Impact of Exercise on Expression of Hsp70 in Individuals at Risk of Peripheral Arterial Disease. A Randomised Trial
Phase: NA
Conditions: Peripheral Arterial Disease
Sponsor: Universidad Popular Autónoma del Estado de Puebla

Article title: “Impact of exercise on expression of Hsp70 in individuals at risk of peripheral 
arterial disease” 
Authors: 
Clara L Perez-Quiroga. MSc UPAEP University, ISSSTEP Specialties Hospital 
Javier Rubalcaba-Priego. MD ISSSTEP Specialties Hospital  
Jose I Diego Perez-Montes.  MD UPAEP University 
Virginia Sedeno-Monge. PhD UPAEP University. 
 
Short title: “Exercise on expression of HSP 70 in arterial risk” 
 
_________________________________________________________________________ 
PALABRAS:----- 
 
Correspondence author:  
Clara Luz Pérez Quiroga.  Address: Street 21 sur #1103, Santiago neighborhood. CP 72410 
Puebla, Mexico. E-mail: claraluz.perez@upaep.mx 
 
 
 
 
 
 
 
 
 
 
 
 
 

Abstract__________________________________________________________________ 
● Objetives To determine the level of serum expression of the HSP70 protein in individuals 
at risk of atherosclerotic peripheral arterial disease, after an intervention of moderate 
exercise.  
● Design randOmized, single blinded, clinical trial. 
● Setting Hospital de Especialidades “5 de Mayo” ISSSTEP in Puebla, Puebla, México. 
● Participants 260 individuals were evaluated by a screening test employing the Ankle-
Arm Index (ABI), 220 of them were not within risk value (0.91 to 0.99 mmHg). In the 
clinical trial, 40 individuals were SELECCIONADOS between February to September of 
2019. 
● Interventions The experimental group started an exercise program to achieve 65% to 80% 
of the maximum heart rate for 40 minutes by using a pulsometer that measured the heart 
rate in order to get to the moderate intensity activity goal, the control group received 
written recommendations to exercise three times per week. 
● Main outcome measures The lowest of the protein level were in the experimental group, 
with significant changes, confirming the results of studies in PAD. The highest serum 
levels of the Hsp70 were found in the group that only received written recommendations 
(control group). 
● Results 32 individuals completed the 12-week follow-up.  Both the control group (P= 
0.004) and the experimental group (P= 0.005) had significant changes in serum levels of 
the protein (95% Confidence Interval).  The risk analysis for PAD using the ABI only 
showed a significant difference (P= 0.001) in the experimental group (95% Confidence 
Interval). 
● Conclusions The risk for PAD can be modified after the intervention of a moderate 
intensity exercise program and is closely correlated with lower levels of Hsp70 
expression. 
Keywords: Hsp70, aerobic exercise, peripheral arterial disease 
 
 
 

Introduction______________________________________________________________ 
The objective of the study is to determine the level of serum expression of the HSP70 protein 
in individuals at risk of atherosclerotic peripheral arterial disease, after an intervention of 
moderate exercise. 
Atherosclerotic Peripheral Arterial Disease (PAD) is a systemic disease that causes 
obstruction of arterial blood flow and is a significant cause of morbidity and mortality 
worldwide 1. 
Patients with PAD have a three times greater risk of myocardial infarction, stroke and death 
than those without the disease. However, more than 50% of patients are asymptomatic; so 
the disease remains underdiagnosed. 2 
It is estimated that 202 million people in the world are affected with PAD,  45 million of 
them die from coronary or cerebrovascular disease in a range of ten years 3, 4 
The disease is strongly related to modifiable risk factors (blood pressure, sedentary lifestyle, 
baseline glycemia, cholesterol, and obesity) and unhealthy lifestyle (tobacco, physical 
inactivity, food and psychosocial stress), which increases the risk of acquiring the disease up 
to 17.2 times  4, 5, 6 
Increased membrane permeability leads to the accumulation and modification of proteins, 
lipids and lipoproteins in the endothelium. The accumulation of pro-inflammatory molecules 
such as chemo-tactical monocyte protein 1 (MCP-1), intracellular adhesion molecules 
(ICAM-1), vascular cell adhesion (VCAM-1) and higher nitro-tyrosine content are also 
generated. 7 
Macrophages are active in response to the spread of the inflammatory response and bind to 
low-density lipoprotein (LDL) receptors that had internalized.  
Macrophages secrete pro-inflammatory cytokines resulting in the recruitment and activation 
of additional immune cells at the site of the lesion, amplifying the immune response and 
promoting the development of complex and advanced plaques that become mature 
atherosclerotic plaques.8 
The Ankle Arm Index (ABI) is the non-invasive gold standard accepted for both the diagnosis 
and evaluation of the severity of this disease. The cut-off point for the diagnosis of PAD is 

an ABI of less than 0.90 at rest, regardless of the Framingham Risk Score, being 95% 
sensitive in the detection of PAD. 9, 10 
To maintain homeostasis on the vessel wall, vascular cells produce a high level of heat shock 
proteins (HSP) 11. These types of stress proteins are cytoprotective agents that promote cell 
survival during stressors. 12 The reduction of HSP intracellular expression is also related to 
PAD, cardiovascular diseases and metabolic syndrome, where they are greatly diminished. 8 
HSP are normally intracellular proteins, but when released at an extracellular level, they exert 
an immune response.13 
The most thermo-sensitive and highly inducible HSP belongs to the 70 kDa family, as 
established by Mizzen et al., In 1998 (figure 1). It is believed that extracellular Hsp70 
(eHsp70) stimulates innate immunity and acts as a danger signal. Serum Hsp70 has been 
detected in the peripheral circulation of apparently healthy individuals and increases in 
response to different stressors, including exercise. 12, 14 
 
 
Figure 1. Schematic representation of the molecular structure of HSP70. Adapted from Liu 
Y et al. 15 
Exercise increases HSP levels primarily through reactive oxygen species (ROS), elevated 
temperature, hormones, calcium fluxes or mechanical tissue deformation.16 

It has been shown that the concentration of Hsp70 increases after performing both acute and 
long-term exercises, reaching levels that remain high, but once its protective action decreases 
both at intra and extra-cellular levels.17 
Exercise exerts beneficial effects against atherosclerosis by increasing circulating endostatin, 
which inhibits the development of atherosclerotic plaque by blocking angiogenesis in plaque 
tissue.18 
Isotonic contractions of the type of resistance exercise (aerobic) tend to lead to increases in 
Hsp70 levels, as opposed to eccentric exercise. With repetitive exercise, an induced increase 
in this protein is maintained, while the initial response of other HSPs to exercise decreases 
as training progresses.16 
Regular exercise initiates long-term processes of adaptation of muscle metabolism, 
cardiovascular system and immuno-modulatory effects that are largely considered beneficial; 
There is evidence that during exercise, Hsp70 is released into human circulation in 
association with exosomes, specifically with aerobic exercise.19, 20 
It has been shown that Hsp70 directly inhibits inflammatory processes through the 
suppression of oxidative stress, directly reduces apoptosis, hyperplasia, as well as decreasing 
the expression of adhesion molecules that lead to leukocyte extravasation and the 
manifestation of inflammatory cytokines.16 
Higher serum levels of Hsp70 are associated with reduced thickening of the atherosclerotic 
intima and a lower risk of coronary artery disease.21 
Méthods__________________________________________________________________ 
Study design: This is a randomized clinical trial, single-blind. 
Individuals who attended the internal medicine service of the ISSSTEP hospital were invited 
to participate voluntarily, from February to September of 2019. In order to detect people at 
risk of PAD, the measurement of the ABI was done. The measure was taken after 10 minutes 
of resting. The measure was taken in the four extremities to obtain systolic BP of the posterior 
tibial artery and brachial artery. The cut point to enter the study was between 0.91 and 0.99 
mmHg. The informed consent was signed by all the individuals. 
 

The sample selection was done randomized for convenience. A total of 260 candidates were 
evaluated, 40 of them were selected  randomly divided in two by using version 4.2 of 
EPIDAT, an experimental and a control group. During the progress of the clinical trial four 
people of each group decided to leave the study by personal decision, thus the groups ended 
with 16 people each. Dietetic recommendations of low sodium foods were provided to all. 
The experimental group started an exercise program to achieve 65% to 80% of the maximum 
heart rate for 40 minutes by using a pulsometer that measured the heart rate in order to get to 
the moderate intensity activity goal. Each exercise session included a warm-up and a cool-
down phase of 10 minutes at 50 - 60% of the maximum heart rate. The exercise session was 
60 minutes long, three times per week, completing 180 minutes per week of moderate 
intensity exercise, for 12 weeks in total.  The control group received written 
recommendations to exercise three times per week, for a period of 12 weeks but they did not 
attend the training sessions. 
There were two examinations, one at the beginning and the second at the end of the study. 
Different physiotherapists did the initial and final evaluation. The physiotherapists stayed 
blind to the group assignation. It was indicated to the participants to not reveal their 
assignations. 
These examinations included anthropometric measurements, including: height, weight, BMI, 
waist circumference, Systolic BP,  Diastolic BP, RHR and the sit and reach test.  
Weight, Height and BMI. A weighing machine (seca 700, seca gmbh & co, Hamburg, 
Germany) was used to measure weight and height. Body Mass Index (BMI) is a simple index 
that compares weight to height and it is commonly used to classify underweight, 
overweight and obesity in adults.  It is defined as the weight in kilograms divided by the 
square of the height in meters (kg/m2). The result of this division is classified as follow: 
Underweight <18.5 kg/m2 , Normal range 18.5 – 24.99 kg/m2 , Overweight ≥25.0 kg/m2, 
Preobese  25.0 – 29.99 kg/m2 , Obese class I 30.0 – 34.99 kg/m2, Obese class II 35.00 – 39.99 
kg/m2, Obese class III ≥40.00 kg/m2. 
According to the classification, the risk comorbidities are: Low for underweight, average for 
normal weight, increased for preobese, moderate for obese class I, Severe for obese class 
II and very severe for obese class III. 22 

Waist Circumference (WC). The WC is a convenient and simple measurement that is 
unrelated to height. It is measured at the midpoint between the lower border of the rib 
cage and the iliac crest. To measure the waist, an anthropometric type Rosscraft TAQ tape 
was used (Rosscraft Innovations, Canada). It classifies people in two groups based on the 
risk of metabolic complications: Increased in >94 cm in men and >80 cm in women, 
Substantially increased >102 cm in men and >88 cm in women. 23, 24 
Waist to Height Ratio (WHtR). It is the quotient between WC and height. A WHtR higher 
than 0.50 is considered to be an indicator of central obesity in adults. 
Ankle-Brachial index (ABI). The ABI is an estimation of the prevalence of Peripheral Arterial 
Disease (PAD). It involves measuring the systolic blood pressure (Systolic BP) in the ankles 
(dorsalis pedis and posterior tibial arteries) and arms (brachial artery) and then calculating 
a ratio. The diagnostic criteria for PAD based on the ABI are interpreted as follows: Normal 
if 0.91–1.30, Mild obstruction if 0.70–0.90, Moderate obstruction if 0.40–0.69, Severe 
obstruction if <0.40, Poorly compressible if 1.30 An ABI value 1.3 suggests poorly 
compressible arteries at the ankle level due to the presence of medial arterial calcification.25 
Systolic and Diastolic Blood Pressure. Blood Pressure (BP) category is defined according to 
seated clinic BP and by the highest level of BP, whether systolic or diastolic, a blood pressure 
monitor (ELITE HEM-7130, © OMRON, Japan) was used to measure the systolic BP and 
the diastolic BP  and it is classified as follow for systolic values: Optimal <120 mmHg, Normal 
120-129 mmHg, High normal 130-139 mmHg, Grade 1 hypertension 140-159 mmHg, Grade 
2 hypertension 160-179 mmhg, Grade 3 hypertension ≥180 mmHg, Isolated systolic 
hypertension ≥140 mmHg and <90 mmHg in Diastolic Blood Pressure. The Diastolic Blood 
Pressure (Diastolic BP) is classified as follow: Optimal <80 mmHg, Normal 80-84 mmHg, High 
normal 85-89 mmHg, Grade 1 hypertension 90-99 mmHg, Grade 2 hypertension 100-109 
mmhg, Grade 3 hypertension ≥110 mmHg.26 
Resting Heart Rate (RHR). It is the lowest heart rate to provide the minimum amount of 
blood for the body to use while a person is not exercising, the blood pressure monitor 
(ELITE HEM-7130, © OMRON, Japan) was also used to measure the RHR. According to 

the American Heart Association (AHA) the normal heart rate is between 60 beats per minute 
to 100 beats per minute.27 
The Sit and Reach test. Is used to measure the hip joint angle or sacrum angle in the 
maximum flexion position while the patients are sit and asked to reach the toes with their 
fingers, always keeping the ankle in a 90° angle.(LhFx). The values for reference are: Normal 
≥2 cm, shortening Grade -3 a -9 cm and shortening Grade II ≤10 cm. 28 
Biomarkers. A commercial kit was used to measure the HSP70 in blood (HSP70 ELISA kit, 
©2020 Enzo Life Sciences, Inc., New York, USA) and a professional software was used to 
read the ELISA by spectrometry with an optic density of 570 ηm. 
Analysis of data: All descriptive statistics of the variables in the study were reported as 
medians, with ± the standard deviation. The non-parametric Mann-Whitney test was used to 
determine the importance of the difference in the medians between the control group and the 
experimental group before and after exercise. The differences are considered statistically 
significant at p values <0.05 with a 95% CI. 
 
RESULTS 
Study sample. There were 260 individuals analized, 40 of them were included and 32 were 
included in the study. Two groups were made, one for control and the other for experimental. 
Four patients left each group the study because of personal decisions thus both groups ended 

with 
16 
patients 
each 
(fig 
1).
 
Figure 1. Progress of the participants along the study. 
Ankle-Brachial Index: ABI, Peripheral Artery Disease: PAD 
 
 
 
 
Anthropometric effects of exercise training. Cardiovascular medication was not used in either 
control and experimental groups. During the follow up, minor modifications were noted on 
anthropometric parameters (Table 1).  
 
 
 
Table 1. Comparison between sample and randomised groups at the beginning and at 
the end of the study. Values are numbers (percentages). 
INITIAL RESULTS      
VARIABLES 
SAMPLE 
CONTROL 
EXPERIMENTAL 
  
 
 
260 were evaluated 
with  the ABI 
screening test 
 
40  
Were at risk of  
PAD (ABI 0.91 a 
0.99)  
 40 signed the consent 
and were randomized 
 
 
 
n=20 
EXPERIMENTAL 
GROUP 
 
16  
Included in the analysis, 
4 voluntary retirement 
 
n=20  
CONTROL  
GROUP 
 
16  
Included the analysis, 2 lost 
tracing, 2 voluntary 
retirement 
 
220 got an ABI 
<0.90 (PAD) or 
>0.99 
  

(n= 32) 
(n=16) 
(n=16) 
Average age (years) 
52.1 (8.5) 
53.3 (9.2) 
50.9 (7.8) 
Normal weight 
5 (16%) 
5 (31%) 
0 
Overweight/ obesity 
27 (84%) 
11 (69%) 
16 (100%) 
No cardiovascular risk 
(according to BMI) 
6 (19%) 
5 (31%) 
1 (6%) 
Presence of cardiovascular 
risk (according to BMI) 
26 (81%) 
11 (69%) 
15 (9.4%) 
No risk of cardiovascular 
complications (according to 
WC) 
9 (28%) 
6 (38%) 
3 (19%) 
Presence of risk of 
cardiovascular  complications 
(according to WC) 
23 (72%) 
10 (62%) 
13 (81%) 
No risk of arteriopathy 
0  
0 
0 
Presence of arteriopathy risk 
32 (100%) 
16 (100%) 
16 (100%) 
Normal BP 
27 (84%) 
13 (81%) 
14 (88%) 
Hypertension 
5 (16%) 
3 (19%) 
2 (12%) 
Diabetes tipo 2 
0 
0 
0 
FINAL RESULTS 
Normal weight 
6 (19%) 
6 (38%) 
0 
Overweight/ obesity 
26 (81%) 
10 (62%) 
16 (100%) 
No cardiovascular risk 
(according to BMI) 
6 (19%) 
6 (38%) 
0 
Presence of cardiovascular 
risk (according to BMI) 
26 (81%) 
10 (62%) 
16 (100%) 
No risk of cardiovascular 
complications (according to 
WC) 
8 (25%) 
5 (31%) 
3 (19%) 
Presence of risk of 
cardiovascular  complications 
(according to WC) 
24 (75) 
11 (69%) 
13 (81%) 
No risk of arteriopathy 
19 (59%) 
9 (56%) 
10 (62%) 
Presence of arteriopathy risk 
13 (41%) 
7 (44%) 
6 (38%) 
Normal BP 
27 (84%) 
13 (81%) 
14 (88%) 

Hypertension 
3 (9%) 
2 (13%) 
1 (6%) 
Hypotension 
2 (6%) 
1 (6%) 
1 (6%) 
 
 
 
DISCUSSION 
The relevant finding of this study is the significant decrease in the levels of the Heat Shock 
Protein Hsp70 in serum, specifically in the group where the exercise was directed, unlike the 
strategy of written recommendations (control group) after 12 weeks follow-up, although both 
groups had significant differences. Hsp70 has been recognized to be present in the peripheral 
circulation of normal individuals, providing the first evidence that Hsp70 can be released into 
the extracellular environment not only in response to stress but also under physiological 
conditions. 18, 29 The activation response of Hsp70 depends on the severity and type of 
stress to which the subject is exposed. 30. In addition to the fact that Hsp70 can modulate 
vascular contractility 16. Since 2000 31, it has been determined that protein increases in 
untrained individuals after moderate aerobic exercise, probably as an adaptation of the 
training load 11, 30, 32. But there are also studies that report its decrease as training 
progresses 8, 11, 16. In the present study, the application of a training program from 65 to 
80% of the maximum heart rate, to achieve the adaptation of cumulative episodes and the 
recovery of the physiological stressor as the training progressed in the experimental group. 
The application of exercise with moderate intensity for 12 weeks was sufficient to restore 
and decrease the expression of the Hsp70. 
The family of 70 kDa Heat Shock Proteins (HSP70) has been widely studied for its 
participation in obesity, inflammation and endothelial dysfunction 33, 34. Since 2000 Wright 
et al.35 have shown that serum levels of Hsp70 increases in patients with PAD, various 
epidemiological studies have confirmed that serum levels of the protein Hsp70 are elevated 
in peripheral arterial disease 36, in more severe arterial calcification and continue to increase 
in patients with peripheral atherosclerosis 34 and the presence of an antiatherogenic status in 
the vasculature 33, 37. In our study group, the highest serum levels of the Hsp70 were found 
in the group that only received written recommendations (control group). The lowest of the 

protein level were in the experimental group, with significant changes, confirming the results 
of studies in PAD. 
Peripheral arterial disease (PAD) increases its prevalence with age and is associated with a 
reduction in life expectancy 38. The presence of PAD has been shown to be a marker of 
cardiovascular disease 39 and is little recognized in primary care practices 40. The present 
study examined patients at risk of PAD in a hospital setting, using the oscillometric arm-
ankle index (ABI), which indicates the degree of risk for PAD. The oscillometric method is 
easy, simple, requires minimal training, and can be used in large and small hospitals, health 
centers, or even at home 39, 41. Transient responses to stress induced by regular and 
moderate exercise tend to down regulate vascular inflammation and protect vessels from 
injury 9, 16. Peripheral resistance decreases during exercise, which increases blood flow to 
peripheral musculature 40. Significant changes were found in the experimental group, with 
an average of end of study out of risk level for PAD; however, there were no significant 
changes in the group of written recommendations, maintaining an average risk for the 
disease, statistically verified. The ABI is the accepted non-invasive gold standard both for 
the diagnosis of PAD and for the assessment of the severity of the disease 41, it has been 
described as a strong predictor of mortality in patients with PAD 38. The measurement of the 
ABI should be performed routinely in patients with intermediate / high risk, allowing early 
intervention and monitoring of populations at risk, 1, 2. Coinciding with the results of our 
study. 
Classic risk factors for the development of PAD,  such as diabetes 4, 5 were not included in 
our study, where no patient had this diagnosis; However, the risk factors for PAD considered 
in this investigation were the level of blood pressure (both systolic and diastolic), the waist 
circumference, the waist-height index; as well as the body mass index, respectively. There 
were no significant changes at the beginning and end of the study in any of the groups. The 
level of the baseline heart rate contemplated in the present investigation had a significant 
change in the experimental group, compared to the control group. Higher resting HR has 
been shown to correlate with an increased risk of cardiovascular disease and death in the 
general population. The heart rate response to stress, exercising or drug injection, has been 
shown to have prognostic and diagnostic power 27. Regular exercise appears to be inversely 

related to life expectancy 42 and positively related to cardiovascular and all-cause mortality 
43, 44. There are intervention studies on physical training, however, to date, according to the 
evidence sought, we have not found clinical trials for PAD that examine these clinical levels 
and their association with physical exercise. 
Lower body flexibility was tested by attempting to reach distance from the subject's fingertips 
and toes with the legs extended. Poor trunk flexibility could be associated with higher arterial 
stiffness in middle-aged and older people, regardless of other fitness components 45, 46. 
Therefore, poor flexibility may be a useful indicator of physical fitness for remodeling 
arterial 47, as studies reveal. In our study there were no significant changes after the 
implementation of an exercise program, this may be due in part to the objective of the study, 
it was found a potential role of moderate intensity exercises to improve the risk of PAD, 
having an improvement in terms of the decrease in distance, but not significantly in the 
experimental group. 
LIMITATIONS 
The characteristics of the subjects in this study limited the generalizability of the results. All 
the participants belonged to the hospital's medical service, middle-aged Mexicans, and our 
findings cannot beapplied to subjects of other ages or ethnicities. There were only two times 
for the application of the tests, at the beginning and at the end of the study, which limits the 
results. The number of participants for the study was small, considering the subjects 
previously evaluated for participation in the study. However, this is the first investigation 
related to the risk of PAD, exercise, and serum Hsp70 levels. More research with different 
degrees of the disease is needed to confirm the new findings. 
CONCLUSION 
This is the first study in humans to investigate the serum level of the  Hsp70 protein in 
association with the risk of PAD, after the implementation of a moderate exercise program. 
Our findings indicate that the risk for PAD changes after the intervention of the exercise 
program and is closely correlated with lower levels of Hsp70 expression. More prospective 
studies are required to establish the possible biomarker role of 

Hsp70 in PAD, in order to clarify whether elevated circulating levels of Hsp70 are a 
consequence of having an increased risk of PAD; as well as the role of moderate exercise in 
the prevention. 
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