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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 -80C

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

Nu

mb

ers

Diameter(nm)

Subject 1

P1

A1

0.008456607

0.004472336

0.002340522

0.000563903 0.000172967 0 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

Nu

mb

ers

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

0.000134388 0 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

Nu

mb

ers

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

0.000382967 0 0 0.000100138 7.86E-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

Nu

mb

ers

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

Nu

mb

er

Diameter (nm)

Female Exosome Level After Exercise

SedentaryFemale

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

Nu

mb

er

Diameter (nm)

Male Exosome Level After Exercise

SedentaryMale

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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9.0 References

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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]