Mitochondrion 48 (2019) 16–30

Contents lists available at ScienceDirect

Mitochondrion
journal homepage: www.elsevier.com/locate/mito

Review

Forms of extracellular mitochondria and their impact in health T

a b,1 c,d,1 a b,d,e,f,⁎
Sophia Miliotis , Bryan Nicolalde , Mayra Ortega , Jackie Yepez , Andrés Caicedo
a
Universidad San Francisco de Quito, The Latitude Zero Ecuador Research Initiative, L0ERI, 17-12-841, Ecuador
b
Universidad San Francisco de Quito, Colegio de Ciencias de la Salud – Hospital de los Valles, Escuela de Medicina, Quito 17-12-841, Ecuador
c
Universidad San Francisco de Quito, Colegio de Ciencias Biológicas y Ambientales, Escuela de Biotecnología, Quito 17-12-841, Ecuador
d
Universidad San Francisco de Quito, Instituto de Investigaciones en Biomedicina, Quito 17-12-841, Ecuador
e
Mito-Act Research Consortium, Quito, Ecuador
f
Sistemas Médicos - Universidad San Francisco de Quito, SIME-USFQ, Quito 17-12-841, Ecuador

ARTICLE INFO ABSTRACT

Keywords: Mitochondria play an important role as an intracellular energy plant and signaling organelle. However, mi-
Mitochondria tochondria also exist outside cells where they could mediate cell-to-cell communication, repair and serve as an
Cell-free-circulating mtDNA activator of the immune response. Their effects depend on the mitochondrial state or the form in which it is
Transplant present, either as a whole functional structure as fragments or only as mitochondrial DNA. Herein, we provide
Regeneration
evidence of why extracellular mitochondria and their varying forms are considered regenerative factors or pro-
Inflammation
Therapy
inflammatory activators. Understanding these aspects will provide the base of their use in therapy or as a bio-
Biomarker marker of disease severity and prognosis.

1. Introduction cellular stress, damage or to preserve other cells' function (Torralba

et al., 2016; Lindqvist et al., 2018; Liu et al., 2015). A high con-
Today mitochondria are considered more than an active energy centration of ccf-mtDNA has been associated with chronic diseases and
plant inside cells, as they can be found in the extracellular space intact, is tested today as a biomarker of prognosis and survival (Lindqvist
in vesicular structures and fragments being able to elicit regenerative et al., 2018; Shockett et al., 2016; Pyle et al., 2015). The effects of
effects, or act as a danger signal when interacting with other cells. platelet mitochondria, before and after being released, is puzzling, as
Forms of extracellular mitochondria can be found free (freeMitos), there is evidence that can trigger inflammatory responses or in contrast
enclosed by a membrane as inside platelets or vesicles, or as cell free be immune-regulatory and have regenerative properties (Zhao et al.,
circulating mitochondrial DNA (ccf-mtDNA). All forms of mitochondria 2017).
outside cells can induce paracrine or endocrine responses in an or- The interactions of these forms of extracellular mitochondria with
ganism. However, the effects of extracellular mitochondria are only others cells open a new field of study in which mitochondria goes
starting to be elucidated, especially their role in a pro or anti-in- further than its role as the powerhouse and becomes a signaling orga-
flammatory immune response, and in determining their fate inside the nelle outside the cell (Torralba et al., 2016; Chandel, 2014; Caicedo
recipient cell. The pro or anti-inflammatory effects of membrane free et al., 2017). Understanding the roles of extracellular mitochondria and
intact or damaged mitochondria are still controversial as recent lit- its different forms in the body will have consequences in how to de-
erature shows a contradictory interaction with immune cells in dif- velop new therapies to maintain health and used as a biomarker of
ferent models (Ramirez-Barbieri et al., 2018; Torralba et al., 2016; disease. The focus of this review is to provide information about the
Boudreau et al., 2014). It is also of great interest to know if exogenous origin of the different forms of extracellular mitochondria (in platelets,
mitochondria when uptaken by recipient cells persist with time, if they in EVs, free or as mtDNA) the interaction with the recipient or reactive
degrade as seen in cells of the nervous system (Davis et al., 2014), or if cells and the induction of physiological changes. This knowledge is
they are able to mix with endogenous mitochondria (Cowan et al., meant to encourage the scientific and medical community to under-
2017). stand the many interactions of mitochondria outside cells and continue
Mitochondrial DNA (mtDNA) can be liberated as ccf-mtDNA or to search for answers that are controversial and puzzling.
packed inside EVs or exosomes in response to many signals such as

⁎
Corresponding author.
E-mail address: acaicedo@usfq.edu.ec (A. Caicedo).
1
Both authors equally contributed to the article, names are in alphabetical order.

https://doi.org/10.1016/j.mito.2019.02.002
Received 19 August 2018; Received in revised form 9 November 2018; Accepted 6 February 2019
Available online 14 February 2019
1567-7249/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S. Miliotis, et al. Mitochondrion 48 (2019) 16–30

Fig. 1. a. Schematic representation of a platelet and its structure and content. b. Schematic representation of a Microvesicles (MVs), Exosomes and Cell of Origin
structure and content. c. Schematic representation of an Exosome, its structure and content.

2. Platelets, complex structures and mitochondrial carriers anucleate, with a discoid form, being produced by megakaryocytes
(MK) in the bone marrow and released in the blood. After activation by
Platelets are the largest and more complex extracellular mitochon- danger signals, they prevent excessive bleeding, facilitate regeneration
dria-carrying structures. With a size between 2 and 5 μm, platelets carry and confinement of pathogens after injury (Koupenova et al., 2018).
approximately 4 mitochondria being located close to the exterior There are nearly one trillion platelets in blood which last from 8 to
membrane of the platelet (Fig. 1a) (Boudreau et al., 2014). They are 10 days in circulation (Chandel, 2014). The processes of production,

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S. Miliotis, et al. Mitochondrion 48 (2019) 16–30

Fig. 1. (continued)

activation and elimination are dependent of mitochondrial state and The increase in size of MKs and mitochondria help them to accumulate
dynamics together with the interaction with other organelles inside intracellular content which is delivered to PPs by motor kinesins con-
platelets. It has been observed that any malfunction or perturbations of nected to the microtubules in the form of cargo that accumulates in the
proteins related to its activity have been linked to the severity of Type 2 granules (Poulter and Thomas, 2015).
diabetes, sepsis, thrombocytopenia, and Parkinson's and Alzheimer's Platelet function is dependent on mitochondria and is crucial for the
disease (Kholmukhamedov and Jobe, 2017; Fišar et al., 2016; Cardenes initiation of the coagulation cascade and injury repair (Yun et al.,
et al., 2014). Many aspects related to the effects and function of mi- 2016). First, platelets adhere to the wounded extracellular matrix, then
tochondria inside or outside platelets and release still need to be un- the Willebrand factor (vWF) stored in a-granules is released in the
derstood and as their status change during platelet life span, activation platelet membrane forming a union between collagen and platelet
and apoptosis. glycoprotein (GP) Ib-IX-V receptor complex (Al-Rekabi et al., 2015;
Platelets perform complex activities as mediators of inflammation or Ruggeri and Mendolicchio, 2015; Springer, T. A. von Willebrand factor,
tissue repair carrying other organelles besides mitochondria, such as 2014) as well to the GP Ia/IIa and GP VI receptors facilitating the
the Golgi apparatus and factors that interact with each other to sustain change in platelet structure and the release of granules. Prothrombotic
its function. Platelets transport mRNA, microRNA, metabolically active factors stimulate platelet secretion of granules through GP Ib-IX-V, GO
substrates, cofactors, chemokines, serotonin, glutamate, calcium among VI, or C-type lectin-like receptor 2 (CLEC-2) activation. ADP and
others (Koupenova et al., 2018; Sunderland et al., 2017; Fejes et al., thromboxane A2 (TxA2) trigger the platelet response through the G
2017; Deutsch and Tomer, 2006). They are mainly constituted by protein-coupled receptors increasing cytosolic calcium and favoring the
granules, skeletal components like microtubules and actin, which play release of intraplatelet content (Yun et al., 2016; Banerjee, 2017).
an important role in platelet physiology and activation. Platelets have a Thrombin induces a strong platelet response through the protease-ac-
complex system of membranes consisting by an Open Canalicular tivated receptors (PAR) via the GPCR, and induces the loss of mi-
System (OCS) and a network of residual endoplasmic reticulum called tochondrial membrane potential (MMP), and a “fast like apoptotic
the Dense Tubular System (DTS) (Fig. 1a). Additionally, platelets have a process” release of cytochrome c and the activity of the caspases-3 and
membrane skeleton consisting of spectrin, an actin cytoskeleton and 9 (Leytin et al., 2009). Other agonists such as epinephrine, pros-
microtubules (Rendu and Brohard-Bohn, 2001; Selvadurai and taglandin, and serotonin activate the platelets by GPCR (Yun et al.,
Hamilton, 2018). Platelets carry glycogen as an energy source for their 2016; Estevez et al., 2015). After the activation signal, calcium released
physiological functions especially after activation and release of in- by the DTS increases to a concentration superior of 100 nM which
traplatelet material. Glycogen particles exist in association with en- triggers the movement of granules from the inner part of the platelet till
zymes involved in their synthesis and degradation sustaining platelet fusing with the plasma membrane and then releasing the content
persistence in blood and activity (A Simple Method to Quantify (Rendu and Brohard-Bohn, 2001). The timing and conditions for the
Glycogen from Human Platelets, 2014). Mitochondria inside platelets activation of apoptosis in platelets, cleaning mature or aged platelets, is
are actively regulated in response to activation signals were its inter- important for mitochondria adaptation to the type of response desired.
action with the complex tubular network and cytoskeleton, granules, Mitochondria inside platelets not only produce energy but also
cytokines could affect their pro-inflammatory or regenerative effects. mediate its activation and regulation in thrombus formation by a tight
MKs reconstitute the platelet pool in response to the hormone equilibrium between ROS production and induction of apoptosis
Thromobopoietin (TPO). These cells increase their DNA content and (Kilkson et al., 1984; Sjövall et al., 2013; Kramer et al., 2014; Wang
size during its maturation from 50 to more than 100 um in order to et al., 2017). After receiving an activation signal, mitochondria increase
produce proplates (PPs) and later platelets. The major pathways re- their membrane potential and ROS production, essential for the release
ported during MKs maturation are JAK-STAT, PI3K-AKT, MAPK and process of granule content. The increase in mitochondria membrane
Wnt all of them related to mitochondrial biogenesis (Undi et al., 2017). potential lead to a collapse in its function over time inducing the

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S. Miliotis, et al. Mitochondrion 48 (2019) 16–30

opening of the membrane transition pore and release of cytochrome c mitochondria actively binds to the pancreatic cells but the inter-
from the mitochondrial matrix to the platelet cytosol. After the release nalization method is not described. CB-platelet-derived mitochondria
of cytochrome c, it binds to the Apoptotic Proteinase Activating Factor has fibronecting, CXC chemokine receptor 2 (CXCR2), CXCR4 and
(APAF-1), activating caspase 9, 3 and the apoptotic downstream chemokine C-C motif receptor 7 (CCR7) and the islet β cells the fi-
pathway (Cardenes et al., 2014; Leytin et al., 2009). Perturbations in bronectin ligand CD29 and the Toll-like receptor 4 which mediates the
the metabolism due to chronic diseases induce pathological platelet interaction. Additionally, authors observed that pancreatic islet B-cell
activation and ROS production which make them very sensitive to function from healthy donors is improved after treatment with platelet-
apoptosis (Ferroni et al., 2004; Karimova et al., 2018; Arthur et al., derived mitochondria. The increase in the number of platelets in the
2017). SCE treated patients could involve mitogenesis by MKs among other
The BCL2 protein family mediates the entry in the apoptotic process effects that drive to the presence of immune-regulatory and stem cells
by the mitochondria in a coordinated response to the organism phy- markers. It is interesting to observe that isolated mitochondria from
siology. The BCL2 family members that induce apoptosis are BAX and non-activated platelets induce regenerative properties in contrast from
BAD by the release of cytochrome C (Leytin et al., 2009; Vogler et al., Boudreau et al., 2014 study in which mitochondria from activated
2011). This apoptotic mechanism is the same between cells and plate- platelets are proinflammatory (Boudreau et al., 2014; Zhao et al.,
lets, but the timing and functional role and response changes. The in- 2017).
hibition of BCL2 antiapoptotic (BCL2, BCL-XL, BCL-w, MCL1 and We have evidence that platelets are reactive extracellular structures
BCL2A1) proteins decrease platelet activation by the depletion of in- secreting pro-inflammatory or healing factors in response to stimuli
tracellular calcium which is of great importance to this process. De- coming after damage or in response to disease. Their complex structure
fining how platelet activation mediates changes in mitochondria or in interact with each other and support the specific delivery of cargo. Even
caspase-dependent apoptosis is still to be completely explained (Vogler though evidence in the type of mitochondria that is being released after
et al., 2011). activation, many questions are still present regarding how different
Boudreau and colleagues, 2014, observed that platelet activation by stimuli modify the delivery and state of mitochondria. Research in this
immunoglobulin, thrombin, collagen or phorbol myristate acetate regard will provide necessary data to understand the dynamic and role
(PMA) during 4 h at ambient temperature triggers the release of mi- of mitochondria transported in platelets as a healing or pro-in-
croparticles containing mitochondria (mitoMPs), active mitochondria flammatory factor.
(freeMitos) and mtDNA (Boudreau et al., 2014). In their model, the
release of mitoMPs and freeMitos was mediated by actin independently 3. The biology of extracellular vesicles as mitochondria carriers
of microtubules. Interestingly, inactive or damaged mitochondria are
detached from the microtubules entering to degradation (Ni et al., Prokaryotic and eukaryotic cells secrete extracellular vesicles (EVs)
2015) and could imply that mitoMPs or freeMitos produced by acti- which are stable structures that carry and protect biological molecules
vated platelets are fragilized promoting process like apoptosis. Authors such as proteins, lipids and RNAs from degradation during transfer from
determined in Osteoarthritis (OA) and Rheumatoid Arthritis (RA) that cell to cell (Aryani and Denecke, 2016). They are secreted by many cell
the synovial fluid in RA contain more CD41+ mitoMPs derived from types and can be classified as apoptotic bodies (ABs), microvesicles
platelets than OA. Also, they put in evidence that platelet concentrates (MVs), or exosomes based on size, contents, and mechanism of bio-
associated with adverse transfusion reaction contain a higher con- genesis (Table 1, Fig. 1b) (Crescitelli et al., 2013; Zappulli et al., 2016).
centration of mitoMPs and freeMitos. They observed that mitochondria EVs are found in a variety of body fluids and play an imperative role in
is a substrate of phospholipase A2 IIA (sPLA2-IIA), specific for the de- normal physiology and intercellular communication via exchange of
tection of foreign bacteria, but they didn't show that it triggers its se- cellular components between cells (Frühbeis et al., 2015). They are
cretion as in the case of Sepsis (Tan and Goh, 2017). Degradation of the responsible for organelle disposal and mediation of fundamental cel-
mitochondrial membrane by sPLA2-IIA trigger inflammatory mediators lular responses such as cell-to-cell signaling and immune reactions
and promote leukocyte activation, being at the end of a pro-in- (Robbins and Morelli, 2014). Larger EVs such as MVs can contain entire
flammatory cascade of events. mitoMPS and freeMito released from mitochondria, and smaller EVs like exosomes can transport mtDNA and
activated platelets seems to be primed to induce a defense response as other nucleic acids to target cells (Berridge and Neuzil, 2017; Sansone
during this process they are induce to pass through apoptosis (Boudreau et al., 2017). Notably, the mechanism by which mtDNA is incorporated
et al., 2014). into the recipient cell after the EV is uptaken is still unknown (Sansone
J. Otero and his team reported in 2017 that human subjects with et al., 2017).
type 1 and 2 diabetes used a Stem Cell Educator Device (SCE) that re- EVs containing mitochondria or mtDNA serve to preserve cellular
versed important aspects of their health. For instance, an improved viability and mediate the induction of danger signals. It has been shown
auto-immunity, regeneration of islet B cells, gain of metabolic control that the therapeutic effect of Mesenchymal Stem Cells (MSCs) depends
and an increase in platelet production for 1 month (Zhao et al., 2017). greatly on their capacity to secrete extracellular bodies like EVs. MSCs
This study was part of two clinical trials of using the SCE. The SCE can transfer mitochondria to damaged cells helping them to recover
device uses Cord Blood Derived Multipotent Stem Cells (CB-SCs) at- from loss of function and stress (Phinney et al., 2015). Interestingly,
tached in a support, connected to a closed system of circulation with a MSCs in standard culture conditions undergo mitophagy, or mi-
cell separator where patient's blood runs, after 9 h of co culture re- tochondrial autophagy, and unload mitochondria into EVs to be up-
covering the educated immune cells of patients. Platelets issued from taken by macrophages, thus increasing their bioenergetics and function
this therapy express immune tolerance-related markers such as pro- (Morrison et al., 2017). Forms of cellular communication such as this
grammed death ligand 1 (PD-L1), CD270 (Herpes virus entry mediator), induce changes in others cells and may even reprogram cell fate and
immune modulation associated transcription factor FoxP3, stem cell metabolism. For this reason EVs are known to be associated with pa-
markers, among others. In order to understand if mitochondria isolated thological processes as they can be secreted by cancer cells during
from patients platelets after being educated induce immune-regulatory tumor progression (Sansone et al., 2017; Zheng et al., 2018; Feng et al.,
effects, they transferred them allogenically to donors PBMCs. They 2017; Chen et al., 2018) or in the development of neurological diseases
observed a repression of cell proliferation of T cells in comparison to even by cells indirectly associated with the nervous system such as
control after being exposed to anti-CD3 and anti-CD28 antibodies and erythrocytes (Matsumoto et al., 2017). Because EVs can play a part in
recombinant human IL-2. Authors of this work proposed that CB-pla- both the healing process and in the progression of disease, under-
telet-derived mitochondria express adhesion molecules and chemokine standing their role in regenerative medicine and in cancer could lead to
receptors that help them fix the pancreatic islet β cells. Meaning that the development of new therapies as well as ways to halt their

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Table 1
Main classes of extracellular vesicles. Key characteristics.
Key characteristics Location Cargo Function

Apoptotic body • 1–5 μm diameter (Aryani and Denecke, • Cells undergoing apoptosis (Arandjelovic and • DNA and RNA (Aryani and Denecke, • Clearance of apoptotic debris (Arandjelovic and
2016) Ravichandran, 2015) 2016) Ravichandran, 2015)
• Heterogeneous shape (Aryani and • Histones (Aryani and Denecke, 2016) • Immune response (Arandjelovic and Ravichandran,
Denecke, 2016) • Signaling molecules (Aryani and Denecke, 2015)
• Membrane-bound (Aryani and Denecke, 2016)
2016) • Sometimes entire mitochondria (Berridge
and Neuzil, 2017)
Microvesicle nm–1 μm diameter (Aryani and
• 20Denecke, • Physiological fluids (blood, lymph, urine, etc.) • DNA, mRNA, miRNA (van Niel et al., • Intercellular communication (Lawson et al., 2016)
2016) (Turola et al., 2012) 2018)
shape (Aryani and Denecke, molecules (van Niel et al.,

20
• Homogenous
2016)
• Signaling
2018)
• Membrane-bound (Aryani and Denecke, • Growth factors (Lawson et al., 2016)
2016) • Sometimes entire mitochondria (Sinha
et al., 2016)
• Soluble molecules (IL-1β) (Turola et al.,
2012)
• Insoluble molecules (membrane antigens)
(Turola et al., 2012)
• Exosomes (Turola et al., 2012)
Exosome • 50–100 nm diameter (Lawson et al., • Physiological fluids (plasma, lymph, urine, etc.) • mtDNA (Sansone et al., 2017) • Long distance transport of nucleic acids between
2016) (Salido-Guadarrama et al., 2014) • Small RNAs (Salido-Guadarrama et al., cells (Aryani and Denecke, 2016)
• Tetraspanin proteins on surface (Aryani 2014) • Antigen presentation (Aryani and Denecke, 2016)
and Denecke, 2016) • DNA (Salido-Guadarrama et al., 2014) • Immune response (Aryani and Denecke, 2016)
• Proteins (Salido-Guadarrama et al., 2014)
Mitochondria-derived • 70–150 nm diameter (Sugiura et al., • Mitochondria (Sugiura et al., 2014) • Mitochondrial proteins and lipids • Mitochondria quality control (Sugiura et al., 2014)
vesicle 2014) (Bragoszewski et al., 2017)
Mitochondrion 48 (2019) 16–30
S. Miliotis, et al. Mitochondrion 48 (2019) 16–30

production in the tumor microenvironment (Salido-Guadarrama et al., 2015; Kowal et al., 2014). Inward budding creates intracellular va-
2014). cuoles that mature into late endosomes, or multivesicular bodies
Biogenesis and excretion of EVs depends greatly on their cell of (MVBs) (Ailawadi et al., 2015; Piper and Katzmann, 2007). The MVB
origin and their specific purpose. For example, the liberation of ABs, the then undergoes inward budding in a similar fashion, generating an
largest type of EV, is tied to the apoptotic process in which the body internal vesicle, the exosome, which envelops its respective cellular
eliminates old or no longer viable or needed cells without representing components (Hessvik and Llorente, 2018).
harm to the tissue or inducing a sustained immune response Mitochondria possess the capacity to produce their own vesicles in
(Arandjelovic and Ravichandran, 2015). ABs are released via external order to transport mitochondrial proteins and lipids to nearby orga-
blebbing of the plasma membrane (Ailawadi et al., 2015). Research has nelles within the cell (Sugiura et al., 2014). These mitochondrial-de-
been shown that ABs are associated with an anti-inflammatory immune rived vesicles (MDVs) either fuse with the late endosome or are de-
response, as they can be detected as “eat me” signals and engulfed by graded via lysosomes or peroxisomes (Sugiura et al., 2014). MDVs
tissue resident phagocytes, especially macrophages (Arandjelovic and destined for lysosomal degradation contain protein kinase PINK1 and
Ravichandran, 2015). During mitophagy, some ABs may engulf entire cytosolic ubiquitin E3 ligase Parkin, and those following the peroxi-
mitochondria with impaired respiratory and metabolic function in some pathway carry the retromer complex and MAPL protein
order to be removed conservatively (Berridge and Neuzil, 2017). (McWilliams and Muqit, 2017). Mitochondria also form vesicles as an
Knowledge of the fate of mitochondria inside ABs when uptaken by alternative means of quality control alongside the action of mitochon-
others cells, especially immune cells, is still lacking as there is no re- drial proteases, ubiquitin-mediated proteasomal degradation, and mi-
sponse for whether or not the mitochondria are primed for apoptosis tophagy (Bragoszewski et al., 2017). As one of the mitochondrion's first
and could elicit a detrimental cellular effect. lines of defense, MDVs expel damaged proteins in order to prevent
The generation of MVs, which constitute the next largest type of EV, complete mitophagy. They are formed through selective incorporation
involves the external blebbing of the plasma membrane and in- of protein cargoes, which can include outer and inner membrane pro-
corporation of cytosolic proteins, among other cargo (Lawson et al., teins as well as matrix content. The mechanisms that dictate cargo se-
2016). Sizeable MVs are also able to contain intact mitochondria lection are still unclear, however proteins are known to be selected
(Berridge and Neuzil, 2017). Once detached from its cell of origin, the based on their target destination and the nature of mitochondrial stress.
MV is released into physiological fluid to deliver its cargo across long Soubannier and colleagues suggest that MDVs induced by stress are
distances (Turola et al., 2012). The MV membrane contains surface selectively enriched for oxidized proteins, and some MDVs contain the
receptors and transmembrane proteins, appertaining to the cell of outer membrane protein MAPL, which shuttles to the peroxisome
origin, that allow for identification and interaction with target cells (Sugiura et al., 2014; Soubannier et al., 2012). The mechanisms for
(Lawson et al., 2016). Cargo is released into the recipient cell cytosol, MDV transport and delivery still remain unclear, yet several lipid
and the MV may remain at the plasma membrane or be endocytosed binding and modifying enzymes such as endophilin B1, Rab GTPases
and degraded by the lysosome (van Niel et al., 2018). MVs release so- and PIP-related microdomains may be involved given their functions in
luble molecules like IL-1β and insoluble molecules (membrane anti- vesicle transport in biosynthetic and endocytic pathways (Sugiura et al.,
gens), and may even release exosomes as a mechanism for removing 2014).
excess or harmful molecules (Turola et al., 2012). Larger MVs carrying EVs are known to be associated with disease, as they can be actively
mitochondria are secreted by different cell types, among them MSCs produced during cellular stress and death. An abundance of research
and astrocytes (Sinha et al., 2016; Hayakawa et al., 2016), and are has shown EVs as cancer or infectious disease agents, yet they also serve
received by epithelial cells, immune cells, and neurons (Torralba et al., in the progression of cardiovascular disease as myocardial function
2016). The transfer of this material has been seen to repair cellular depends on a controlled communication system provided by EVs
stress and damage together with the decrease of ATP and overall (Ailawadi et al., 2015). High levels of circulating EVs in plasma is a sign
bioenergetics (Sinha et al., 2016). For example, a neuroprotective me- of cardiovascular disease and obesity, as observed in patients with type
chanism was observed in a mouse model of transient focal cerebral 2 diabetes mellitus, insulin resistance, and atherosclerosis as well as
ischaemia in which the release of MV-bound mitochondria derived from those who have suffered from stroke or myocardial infarct (Lawson
astrocytes were then uptaken by neurons (Berridge and Neuzil, 2017; et al., 2016). However, EVs may also exhibit potential cardio-protective
Hayakawa et al., 2016). Whether the MVs entered the lysosomal effects (Ailawadi et al., 2015). EVs also mediate juxtacrine and para-
pathway or were endocytosed to release mitochondria into the recipient crine signaling in metastatic tumor progression and therapy resistance
neuron is unclear. Another experiment performed by electron micro- by inducing signaling cascades and epigenetic changes via transfer of
scopy revealed that MVs package injured mitochondria from the optic their cargo from cell to cell (Sansone et al., 2017). It has been reported
nerve head of retinal ganglion cell axons and transfer them to nearby that EVs can transport the entire mtDNA chromosome from one cell to
astrocytes to undergo lysosomal degradation (Berridge and Neuzil, another, rescuing cancer cells after hormone therapy, which greatly
2017; Hayakawa et al., 2016). Thus, MVs are useful in cell maintenance affects the endogenous mitochondrial pool (Sansone et al., 2017). EV-
and repair. mediated mtDNA transfer can restore metabolic activity of cells with
It was originally hypothesized that exosomes, the smallest EV, impaired metabolism (Sansone et al., 2017). The ability of the exosome
functioned to remove unneeded membrane proteins from reticulocytes; to transverse the blood-brain barrier is employed so as to engineer
however it is now apparent that they serve as vehicles of long-distance exosomes as drug delivery vehicles (Sarko and McKinney, 2017). In
molecular transport of nucleic acids and proteins between cells in- addition, because circulating EVs are present in plasma of patients with
cluding mtDNA (Aryani and Denecke, 2016; Guescini et al., 2009; cardiovascular disorders and cancer, they can be collected and profiled
Urbanelli et al., 2015; Dani and Dani, 2010). The quantity and content with a patient's blood sample, presenting a less invasive approach to
of exosomes depends on environmental conditions, cellular stress, and disease diagnostics (Jia et al., 2014). Even if EVs have been associated
parent cell maturity (Aryani and Denecke, 2016). Exosome biogenesis with a damage response or diseases like cancer, knowing if one process
occurs in a series of steps. It is observed that physical exercise, calcium or the other leads to a higher or less mtDNA or mitochondria cargo is
level and p53 activation in response to DNA damage stimulates exo- still under question.
some production (Frühbeis et al., 2015; Yu et al., 2006; Hessvik and EVs are part of several processes involved in health and disease,
Llorente, 2018; Cooks et al., 2018). It is known that the first stages of ranging from inflammation to cell-to-cell communication to tumor-
biogenesis are controlled by the Endosomal-Sorting Complex Re- igenesis. Understanding how and in which context the different forms
sponsible for Transport (ESCRT), which signals membrane receptor of EVs are secreted will provide clues so as to manipulate and customize
recycling and inward budding of the plasma membrane (Ailawadi et al., their cargo. The particular characteristics of EVs regarding their

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S. Miliotis, et al. Mitochondrion 48 (2019) 16–30

components and the possibility to track their cellular origin by their role in patients with severe trauma, as it has the ability of activating
membrane markers opens the possibility to use them as biomarkers of innate immune responses via different pathways (Fang et al., 2016).
disease or even to study physical performance and recovery (Chen mtDNA has both extracellular and intracellular immune activation of
et al., 2018; van Niel et al., 2018; Safdar et al., 2016). Patient-derived toll-like receptor 9 (TLR-9), Nod-like receptor family pyrin domain-
EVs could be also used as therapeutic agents due to their reduced ca- containing 3 (NLRP3), and the STING pathway (Itagaki et al., 2015). In
pacity to induce an immune response or even to transfer regulatory particular, mtDNA has been found to bind with TLR9, which can result
nucleic acid sequences that can be delivered to the tumor to induce cell in activation of neutrophils. Beneficial effects of these immune re-
cycle arrest, among other effects (van Niel et al., 2018; Cătană et al., sponses could be seen when neutrophil extracellular traps (NETs) are
2017). MSC-derived EVs containing mitochondria have shown to formed. NETs are networks of extracellular structures composed of
modulate macrophage function by enhancing their respiration and mtDNA and released by neutrophils that are used to bind and kill mi-
phagocytic activity (Morrison et al., 2017), but their role in many of the croorganisms (Thurairajah et al., 2018). On the other hand, TLR-9
regenerative effects of MSCs still needs to be elucidated to fully re- mediated inflammatory responses also have the ability to induce
plicate and standardize their results for use in future novel therapies myocarditis when mtDNA escapes autophagy in cardiomyocytes due to
(Matthay, 2017). hemodynamic stress (Circulatory Mitochondrial DNA, 2018). Further-
more, levels of ccf-mtDNA have been observed to be much higher
4. Cell free circulating mitochondria DNA (ccf-mtDNA) a danger during severe trauma in patients with multiple organ failure due to
signal mtDNA-induced inflammation (Thurairajah et al., 2018). Thus, the
liberation of mtDNA to the bloodstream can severely affect the body's
The mitochondrion has its own DNA that interacts with the cell organ systems. High concentrations of ccf-mtDNA have not only been
nucleus to produce new mitochondrial components and regulate its observed in trauma and post-surgical interventions, but also in patients
own function. Inside the cell, mitochondria contain 100 to 1000 copies with diabetes, cancer, myocardial infarction, and other medical con-
of mtDNA that are maternally inherited (Li et al., 2016). The mi- ditions. For this reason, cff-mtDNA levels can possibly be used as a
tochondrial genome consists of 37 genes, and 13 of these are funda- biomarker and predictor of outcome in these diseases (Lindqvist et al.,
mental for the production of enzymes related to oxidative phosphor- 2018).
ylation to produce ATP The rest produce transfer RNAs (tRNAs) and Studies have been performed to provide evidence of differing levels
ribosomal RNAs (rRNAs) (Kesner et al., 2016). Changes in the mtDNA of ccf-mtDNA among patients with varying diseases (Gonzalez-Hunt
sequence can accumulate with age and partake the evolution of serious et al., 2016). Nakahira et al. analyzed blood samples of two distinct ICU
clinical disorders. These double-stranded circular structures lack the patient groups by measuring the copy number of the NADH dehy-
complex repair mechanisms and protective histones that are seen in drogenase 1 gene, a specific mitochondrial gene, through quantitative
nuclear DNA, causing mtDNA to be more highly susceptible to muta- real-time PCR (qPCR), a laboratory technique used to measure mtDNA
tions (Li et al., 2016). Any change or distortion to the mtDNA in con- damage and copy number. Among those patients in the medical ICU,
junction with deletions, duplications, and inversions will develop mi- ccf-mtDNA levels were elevated (more than 3200 copies per microliter
tochondrial defects, produce ROS, and/or damage the cell, inducing of plasma) in which case the risk of death within 28 days of admission
apoptosis and liberating mtDNA. mtDNA can be liberated by active, increased 7-to-8 fold when compared to those with less than 3200 co-
stressed, apoptotic, or necrotic cells becoming cell free circulating mi- pies of NADH dehydrogenase 1 gene. Although their findings do not
tochondrial DNA (ccf-mtDNA) (McCully et al., 2009). indicate the cause behind the elevated levels of cff-mtDNA, it still
In normal physiology, cells and mitochondria prevent or favor the proved to be a vital predictor to those patients in medical ICUs
clearance of mutated mtDNA as it can accumulate with age or disease (Nakahira et al., 2013). Additionally, Zachariah et al. used both cir-
due to the damaging effects of ROS or cumulative mutations due to culating cell free nuclear DNA and ccf-mtDNA as biomarkers to detect
sustained proliferation (Caicedo et al., 2015). The accumulation of ovarian cancer. They used multiplex polymerase chain reaction (PCR)
mtDNA damage can favor the liberation of ccf-mtDNA as has been seen to analyze the levels of circulating cell free nuclear DNA and circulating
in patients with sepsis, diabetes, and cancer. The mitochondria quality cell free mitochondrial DNA in serum and plasma among patients with
control system eliminates damaged mitochondria together with their epithelial ovarian cancer, benign epithelial tumors, or endometriosis.
DNA by fusion or fission mechanisms. Fusion allows for the mix of The patients' levels were then compared to healthy individuals. The
damaged and healthy mitochondria favoring heteroplasmy, when bal- difference between healthy individuals and those with epithelial
ance towards the healthy mtDNA is conserved and damaged mtDNA is ovarian cancer was noticeable as they appeared to have much higher
eliminated by polarization facilitating its degradation by fission and levels of circulating nDNA and mtDNA. Unlike circulating nDNA, cir-
mitophagy (Popkov et al., 2017). It has been observed that mitochon- culating mtDNA was found to also have substantially higher con-
dria can secrete vesicles in times of stress but the understanding of the centrations in ovarian cancer patients than in endometriosis patients
entire process, such as transporting intra-mitochondrial material for (Zachariah et al., 2008).
degradation, recycling, or expulsion, is still missing (Shutt and McBride, Ccf-mtDNA is known to contribute to activation of inflammation
2013). and immune responses. Elevated ccf-mtDNA levels have been sig-
Mitochondria that accumulate damage become a trigger for cell nificantly correlated to the development of diseases. Further research is
senescence, apoptosis or the rapid elimination of cells via necrosis vital to fully understand the biological role of ccf-mtDNA in order for it
(Laberge et al., 2013). In all of these cases, mtDNA can be liberated to be used as a diagnostic tool in clinical settings. What is more, ccf-
acting as an activation signal for the immune cells to stimulate the mtDNA has been most commonly associated with a danger signal
clearance of damaged cells and pro-inflammatory signals (Randow and working as a Damage Associated Molecular Pattern (DAMP) or death-
Youle, 2014). The context in which mtDNA can be liberated or actively associated molecules. These molecules are released and exposed to the
secreted has been profoundly studied as it can induce pro-inflammatory circulatory system as a response to cell injury or death due to a pa-
responses (Cowan et al., 2017; Lindqvist et al., 2018; McCully et al., thological attack or cellular stress (Hayakawa et al., 2016; Gerdes et al.,
2009) or acts as a source for recombination and mixture with the host 2007). DAMPs, besides being related to mitochondria fragments or their
cell when transported in EVs (Torralba et al., 2016; Lawson et al., 2016; DNA, can derive from several organelles or cell structures in the cytosol,
Weiss and Schaible, 2015). plasma membrane, and endoplasmic reticulum (Hayakawa et al.,
For a long period of time, ccf-mtDNA in clinical settings remained 2016). DAMPs other than mtDNA have specific molecular conserved
irrelevant until its particular similarities to bacterial DNA were detected patterns: N-formyl peptides, cytochrome C, mitochondrial ATP, cardi-
(Thurairajah et al., 2018). ccf-mtDNA is now considered to play a major olipin (CL), Carbamoyl phosphate synthetase I (CPS1) and

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mitochondrial reactive oxygen species (ROS) that work as activators for In this context, because the release of DAMPs into circulation alters
the immune cell clearance response. Likewise, pathogen-associated homeostasis, an alarm to the immune system is generated (Clark and
molecular patterns (PAMPs) and DAMPs are often recognized by the Shay, 1982). For instance, DAMPs that are liberated take part of the
same cellular receptors, denoting a resemblance between a non-in- activation of the macrophage inflammasome, a multiprotein complex
fectious inflammatory response with a pathogen -induced one; their responsible for the onset of inflammation (Margus et al., 2012). In
distinguishing relay on specific pattern-recognition receptors (PRRs): addition, some of these molecular patterns such as ATP activate the
Toll-like receptor (TLR), RIG-I-like receptors (RLRs) and NOD-like re- NLRP3 inflammasome designated as pyroptotic cell death (Shi et al.,
ceptors (NLRs); purinergic receptors (Sluijter et al., 2014); and Formil 2017), a cell death pathway activated by microbial infections and non-
Peptide Receptor (FPR) (Clark and Shay, 1982). Nevertheless, the infectious stimuli (Marín-García, 2013). In the case of neutrophil-
evolutionary connection among DAMP and PAMP signaling pathways mediated organ injury, Zhang and his team found that by intravenously
are not clearly recognized (King and Attardi, 1988). injecting mitochondrial DAMPs (formyl peptides and mitochondrial
DAMPs can be divided into subgroups according their localization DNA) into rats, the DAMPs did generate systemic inflammation in nu-
or the place they were released: they can be exposed 1) on the plasma merous tissues (Shi et al., 2018). However, more studies using nuclear
membrane, 2) extracellularly, into the systemic circulation as mtDNA and mtDNA its capacity to induce a pro-inflammatory should be ad-
(Krysko et al., 2011; Kuck et al., 2015), 3) and in the final stages of dressed in-vitro and in-vivo. The mitochondrial genome fragments re-
products degradation, such as uric acid (Krysko et al., 2011). The sig- leased after injury into circulation work as intercellular signals
nals presented by mitochondrial-derived are chemotactic, immune-sti- spreading damage via the Toll-like-receptor from the injury itself to
mulatory, phagocytotic, and regenerative. The type of DAMP secreted, distant organs, contributing to the evolution of multiple organ dys-
their pathway, and function are directly involved and ruled by the type function syndrome (MODS) (Fillmore and Lopaschuk, 2013). Moreover,
of cell that was injured or dying (Sluijter et al., 2014), also known as the DAMPs can modulate the role of other types of cells as mast cells,
“Danger Model”, which implies that the activation of the im- neutrophils and eosinophils, and have the ability to disturb the antigen-
munological response depends on the presence of potentially or dan- presenting cells (APCs), such as macrophages, by modifying cell ma-
gerous stimuli to the host and works as a warning for cell damage or turation and altering the process of antigen presentation (Sluijter et al.,
death, such as DAMPs (Wu et al., 2016; Margus et al., 2012; Kim et al., 2014). The released quantity of mitochondrial DAMPs after trauma
2018). For instance, mtDNA DAMPs, through the stimulation of neu- induce a pro-inflammatory response, not only affecting the clinical
trophils, activate supplementary immune cells which release pro-in- outcome but also reflecting a poor prognosis (King and Attardi, 1988;
flammatory cytokines hence producing an inflammatory response; and Fillmore and Lopaschuk, 2013; Boyapati et al., 2017; Court et al.,
in some cases propagating the injury to distant organs (Simmon et al., 2018).
2013). Despite there being an abundance of knowledge surrounding the
Polly Matzinger, in 1994, suggested the possibility of an immune function and the importance of ccf-mtDNA and mtDNA DAMPs, a better
response to intracellular alarms, activated the moment an endogenous understanding of their roles in different diseases as well as their de-
molecule is freed into the body; similar to a pathogenic immune re- tection and diagnostics could help in comprehending the pro in-
sponse (Pacak et al., 2015). It was experimentally shown, in the sub- flammatory response, developing anti-inflammatory therapies, and fa-
sequent years, that DAMPs have the capacity of originating an in- cilitating tissue repair (Fig. 2).
flammatory response similar to PAMPs. Qin Zhan and colleagues aimed
to discover why the signaling pathways are so alike between internal 5. Extracellular forms of mitochondria as a biomarker of disease
and external triggers, and taking into consideration the evolution of the and therapeutic agent
mitochondria, they established a new hypothesis: both mtDNA and
proteins may function as DAMPs, activating identical PAMP pathways. The normal function of mitochondria can be affected during many
This research team confirmed this hypothesis by in vivo studies, dis- pathological processes such as Alzheimer's, Parkinson's, Diabetes, acute
covering that in major trauma and bone fracture patients, the release of myocardial infarction or stroke for which providing new mitochondria
mtDNA levels were high compared to patients without injuries. More- or renewing its pool by its artificial transfer could induce a therapeutic
over, Zhan's group studied and confirmed the role of the mitochondria- effect. Mitochondria in response or as a result of pathology change their
derived DAMPs in the immune response by activating FPR1 and MAP shape, energy production, and nucleus coordination of gene expression
kinases using mitochondria-derived DAMPs. The identified sources of to preserve cellular health or accompany cell elimination by apoptosis.
N-formyl peptides are bacteria and mitochondria; a formyl peptide can The release of mitochondria inside MVs has been linked to the halt of
call neutrophils or activate them by specific binding to its receptor tissue degradation or in a systemic effect, the increase in an in-
(FPR1), promoting the inflammatory response by the release of che- flammatory response. For example, healthy MSCs activated by in-
mical mediators and by triggering the MAP kinase enzymes. It was flammatory signals transfer mitochondria inside MVs to stressed or
determined that due to the mitochondrial DAMPs, the immune response damaged cells rescuing their viability and function. It has also been
to injury imitates the sepsis response normally activated by PAMPs (Shi shown that activated platelets, essential components of the in-
et al., 2018). flammatory response, release functional mitochondria encapsulated in
The trigger by which mtDNA DAMPs are created and released is still microparticles or as free organelles promoting leukocyte activation.
unknown, however it functions as an intercellular signal. These DAMPs Increasing evidence has shown that using mitochondria to treat
act on the formyl peptide receptor-1 (Walko et al., 2014) and Toll-like heart ischemic events improved tissue recovery and open the possibility
receptor 9 (TLR-9), which have been found in endosomes and also on for its application in other pathologies (Picard et al., 2016; McCully
the plasma membrane of the majority of cells that are associated with et al., 2016). Since the discovery of artificial mitochondrial transfer in
the acute respiratory distress syndrome (ARDS). Additionally, mtDNA 1982, different experiments have been conducted to see the benefits or
has been associated with the mitochondrial transcription factor A risks of this method as a therapeutic agent (Caicedo et al., 2017). Ad-
(TFAM), an mtDNA-binding protein (Kuck et al., 2015). This protein ditionally, emphasis has been placed on the discovery of new mi-
has shown to be an amplifier of TNF-α release when it is bound to tochondrial transfer techniques. Particularly, transfer of extracellular
mtDNA, via TLR9 in the signaling pathway (Julian et al., 2013). Mi- mitochondria could be a standard therapeutic option in the future
tochondrial DNA DAMPs have been found, at high levels, in the plasma especially in cases where tissue mitochondria is rapidly affected such as
of patients with systemic inflammatory response syndrome, as in pa- in the case of ischemic events (McCully et al., 2016; Cowan et al., 2016;
tients with multiple organ dysfunction syndrome (MODS), ARDS, and Masuzawa et al., 2013). Many groups are interested in and working on
with sepsis (Kuck et al., 2015). developing mitochondrial transfer/transplant approaches and

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Fig. 2. DAMPs derived from mitochondria and its response in the immune system.

applications with names that reflect the particularity of their adaptation to the cytotoxic agents chloramphenicol and efrapeptin. Mitochondria
or the conception of the process. Examples include Mitochondrial carrying genes of resistance in their mtDNA helped their parent cells to
Transformation (Kesner et al., 2016), Magnetomitotransfer (Macheiner survive in this selective environment. The transfer of mitochondria is-
et al., 2016), MitoCeption (Caicedo et al., 2015), and Mitochondrial sued from resistant to susceptible cells also helped the susceptible cells
Invasion (Popkov et al., 2017). The Mitochondrial Invasion proposal to survive in the presence of cytotoxic agents. For the first time, this
has interesting homologies with the bacterial origin of the mitochon- transfer provided a functional advantage to the cells that received
drion as its passage from cell-to-cell could be similar to other in- exogenous mitochondria and exchanged their mtDNA (Clark and Shay,
tracellular bacteria (Popkov et al., 2017). However, the identification of 1982). Since then, many other transfer techniques appeared, such as
foreign bacteria versus intracellular endosymbiosis such as the mi- that of King and Attardi in 1988, where the direct microinjection of
tochondria is very precise leading to the elimination by autophagy of mitochondria was used to re-populated the cell with a new pool (King
the parasite (Randow and Youle, 2014; Weiss and Schaible, 2015). In and Attardi, 1988). In 2016, the transfer of mitochondria through a
this section we will discuss the different methods of artificial mi- photodermal nanoblade, make this process independent of the biolo-
tochondrial transfer that currently exist and about the usefulness of gical interaction that could be happening between the isolated orga-
mitochondria as a therapeutic agent or biomarker. Emphasis will be nelle and the recipient cells. Even, if this process could result ad-
placed on the nervous system and cardiovascular system as these tissues vantageous when forcing the transfer could be needed a high
rely heavily on mitochondrial function, hence the transfer of this or- throughput application could seem challenging as the photodermal
ganelle could become a therapy of choice (Hayakawa et al., 2018; nanoblade is only acting in a few cells at the time (Caicedo et al., 2017;
McCully et al., 2017). Wu et al., 2016).
Mitochondrial transfer from a donor cell can occur as a physiolo- Further steps were developed to improve co-incubation and the
gical phenomenon in situations of stress to protect the recipient cells efficacy of the mitochondria transfer. Pep-1 is a delivery peptide de-
from further harm and malfunction (Hayakawa et al., 2016). This signed to induce pores in the cell membrane and facilitate the delivery
transfer can occur in the form of microvesicles or nanotubes (Gerdes of mitochondria (Margus et al., 2012). The use of this technique seemed
et al., 2007; Sluijter et al., 2014). In 1982 Clark and Shay developed the to improve the introduction of mitochondria compared to coincubation
first technique for artificial mitochondrial transfer called “Mitochon- alone (Caicedo et al., 2017; Margus et al., 2012). Another improvement
drial Transformation”. In this technique, they exposed a group of cells to the coincubation method is the MitoCeption technique where, in

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addition to the coincubation, thermal shock and a centrifugation were function of the heart tissue.
performed, causing mitochondrial uptake by the recipient cells pro- Many studies regarding the use of the mitochondrial transplant in
portional to the added mitochondria (Caicedo et al., 2015). Even animal models and in humans show a favorable outcome with minimal
though a proportional uptake with a high performance was observed, adverse effects such as a negative immune reaction or arrhythmia. The
further improvements are needed to decrease the time of the transfer multiple techniques of isolation, purification, and mitochondrial
and minimize the stress in the recipient cells. New techniques are being transplantation require a procedure duration of more than 120 min
introduce in which the quantity of mitochondria needed to induce a (Cowan et al., 2016; Masuzawa et al., 2013; McCully et al., 2017).
change in the phenotype is optimized, including the technique pro- However, in medical practice it is necessary to obtain viable mi-
posed by Mi Jin Kim and colleagues, who merely centrifuged after tochondria in a shorter period because the majority of cardiac surgical
putting exogenous mitochondria and recipient cells together (Kim et al., procedures take approximately 60 min and prolonging surgeries is
2018). It will become necessary to adapt the transfer protocols to dif- counterproductive. The method described by James D McCully and his
ferent cell types as they have been applied only to attached cells. These team in rabbit hearts, rabbit in vivo and, pig hearts during ischemia
protocols will support today's efforts to apply the transfer/transplant of reperfusion exercises seems to be promising for clinical uses as it re-
mitochondria in the clinic and open the spectrum of its therapeutic quires only 30 minutes (McCully et al., 2017). McCully's method calls
effects. for two samples of skeletal muscle (pectoral or rectus abdominis) of
Determining which is the most appropriate route of application of approximately 0.1 g by surgery. Then this tissue is homogenized by a
exogenous mitochondria in-vivo to harmed tissues is necessary for the Miletenyi gentleMACS Dissociator. The homogenate is digested with
validation of their clinical applications. Different forms for this process subtilisin A, a proteinase, then filtered by a sterile mesh. The obtained
have been used, such as the direct injection of mitochondria at the mitochondria can be used directly or concentrated by means of cen-
damaged site or injection into the circulatory system near the area of trifugation (9000 rpm at 4 °C for 10 min). Finally, the total concentra-
interest. Both approaches rescued the functionality of the damage site; tion is obtained with a reference sample of 1 × 10 (Zhao et al., 2017)
however, while the in-situ injection had better results for delay in mitochondria. This procedure provides functional mitochondria, as the
reaching the damaged site, it also presents the possibility of decreased membrane potential, viability, and oxygen consumption and respiration
mitochondria concentration and a loss of mitochondria integrity when is maintained. It has been shown that mitochondrial transplantation can
applied systemically. This opens up the possibility of mitochondria to be done through direct injection of mitochondria or through vascular
be detected by the immune system and recognized as a pro-in- delivery during reperfusion (McCully et al., 2017). Multiple experi-
flammatory trigger (Caicedo et al., 2017; Masuzawa et al., 2013; Pacak ments in animal models reveal that two enzymes that reflect myocardial
et al., 2015). damage (creatinine kinase isoenzyme MB and sensitive troponin I) are
Mitochondrial transfer/transplant techniques have become an area significantly reduced when mitochondrial therapy is used. Additionally,
of interest in translational medicine due to the possible benefits of their it was observed that myocardial contractile function improves after
application in the treatment of human diseases. Currently, mitochon- 10 min and it is maintained during the next 28 days (McCully et al.,
drial therapy has shown to have promising effects in different pathol- 2017). The mechanism of action proposed for mitochondrial trans-
ogies ranging from myocardial ischemia, to neurodegenerative diseases plantation seems to involve the uptake by the recipient cells through an
such as Parkinson's disease, to fulminating hepatitis (Shi et al., 2018; actin-dependent mechanism, leading to the synthesis of new ATP, an
Shi et al., 2017). Particular emphasis has been placed on myocardial increase in proteomic activation pathways, the up-regulation of cardi-
ischemic disease and is probably the area of study where most research oprotective cytosine and the replacement of damaged mitochondrial
has been conducted. DNA with new copies. Interestingly, cardioprotective cytokines stimu-
The heart is a vital organ with constant energy consumption to late cell growth and increase cardiac function and cardiac remodeling
guarantee the transport of blood to all systems (Marín-García, 2013). (McCully et al., 2017). Other studies have shown that there is no sig-
This organ is made up of specialized muscle fibers with a large number nificant improvement in cardiac function when ATP is administered to
of mitochondria to satisfy its metabolic demands (Marín-García, 2013). the ischemic tissue. This implies that the transfer of mitochondria could
Mitochondria constitute 30% of the cell volume of the heart's muscle induce regenerative effects, more that the artificial compensation of the
fibers providing the greatest amount of energy (McCully et al., 2017). affected constituents in heart ischemic events. (McCully et al., 2017)
The patency of the coronary arteries provide a continuous blood flow The mitochondrial transplant with regenerative and protective
full of oxygen to the cardiomyocytes. It has been observed that in purposes have advanced very quickly from in-vitro and in-vivo to
equilibrium conditions, the cardiac mitochondria extract more than clinical trials. Five pediatric patients at Boston Children Hospital pre-
79% of the oxygen from the blood provided by the coronary arteries sented a diagnosis of cardiovascular malformations: transposition of
(Fillmore and Lopaschuk, 2013). During an ischemic event due to the large vessels (4 days and 25 days), left hypoplastic heart (6 days), left
occlusion of the coronary arteries, mitochondrial dysfunction occurs, flow obstruction (6 months) and tricuspid atresia (2 years). They un-
thereby producing a decrease in ATP synthesis, increase ATP hydro- derwent different surgical techniques but suffered ischemia events for
lysis, alterations in homeostasis of ionic balance, formation of reactive which they were supported by extracorporeal membrane respiration
oxygen species, alteration in mitochondrial volume, accumulation of (ECMO) and inotropic drugs. To treat these patients, mitochondria were
calcium inside of the mitochondria and activation of proapoptitic pro- obtained from their rectus abdominis by McCully's method in sterile
teins (McCully et al., 2016; McCully et al., 2017; Marín-García, 2013). conditions. They obtained 1 × 108 ± 1 × 105 mitochondria from this
Additionally, once the tissue has been reperfused, many debris related procedure and these mitochondria were suspended in 1 ml of solution
to mitochondrial impairment such as free radicals are released from buffer. Ten injections containing 1 × 107 ± 1 × 104 mitochondria
dead tissue. This can destabilize the membrane potential of cardiac cells were applied in tissue that proved to be hypokinetic or akinetic by
and can cause potentially fatal post-reperfusion complications such as echocardiography. The results were surprising and favorable since in
cardiac arrhythmias (Marín-García, 2013). Many of these alterations the echocardiographic records after 24, 48, 96–144 and 240 h demon-
occur during the ischemic event, however they could persist after the strated an improvement in cardiac function. Patients with the diagnosis
blood flow has returned leading to a decrease in contractility and car- of severe or moderate myocardial dysfunction showed a decrease in the
diac output (McCully et al., 2017). The level of mitochondria dys- severity of hypokinetic tissue to mild or even null. It is necessary to
function determines the outcome as it represents an important part of mention that two patients in this study did not survive after other
the pathophysiology of cardiovascular ischemic events. It has been complications unrelated to the mitochondrial transplantation. One of
proposed that mitochondrial transfer/transplant in therapy could lead the these patients recovered his cardiac function but died after hepatic,
to the replacement of damaged mitochondria and the repair of normal renal and pulmonary insufficiency while the other patient regained his

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cardiac function from severe to slight damage but died due to re- observed in patients with better prognosis at 3 months. Additionally,
spiratory failure. It is noteworthy that in an autopsy of one of these during this study, the MitoTracker Red CMXRos tool used by flow cy-
patients there were no features of rejection or inflammation at the place tometry defined the origin of the mitochondrial flow showing that pa-
of injection (Emani et al., 2017; McCully et al., 2017). This study clearly tients with the best prognoses had mitochondria of astrocytic origin
reveals the successful applicability of mitochondrial transplantation in when compared with those of microglial, endothelial or platelet origin
pediatric patients after an ischemic event, and it is likely that the same (Chou et al., 2017). Clearly, these assays revealed that quantification of
protocol can be used in adult patients. However, it is necessary to note extracellular mitochondria could be a marker for prognosis of SAH.
that more studies are required in order to identify the safety, efficacy, Future studies are necessary to validate the effectiveness of this tool,
optimal dose of this technique to use mitochondria as a therapy of particularly to compare the sensitivity and specificity of the single use
choice within the schemes of cardiac reperfusion therapy. of JC1, JC1 vs clinical scales such as Hunt and Hess or the current use of
Recent evidence suggests that the mitochondria isolated from MSCs JC1 and clinical scales to establish the prognosis of this type of cere-
and muscle tissue do not exacerbate the immune response when brovascular disease (Hayakawa et al., 2018; Chou et al., 2017). Chou
transferred/transplanted in-vitro or in-vivo. M. Khoury and his team in and Hayakawa evidenced that the exchange and presence of extra-
2018 used a Xeno-Graft-Versus-Host-Disease (GVHD) mouse model to cellular mitochondria outside neurons and its activity reflects the ad-
observe if the artificial transplant of mitochondria could improve the vancement of disease which could be reinforced by the estimation of
survival of the tested animals. They evidenced in-vitro that the transfer ccf-mtDNA. The great challenge today is to define if the presence of
of human MSCs mitochondria to different donor CD45+ CD3+ Human mtDNA as ccf-mtDNA in many diseases (Lindqvist et al., 2018; Shockett
T-cells by MitoCeption induced cell anergy as a decrease of the 25% in et al., 2016) exists inside viable or defective mitochondria and if their
the division index of the cells that received the mitochondria (Court uptake leads to the recycling of the organelle in the receiver cells to
et al., 2018). Additionally a reduction of the Th17 cell populations in promote cell survival as previously seen by Hayakawa (Hayakawa et al.,
contrast to an increase of the Tregs was observed. In-vivo the transplant 2016) and other authors such as Davis et al., in 2014 (Davis et al.,
of human mitochondria to mice GVHD model improved its survival and 2014).
body weight together with a decrease in the proliferation rate of CD4+ The studies of Hayakawa (Hayakawa et al., 2016) and Davis (Davis
cells by 20% and IFNγ in both CD4+ and CD8+ cell populations (Court et al., 2014) regarding the nervous system and how the released mi-
et al., 2018). To test if exogenous and viable mitochondria could be tochondria is associated to a mechanism of transmitophagy points out
recognized as DAMPs, James McCully and his team in 2018 inject to evidence that the uptake of extracellular mitochondria could be re-
syngenic and allogeneic mitochondria to fully MHC-mismatched skin lated to a endocytic recycling process associated to a danger signal or
allografts in mice observing no alloreactivity or allorecognition and damage. Even if Hayakawa demonstrates that the astrocytic support of
DAMP reaction. In their assays no increase in the levels of Il-2, IFNγ, IL- neuronal functions by mitochondrial transfer is dependent of the ac-
1, IL-4, IL-6, IL-12, IL-18, IP-10, macrophage inflammatory protein tivity of CD38, this process still needs further corroboration as men-
MIP-1α and MIP- 1β1 were observed. This line of evidence in which the tioned by Berridge et al., 2016 (Berridge et al., 2016). The use of
allogeneic and xenogeneic transplant of mitochondria is tested support fluorescent dyes such as MitoTracker Red employed by Hayakawa have
the notion that viable and isolated mitochondria do not induce an im- the tendency to damage the mitochondria structure and leak out of the
mune response but rather immunoregulation. labeled cells forming particles in suspension when used at high con-
The usefulness of mitochondria within translational medicine can centrations (Berridge et al., 2016). Further analysis to define how other
not only be exemplified by artificial mitochondrial transplantation cells respond to extracellular mitochondria and to use other approaches
therapy, but can also be evidenced in other areas such as the use of the that are not dye dependant are necessary to fully understand the bio-
mitochondria or the mtDNA as a biomarker of the severity of disease. logical context of the internalization process. Another questions comes
Currently, being able to establish the prognosis of vascular neurological if differences exist in the up-take when the exogenous mitochondria is
diseases is challenging due to the multiple clinical scales that define artificially isolated, free by cells, or other subcellular components such
mortality or disability after an event and its lack of accuracy as MVs or platelets. Other work has shown that isolated mitochondria
(Hayakawa et al., 2018). Multiple investigations are conducted to de- artificially transferred to other cells can be endocytosed or uptaken by
fine the utility and quantification of mitochondria or their DNA as a micropinocytosis like mechanisms (Kesner et al., 2016; Kitani et al.,
biomarker in several diseases. The studies of Chou SH and colleagues in 2014). Once inside the cell, the exogenous mitochondria depending on
2007 evaluated not only the presence of mitochondria but also their its membrane constituents and cell can fuse with the endogenous net-
activity by fluorescence markers (Chou et al., 2017). Hayakawa showed work, and that this process is dependent on the presence of fusion-as-
that during stroke, neurons can release their damaged mitochondria sociated proteins (e.g. MFN1, MFN2, and OPA1) (Cowan et al., 2017;
and be uptaken by astrocytes, revealing a possible mode of cell-to-cell Cowan et al., 2016).
signaling in the central nervous system (Hayakawa et al., 2016). In Decrypting if the internalized mitochondria will persist or if its
these experiments the prognosis worsens if the release and recycling of mtDNA is replicating, is still a question that hasn't been fully answered
mitochondria is blocked (Hayakawa et al., 2016). Chou and colleagues from the in vitro and in vivo assays detailed in this manuscript.
measured the quantity and activity of extracellular mitochondria in However, insights to answer these questions comes from the seminal
cerebrospinal fluid (CSF) by the fluorescent probe JC1, which showed paper from Clark and Shay (Clark and Shay, 1982) where the resistance
to be an effective prognostic factor after a subarachnoid hemorrhage to antibiotics that specifically target the mitochondria, such as chlor-
(SAH) in mice and patients (Chou et al., 2017). The JC1 assay enables amphenicol and efrapeptin, was transferred to sensitive cells by the
the labeling of mitochondria according to their membrane potential, incorporation of exogenous mitochondria carrying the DNA mutations
where red and green fluorescence indicate active and inactive, respec- necessary to survive the antibiotic exposure. The cells resulting from
tively In rats, it has been shown that the red/green ratio at 24 h of the the incorporation of exogenous mitochondria, named mitochondrial
subarachnoid hemorrhage was extremely low and there was an increase transformants, carrying the resistance through the mtDNA mutations
at 72 h demonstrating a mitochondrial continuous flow outside cells. It seems to stably carry the new mitochondria, leading to the possibility of
was also determined in this study that rats with a greater neurological an active replication process of the exogenous mtDNA. More studies
deficits had the lowest red/green ratio, in contrast rats with a greater regarding the conditions in which the receptor cell accept and replicate
recovery have more active mitochondria released in the CSF. Ad- exogenous mitochondria are necessary to fully understand the biolo-
ditionally, the lowest red/green ratio corresponded with a poorer out- gical significance and further applications of the artificial mitochondria
come by the Hunt and Hess clinical scale Classification of Subarachnoid transfer/transplant.
Hemorrhage (SAH). On the other hand, higher red/green ratio was Methods of mitochondria transfer or transplant include confocal

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imaging besides the Flow Cytometry Analysis as a test to verify the immune cells has shown that mitochondria when isolated from healthy
internalization of the mitochondria (Macheiner et al., 2016; Caicedo cells lack of an pro-inflammatory immune response (Emani et al., 2017;
et al., 2015; Pacak et al., 2015; Kitani et al., 2014). Evidence by con- Jackson et al., 2016). Platelets are enucleated structures reactive to
focal imaging shown that cells incorporate mitochondria differently injury which can trigger an immune response. They can carry mi-
among them (Kesner et al., 2016; Caicedo et al., 2015). Future research tochondria that are also activated by the same stimuli to produce ROS
could provide answers to know if the internalization process depends or induce the apoptosis signaling cascade (Leytin et al., 2009; Sjövall
on the activation of micropinocytosis related mechanisms which re- et al., 2013). After platelet activation, mitochondria become primed for
spond to specific mitochondria membrane proteins or activity which apoptosis, and once outside of the cell, can be strongly immunogenic
could vary in the isolate. Identifying exogenous mitochondria in the (Boudreau et al., 2014). In contrast, non-activated platelets and their
recipient cells involves the use of other methods besides the use of dyes mitochondria appear to induce a regulatory effect upon contact with
due the possibility of leakage of fluorescent dyes from mitochondria, as immune cells (Zhao et al., 2017). So far, the type and state of mi-
mentioned before in the analysis of Berridge et al. (2016). Leakage tochondria seems to mediate their pro-inflammatory, regulatory or re-
could stain endogenous mitochondria instead of being specific of the generative properties.
isolated organelles. Functional assays regarding the acquisition of a EVs, as ABs or MVs, can carry mitochondria and other intracellular
phenotypic advantage acquired by the exogenous mitochondria, de- content through the body in response to harm, exercise, disease and a
tection of specific sequences of donor mtDNA or evidence of the of the variety of other inducers. EVs from MSCs, immune cells and even as-
genetic replication should be part of the characterization of the process trocytes have been shown to induce survival effects in other cells
and future understanding of the natural or artificial mitochondrial (Hayakawa et al., 2016; Favaro et al., 2016). Even if a regenerative
transfer or transplant process. effect has been observed by EVs that transport mitochondria to other
Within the physiology and pathophysiology of different diseases, cells (Morrison et al., 2017), the role of EVs and their internal mi-
the mitochondrion plays a fundamental role, even if the etiology is not tochondria in regards to cancer and chemotherapy survival must be
mitochondrial. Mitochondria are able to respond to cellular and tissue further understood to generate effective methods to halt their pro-
damage compensating for harm, but eventually they can accumulate gression. mtDNA can be also transported through EVs, favoring cell
injuries and become irreversibly affected, such as in type II diabetes and viability and survival (Duregotti et al., 2015), however unprotected and
many other diseases (McCully et al., 2017; Wang et al., 2015; Dos free mtDNA (ccf-mtdna) has been shown to induce a pro-inflammatory
Santos et al., 2018). Several pathologies affect mitochondrial function response due its similarities with bacterial DNA (Boyapati et al., 2017).
and structure like sepsis, pancreatitis, fulminant hepatitis, among Preserving mitochondria and mtDNA from circulatory harm by a vesicle
others, being potential candidates for the use of mitochondrial therapy casing can favor its mobility and therapeutic effects, but when mi-
(Yamanouchi et al., 2013; Kung et al., 2012; Shi et al., 2017; Shi et al., tochondria are harmed or their mtDNA is freed into circulation, they
2018). Although the benefit of the therapy has been demonstrated can elicit strong immune activation.
especially in cardiac ischemia, there are still questions surrounding the Today the methodology to detect ccf-mtDNA involves a DNA iso-
most suitable method to obtain mitochondria, the dosage and the ef- lation step from plasma or serum (Lindqvist et al., 2018; Ajaz et al.,
fectiveness of the route of administration depending on the disease. 2015), but how to fully establish if this isolated DNA comes from mi-
Additionally, more studies regarding the measurement of the quantity, tochondria, freeMito or vesicle-bound mitochondria is still under
activity or content of extracellular mitochondria in different disease question. The detection of large quantities of ccf-mtDNA has been as-
scenarios or body fluids could support their use as a wide spectrum sociated with a grave prognosis in the case of sepsis and mental dis-
biomarker of disease or be informative together with other parameters. orders (Lindqvist et al., 2016; Kung et al., 2012), however determining
Both, using the mitochondrial transfer/transplant and measuring their its levels in healthy individuals or during exercise is controversial
presence by detecting mtDNA, mitochondria number or activity could (Shockett et al., 2016; Stawski et al., 2017; Beiter et al., 2011). Un-
have a positive impact in improving current clinical practices (Ellinger derstanding specifically which forms of extracellular mitochondria
et al., 2012; Blanco et al., 2018). could be acting in conjunction with pro-survival, regenerative or de-
leterious effects is key to knowing which form to reinforce ther-
6. Conclusion apeutically for potential medical applications.
Eukaryotic cells have uptaken mitochondria at the very beginning of
The understanding of the role of mitochondria inside cells has multi-cellularity, and today this process can happen simply by co-in-
evolved from merely being considered a producer of ATP and inter- cubation between isolated mitochondria and recipient cells. The ability
mediary metabolite to an active component of cell signaling (Chandel, of a cell to internalize mitochondria seems to be a conserved process by
2014). Recently, a new concept has arisen acknowledging extracellular evolution which has beneficial results for damaged cells when the mi-
mitochondria as an active player in health and disease with effects in tochondria come from healthy cells or tissue. New protocols have im-
tissue regeneration or inflammation. Comprehending why mitochon- proved the intake of mitochondria with a trend to make them more time
dria in one case produce pro-inflammatory (Boudreau et al., 2014) or and material efficient. McCully and his team provided clinical evidence
immune regulatory effects in others (Ramirez-Barbieri et al., 2018; of the use of mitochondria to treat affected tissue by ischemic events
Deutsch and Tomer, 2006; Jackson et al., 2016) is key to determining with positive results. Today, mitochondria are not simply considered a
their role in therapy and their cause or participation in disease. Ex- power plant thanks to the latest advancements in the study of their role
tracellular mitochondria, once in circulation or as ccf-mtDNA, can serve inside and outside cells. They have now become a signaling organelle
as a biomarker of disease or damage prognosis as many studies have and biomarker of disease and damage prognosis. Mitochondria are
explored its presence in correlation with positive or negative outcomes moving very fast from a passive role to being an active therapeutic
(Lindqvist et al., 2018; Liu et al., 2015; Shockett et al., 2016). This agent with potential effects in the treatment of cardiac, neurological
review provided evidence to understand how the varying forms of ex- and metabolic diseases.
tracellular mitochondria, freeMitos, inside platelets and vesicles, as
mtDNA inside exosomes or as ccf-mtDNA, interact with other cells as a Contributions
base to developing further research.
Mitochondria and their contents were strictly considered an acti- Sophia Miliotis contributed to this work as they wrote the part
vator of the immune system (West and Shadel, 2017; Weinberg et al., centered in microvesicles as carriers of mitochondria together with the
2015). However, the use of mitochondria as a treatment to cardiovas- analysis of the bibliography. Bryan Nicolalde wrote the last part of the
cular diseases and recent evidence regarding its interaction with review and contributed to the revision, Mayra Ortega analyzed the

27
S. Miliotis, et al. Mitochondrion 48 (2019) 16–30

literature corresponding to the ccf-mtDNA and elaborate the figures, Clark, M.A., Shay, J.W., 1982. Mitochondrial transformation of mammalian cells. Nature
both authors equally contributed to the manuscript. Jackie Yepez par- 295, 605–607.
Cooks, T., et al., 2018. Mutant p53 cancers reprogram macrophages to tumor supporting
ticipated in the ccf-mtDNA part and collaborate in the revision of all the macrophages via exosomal miR-1246. Nat. Commun. 9, 771.
manuscript. Andrés Caicedo conceptualize the article; participate in the Court, A., et al., 2018. Mitochondrial transfer from MSC to human T cells: a first evidence
literature analysis and writing of all segments of this work. of a stem cell-mediated reprogramming of multiple immune cell function.
Cytotherapy 20, S94.
Cowan, D.B., et al., 2016. Intracoronary delivery of mitochondria to the ischemic heart
Competing interests for cardioprotection. PLoS One 11, e0160889.
Cowan, D.B., et al., 2017. Transit and integration of extracellular mitochondria in human
heart cells. Sci. Rep. 7, 17450.
The authors declare no competing financial and no-financial inter- Crescitelli, R., et al., 2013. Distinct RNA profiles in subpopulations of extracellular ve-
ests. sicles: apoptotic bodies, microvesicles and exosomes. J. Extracell. Vesicles 2.
Dani, M.A., Dani, S.U., 2010. Improving upon nature's somatic mitochondrial DNA
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Dos Santos, J.M., de Oliveira, D.S., Moreli, M.L., Benite-Ribeiro, S.A., 2018. The role of
Ecuadorian and International students to generate this work. This re- mitochondrial DNA damage at skeletal muscle oxidative stress on the development of
view was supported by the Collaboration Grant # 5501 “Initiation of type 2 diabetes. Mol. Cell. Biochem. 449, 251–255.
the Mito-Act Research Consortium, Mitochondria as a therapeutic agent Duregotti, E., et al., 2015. Mitochondrial alarmins released by degenerating motor axon
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in Immune-Regulation, Tissue Repair and Fertility” from the E497–E505.
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