction integrity in the recipient cells.

Taken together, the oxidatively stressed RPE
release exosomes which package stress cargos and communicates stress message to
the neighboring healthy cells. These exosomes release its cargo (HDAC6 in the
present study) in the recipient cells. HDAC6 deacetylates ?-tubulin which disrupts tight junction integrity in
the recipient cells and induces RPE dysfunction.