Exosomes TER in recipient cells when stressed donor

Exosomes are 30–150 nm vesicles thatare secreted by almost every cell type in the human body, which occurs viafusion of multivesicular bodies (MVBs) and the plasma membrane and subsequentrelease by their parent cells (Tonget al., 2016). Exosomes were initially consideredwaste disposal material but recent evidence has progressively changed thisview, and exosomes are currently considered as a broad intercellularcommunication system that can internalize, transport and transfer all types ofbiomolecules, from nucleic acids to peptides and proteins (Soriaet al.

, 2017). Thus exosomes play a fundamentalbiological role in the regulation of normal physiological as well as aberrantpathological processes. In this manuscript, we are elucidating the role ofexosomes derived from oxidatively stressed RPE in the development of agerelated macular degeneration (AMD). Our hypothesis is that the dry AMDpathology occurs in patches, such that damage occurs in multiple locations inthe area of the macula, that slowly coalesce. Do these small areas of damageoccur randomly, or does this phenomenon involve long-distance communicationbetween a damaged and a healthy part of the RPE? And if so, what would mediatethis long-distance communication-Exosomes? The main results of this study wereas follows- (a) The concentration of Exosomes released apically weresignificantly increased in stressed ARPE-19 cells as compared to control; (b)Exosomes were released from the donor cells and uptaken by the recipient cellsas seen by live imaging.

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The exosomes released from oxidatively stressed cellssignificantly reduces transepithelial resistance (TER) which is the measure oftight junction integrity in the recipient cells; (c) This reduction in TER israpid and partially dependent on VEGF pathway; (d) Mass spec analysisidentified 1248 unique protein sequences present in exosomes. We utilizedpathway analysis tools to identify proteins involved in tight junctionfunction. One of the proteins increased in exosomes released from H2O2stressed cells was Histone deacetylase 6 (HDAC6).

HDAC6 is known to beinvolved in endothelial cell barrier function (Chistiakovet al., 2015, Yu et al,2016). HDAC6 was further evaluated in thisstudy and was found to be involved in reduction of TER in recipient cells whenstressed donor exosomes (with increased HDAC6 expression) were transferred tohealthy recipient cells. Taken together, our data suggest that exosomes fromoxidatively stressed donor cells were taken up much faster by recipient cellsas compared to control exosomes and communicate stress message to the healthycells making neighboring healthy cells sick.

It is currentlybelieved that exosomes mediate information exchange between cells through fourways: (1) Exosomes are used as a signal complex directly stimulating recipientcells through binding to a cell surface ligand; (2) Exosomes transfer receptorsbetween cells; (3) Exosomes deliver functional proteins or infectious particlesto recipient cells; and (4) Exosomes transfer genetic information to recipientcells through mRNAs, microRNAs, or transcription factors (Camussi et al., 2010). Onceexosomes are absorbed by the recipient cell, stored lipids, proteins, mRNAs,miRNAs, and other molecules can then affect the function and cellular phenotypeof the recipient cell by regulating signal cascade pathways, key enzymereactions, cellular homeostasis, or other mechanisms. However, thephysiological and pathological status of the source cell and the type of sourceand recipient cells determine the mechanism used and its effect.

A majorbreakthrough in exosome research was the finding of their nucleic acid contentssuch as messenger RNAs (mRNAs), small non-coding microRNAs (miRNAs) andmitochondrial DNA (mtDNA) which can be transported to other cells (Valadi etal., 2007) (Guescini etal., 2010). Since then, exosomes became more and moreinteresting in many research fields indicating a novel role as regulators incell–cell communications during diverse biological processes (Thery et al.

,2002a) (Simons andRaposo, 2009) (Gupta et al.,2010) (Cocucci etal., 2009) (Simpson etal., 2008) (Bang andThum, 2012).Recent studies have shown that exosomesreleased from donor cells have influence on the neighboring cells.

It has beendemonstrated that stressed RPE cells release high quantity of exosome whichpackaged VEGF receptors in their membrane, and VEGFR?1 and ?2 mRNA within the exosome and thus promote vasculogenesis/angiogenesiswhen they interact with neighboring endothelial cells (Atienzar-Arocaet al., 2016). Hajrasouliha et. al. reported that exosomesreleased from retinal astroglial cells (RACs) suppress retinal vessel leakageand inhibit CNV but not RPE exosomes (Hajrasoulihaet al., 2013).

Wang et al proposed that exosomes releasedfrom aged RPE contribute to the formation of drusen (Wang et al.,2009). Apart from RPE, influence of exosomes onneighboring cells have been shown in many other cell types. In mammalian heart,it is well recognized that endothelial cells play a critical role incardiomyocyte survival and myocardial contraction (Record etal., 2014).

Aliwadi et. al. have observed that exosomesderived from cardiomyocytes harbor a variety of mRNAs, miRNAs and proteins,which may be transferred to the adjacent endothelial cells and modulate theirfunction (Kharaziha etal., 2012) (Kalani etal., 2014) (Ailawadi etal., 2015) (Kowal et al.,2014) (Akers et al.,2013).

In this study we have shown that whenexosomes from normal RPE cells were transferred to healthy RPE monolayer, therewas no significant effect on the recipient cells but when exosomes from stressed(H2O2 or Rotenone) RPE cells were transferred to heathyrecipient cells, the integrity of tight junctions were affected as seen byreduced transepithelial resistance (TER) (Fig 2). This indicates that theseexosomes package stress message in their cargo and transfer it to the healthycells, thus making recipient cells sick and communicating disease to thehealthy cells. It has been reported that Microglia spread tau via exosomesecretion and depletion of microglia or inhibition of exosome synthesissignificantly reduced tau propagation in vitro and in vivo (Hsu et al.,2010; Xiao et al., 2017).

We have shown by live imaging that exosomeswere taken up by the recipient RPE cells. The oxidatively stressed exosomeswere taken up rapidly in as less as 10 to 20 mins which was 5-10 times fasterthan the control exosomes (Fig 5). The uptake of exosomes was believed to bemediated by endocytosis as uptake of exosomeswas interfered when cells were treated with dynasore (Fig 4B), a GTPase inhibitorthat targets dynamin and completely blocks endocytosis in hippocampal neuron (Newton et al., 2006). Similarly, exosomes from RPE cells lacking anessential ligand, annexin II for exosome generation by donor cells were foundto interfere with uptake mechanism (Fig 4A). This endocytosis process was furtherconfirmed by transepithelial resistance which was not reduced by exosomesderived from annexin II knockdown cells or in recipient cells treated withdynasore as exosomes were not endocytosed or uptaken by the recipient cells toinduce its effect (Fig 4).  Both direct and indirect evidence exists tosuggest that exosomes are internalized into the recipient cells. Exosomes havebeen shown to transfer functional cargo into the recipient cells {Gutierrez-Vazquez,2013 #68}{Pulliam, 2015 #69}{Villarroya-Beltri, 2014 #70}.

A number of researchgroups have provided evidence to suggests that exosomes are usually taken upinto endosomal compartments via endocytosis (Morelli etal., 2004) (Mulcahy et al., 2014).Mass Spec analysis identified 1248 unique protein sequencespresent in exosomes. To reduce the number of proteins and pathways to analyze,we utilized pathway analysis tools to identify proteins involved in tightjunction function.

One of the proteins increased in apical H2O2was Histone deacetylase 6 (HDAC6). HDAC6 is known to be involved in endothelialcell barrier function  (Chistiakov etal., 2015) and HDAC6 inhibitor, TSA was reported toimprove tight junction stability in various endothelial (Edelman etal., 2005) (Stewart,2014) and epithelial tissues (Ablonczy etal., 2011; Desjardins et al., 2016a). Bordin et al.

showed that histonedeacetylase inhibitors up-regulate the expression of tight Junction proteinsincluding cingulin, ZO-1, and ZO-2 in Rat-1 fibroblasts (Bordin etal., 2004). Our data show increased activity of HDAC6in stressed exosomes released from the apical side (Fig.

7). When recipientcells were treated with HDAC6 inhibitors TSA or Tub A, the transfer of stressedexosomes (H2O2 or Rotenone)  to the recipient cells didn’t induce anydamage to the tight junction integrity of these cells. This is due to the factthat the effect of increased level of HDAC6 from these stressed exosomes wasblocked by HDAC6 inhibitors in the recipient cells. This is further confirmedby binding assay where H2O2 apical exosomes wereincubated with TSA to block effect of HDAC6 in these exosomes and when thiscomplex (with no HDAC6 activity) was used for transfer or cell-communicationassay, there is no reduction in TER in the recipient cells (Fig. 6) whichconfirms that the reduction of transepithelial resisitance (TER) or damage totight junction is due to the effect of HDAC6 from stressed exosomes in therecipient cells.

 ?-tubulin is a endogenous substrate ofHDAC6 {Yu, 2016 #72}{Zhang, 2003 #71}). Acetylation of the ?-tubulin at Lys40 stabilizes microtubule structure {Asthana,2013 #73}{Yu, 2016 #72} and overexpression of HDAC6specifically reduces the acetylation levels of ?-tubulin {Hubbert, 2002 #75}. Our data confirms that H2O2 stressedexosomes 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 equallyand RPE cells respond differently to specific cellular stressors {Kunchithapautham, 2011 #52}. Smoke and FAC exosomes didn’t reduce TERsignificantly as compared to control exosomes (Fig. 9).

As seen in earlierexperiments, increase in HDAC6 is linked to decrease in TER. So, we checkedHDAC6 activity in these exosomes and as expected, these exosomes didn’tpackaged increased level of HDAC6 as compared to control exosomes. Thisexplains the inability of exosomes released from smoke and FAC stressed cellsto damage tight junction integrity in the recipient cells.Taken together, the oxidatively stressed RPErelease exosomes which package stress cargos and communicates stress message tothe neighboring healthy cells. These exosomes release its cargo (HDAC6 in thepresent study) in the recipient cells.

HDAC6 deacetylates ?-tubulin which disrupts tight junction integrity inthe recipient cells and induces RPE dysfunction.