Examining how Inactivity Alters Circulating Microvesicles and Impacts the Proangiogenic
Potential of Endothelial Cells: Literature and Meta-Analysis Review
Phakchanok (Pugun) Sapphansaen and Dr. Mark Rakobowchuk

Abstract
Objective
Performing a meta-analysis using Bayesian statistics to review the association between physical
inactivity and circulating platelet-derived microvesicles (PMVs) and endothelial-derived
microvesicles (EMVs) to promote or hinder blood vessel formation.
Methods
The research examined the impact of reduced physical inactivity on PMVs and EMVs. production
response compared to the microvesicles before being physically inactive as an intervention were
selected for the analysis. Studies were identified through searches of the PubMed and Google
Scholar databases. Furthermore, the process of selecting quality papers included searching
reference lists of relevant studies. Study quality was assessed using the Downs and Black Checklist
and Cochrane’s criteria.
Results
Three studies met the inclusion criteria for the meta-analysis for the systematic review. One study
assessed EMPs on two different types of EMVs, thus both sets of data were included, giving 4
total trials included in the analysis. However, the change in SD in each study was not given, nor
the exact t-values and SD at each time point under control, or the experimental conditions.
Therefore, the meta-analysis could not be further calculated.

Conclusion
Reduced physical activity increased PMV and EMV production against the lower of EMVs before
the intervention. More research is needed on the topic for the correlation between reduced physical
activity and microvesicle production to be fully understood.
1. Introduction
Microvesicles/microparticles are plasma membrane-derived vesicles that vary in size, lipid
content, and protein composition [4]. They are shed by different cells and express different subsets
of cell surface proteins, thus enabling one to identify their cell of origin. They were first identified
when platelets were stimulated with thrombin during the process of clot activation, but later studies
demonstrated that they are also released by other stimuli like collagen, and shear stress (blood
flowing within arteries and veins) [5].
A relationship between health and the presence of circulating microvesicles has been shown in
numerous populations [1,2,7,8]. Furthermore, they can promote angiogenesis, the development of
new blood vessels. This has been shown both in vitro and in vivo when platelet microvesicles
(PMVs) are isolated and applied to cells. This was done by injecting them under the skin of mice
and into heart muscle after an experimental heart attack [1]. The latter showed extensive restoration
of the heart muscle [1]. This proangiogenic potential has also been demonstrated with human cells.
PMVs can stimulate human endothelial, cells that line blood vessels, to proliferate, migrate, and
form tubule-like structures. These are essential steps in the process of angiogenesis [6]. Regarding
the impacts of exercise on the microvesicles, strenuous exercise enhanced the production of PMVs
and enhanced endothelial cell migratory and morphogenic potential. This indicates the possible
enhancement of angiogenesis and proliferation after exercise [6].

Conversely, elevated circulating microvesicles have been linked to negative health effects. For
instance, some varieties of PMVs microvesicles have procoagulant potential, which may promote
thrombus formation. Thrombus formation can be found in plaques that build-up over time in
arteries [7]. This can result in hardening plaques, narrowing blood vessels, and eventually a heart
attack. In the case of the precursor to Type 2 Diabetes Mellitus, a process of insulin resistance
exists where the body no longer responds correctly to increases in blood glucose. In this
population, PMVs may be elevated and contribute to insulin resistance by causing dysfunction of
the endothelial cells which ultimately leads to blood vessel destruction [7].
Given that there are different varieties of platelet microvesicles that can 1) cause increased
thrombus formation or 2) new blood vessel formation, identifying their concentrations in the blood
of active and very inactive people is necessary for determining platelet-microvesicle relationships
to the health of an individual. Therefore, research is needed to investigate the differences in
circulating microvesicles between active and inactive people and whether active or inactive
derived microvesicles will help promote or hinder blood vessel formation.
2. Methods
2.1 Population
This review included: studies of healthy men undergone through intervention, studies with patients
who were diagnosed with venous thromboembolism and healthy controls, and studies with
untreated patients who were diagnosed with venous thromboembolism and hypercholesterolemia
and healthy controls. This allowed various types of microparticles to be measured so experimental
values could be compared with the control treatments.
Studies excluded smoking, a recent change in body weight, or being involved in competitive
endurance events. Eligible participants did not have cardiovascular, pulmonary, or kidney disease

and were not receiving drug treatment with anti-platelet, anti-inflammatory, hypolipidemic agents,
or hormone replacement therapy. Also, eligible studies must not have included participants with
known conditions suspected to independently increase levels of EMVs and PMVs; such as sepsis,
acute infection, pregnancy, acute coronary syndromes, heparin-induced thrombocytopenia, severe
hypertension, recent cardiopulmonary bypass, or multiple sclerosis [13].
2.2 Intervention and Comparator
For implementing an intervention, studies must reduce participants' physical activity. This
included reducing daily physical activity, avoiding any planned exercise sessions to achieve less
than 5,000 steps a day (Boyle studies), or undergoing dry immersion protocol. For comparator, the
number of PMVs and EMVs in participants who had undergone the intervention were compared
with EMVs from pre-intervention. Also, the number of EMVs in patients with VTE and
hypercholesterolemia were compared with EMVs from healthy controls.
2.3 Outcomes
The studies were required to report the impact of increased physical inactivity or report patients
with VTE and hypercholesterolemia by the number of PMVs and EMVs compared with healthy
controls. For increased physical inactivity studied, the numbers of EMVs at baseline (preintervention) and after experimental treatment (post-intervention) were reported to calculate the
change of EMVs.
2.4 Search Strategy and Study Selection
PubMed website was utilized to do a systematic literature search from May to August 2020. The
studies were not limited to the date of publication. The following search terms were used to identify
potential studies:

“Sedentary Behaviour & Microparticle AND Sedentary Behaviour & Microparticles” or
“Inactivity & Microparticle” or “Step Reduction & Microparticle” or “Bed Rest & Microparticle”
or “Immobilization & Microparticle” or “Space flight & Microparticle” or “Detraining &
Microparticle” or “Prolonged sitting & Microparticle” or “Dry immersion & Microparticle” or
“Prolonged immersion & Microparticle” or “Hind-limb unweighting & Microparticle” or
“physical inactivity and microvesicle” or “physical Inactivity and microparticle” or “physical
reduction and microparticle” or “physical reduction and microvesicle” or “sedentary Behaviour &
Exosome* or “Physical Inactivity & Exosome” or “Inactivity* & Exosome” or “Bed Rest* &
Exosome” or “Step reduction* & Exosome” or “immobilization & exosome” or “Space flight* &
Exosome” or “Detraining & Exosome” or “Prolonged sitting & Exosome” or “Dry immersion &
Exosome” or “Prolonged immersion* & Exosome” or “Hind-limb unweighting & Exosome” or
“Sedentary Behaviour & Microvesicle” or “Physical Inactivity & Microvesicle” or “Inactivity* &
Microvesicle” or “Bed Rest & Microvesicle” or “Step reduction & Microvesicle” or
“immobilization & microvesicle” or “space flight* & microvesicle” or “Detraining &
microvesicle” or “Prolonged sitting* & microvesicle” or “Dry immersion* & microvesicle” or
“Prolonged immersion & microvesicle” or “Hind-limb unweighting & microvesicle”.
Following Cochrane’s criteria, the titles and abstracts of articles found by the listed keywords were
screened for potential relevance. A full-text review was done on the potentially relevant articles.
The reference list of those studies which underwent a full-text review was examined for other
manuscripts, which may have been missed in the initial database search.
2.5 Quality of Evidence
The Downs and Black Checklist [9] was used as criteria to evaluate the quality of each study. This
included assessing whether the study had biases and the validation of the evidence of each study.

This checklist included 27 questions designed to evaluate the strength of reporting, external
validity, internal validity, and power. Some questions in the checklist were worth more than one
point, so the maximum score a study could receive was a 32.
2.6 Data Extraction
Data extraction was performed by one author and verified by Dr. Rakobowchuk. Studies that
assess the intervention between inactivity and the production of PMVs were not identified.
Therefore, only the level of EMVs concentrations with inactivity assessment was reported in all
studies and included in the meta-analysis. Thus, no calculations were required for this data. The
data was extracted as a mean and standard deviation. However, all of the chosen articles were
reported in standard error, so standard deviations were calculated. Other critical variables such as
paired t-values were extracted from the articles as well.
2.7 Meta-Analysis
Studies were included in the meta-analysis if 1) they compared concentrations of EMVs and PMVs
before the intervention with EMV and PMV concentrations after being physically inactive or 2)
the concentration of EMVs or PMVs of patients’ groups were compared with healthy controls.
Studies included were of crossover design and collected data pre and post-treatments. However,
no studies were assessing PMVs with a physical inactivity intervention, so no data of PMVs
concentration were included in the meta-analysis. In our meta-analysis, we wanted to study the
change in EMVs concentration from the experimental to the control group. However, every study
reported the standard errors of the pre and post manipulation data, but not the standard deviation
of the change, which is needed to calculate the effect size. Thus, the method in the Cochrane’s
Handbook chapter 16.1.3.2 which describes a process of imputing standard deviations for changes
from the baseline to after post-treatment were utilized.

3. Result
3.1 Narrative Synthesis
A total of 450 studies were found to be potentially relevant during the database search process
after removing duplicate results. After screening the title and abstract, 66 articles were selected for
full-text review. Of these articles, 62 were excluded from use in the review and meta-analysis due
to ineligible interventions, such as no physical activity reduction. Studies were also excluded due
to the ineligible of their findings, such as no measurement of EMVs and PMVs concentrations nor
exact t-values for each treatment. Also, no studies were assessing PMVs with physical inactivity
interventions. The remaining 4 articles met the requirements of the meta-analysis, and one of the
articles acquired two data sets of EMVs concentration [13]. Therefore, a total of five data sets
could be used towards meta-analysis. However, a meta-analysis could not be further analyzed as
all of the articles reported only standard error for each treatment and did not include the change in
standard deviation or the exact T-statistics required to complete the analysis (Figure 1).

Records identified through
database searching (n = 810)

Records screened after the
duplicates removed (n = 450)

Full-text articles review
(n = 66)

Articles for systematic review
(n= 4)

Full-text articles excluded due to
Ineligible intervention or study design
(n= 62)

Articles for potential meta-analysis
systematic review (n= 5)

Figure 1. Flow chart for selection and inclusion of the eligible studies
The number of participants in each study ranged from 8 [14] to 25 [13]. Of the 4 articles included,
one included only male participants [14], and the other 2 included both male and female
participants. The level of physical activity reduction ranged from 5 days of reduced physical
activity [15] and 11 days of being in a supine position on a dry immersion setting [14] The other
2 studies analyzed the EMVs concentration of the patients diagnosed with VTE [13] or
hypercholesterolemia [16] and healthy controls.
3.2 Quantitative Synthesis
With quality studies that were selected for systematic reviews and meta-analysis reviews, the
experimental and the control groups were compared. Specifically, the comparison happened
between pre and post-treatment for the intervention method and between patients and healthy
controls for the comparator method. Although mean values, SE/IQR values and N values of control
and experiment were provided, the correlation coefficient could not be calculated for several
reasons. A change in SD was not able to be calculated if raw data were not given; a change in SD
is needed to calculate the effect size. Effect size required N-values, standard deviation, and exact
t-values for each treatment. Another alternation was to convert standard deviation from standard
error values to be input on the effect size calculator program. However, standard deviations at each
time point under each treatment were not given; only the final standard deviations of each
treatment were stated. Also, exact paired t-values were not given, which were also required for
calculating the effect size. Therefore, the meta-analysis could not be completed.

4. Discussion
Meta-analysis may proceed if the change in SD value was known by obtaining raw data from
the authors of each study. Although meta-analysis could not be completed, the selected quality
studies associated increased concentrations of PMVs and EMVs with being physically inactive. A
level of change in EMVs production was assessed when there were alterations in physical activity.
In Boyle's study, 5 days of reduced physical activity increased levels of CD31+/CD42b- EMVs 5fold. Similarly, extreme physical inactivity in a dry water immersion setting, such as Navasiolava’s
study, also had a 54.9% increase in CD31+/CD41- EMVs.
Also, EMVs populations were elevated in a variety of patient populations. For example, the
presence of elevated EMVs concentration of CD31+/CD42b- and E-selectin (62E) EMVs was
found in patients with VTE compared with healthy controls. Patients diagnosed with
hypercholesterolemia also presented a significantly higher number of CD31 +/CD42+ EMVs than
in controls 11.7% [16].
Endothelial damage could correlate with physical inactivity which leads to several diseases
related to endothelial dysfunction and the related cardiovascular risk. Endothelial dysfunction at
the microcirculatory level might contribute to several inactivity related deficiencies, such as a
decrease in exercise capacity, muscle atrophy, and cardiovascular deconditioning. The endothelial
dysfunction at the microcirculatory level seems to be an early consequence of hypokinesia [14].
EMVs have been shown to correlate with measures of Flow Mediated Dilation (FMD) [14].
The measurements of EMVs concentration and % of FMD are associated with several
cardiovascular disease risk factors [14] due to their pro-inflammatory effects and their ability to
promote thrombosis (hyperthermia). For example, circulating EMVs levels are associated with
reduced FMD and increased aortic stiffness in patients with end-stage kidney failure [14].

Microvesicles might be associated with reducing the vitality of endothelial progenitor cells
(EPCs). Microparticles from hypercholesterolemic patients caused a significant in vitro EPCs
apoptosis [16]. Moreover, a correlation was found between increasing CD31 +/CD42− MVs,
CD31+/CD42+ MPs, and the reduced number of circulating EPCs. Hence, the presence of
microvesicles might induce endothelial dysfunctions and the reduction EPCs which could trigger
the mechanisms of cholesterol-induced endothelial damage and impaired vascular reparation,
which in turn may contribute to the increased vascular tone and arterial stiffness.
The presence of elevated EMVs was also associated with the increased endothelial
activation in VTE [13]; this finding is consistent with previous studies [11, 12]. Under
physiological conditions, endothelial activation is shifted toward a prothrombotic state [13] and
Endothelial Cells (EC) activation is associated with EMV release [2]. Hence, EMVs provide a
source of tissue factor (TF) as well as a catalytic surface for the assembly of the
prothrombinase complex [16]. Therefore, these considerations suggest that EMVs are not only
markers of endothelial activation in VTE but also play an active role in the thrombotic process.
Many quality studies illustrated that increased concentrations or ratios of EMVs correlated
with physical inactivity and can be used as systemic markers for alterations in physical activity.
EMVs could cause endothelial dysfunction, causing a series of deleterious effects leading to
several diseases related to endothelial dysfunction and the related cardiovascular risk.
Nevertheless, research should focus on the impact of physical activity reduction on the endothelialderived microvesicles (EMVs), so more meta-analyses could be conducted with large sample sizes.
In summary, most studies have examined the acute effects of endurance exercise on
circulating microvesicle dynamics and their impact on surrounding endothelial cells [3,9];
however, they mainly analyze the extreme scenarios such as the impact of intense exercise versus

absolute resting on circulating microvesicles. Thus, the chronic effects of physical activity and
inactivity upon microvesicle in the circulation and their effects on endothelial cells is limited,
especially whether they induce proangiogenic potential. Also, the mechanisms by which inactivity
bring about endothelial adaptations not directly dependent on shear stress, are not fully understood
and may relate to a variety of still unidentified factors.
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