This document summarizes a study examining the effect of exercise on exosome levels in trained and untrained individuals. Blood samples were taken from 4 subjects (2 male, 2 female) - one active and one sedentary of each gender - before and after an exercise test. Exosome levels were analyzed using the DelsaTMNano C machine. The study found that exosome levels increased more in the active subjects compared to the sedentary ones after exercise. This suggests exercise plays a role in exosome production and levels may be related to cardiovascular health, though more research is needed.

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‘THE EFFECT OF EXERCISE
ON EXOSOME LEVELS
BETWEEN TRAINED AND
UNTRAINED INDIVIDUALS
AND THEIR RELATIONSHIP
WITH CARDIOVASCULAR
DISEASE’
Stephen Reidy
Dr. Ronan P. Murphy PhD FCATH
Group Members: Fiontan O’Curraoin, Vincent Nally, Jason
O’Toole, Hannah Trehy, Sandra McGrath and Julie Rogers

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DCU Science & Health
Assignment Submission
Student Name(s): Stephen Reidy
Student Number(s): 10332463
Programme: PEB4 - B.Sc Physical Education and Biology
Project Title: Final Year Project
Module code: SS419
Lecturer: Dr. Ronan Murphy
Project Due Date: 28-Jan-2014
Declaration
I the undersigned declare that the project material, which I now
submit, is my own work. Any assistance received by way of
borrowing from the work of others has been cited and
acknowledged within the work. I make this declaration in the
knowledge that a breach of the rules pertaining to project
submission may carry serious consequences.
I am aware that the project will not be accepted unless this form
has been handed in along with the project.
Signed:_________________________

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CONTENTS
1.0 Acknowledgements
2.0 Abstract
3.0 Introduction and Justification
4.0 Methodologies
4.1 Ethical Clearance
4.2 Subject Selection
4.3 Study Protocol
4.4 Blood Analysis And Centrifuging
4.5 Analysing the Plasma Samples
5.0 Results
5.1 Subject 1
5.2 Subject 2
5.3 Subject 3
5.4 Subject 4
5.5 Female Exosome Level after Exercise Comparison
5.6 Male Exosome Level after Exercise Comparison
6.0 Discussion
6.1 Cardiovascular Disease and Exercise
6.2 Cardiovascular Disease and Exosomes
6.3 miRNA in Exosomes and Relationship with CVD
6.4 Stress, Heat Shock Proteins in Exosomes and Relationship with
CVD
7.0 Conclusion
8.0 Future Research
9.0 References

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1.0 Acknowledgments
Firstly I would like to offer my sincerest gratitude to my Final Year Project supervisor Dr.
Ronan Murphy. Dr. Murphy has been a pleasure to work with over the last year and has been
more than helpful in guiding us, encouraging us and providing us with vital information
throughout the process.
Secondly I would like to thank all PhD students and scientists in the laboratory that assisted
us during our research. They were more than helpful to us and were very patient and
welcoming as we worked around them in the laboratory.
Finally I would also like to thank the four participants that took part in study for their
willingness and cooperation as without them the research would not have been carried out.
2.0 Abstract
Exosome background
Exosomes are cell-derived vesicles that are found in various biological fluids such as blood,
urine, cerebrospinal fluid and cultured medium of cell structures. It is believed that exosomes
range in size from 30nm to 100nm in diameter and contain high levels of cholesterol proteins,
RNAs and miRNAs in regards to their composition. Specialised functions that are associated
with exosomes are cell-cell communication, transfer of proteins between cells and delivery of
toxic agents. Exosomes are being studied very closely lately and findings have shown that
they can potentially be used for prognosis, therapy, and biomarkers for various diseases.
Methods:
Testing for our research project was completed using four subjects, two male (one active, one
sedentary) and two female (one highly trained, one sedentary). On the 19th
of November 2013
blood samples were taken from each subject pre and post incremental exercise (Bleep Test). 2
hours post exercise blood samples were taken from one participant in each group (one

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physically active and one sedentary). All blood samples were centrifuged and frozen. O
detect exosome levels in the blood the Delsa™Nano C machine was used.
Results:
From analyzing the blood samples and identifying exosome levels in each participant it was
shown that exosome levels increase post exercise. Both active male and female subjects had a
considerably larger increase in exosome levels post exercise when compared to the sedentary
individuals. When comparing genders we noted a considerable difference in exosome levels
in males post exercise irrespective of fitness levels in comparison to our female participants.
Findings & Conclusion:
Following the analysis of our results we found that there as an increase in exosome levels
regardless of fitness level. Although this is true we found that exosome levels increased much
more dramatically in the active subjects in comparison to the sedentary participants. After
research on these patterns we can only postulate as to why there is such considerable
increases in exosome levels after exercise. (i) Exosomes contain miRNA and some have been
found to protect against cardiovascular disease. This suggests that healthy individuals that
exercise regularly produce higher levels of exosomes which results in a higher number of
miRNA. (ii) Exosomes are involved in the transport of heat shock proteins when the body is
exposed to stress. However further research is required in this area to accept/reject these
hypotheses.

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3.0 Introduction and Justification
3.1 Aim of Study
The direction of our study has changed from the literature review that was completed as part
of the SS326 module. Instead of examining microparticles and cardiovascular disease we
have shifted our attention to exosomes and cardiovascular disease.
The aim of this study was to investigate the impact of exercise in relation to exosome levels
in sedentary and active individuals before and after an aerobic incremental test. The
hypothesis under investigation was that after a bout of aerobic incremental exercise, exosome
levels would increase more drastically in active/fit individuals compared to sedentary/unfit
individuals. A second hypothesis estimated that there would be a direct correlation between
exosome levels and the risk of cardiovascular health e.g. high exosome levels play a role in
preventing cardiovascular disease.
3.2 Exosomes
Exosomes are cell derived vesicles that can be found in numerous biological fluids such as
urine, blood, ascites and cerebrospinal fluid (Van der Pol et al. 2012). Recently, research has
shown that exosomes are known to be secreted from a number of cell types such as B cells,
dendritic cells, T cells, platelets, Schwann cells and sperm (Lai et al 2011). It is believed that
the sizes of exosomes are between 30 and 100nm in diameter, with densities ranging between
1.13 and 1.19 g/ml and are isolated by the process of ultracentrifugation (100,000g –
200,000g). In comparison with other secreted vesicles, research has shown that exosomes
have superior defined biophysical and biochemical properties. Regarding their biological
composition, exosome membranes are enriched in very high levels of cholesterol,
sphingomeylin, ceramide and lipid rafts while it is also reported that they contain large
amounts of proteins, RNAs and miRNAs (Wubbolts et al. 2003; Simons and Raposo 2009).

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Fig 3. Composition of an exosome. (Visembryo 2012)
Exosomes are released into the extracellular environment when multivesicular bodies (MVB)
merge with the plasma membrane of the cell (Valaldi et al. 2007). The formation of
exosomes can occur in two different ways and is slightly different than the formation of other
microparticles. The first technique which is called the ‘direct pathway of exosome formation’
occurs when the exosome buds directly from the plasma membrane of the cell in the same
process as a microparticle. The second technique which is called the ‘classic pathway of
exosome formation’ involves the development of intraluminal vesicles. These intraluminal
vesicles bud into early endosomes and in turn form micro vesicle endosomes (MVEs).
MVEs then combine with lysosomes for cargo degradation or with the plasma membrane to
secrete intraluminal vesicles within, which are released as exosomes. (Raposo and Stoorvoge
2013; Van der Pol et al 2012).

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Fig 2. The classic and direct pathways of exosome formation (Van der Pol et al 2012)
Up until a short time ago, there has been very little research done on the functions of
exosomes as there were no publications released on them. Now researchers find the
physiological and pathological roles of exosomes to be very intriguing. Three biological
processes that are believed to be associated with exosomes are cell-to-cell communication via
trans-cellular signaling, the transfer of membrane receptors, proteins, mRNA and microRNA
(miRNA) between cells and the distribution of transmittable and toxic agents into cells (e.g
agents that fight cancer) (Rani et al. 2011). Since exosomes contain a wide variety of proteins
that reflect the originating host cell, they possess vital and important information in relation
to physiological processes in the body including details on future cardiovascular events (De
Kleijn 2012).
4.0 Methodology and Analysis
4.1 Ethical Clearance
Informed consent was received from the subjects while ethical clearance was approved by the
Dublin City University committee.
4.2 Subject Selection
The study comprised of 4 participants, 2 of which were male and 2 of which were female.
Participants were recruited from the DCU population. Two of the participants (One male and

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one female) were classed as sedentary - participating in little of no physical activity each
week. The other two participants (One male and one female) were classed as physically
active – competing either competitively or training four or more times each week. They were
informed of the study and what they would have to do in order to complete it. If happy to
participate they then were asked to provide the required information. Those that were
interested were contacted and a date was arranged to complete the testing. The day/night
prior to testing participants were asked to eat as best they could and avoid any activity which
may hinder the findings e.g. consumption of alcohol. All participants arrived on the morning
of testing in sports gear.
4.3 Study Protocol
Testing began on 19th
of November 2013 Blood sampling was carried out by a trained
professional (Paul O’Connor). Before the subjects carried out the bleep test, one tube of
blood was taken for analysis. Blood was drawn using a 19G needle or larger (to avoid cell
activation or damage) into a 3ml citrate tube. These tubes were labelled with the subject’s
name, the time the blood was taken, and the analysis type it was due to undergo. The tubes
were sealed and transported to the laboratory to be analyzed.
The samples taken were placed in a centrifuge for eighteen minutes to obtain platelet-free
plasma, these were then frozen for future analysis. This procedure was then repeated
immediately after the subjects had participated in the bleep test, samples were taken from two
of our participants two hours after the bleep test also. Latex gloves were worn when handling
blood, all work tops were disinfected and equipment that was in direct contact with blood was
discarded appropriately.
The beep test involved running continuously until exhaustion, between two points that were
20m apart these runs were synchronized with an audio tape with set intervals. The test was
supervised by four of the testers and lasted between six and fifteen minutes depending on the
subject’s fitness levels. We chose the bleep test as it is the gold standard field rest for analysing
participants VO2 max capacity when undertaken correctly.
All participants’ blood was taken pre/post a bleep test. One participant from each group (One
physically active and one sedentary) was asked to stay back after testing to have their blood
taken again two hours post bleep test.

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4.4 Blood analysis Centrifuging
 Blood drawn using a 20G needle, into a 3ml citrate (0.32% final concentration)
vacuette/tube. 3 blood draws are taken with the 1st
tube being discarded avoid cell
fragments from venepuncture being collected (may skew results).
 Blood centrifuged within 15 minutes of blood collection.
 Plasma (Platelet Free Plasma-PFP) containing the MPs, carefully aspirated, leaving a
layer of about 0.5-1 cm undisturbed on top of the cells.
 Collected platelet free plasma is again centrifuged at 13,000g for 2 minutes to remove
any contaminating cells.
 Platelet free blood is then extracted from tube into smaller eppendorf tubes leaving
about 20% of the PFP at the bottom of the tube.
 Freeze approximately 250-300 ul in each eppendorf (250 ml portions, using the
Sarstedt screw cap tubes). Prepare number of aliquots as cannot freeze thaw these. 3
per sample)
 Cell free plasma is then snap frozen using liquid nitrogen. Place tubes in liquid
nitrogen using tweezers for 10 seconds until bubbles stop appearing.
 Samples stored at -80C
4.5 Analysing the plasma samples
To analyse the blood samples the The Delsa™
Nano C was used .This uses photon correlation
spectroscopy (PCS), which determines particle size of samples in suspension ranging from
0.6 nm to 7 µm. Prior to testing the machine must be switched on and given thirty minutes to
heat up. 1ml of plasma free sample is then placed into a cuvette. Cuvette is placed into the
machine by opening the instrument hatch. This hatch must be closed before continuing
testing. To begin testing double click on the Delsa Nano C software then click “start”. Each
sample takes approx. 5 –min to analyse. The results obtained from the Delsa Nano C must be
converted to an excel format. For the purpose of our research project we plotted diameter
(nm) against numbers with particular emphasis on exosomes (diameter ranges between 60-
100nm.

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5.0 Results
Incremental Exercise Results (Bleep test)
The following tables provide information on how to read the graphs in the results section:-----
Graph Label Description (Exosome Levels)
P Pre Exercise
A After Exercise (immediately)
X 2 Hours Post Exercise (only values for
subject 2 & 3)
Subject Description
Subject 1 Sedentary Female
Subject 2 Sedentary Male
Subject 3 Active Female
Subject 4 Active Male
Each subject will have a graph that will indicate the difference in resting exosome levels in
comparison to their exosome level immediately post exercise. For subject 2 and subject 3 the
graph will also show the results of exosome levels 2 hours post exercise. The results that we
acquired were got by using the Delsa Nano C made by Beckman Coulter. This machine uses
photon correlation spectroscopy (PSC) to determine size of particles by analysing the rate of
laser light intensity fluctuations which are scattered by particles as they move through a fluid.
Subject Bleep Test Result
Subject 1 4.7
Subject 2 9.1
Subject 3 10.8
Subject 4 11.8

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5.1 Subject 1 (Sedentary)
Exosome Levels: P = Pre-Exercise and A= Immediate Post-Exercise
For subject 1 there was a major elevation in exosomes that fall between 30nm and 40nm from
pre to post exercise. From 40nm to 60nm in diameter exosomes level rises very minutely post
exercise. Levels from 60nm to 100nm have a slightly larger increase due to the bout of acute
exercise. Pre exercise levels of exosomes in the blood appear to be almost non-existent at all
sizes.
5.2 Subject 2 (Sedentary)
Exosome Levels: P = Pre-Exercise, A= Immediate Post-Exercise and X= Two Hours Post-Exercise
0.001589489
0.000500862
0 0 0 4.44E-05 5.84E-05 2.49E-05
-2.00E-04
0.00E+00
2.00E-04
4.00E-04
6.00E-04
8.00E-04
1.00E-03
1.20E-03
1.40E-03
1.60E-03
1.80E-03
20 30 40 50 60 70 80 90 100
Numbers
Diameter(nm)
Subject 1
P1
A1
0.008456607
0.004472336
0.002340522
0.000563903
0.0001729670 0
-1.00E-03
0.00E+00
1.00E-03
2.00E-03
3.00E-03
4.00E-03
5.00E-03
6.00E-03
7.00E-03
8.00E-03
9.00E-03
10 30 50 70 90 110 130 150
Numbers
Diameter(nm)
Subject 2
P2
A2
X2

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For subject 2 there is a there is an extensive rise in exosomes post exercise (A2) that range
from 40nm to 60nm. There is also a smaller rise in exosome levels that range from 60nm to
90nm which is also important to note. As exosomes generally fall within the range of
30/40nm to 100nm these results show a dramatic rise in all sized exosomes post exercise.
Subject 2 shows that there was very few exosomes present in the blood pre-exercise (P2). The
results for 2 hours post exercise also show small levels of exosmes in the blood which
indicates that blood levels return to their normal state after exercise.
5.3 Subject 3 (Active)
Exosome Levels: P = Pre-Exercise, A= Immediate Post-Exercise and X= Two Hours Post-Exercie
Subject 3 shows different results in regards to exosome levels in comparison to subjects 1, 2
and 4. The graph shows that subject 3 had moderately raised exosome levels ranging from
30nm to 60nm prior to exercise. After researching to find out the meaning of this, we have
concluded that the high exosome levels pre-exercise were due to a handling error when
analysing the blood which resulted in the activation of exosomes.
Again similar to other subjects after exercise, exosome levels ranging in diameter from 30nm
to 60nm are dramatically elevated. As with subject 2 the results for 2 hours post exercise also
show small levels of exosomes in the blood which indicates that blood levels return to their
normal state after exercise.
0.004128012
0.003845589
0.002321137
0.000692054
0.0001343880 0 0 0 0 0
-0.0005
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0.004
0.0045
-10 10 30 50 70 90 110 130 150
Numbers
Diameter(nm)
Subject 3
A3
P3
X3

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5.4 Subject 4 (Active)
Exosome Levels: P = Pre-Exercise and A= Immediate Post-Exercise
The results for subject 4 show that exosome levels are very low pre-exercise which is similar
to the levels in subjects 1 and 2. Subject 4 demonstrates a raise in post exercise exosome
levels that range between 30-40nm in diameter. However this raise in exosome levels does
not replicate the dramatic rise in levels in the other subjects as the rise only occurs in the
smaller exosomes. This rise of exosomes from 30nm to 40nm is similar to the results in
subject 1 nevertheless.
The following graphs represent exosome levels in correspondence to gender and activeness.
Research indicates that there is a significant difference between male and female exosome
levels in the blood
5.5 Female Exosome Level After Exercise Comparisons
0.03935546
0.01839531
0.008287488
0.0003829670 0 0.0001001387.86E-05 3.52E-05 2.30E-05
-0.005
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
20 30 40 50 60 70 80 90 100
Numbers
Diameter(nm)
Subject 4
A4
P4
-0.0005
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0 20 40 60 80 100 120 140 160
Number
Diameter (nm)
Female Exosome Level After Exercise
Sedentary
Female
Active Female

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Active Female v Sedentary Female:
The above graph shows a comparison in exosome levels in the sedentary v active female
subjects. It is evident that there an increase in exosome levels in the range of 20nm to 60nm
for both subjects however there is a much larger rise in exosome levels of the active
participant. Almost double the number of exosomes of 30nm diameter is evident in the active
individual in comparison to the sedentary. From the graph you can also see that in the range
of 40nm to 60nm the sedentary individual shows no raise in exosome levels in comparison to
the active individual who displays an increase.
5.6 Male Exosome Level After Exercise Comparisons
Active Male v Sedentary Male:
The above graph shows a comparison in exosome levels in the sedentary v active male
subjects. From the graph it is evident that the active male has dramatically higher exosome
levels at certain diameters in comparison to the inactive male. The overall number of
exosomes in the blood inclusive of all diameters is radically higher in the active male than the
inactive male. An interesting find from the results is that the active male has raised numbers
in the diameter of 20nm to 40nm while sedentary males exosome rise is present in the range
of 40-60nm in diameter.
-0.005
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0 20 40 60 80 100 120 140
Number
Diameter (nm)
Male Exosome Level After Exercise
Sedentary
Male
Active Male

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6.0 Discussion
6.1 Cardiovascular Disease and Exercise
The term Cardiovascular Disease (CVD) refers to conditions that involve narrowed or
blocked blood vessels that can lead to a heart attack, chest pain or stroke. (Mayo Clinic,
2013). Myers (2003) states that over the last 40 years there has been numerous studies that
have examined the relationship between exercise, physical activity and cardiovascular health.
A main finding that prevailed in these reports was that the more active and individual was the
less likely they were going to develop CVDs such as coronary heart disease. If exercise is
carried out on a regular basis it will reduce an individual’s chance of developing many of
chronic disease such as CVDs (Warburton et al 2006). For example, exercise promotes
weight reduction and relives high blood pressure and cholesterol which are closely related to
the development of CVDs (Myers 2003). Physical activity has also been proven to have
significant benefits on the heart and coronary vasculature such as endothelial function.
Coagulation, automatic tone and clotting factors and inflammatory markers, vascular wall
function, the ability to provide oxygen and capacity of blood vessels to dilate all increase as
one exercises and promotes a healthy body against CVDs. (Hambrecht et al, 2000; Myers
2003). For one to increase their cardiovascular health and reduce the risks of forming CVDs
they must engage in specified physical activity daily such as dynamic aerobic exercise (Kraus
and Haskell, 2010).
6.2 Cardiovascular Disease and Exosomes
Recently there has been a lot of speculation that exosomes and cardiovascular disease have a strong
relationship with each other as research has shown that exosomes can be biomarkers for
predicting cardiovascular disease. De Kleijn et al. (2012) carried out a study were plasma
exosome samples were taken form subjects that have suffered from a cardiovascular event
and subjects who have not suffered from one. Findings showed that the protein composition
of the plasma exosome samples in the group that suffered from a cardiac event differed from
the other group. It’s now believed that this difference can be used for prediction of patients
that are susceptible to cardiovascular disease. Exosomes have now been found in many body
fluids such as urine, amniotic fluid, broncho-alveolar lavage fluid, saliva and blood where
they express a wide range of proteins. These proteins reflect the host cell and possess
valuable information about the body’s (patho) physiological processes including information

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on future cardiovascular events. From this find, the detection of plasma exosome samples are
biomarkers for predicting the risk of cardiovascular disease in a patient.
6.3 miRNA in Exosomes and Relationship with CVD
After doing research on exosomes, I have discovered that they contain a large amount of
proteins, RNAs and microRNAs (miRNAs). There have been many research studies that have
associated miRNA levels in exosomes with cardiovascular disease and that will be the focal
point of this discussion section. MiRNA are small, noncoding RNAs that control the
expression of complementary target RNAs. The impairment of intracellular miRNA
expression and the role of miRNAs have been described in a many cardiovascular conditions
such as cardiac fibrosis, coronary heart disease, myocardial infraction and heart failure
(Catalucci etal. 2009). MiRNAs are protected against RNases when in a serum which is why
they are found in uncommon levels in exosomes. Cells that have been confirmed as secreting
miRNAs through exosomes include mast cells and embryonic stem cells (Valaldi et al. 2007).
As there are high levels of miRNA secreted from exosomes they have been gradually
becoming a great interest to researchers as they postulate that they can be used as a biomarker
in CVDs (Gupta et al. 2010). Exosomes secrete various types of miRNA in different amounts
which can be classified a good and bad miRNA. In relation to coronary artery disease (CAD),
good and bad miRNAs have been found in various levels. It is reported that reduced levels of
miR-126, members of the miR-17-92 cluster, inflammation-related miR-155, and smooth
muscle-enriched miR-145 were found in patients with CAD compared with healthy patients
(Fichtlscherer et al. 2010). This suggests that healthy individuals that exercise regularly
produce high levels of exosomes which results in a high number of these miRNA. It further
suggest that these miRNAs found in healthy individuals play a role in preventing CVDs such
as coronary heart disease.
There is also evidence that cardiac risk factors such as diabetes affect miRNA levels. A study
showed that patients that suffered from diabetes had significantly decreased levels of miR-
20b, miR-21, miR-24, miR-15a, miR-126, miR-191, miR-197, miR-223, miR-320, and miR-
486 (Zampetaki et al. 2010). Mikus et al. (2012) states that not partaking in regular physical
activity impairs glycemic control (control of blood sugar levels) and suggest that this
inactivity can result in the development of type 2 diabetes. This evidence proposes that if one
does not partake in regular exercise, they will most likely have reduced levels of these

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miRNAs. On the contrary, taking part in regular physical activity, not only reduces the risk of
developing diabetes, but results in higher levels of these miRNAs which means there are
higher levels of exosomes.
Our hypothesis at the begin of this study was that was that high exosome levels play a vital
role in preventing CVDs. Evidence has shown that specific types of miRNAs have been
found at reduced levels in patients with a CVD or that have a cardiac risk factor. We
postulate that these low levels of miRNA are due to low levels of exosomes which effectively
is a result of low levels of physical activity. We also hypothesize that when there are high
levels of exosomes they also secrete high levels of miRNA that are beneficial for CVD
prevention. Research on this evidence is only in the minority and is an ongoing process but
there are signs that these hypotheses are correct.
6.4 Stress, Heat Shock Proteins in Exosomes and Relationship with CVD
There is a strong relationship between exosomes and heat shock proteins (HSPs) when
exposed to stress. When the human body is exposed to stress it produces bigger quantities of
exosomes which in turn speculate that they protect from intercellular and extracellular stress.
(Clayton et al. 2005). This protective feature occurs as exosomes can influence the response
of other cells to stress by providing cells with a resistance to the stress. By sending this signal
to other cells that are exposed to stress there is a subsequent reduction in cell death (Eldh et
al. 2010). This signal from cell to cell that causes protection is done by proteins. When the
body is exposed to stress there is damage to cellular proteins which results in a disturbance of
homeostasis and can lead to cell death. To counter this disruption exosomes in cells produce a
group of stress proteins called heat shock proteins (HSPs). Under conditions such as elevated
temperature, oxidative stress and pH alterations these HSPs protect the cell against injury
(Knowlton 1997). Exercise induces stress on the body and cellular stress in many tissues
which indicates that exercise promotes the production of these HSPs. When an individual
partakes in prolonged exercise, this results in an increase in the expression of HSPs in many
organs such as the cardiac muscle (Locke 1997). Other evidence also exists that suggests
HSPs play a role in myocardial protection If an individual participates in endurance exercise
training there will be a rise in myocardial HSP72. This HSP plays a vital role in protection
against CVDs and heart failure as it is associated with a reduction in I-R injury in the heart
(Powers et al. 2001). In relation to the study that we carried out, this shows us that when an

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individual trains at higher level, it will result in a rise of exosomes which also leads to the
production of more HSPs. These HSPs reduce cell death and can protect against CVDs and in
the case of HSP72 play a role in myocardial protection. The more active an individual is
means that during exercise there will be higher levels of exosomes. These exosomes will
produce higher levels of HSPs which results in a higher degree of protection and a lower
chance of developing a CVD.
7.0 Conclusion
This study aimed to examine the levels of exosomes in the body between sedentary and
trained individuals following a bout of exercise. After analyzing our results we have found
that circulating exosomes levels increased considerably in active individuals in comparison to
their sedentary counterparts. The study also shows that when comparing gender exosome
levels in the male participant’s peak much higher than in female for both sedentary and
trained individuals. These rises in levels have never been explained and further research will
be needed to explain these findings. In relation to cardiovascular diseases we believe that
exosomes have the potential to play a huge role in preventing CVDs. Due to the fact that
there is little research on this area we can only postulate to why exosome levels are related to
CVDs. From completing a vast amount of research we believe that (i) Exosomes contain
miRNA and some have been found to protect against cardiovascular disease. This suggests
that healthy individuals that exercise regularly produce higher levels of exosomes which
results in a higher number of miRNA. (ii) Exosomes are involved in the transport of heat
shock proteins when the body is exposed to stress. These heat shock proteins play a role in
preventing CVDs. To fully accept or reject these hypothesis further research is required in
this area.
8.0 Future Research and Limitations
8.1 Sample size and selection of sample
For future research we recommend that a larger sample size be examined. The main reason
for this would be that it would more representative of the population and it would limit the
influence of extreme outlier thus reducing sample error. This would in turn increase the
accuracy of the data. Also, it would be important to devise a recruitment strategy which could

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involve screening and selecting the appropriate target group. From this target group
individuals should be selected at random.
8.2 Use of high-frequency Ultrasonographic imaging of the brachial artery flow
mediated dilation
A technique using high-frequency Ultrasonographic Imagine was created in the 1990s in
order to assess vasculature health. This technique was used to look at the brachial artery to
assess endothelium-dependent flow-mediated vasodilation. Briefly, this technique can
identify an index of vasomotor function by eliciting the release of nitric oxide which in turn
results in vasodilation. (Corretti et al., 2002)
This is a non-invasive procedure and with the correct personnel and equipment could easily
be implemented into a study similar to ours to identify vascular health. This information
would correlate to being able to assess some of the risk factors for cardiovascular disease
depending on the health of the vasculature. In turn our research on exercise influence on
exosome production could be combined with information on our subjects vascular health in
order to have a more extensive research project taking in all angles of this interesting area.

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8.3 Flow cytometer analysis and T-cadherin
This research project solely looked at the changes in levels of exosomes before after exercise.
However we had hoped to also carry out flow cytometer analysis but due to time constraints
we were unable to achieve this. Flow cytometer is the most commonly used method for
analysing number, size and properties of small structures. Flow cytometer analysis would
have enabled us to identify specific subpopulations of MPs and establish specific
phospholipid characteristics (Ardoin, Shanahan and Pisetsky 2007). This would have allowed
us to compare both MPs and exosome levels before and after exercise and if their
characteristics are in some way contributing or preventing CVD.
Another area for further research would be the recognition of T-cadherin on the surface of
endothelial MPs. Recent research has shown the recognition of elevated T-cadherin on the
surface of endothelial MPs as a possible bio-marker for early stage atherosclerosis and its
expression correlates with endothelial dysfunction (Philippova et al. 2011).
8.4 Use different types of Exercise.
For future research we would recommend that different types of exercise be examined to
investigate what effects each have on exosome levels as there is limited to catagorise the
various types of exercise. Endurance, strength, balance and flexibility will all have different
types of effects on exosomes in the human body. An interesting study would be to compare
two sedentary groups who under take different types of exercise interventions; one group pre
and post a weight lifting training program and one group pre and post an aerobic activity
training program.

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Images
1. Van Der Pol, E., B"Oing, A. N., Harrison, P., Sturk, A. and Nieuwl. 2012. Classification,
functions, and clinical relevance of extracellular vesicles. Pharmacological Reviews, 64
(3), pp. 676--705.
2.
3. Visembryo 2012. Welcome to The Visible Embryo. [online ] Available at:
http://visembryo.com/story1133.html [Accessed: 27 Jan 2014]