Vascular Dysfunction in Black Individuals: Roles of Nitric Oxide and Endothelin-1
Phase: EARLY_PHASE1
Conditions: Vascular Function, Racial Disparity
Sponsor: University of Mississippi Medical Center

Vascular Dysfunction in Black Individuals: Roles of 
Nitric Oxide and Endothelin-1 
NCT04770155 
08/10/2021 

SPECIFIC AIMS 
Cardiovascular diseases (CVD) are the leading causes of mortality in the U.S., costing about $350 billion 
annually in healthcare1. However, while affecting a large portion of the U.S. population, the prevalence of CVD 
is disproportionately higher in Black (i.e., African American) men and women than in other racial groups1,2. 
Intensive research on the physiological basis for this elevated risk3-5 has demonstrated a prominent role of 
deleterious alterations in vascular function starting early in life in Black individuals. Despite no symptoms of overt 
CVD, young Black individuals present less vasodilation6,7, more vasoconstriction7,8, and impaired vascular 
control of blood pressure8,9 at rest and during exercise models10-12 in comparison with White counterparts. These 
findings support the idea that healthy young Black individuals must cope with subclinical impairments in 
vascular regulation that place continuous stress on the cardiovascular system and predispose to 
vascular remodeling and dysfunction. Since vascular dysfunction is a predictor of CVD15-18, research is 
needed to understand the mechanisms driving these racial differences and identify interventions that can 
improve vascular function, supporting future clinical practices to prevent CVD in the Black population. 
Among multiple mechanisms, nitric oxide (NO) and endothelin-1 (ET-1) appear as key targets for 
improving vascular regulation in Black individuals. Treatments to increase NO bioavailability improve 
survival and health outcomes in Black patients with heart failure19, demonstrating a critical role of NO signaling 
dysfunction on the CVD risk in this population. Indeed, studies indicate that impaired vasodilation in Black 
individuals is largely due to low NO signaling20-25 compared with White counterparts. However, interventions 
to increase NO signaling aimed at improving vasodilation in peripheral macro- and microvascular beds 
in Black individuals have not been tested. Furthermore, NO mitigates the actions of ET-1, which is an acute 
vasoconstrictor and mediator of chronic vascular remodeling26. Accordingly, low NO signaling may lead to 
exaggerated ET-1 signaling in Black individuals. Indeed, ET-1 is associated with the pathophysiology of 
hypertension27-29 and with impaired vascular control of blood pressure30 in Black individuals, supporting a 
differential dysfunctional mechanism compared with White counterparts. Since ET-1 acutely restrains vasodilator 
responses31-33, it is conceivable that impaired vasodilation observed in Black individuals may be, in part, 
due to exaggerated ET-1 signaling compared with White counterparts. However, interventions to decrease 
ET-1 signaling and improve vasodilation in Black individuals have not been tested. 
We postulate that the peripheral micro- and microvasculature of 
Black individuals express constitutively lower NO signaling and higher 
ET-1 signaling than that of White individuals, which manifests as 
impaired vasodilation. Accordingly, acute interventions to increase NO 
signaling and decrease ET-1 signaling will greatly improve 
vasodilation in Black individuals but not in White individuals. 
Therefore, the research aims of this proposal are: 

Specific Aim 1: To test whether an increase in NO signaling
can increase vasodilator responses in young Black
individuals. We will use oral administrations of beetroot juice (Aim
1a), L-citrulline (Aim 1b), and Sildenafil (Aim 1c) to stimulate NO-
mediated signaling. We hypothesize that each of these
interventions will improve NO-mediated vasodilation in Black
individuals but not in White individuals.

Specific Aim 2: To test whether a decrease in ET-1 signaling
can increase vasodilator responses in young Black
individuals. We will use an acute oral administration of Bosentan
to block ETA and ETB receptors. We hypothesize that this blockade
of ET-1 signaling will reduce the vasoconstrictor tone and improve
vasodilation in Black individuals but not in White individuals.
With this investigation, we strive to provide evidence on the roles
of NO and ET-1 underlying the racial differences in vascular function 
and generate preliminary data on tailored novel interventions to 
improve 
vascular 
regulation 
in 
Black 
individuals, 
ultimately 
contributing to reduce mortality and improve cardiovascular health in 
this underrepresented group of the American population (Figure 1).
Figure 1. Proposed interventions and expected
outcomes in Black and White individuals. 

RESEARCH STRATEGY 
Background 
We and others have identified several subclinical deleterious alterations (e.g., impaired vascular control of 
blood pressure8,9,34,35, reduced dilation6,7,25, increased constriction7,8, attenuated muscle contraction hyperemia10-
12, etc.), occurring at an early age before overt comorbidities in Black individuals in comparison with White 
counterparts. These findings indicate that the onset of CVD risk in Black individuals manifests early in life as 
subclinical impairments in vascular function. However, despite these well-documented alterations, our findings 
also suggest that some regulatory mechanisms (e.g., functional sympatholysis11) may be preserved in young 
Black individuals. Such unexpected results emphasize the need for identifying the exact physiological 
mechanisms underlying the racial differences in vascular function, aiming at developing tailored novel 
interventions to improve vascular regulation in Black individuals, a population at a higher risk of developing CVD 
than other racial groups in the U.S.1,2. 
NO is a signaling molecule with a well-known role in promoting vasodilation in several conditions36,37. 
Research has associated low NO signaling with impaired vasodilation in Black individuals compared with White 
counterparts24,25, which is concerning because low NO-dependent vasodilation is a mark of CVD38,39. Some 
experimental models using local infusions of L-arginine6,38 support the hypothesis that interventions to increase 
NO signaling can improve vascular responses in Black individuals in comparison with White counterparts. 
However, systemic interventions to test this hypothesis have not been performed. 
ET-1 is a vasoconstrictor peptide produced by the endothelium that binds to ETA and ETB receptors on 
smooth muscle14. NO inhibits ET-1 actions26 by lowering its expression40, release41, and binding to receptors42. 
It is possible that low NO signaling in Black individuals may lead to exaggerated ET-1 signaling, which is indeed 
supported by an association of ET-1 with the pathophysiology of hypertension27-29 and with impaired vascular 
control of blood pressure30 in Black individuals. This is concerning because, besides attenuating acute 
vasodilator responses31-33, prolonged exposure to exaggerated ET-1 signaling can induce irreversible vascular 
remodeling and dysfunction26,43. Nevertheless, systemic interventions to lower ET-1 signaling and improve 
vasodilation in Black individuals have not been investigated. 
Significance 

Our research focuses on Black individuals, an understudied
American population despite being at a higher risk for
developing CVD than other racial groups.

Our study includes healthy young individuals to allow for
better identification of CVD risk factors associated with race
while avoiding comorbidities and functional deterioration due
to aging. We will shed light on the role of early specific
subclinical alterations in young Black individuals that can,
later on, lead to life-threatening diseases in this population.

Low NO signaling appears to be a major contributor to the
impaired vasodilation observed in Black individuals. Thus, we
will directly target different steps of the NO pathway (Figure
2) to elucidate molecular aspects that may be particularly
dysfunctional in Black individuals.

Studies suggest that poor vascular control of blood pressure
in Black individuals may be associated with exaggerated ET-
1 signaling, but no direct evidence linking ET-1 with impaired
vasodilator responses has been shown. We will directly
examine a potential differential role of ET-1 on acute vascular
regulation between Black and White individuals (Figure 3).

In addition to elucidating molecular mechanisms, our
research will likely generate supporting evidence on systemic
interventions for the improvement of vascular regulation in
Black individuals.
Figure 2. NO signaling13 as a mechanism of racial 
differences 
in 
vascular 
function. 
By 
using 
oral 
administrations of beetroot juice (Aim 1a), L-citrulline (Aim 
1b), and Sildenafil (Aim 1c), we expect to increase 
vasodilator responses in Black individuals, attenuating the 
impairment in comparison with White individuals. 

Research Strategy 
Innovation 
CONCEPTUAL: 

We will identify acute physiological benefits with oral drugs
and supplements in young Black individuals, an understudied
group with early impairments in vascular function.

We will establish that such initial vascular alterations
represent a novel research target for CVD prediction and
early prevention in Black individuals and possibly in other at-
risk populations.

We will define exaggerated ET-1 signaling as a potential
mechanism of the impaired vasodilator responses in young
Black individuals.

Our research model will show the feasibility of oral
interventions and may set the stage for future clinical trials to
evaluate novel treatments for this at-risk population.
TECHNICAL: 

We will use various techniques to stimulate and examine
vasodilator responses, achieving several goals:
o
Previous research on racial differences has almost
exclusively examined upper limb vasculature, although
impairments in vascular function are typically more
severe in the legs than in the arms44-48. In fact, lower limb
vascular diseases enhance the risk for various CVD49,50
and are more prevalent in the Black population than in
other racial groups1,51-53. Our proposal will perform a comprehensive assessment of vascular function in 
the upper and lower limbs. 
o
We will combine a variety of well-established procedures (flow-mediated dilation, passive leg movement,
rhythmic handgrip exercise, knee extension exercise) with four interventions (oral administrations of
beetroot juice, L-citrulline, Sildenafil, and Bosentan), and examination on two vascular beds (arm and
leg). This approach will generate integrated information on the differential roles of NO and ET-1 on
peripheral macro- and microvascular regulation between Black and White individuals.
Approach 
PARTICIPANTS: Healthy young Black and White men and women of age between 18 and 40 years will be 
recruited from the University of Mississippi Medical Center (UMMC) and the city of Jackson community. Subjects 
will qualify for the study if they identify their race and the race of their biological parents as being only Black (i.e., 
African American) or only White (i.e., Caucasian American). Individuals whose parents are mixed Black and 
White, or of other races (Hispanic or Latino, Asian, Native American, etc.) will not qualify for the study. To avoid 
confounding factors associated with lifestyle, only individuals born and raised in the U.S. will be included. 
Exclusion criteria are any chronic or ongoing disease, prescribed pharmacological treatment, smoking or tobacco 
use, and obesity (BMI > 30 kg / m2). Also, specific exclusion criteria will be considered before participation in the 
visits with the administration of Sildenafil or Bosentan. On a preliminary visit, subjects will sign the Informed 
Consent Form, answer questions about their medical history, be familiarized with the measurements and 
procedures, and perform a maximal knee extension exercise test. 
MEASUREMENTS: Arterial diameter and blood velocity will be measured on the right leg or right arm via 
duplex Doppler ultrasound (Logiq P9, GE Medical Systems, Milwaukee, WI). A linear array transducer (12L-RS) 
will be placed on the popliteal artery immediately distal to the popliteal fossa, on the common femoral artery ~2 
cm proximal to the bifurcation, or on the brachial artery ~5 cm proximal to the antecubital fossa. The position will 
be optimized for the visualization of arterial wall edges and sharpest blood velocity tracings. The skin will be 
marked and photographed to ensure probe placement in the same location throughout the study visits. The 
arteries will be imaged at a frequency of 10-13 MHz. Velocity will be measured at a frequency of 5 MHz, corrected 
to an insonation angle of 60°, with the sample volume encompassing the entire lumen without extending beyond 
the arterial wall edges. Ultrasound data will be recorded and saved on a computer system (Cardiovascular 
Suite, 
Figure 3. ET-1 signaling14 as a mechanism of racial
differences in vascular function. By using oral administration
of Bosentan (Aim 2), we expect to reduce the 
vasoconstrictor tone and increase vasodilator responses in 
Black individuals, attenuating the impairment in comparison 
with White individuals.

Research Strategy 
Quipu, Pisa, Italy). Arterial blood flow will be calculated as [(diameter/2)2 × velocity × π]. Beat-to-beat mean 
arterial pressure (MAP) will be measured using finger photoplethysmography (Human NIBP, ADInstruments, 
Colorado Springs CO) on the left hand. Conductance will be calculated as [blood flow / MAP]. Heart rate (HR) 
will be determined using a standard lead II electrocardiogram (Bioamp, ADInstruments). 
Blood samples will be collected from a venous catheter in the antecubital area. For nitrate and nitrite 
concentrations, blood samples will be separated into Eppendorf tubes containing heparin and centrifuged. 
Plasma will be pipetted into separate Eppendorf tubes, flash-frozen in liquid nitrogen, and stored at -80°C. 
Analyses will be made via chemiluminescence with a NO analyzer (NOA 280i; Sievers Instruments, Boulder, 
CO) by addition to potassium iodide in acetic acid at room temperature, and to vanadium chloride in hydrochloric 
acid at 95°C54. For L-arginine and L-citrulline concentrations, aliquots of plasma will be spiked with stable isotope-
labeled L-arginine and L-citrulline, which will serve as internal standards. Protein will be precipitated, filtrated, 
derivatized, and analyzed by liquid chromatography-tandem mass spectrometry55. For ET-1 concentrations, 
blood samples collected using EDTA as an anticoagulant will be centrifuged, aliquoted, and stored at -20°C for 
later assessment by quantitative enzyme immunoassay (R&D Systems, Minneapolis, Minnesota, USA)56. 
STUDY DESIGN: Studies will be conducted in the Clinical Research and Trials Unit at UMMC. Medical 
doctors and clinical researchers will provide oversight, technical assistance, and safety monitoring. All study 
visits will follow the same procedures (see Experimental Visits), except for the drug or supplement being 
administered. 
For Aim 1, double-blind, randomized, placebo-controlled crossover designs will be used to examine the 
effects of increasing NO and cGMP bioavailability on vascular function of young Black and White individuals. 
Three sets of two visits will be performed as follows: 

Aim 1a: At the beginning of each visit, participants will ingest either a nitrate-rich beetroot juice or a nitrate-
depleted beetroot juice (serving as placebo) in their liquid commercial form, which has a distinct color and
flavor. The nitrates will be absorbed and reduced in the plasma to nitrite and NO, thereby increasing
endothelium-independent NO bioavailability. The nitrate-rich beverage is commercially available as a sports
supplement, while the nitrate-depleted beverage is manufactured-on-demand for research.

Aim 1b: Participants will receive a 7-day supplement of L-citrulline or placebo before each study visit, with a
washout period of 7 days between visits. The activity of the eNOS converts L-arginine into NO and L-citrulline,
which is normally recycled back into L-arginine57. Unlike L-arginine, oral ingestion of L-citrulline is not
catabolized in the gut, nor is it extracted from systemic circulation through hepatic first-pass metabolism.
Thus, ingested L-citrulline becomes available in large quantities in the plasma for enzymatic conversion into
L-arginine55, thereby increasing endothelium-dependent NO bioavailability. The 7-day L-citrulline
administration is effective to increase L-arginine plasma levels and NO signaling in a dose-dependent
manner55, while a single administration has failed to do so58.

Aim 1c: At the beginning of each visit, participants will ingest a liquid mixture prepared by Investigational
Drug Services at UMMC containing Sildenafil or placebo. Sildenafil inhibits phosphodiesterase 5 (PDE5), an
enzyme that degrades cGMP in the vascular smooth muscle cells inactivating the NO-mediated signal; thus,
Sildenafil will prolong the availability of cGMP, enhancing the NO-mediated intracellular cascade.
For Aim 2, a double-blind, randomized, placebo-controlled crossover design will be used to examine the role
of ET-1 on the vascular function of young Black and White individuals. Two visits will be performed, with a 
minimum of 7 days between them. At the beginning of each visit, participants will ingest a liquid mixture prepared 
by Investigational Drug Services at UMMC containing Bosentan or placebo. Bosentan blocks ETA and ETB 
receptors, leading to a reduction in vasoconstrictor tone and a greater magnitude of vasodilator responses. 
EXPERIMENTAL VISITS: Figure 4 displays the design of the experimental visits. Participants will abstain 
from alcohol consumption, caffeine, and exercise for 24 h before all study visits, and will come after fasting 
overnight. Experiments will be conducted in a room with temperature tightly controlled at 20-21°C to minimize 
the influence of skin blood flow on the Doppler ultrasound measures. Women will participate in the study during 
the early follicular phase of the ovarian cycle to minimize variability in physiological responses across the 
menstrual cycle. 
For Aim 1a, upon arriving at the laboratory, subjects will ingest 140 mL of a beetroot juice containing either 
a high (~12.8 mmol) or a low concentration (~0.0055 mmol) of nitrates (James White Drinks, Suffolk, UK)59,60, in 
random order over two consecutive visits. Ninety minutes will be allowed after beverage ingestion to 
start 

Research Strategy 
instrumentation to ensure that data collection occurs in the period of peak nitrate plasma concentration (i.e., 120 
min)60. 
For Aim 1b, participants will receive pills containing 3 g of L-citrulline (Superior Labs, Park City, UT) or 
placebo, to be taken twice daily for 7 days each, before each study visit. The washout period will be 7 days. On 
the day of the study visit, subjects will ingest the last supplement or placebo pill upon arriving at the laboratory. 
Sixty minutes will be allowed before start instrumentation to ensure that data collection occurs in the period of 
peak L-arginine plasma concentration (i.e., 90 min)55. 
For Aim 1c, upon arriving at the laboratory, subjects will ingest a liquid mixture containing the PDE5 inhibitor 
Sildenafil (100 mg)61, or placebo in random order. Thirty minutes will be allowed after drug ingestion to start 
instrumentation to ensure that data collection occurs in the period of peak plasma concentration of Sildenafil 
(i.e., 60 min)62. 
For Aim 2, upon arriving at the laboratory, subjects will ingest a liquid mixture containing the non-selective 
ETA and ETB receptors antagonist Bosentan (125 mg)33, or placebo, in random order over two consecutive visits. 
Ninety minutes will be allowed after drug ingestion to start instrumentation to ensure that data collection occurs 
in the period of peak plasma concentration of the drug (i.e., 120 min)63. 
EXPERIMENTAL PROCEDURES: Subjects will receive instrumentation for HR, MAP, and blood sampling 
in the supine position with the right leg and arm supported for assessment of the popliteal and brachial artery. A 
rapid-inflation cuff (Hokanson, Bellevue, WA) will be placed ~5 cm distal to the right fibular head or ~3 distal to 
the right antecubital fossa. Subjects will rest for a minimum of 20 min, and blood samples will be collected. Then, 
flow-mediated dilation (macrovascular function) and reactive hyperemia (microvascular function) will be 
measured separately in the arm and leg as described previously64,65. Briefly, diameter and blood velocity will be 
recorded for 2 min, following which the cuff will be inflated (220 mmHg) for 5 min. At the last 30 s of cuff inflation, 
recordings will be resumed and continue for 3 min after cuff deflation. The macrovascular function will be 
measured as the percent change in arterial diameter after cuff deflation normalized to arterial shear rate. The 
microvascular function will be measured as the blood velocity area-under-the-curve from cuff deflation to return 
to resting values using the sum of trapezoids. 
After that, a trial of passive leg movement will be performed as described previously66,67. Briefly, a 
researcher will move the subject’s right lower leg (knee extension) through the range of motion of 90° to 180° 
and back to 90°, at a rate of 60 / min for 2 min. 
Following that, subjects will perform a trial of rhythmic handgrip exercise10 (Biometrics, Ladysmith, VA), 
consisting of 3 min of hand contractions at 30% maximal voluntary contraction with a duty cycle of 1 s contraction 
/ 2 s relaxation. Measures will be recorded continuously for 2 min at rest and throughout the trial. 
Subjects will then move to a seated position on a custom ergometer (Technavance, Austin, TX) with the arms 
supported by side tables, and receive instrumentation for HR, and MAP. The right foot will be attached to a boot 
connected to the ergometer68. Subjects will perform three trials of knee extension exercise, consisting of 3 min 
of voluntary knee extension at a rate of 50 / min at 15%, 30%, and 45% of their maximal workload. Measures 
will be recorded continuously for 2 min at rest and throughout each trial. All trials will be separated by 15 min 
intervals. After completing all trials, another set of blood samples will be collected.  
Figure 4. Protocol of the experimental visits. Visits will be identical except for the oral drug or supplement administration before any procedure.

Research Strategy 
STATISTICAL ANALYSES: Sample size estimation (statulator.com) is 11 individuals per group, based on 
the femoral artery blood flow response to knee extension exercise from our preliminary data (see below), using 
Student’s t-test for independent samples, and considering a two-tailed alpha of 0.05, and power of 0.8 as 
parameters. This estimated sample size is adequate relative to our previous investigations10,11. Due to potential 
influence of sex and race on vascular function69, we will duplicate the estimated sample size to recruit an equal 
number of men and women. To account for incompletion and exclusion of data during analyses, additional 
participants will be recruited, leading to an estimate of 30 individuals enrolled in each racial group. We will use 
a mixed-model ANOVA to compare vascular responses according to the effects of drug administration (Placebo 
vs. Drug), group (Black vs. White), and interaction. Subjects’ characteristics will be compared using Student’s t-
test for independent samples and, in case of significant differences, such characteristics will be used as 
covariates in the ANOVA. Additionally, while men and women will be primarily grouped according to race, the 
large and balanced sample size will allow for sub-analyses comparing the effects of race on vascular responses 
separately in men and women. 
PRELIMINARY STUDY: We recently compared common femoral artery blood flow (FABF, Doppler 
ultrasound) between White (n = 4) and Black (n = 8) healthy young men during knee extension exercise at 10 
W, 20 W, and 30 W. Each exercise trial lasted for 3 min with a minimum rest interval of 5 min. Knee extension 
cadence was 50 repetitions per min dictated by a metronome and by real-time visual feedback. The participants 
performed an incremental knee extension exercise test to 
determine their maximal workload on a separate visit. The 
groups were of similar age (White men: 21 ± 2 years vs. 
Black men: 21 ± 1 years; P = 0.905), body mass index 
(White men: 21.3 ± 2.1 kg/m2 vs. Black men: 23.2 ± 3.4 
kg/m2; P = 0.319), and achieved similar peak workloads in 
the maximal exercise test (Whites: 48 ± 15 W vs. Blacks: 
52 ± 9 W; P = 0.542). FABF response to exercise was 
lower in Black men compared with White men across all 
workloads (Group P = 0.038; Figure 5). In this preliminary 
study, we have been able to demonstrate the necessary 
skills 
to 
establish 
the 
proposed 
comprehensive 
assessment of racial differences in vascular function and 
generated data to support our hypothesis of reduced leg 
blood flow response to knee extension exercise in Black 
men compared with White men. 
Figure 5. Individual and mean group changes in femoral artery blood
flow from rest to knee extension exercise at 10W, 20W, and 30W in 
White men (light grey bars, white circles) and Black men (dark grey 
bars, black circles). 

EXPECTED RESULTS AND FUTURE PLANS TO SECURE EXTRAMURAL FUNDING 
Black individuals manifest impaired vascular responses compared with White individuals. We postulate that 
this is due, at least in part, to a low NO dilator signaling and a high ET-1 constrictor signaling. Accordingly, we 
anticipate that: 

Increases in conductance and arterial diameter in response to the experimental procedures (flow-mediated
dilation, passive leg movement, rhythmic handgrip, and knee extension) will be lower in Black than in White
individuals in the placebo visits.

Increase in NO bioavailability with beetroot juice and L-citrulline, and the increase in cGMP with Sildenafil
will augment the dilator signal and increase vascular responses in Black individuals, attenuating the
impairment in comparison with White individuals.

Reduction in the ET-1 constrictor signal with Bosentan will increase vascular responses in Black individuals,
attenuating the impairment in comparison with White individuals.
Some alternative approaches to be attempted in future studies include:

Direct intra-arterial infusions of vasoactive compounds (e.g., L-arginine, L-NMMA, selective blockers of ETA
and ETB receptors, etc.) to evoke quick local responses with lower concentrations, preventing systemic
cardiovascular effects70, and allowing for deeper mechanistic insight.

Chronic oral administration of beetroot juice, L-citrulline, Sildenafil, and Bosentan with multiple follow-up
assessments, to evaluate the persistence of improvements on vascular responses, whether maladaptive
mechanisms become expressed and prevent vascular improvements, effects on blood pressure control, etc.

Harvesting of endothelial cells via catheterization to establish correlations between vascular responses and
cellular ET-1 levels (higher specificity than plasma measurement), expression of ETB receptors (known to
evoke NO production in the endothelium), eNOS, etc.

Use of the stored blood samples collected in this study to examine molecular markers of other vasoactive
mechanisms (e.g., reactive oxygen species, ADMA, ATP, angiotensin II, catecholamines, etc.) to establish
correlations with the vascular responses, as well as for the design of novel hypotheses and future studies.

The large and balanced sample size of men and women will allow for secondary analyses that may
corroborate, as previously suggested69, a differential effect of race on vascular function between men and
women.
External funding opportunities will be pursued (see below) to support the completion of the current specific
aims, as well as new studies in the field as new questions arise: 

NIH Research Project Grant (R01): Parent Funding Opportunity Announcement (PA-19-091), submitted to
National Institute on Minority Health and Health Disparities (NIMHD).

NIH Notice of Special Interest (NOT-HL-19-711): To support epidemiological research to assess
cardiovascular health during childhood and its relationship to adult disease in underrepresented populations.
Submitted through the R01 mechanism (PA-19-056) to National Heart, Lung, and Blood Institute (NHLBI).

AHA Career Development Award: To support highly promising academic professionals in the first
professional appointment to explore innovative questions or pilot studies that will provide preliminary data
and training.

AHA-VIVA Physicians Research Award: A research partnership program to offer training and mentorship to
potential future leaders in the domain of vascular research. Submitted through the AHA Career Development
Award mechanism.
I am an author of 5 articles8-12 about vascular physiology in young Black individuals. The current proposal
will advance our previous findings by examining the differential roles of two molecular mechanisms on the macro- 
and microvasculature of Black individuals. Furthermore, by examining arms and legs with several techniques, 
we will establish that such subclinical alterations affect multiple vascular beds in Black individuals and, 
consequently, impose early and continuous stress on the cardiovascular system and a health risk over time. The 
administration of commercially-available drugs and supplements via the oral route will support and facilitate the 
design of future clinical trials aiming at evaluating long-term responses. By providing a deeper and broader 
knowledge of vascular function in young Black individuals, elucidating CVD risk factors occurring early in life, 
and offering an insight into potentially available treatments, this proposal will build a foundation for the design of 
greater clinical studies with longer observations.

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Malhotra A, Cohen D, Syms C and Townsend RR. Blood pressure changes in the leg on standing. J Clin
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Newcomer SC, Sauder CL, Kuipers NT, Laughlin MH and Ray CA. Effects of posture on shear rates in
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Wu SP, Ringgaard S, Oyre S, Hansen MS, Rasmus S and Pedersen EM. Wall shear rates differ between
the normal carotid, femoral, and brachial arteries: an in vivo MRI study. J Magn Reson Imaging. 2004;19:188-
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cardiovascular disease associated with the ankle-brachial index: Systematic review. Atherosclerosis.
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Park DW, Kang SJ, Kim YH, Lee CW, Park SW and Park SJ. Prevalence and clinical implications of newly
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Aboyans V, McClelland RL, Allison MA, McDermott MM, Blumenthal RS, Macura K and Criqui MH. Lower
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Craig JC, Broxterman RM, Smith JR, Allen JD and Barstow TJ. Effect of dietary nitrate supplementation
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Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F and Boger
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Gmelch BS, Bledsoe AD, Richardson RS and Wray DW. Endogenous endothelin-1 and femoral artery shear
rate: impact of age and implications for atherosclerosis. J Hypertens. 2016;34:266-73.
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and KIR channels in humans. J Physiol. 2017;595:5175-5190.

UNIVERSITY OF MISSISSIPPI MEDICAL CENTER 
2500 North State Street  
Jackson, Mississippi 39216-4505 
Institutional Review Board 
DHHS FWA # 00003630
Telephone (601) 984-2815  
IORG #0000043 
Facsimile   (601) 984-2961  
IRB 1 Registration #00000061 
IRB 2 Registration #00005033 
Approval Notice 
Initial Application 
09/01/2020 
Thales Barbosa, MD 
SOM – Physiology and Biophysics 
University of Mississippi Medical Center 
2500 North State Street 
Jackson, MS 392164505 
RE: 
IRB File #2020V0181 
 Racial Differences in Cardiovascular Function: The Role of Endothelin-1 
Your Initial Application was reviewed and approved by the Expedited Review (revisions 
required) process on 8/28/2020 after the initial review by the Convened Board on 
8/11/2020. You may begin this research. 
Please note the following information about your approved research protocol: 
•
Protocol Approval period:
•
Approved Enrollment  #:
•
Performance Sites:
•
Funding:
09/01/2020 – 08/31/2021
50 
UMMC, Clinical Research and Trials Unit 
NIH 
Documents/ Materials: 
Type 
Description 
Version # 
Date 
Protocol 
TCB_Racial Differences ET-
1_Study Protocol 
1 
09/01/2020 

Consent 
TCB_Racial Differences ET-
1_Informed Consent Form 
1 
09/01/2020 
Recruitment Flyer 
TCB_Racial Differences ET-
1_Recruitment Flyer 
1 
09/01/2020 
Data Collection Sheet Medical History Form 
1 
09/01/2020 
Please remember to: 
- Use the IRB file number (2020V0181) on all documents or correspondence with the
IRB concerning your research protocol.
- Review and comply with all requirements on the enclosure, UMMC Investigator
Responsibilities, Protection of Human Research Participants.
The IRB has the prerogative and authority to ask additional questions, request further 
information, require additional revisions, and monitor the conduct of your research and 
the consent process. 
Please note, if this study involves an intervention (whether or not it involves a drug or 
device) you (or the "responsible party") must register the study before enrollment begins 
and report results within 12 months of study closure through Clinicaltrials.gov 
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We wish you the best as you conduct your research. If you have questions or need 
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IRB 1 
Enclosure(s): (1) Investigator Responsibilities, Protection of Human Research 
Participants 

UMMC Investigator Responsibilities
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If you have questions or need assistance, please contact the Human Research Office at 
601 984-2815.

2021-06-11 
RESEARCH PROTOCOL
Title 
Racial Differences in Cardiovascular Function: The Role of Endothelin-1 
Principal Investigator 
Thales C. Barbosa, Ph.D. 
Co-Investigators 
Licy L. Yanes Cardozo, M.D. 
Joshua S. Speed, Ph.D. 
Vishnu V. Garla, M.D. 
John E. Hall, Ph.D. 
Abstract 
The current study will examine the role of Endothelin-1 (a potent 
endogenous vasoconstrictor; ET-1) on brachial and popliteal artery flow-
mediated dilation, reactive hyperemia, and blood flow responses to passive 
leg movement, rhythmic handgrip exercise, and rhythmic knee extension 
exercise in Black and White individuals. In two separate visits, we will 
administer an acute oral dose of Bosentan (antagonist of ET-1 receptors ETA 
and ETB) or Placebo (randomized double-blind cross-over design), followed 
by examination of brachial artery flow-mediated dilation and reactive 
hyperemia at rest, as well as blood flow responses to passive leg movement, 
low, moderate, and high-intensity rhythmic handgrip exercise and rhythmic 
knee extension exercise in Black and White individuals. 
Background 
Black (i.e. African American) individuals develop cardiovascular diseases 
earlier in life and at greater rates than other racial groups1-3. Because of this 
greater risk, studies assessing racial differences in cardiovascular physiology 
are needed. It has been demonstrated that, compared with White individuals 
(i.e. Caucasian American, the largest racial group in the U.S. population), 
healthy young Black individuals have impaired vascular responses to various 
stressors, characterized by augmented vasoconstriction and reduced 
vasodilation4-8. However, much remains unknown regarding the mechanisms 
by which these individuals demonstrate vascular dysfunction. Techniques to 
assess acute vascular function consist of applying stimuli to evoke dilation in 
the macro- and microvasculature. For example, the prolonged inflation and 
subsequent deflation of a pneumatic cuff on the forearm or calf evoke 
brachial or popliteal artery flow-mediated dilation and forearm reactive 
hyperemia, which have been associated with cardiovascular health9. 
Furthermore, the vasodilator responses to passive leg movement, rhythmic 
handgrip exercise, and rhythmic knee extension exercise are also considered 
sensitive for the detection of vascular dysfunction10-12. Multiple mechanisms 
interact and determine the magnitude of these dilator responses, with 
vasoconstrictor factors (ET-1, adrenergic receptors, angiotensin II, etc.) 
causing attenuation, and vasodilator factors (NO, intravascular ATP, 
adenosine, K+ channels, EDHF, etc.) causing an increase in vascular 
responses13. 

2021-06-11 
Purpose 
To begin to elucidate the alterations in vascular function in Black individuals, 
we chose to target ET-1, a peptide produced by the vascular endothelium 
that mediates vasoconstriction by binding ETA and ETB receptors on smooth 
muscle14,15. ET-1 seems to play a prominent role in the pathophysiology of 
hypertension specifically in Black patients16,17. Also, it is suggested that ET-1 
may be associated with impaired vascular control of blood pressure in young 
normotensive Black men18. Vascular responsiveness to ET-1 is also 
upregulated in adults exposed to insults during their fetal life (e.g., poor 
maternal nutrition, chronic stress, no prenatal care), psychological stressors 
during childhood (e.g., physical and emotional abuse, household 
dysfunction), and high ethnic discrimination19-24—all of which are more 
prevalent among Black than White individuals, possibly contributing to racial 
disparities in vascular function25-28. We hypothesize that Black individuals 
have reduced vasodilator responses as a consequence of a greater 
vasoconstrictor tone mediated by ET-1. Therefore, we hypothesize that the 
antagonism of ETA and ETB receptors will reduce the vasoconstrictor tone and 
augment the vasodilator responses in Black but not in White individuals. 
Specific Aim 
We will examine the role of ET-1 on brachial and popliteal artery flow-
mediated dilation, reactive hyperemia, and blood flow responses to passive 
leg movement, rhythmic handgrip exercise, and rhythmic knee extension 
exercise between Black and White individuals. Using an acute oral 
administration of Bosentan, we will antagonize ETA and ETB receptors to 
reduce the vasoconstrictor tone mediated by ET-1 and, consequently, allow 
for greater flow-mediated dilation, reactive hyperemia, and blood flow 
responses to passive leg movement, handgrip, and knee extension. We 
anticipate that this effect of Bosentan will be more prominent in Black 
individuals than in White individuals. In a separate visit, the participants will 
receive a Placebo to serve as a control. We will also examine prenatal and at-
birth medical records, and apply questionnaires of adverse childhood 
experiences and perceived discrimination to assess whether these early-life 
risk factors are associated with racial disparities in vasodilation between 
Black and White individuals. 
Study Period 
3 years 
Anticipated start date: 09/01/2020 
Anticipated end date: 09/01/2023 
Data Generation 
Period 
N/A 

2021-06-11 
Study Design 
The study will consist of three visits to the research clinic (Clinical Research 
and Trials Unit at the UMMC campus) over 4 months. If a participant misses 
an appointment, rescheduling will be attempted twice. Women will provide 
a urine sample for a pregnancy test and answer questions about the most 
recent menstrual period before any research measurement or procedure be 
performed in any visit. 
Visit 1: Informed Consent Form, written consent for access to their prenatal 
and at-birth medical records, assessment of medical history, application 
of the questionnaires “Adverse Childhood Experiences (ACE)” and “Scale 
of Ethnic Experiences (SEE)”, collection of blood samples and endothelial 
cells from the antecubital vein, familiarization with measurements and 
procedures that will take place in the next visits, and a rhythmic knee 
extension maximal exercise test. Visit 1 will last for about 2 hours. 
Visit 2: Qualifying participants will participate in Visit 2 within 4 weeks after 
Visit 1. At the beginning of the visit, we will randomly administer either 
a single oral dose of Bosentan (125 mg) or Placebo. Two hours after 
administration, we will perform the research measurements and 
procedures as explained below. This visit will last for about 4 hours. 
Visit 3: Those who complete Visit 2 will participate in Visit 3, which will take 
place 1 to 12 weeks later. All research measurements and procedures 
will be identical, except for the drug administration. At the beginning of 
the visit, we will administer either Bosentan (125 mg) or Placebo, 
whichever was not administered in Visit 2. 
Summary of the Research Measurements: 

Anthropometrics: Height, weight, waist, and hip for characterization.

Electrocardiogram: Adhesive patches placed on the chest and abdomen
for heart rate.

Respiration: An elastic belt placed around the abdomen for respiratory
movements.

Blood pressure: Brachial blood pressure will be measured throughout the
study with an automated blood pressure monitor for validation of the
finger blood pressure values.

Finger blood pressure: A small cuff placed around one of the fingers for
continuous recording of beat-to-beat blood pressure.

Arm blood flow: Duplex Doppler ultrasound will be used for
measurements of brachial artery diameter and blood velocity, used for
calculation of blood flow.

Leg blood flow: Duplex Doppler ultrasound will be used for
measurements of popliteal and femoral artery diameter and blood
velocity, used for calculation of blood flow.

2021-06-11 

ET-1 expression: Plasma and endothelial lysates will be collected from
the antecubital vein using a catheter and J-wires. ET-1 peptide will be
measured using an ELISA kit. RNA from endothelial cells will be isolated
and reverse transcribed. The resulting cDNA will be used to quantify ET-
1 mRNA copy count by Droplet Digital polymerase chain reaction using
TaqMan primer gene expression assays with human preproET-1 primers.

Insults during fetal life: Prenatal and at-birth medical records of each
participant will be obtained from the UMMC and local hospitals database
in paper copies and digital formats (e.g., EPIC, DataArk). We will assess
maternal comorbidities, such as over- or undernutrition, pre-eclampsia,
diabetes, obesity, smoking, and alcohol consumption. Gestational age at
birth will be assessed and classified as preterm (less than 37 weeks),
early-term (37 weeks to 38 weeks 6 days), full-term (39 weeks to 40
weeks 6 days), late-term (41 weeks to 41 weeks 6 days), or post-term
(over 42 weeks)29. Also, the birth weight will be assessed, and birth
weight of fewer than 2500 grams will be a marker of impaired fetal
growth26.

Adverse childhood experiences: The questionnaire ACE will measure the
prevalence of 10 types of trauma over the first 18 years of life: emotional
abuse, physical abuse, sexual abuse, emotional neglect, physical neglect,
household substance abuse, domestic violence against mother
(stepmother), household incarceration, household mental illness, and
parental divorce27.

Perceived discrimination: The questionnaire SEE will measure the
magnitude of perceived discrimination, ethnic identity, mainstream
comfort, and social affiliation28.
Summary of Research Procedures 

Bosentan or Placebo: A single oral dose of Bosentan (125 mg; Tracleer,
Actelion Pharmaceuticals US, San Francisco, CA) or Placebo will be
administered in separate visits in a randomized, double-blind, crossover
design. Bosentan is an FDA-approved medicine used for the treatment of
cardiovascular diseases such as pulmonary hypertension. Pharmacists of
the Investigational Drug Services at UMMC will prepare Bosentan and
Placebo in liquid suspensions.

Arm blood vessel response: Participants will lie down on a bed with the
arm abducted and elbow extended on a bedside table. A cuff will be
placed around the forearm. After baseline arm blood flow
measurements, the cuff will be rapidly inflated (< 1 s) and remain inflated
for 5 min. Measurements will resume at the last 30 s of cuff inflation and
continue for 3 min after rapid cuff deflation.

Leg blood vessel response: Participants will lie down on a bed with the
legs extended and slightly rotated medially. A cuff will be placed around

2021-06-11 
the calf. After baseline popliteal blood flow measurements, the cuff will 
be rapidly inflated (< 1 s) and remain inflated for 5 min. Measurements 
will resume at the last 30 s of cuff inflation and continue for 3 min after 
rapid cuff deflation. 

Passive leg movement: Participants will lie down with their upper body
and thighs on a bed, and the lower legs off the bed. One lower leg will be
extended on a bedside table, and the other lower leg will hang
downwards with the knee flexed at a 90o angle. After baseline femoral
blood flow measurements, a researcher will move the participant’s right
lower leg (knee extension) through the range of motion of 90°–180°–90°
for a maximum of 2 min, with continuous measurements of
electrocardiogram, respiration, finger blood pressure, and leg blood
flow.

Handgrip exercise: Participants will lie down on a bed with the arm
abducted and elbow extended on a bedside table. Participants will
perform rhythmic handgrip exercise by squeezing a handgrip
dynamometer. Each handgrip exercise trial will last a maximum of 6 min
or until fatigue. Participants will perform three to six handgrip exercise
trials with continuous measurements of electrocardiogram, respiration,
finger blood pressure, and arm blood flow.

Knee extension exercise: Participants will sit on a custom ergometer with
the arms supported by side tables and one foot on a boot connected to
the ergometer. Participants will perform rhythmic knee extension
exercise by extending their lower leg through the range of motion of 90°–
180°–90°. Each knee extension exercise trial will last a maximum of 6 min
or until fatigue. Participants will perform three to six knee extension
exercise trials with continuous measurements of electrocardiogram,
respiration, finger blood pressure, and femoral blood flow.

Ischemic preconditioning: Participants will lie down on a bed with the
arms and legs extended. A cuff will be placed around the upper arms or
thighs. The cuffs will be rapidly inflated (< 1 s) and remain inflated for 5
min, then deflated for 5 min. This cycle will be repeated up to 4 times.

Ischemia–reperfusion: Participants will lie down on a bed with the arms
and legs extended. A cuff will be placed around the upper arms or thighs. 
The cuffs will be rapidly inflated (< 1 s) and remain inflated for a 
maximum of 20 min continuously. 
Inclusion Criteria 

Black (i.e. African American) or White (i.e. Caucasian American)
individuals

18 to 35 years of age

Born and raised in the United States

Biological parents of the same race (i.e. either Black or White)

2021-06-11 
Exclusion Criteria 

Known chronic or ongoing disease, or on prescribed pharmacological
treatment

History of liver disease

Pregnant, trying to become pregnant, or breastfeeding women

Use of hormonal contraceptives

Smoking or vaping in the last 6 months

Body mass index > 35 kg/m2
Number of 
Participants 
(anticipated) 
50 participants (i.e. 25 on each racial group). 
Based on data from Schreuder et al.30, taking the improvement of blood flow 
response to rhythmic handgrip with Bosentan as the primary outcome, the 
estimated sample size is 42 participants (i.e. 21 per group). We anticipate 
having to recruit about 20% more individuals to account for possible loss of 
data (e.g. participants who cannot complete all visits, problems during 
analyses, etc.), therefore needing to enroll 50 participants. 
Screening and 
Consent Process 
Announcements will be made via UMMC e-mail listserv, UMMC intranet, 
Research Participant Recruitment app, flyers on campus, and word of mouth. 
Potential participants will contact the study personnel via e-mail or phone, 
and answer screening questions. If they meet the inclusion criteria, Visit 1 will 
be scheduled. Consenting will be conducted in Visit 1 at the research clinic by 
Dr. Barbosa or by a study person after supervision and adequate training with 
Dr. Barbosa. Following consenting, there will be assessments of medical 
history, and familiarization with the research measurements and procedures. 
After that, those who qualify for the study will be scheduled for Visit 2 and, 
subsequently, Visit 3. 

2021-06-11 
Risks and benefits 
Risks: 

Electrocardiogram: Minor local skin irritation momentarily.

Blood pressure: Minor temporary discomfort, numbness, and/or tingling
at the fingertips during cuff inflation, which will subside after cuff
release. Bruising at the area where the cuff is inflated, which will
disappear within a few days.

Finger blood pressure: Minor numbness, tingling, and bluish coloration
at the fingertip. The finger cuff will be turned off between the research
procedures, and these symptoms will subside.

Blood vessel response, ischemic preconditioning, and ischemia–
reperfusion: Minor temporary discomfort, numbness, and/or tingling at
the fingertips and toes during cuff inflation, which subside after cuff
release. Bruising at the area where the cuff is inflated, which will
disappear within a few days.

Handgrip and knee extension exercise: Minor fatigue and exhaustion
momentarily.

Bosentan: We will administer a single dose of 125 mg of Bosentan orally.
These dose and route are effective to improve blood flow response to
exercise30. Furthermore, Bosentan is well tolerated up to 2400 mg31.
Therefore, we do not expect any adverse events caused by participation
in the study. Minor effects that may be expected are headache and
swelling. Nevertheless, we will consider the following32: Chronic use of
Bosentan has been associated with alterations in liver function.
Additionally, studies in animals indicate that Bosentan may cause harm
to an unborn fetus. Furthermore, there is a possibility of failure in
contraception when Bosentan and hormonal contraceptives are co-
administered. Therefore, out of an abundance of caution, we will exclude
from participation in the study individuals with a history of liver disease,
women who are pregnant, trying to become pregnant or breastfeeding,
and women using hormonal contraceptives. Also, male participants will
be informed about a possible risk of a decrease in sperm count due to
using Bosentan.

Blood and endothelial cell sampling: Intravenous collection of blood and
endothelial cells may include minor risks for local infection, local
bruising, or occasional light-headedness. These risks are minimized by
being performed under sterile conditions only by research personnel
with the required training and experience with this technique. Blood
drawn will not exceed 150 milliliters on any experimental testing day. To
run the assays all at one time and avoid variability, samples will be stored
in a –80o C freezer until analysis. After analysis, samples will be discarded
into a container labeled with a biohazard sign and stored in secondary
waste containers. All stored samples will be coded to protect
confidentiality. The code-key will be kept in the Principal Investigator’s

2021-06-11 
office in a locked cabinet. Only the Principal Investigator and study 
personnel will have access to the files via key. No other students or 
faculty will have access. 

ACE and SEE questionnaires: Given the sensitive nature of the topics
assessed with these psychometric tests, participants will be informed
that they may experience some distress or discomfort, may decline to
answer any question, and may request to interrupt or end the
assessment at any time. Our research personnel will observe the
participants and may also interrupt or end the assessment if it seems
necessary. In the event of an emergent psychiatric crisis, Dr. Barbosa
and the research personnel will take appropriate steps including
calling an emergency contact person indicated by the participant in
the Medical History Form, and facilitating transportation to
emergency health care. Furthermore, all participants will receive
referral information and telephone numbers for local and national
emotional support services, such as the Student Counseling and
Wellness Center, the Center for Integrative Health, the Behavioral
Health Specialty Clinic, Crisis and Suicide Prevention lines.
Benefits: 

There will be no direct benefit from participating in this research study.
We hope to learn information that may help others in the future.
Randomization and 
blinding 
We will use a Randomization Model tool in the REDCap system to randomly 
select between Bosentan and Placebo to be administered on Visit 2 for each 
individual, aiming at achieving a balanced distribution between Black and 
White individuals, and between men and women. Once the random drug 
selection is made for Visit 2, selection for Visit 3 will automatically be the 
alternative drug. Therefore, participants who complete Visits 2 and 3 will 
have received both Bosentan and Placebo. The participants and study 
personnel will be blind to the administration of Bosentan or Placebo on any 
visit. Pharmacists of the Investigational Drug Services at UMMC will retain 
the information about the drug selection per visit and will reveal it to the 
study personnel after completing data collection and analyses for that study 
participant or in case of emergency health care.  

2021-06-11 
Outcome Measures 
The primary measurements obtained by duplex Doppler ultrasound are: 1) 
brachial artery flow-mediated dilation (i.e., the increase in arterial diameter 
in response to the increased shear stress following forearm cuff release); 2) 
forearm reactive hyperemia (i.e., the increase in blood velocity following 
forearm cuff release); 3) blood flow responses to passive leg movement; 4) 
blood flow responses to low, moderate, and high-intensity rhythmic handgrip 
exercise; 5) blood flow responses to low, moderate, and high-intensity 
rhythmic knee extension exercise. Other measurements are beat-to-beat 
heart rate and beat-to-beat blood pressure, which can be used for further 
analyses of heart rate variability, blood pressure variability, and baroreflex 
sensitivity. 
Study Endpoint 
To determine whether there is an augmented contribution of ET-1 to the 
basal vascular tone in Black individuals compared with White individuals. We 
anticipate that the antagonism of ET-1 receptors will improve vasodilator 
responses in Black individuals but not in White individuals. 
Data Safety 
Monitoring Plan 
Dr. Gailen D. Marshall Jr., Professor of the Department of Medicine, will serve 
as an independent Data Safety Monitor for this study. The study personnel 
will conduct semi-annual meetings with the Monitor to present the research 
progress and discussion of observed events upon drug administration. In case 
of a participant’s emergency health care that could be associated with the 
study, the study personnel will notify the Monitor within 24 h. 
Protected Health 
Information (PHI) 
The information collected in this study includes name, telephone, home 
address, e-mail address, date of birth, age, race and ethnicity, medical 
history, and pictures. Each study participant will be given a study ID and the 
data will be associated with this ID. All data will be accessed via HIPAA 
compliant methods and stored using REDCap. 
Statistical 
Methodology 
Flow-mediated dilation and reactive hyperemia will be compared between 
groups (Black vs. White) and between drugs (Bosentan vs. Placebo) using a 
mixed model ANOVA (two-way). Responses to rhythmic handgrip exercise 
will be compared between groups (Black vs. White), between drugs 
(Bosentan vs. Placebo), and between intensities (low, moderate, high) using 
a mixed model ANOVA (three-way). 

2021-06-11 
References 
1. Virani et al. Circulation. 2020; 141:e139-e596
2. Rosamond et al. Circulation. 2007; 115:e69-171
3. Van Dyke et al. MMWR Surveill Summ. 2018; 67:1-11
4. Stein et al. Clin Pharmacol Ther. 1997; 62:436-43
5. Stein et al. Hypertension. 2000; 36:945-51
6. Kim et al. Microvasc Res. 2018; 118:1-6
7. Vranish et al. Hypertension. 2018; 71:192-198
8. Barbosa et al. Am J Physiol Heart Circ Physiol. 2018; 315:H1316-21
9. Yeboah et al. Circulation. 2007; 115:2390-7
10. Barrett-O'Keefe et al. Am J Physiol Heart Circ Physiol. 2014; 307:H1512-
20
11. Findlay et al. Vasc Med. 2013; 18:63-71
12. Trinity and Richardson. Sports Medicine. 2019; 49:1365-1381
13. Holwerda et al. Auton Neurosci. 2015; 188:24-31
14. Ergul et al. Hypertension. 2000; 36:62-7
15. Haynes et al. Circulation. 1995; 92:357-63
16. Ergul et al. Hypertension. 1996; 28:652-5
17. Campia et al. Circulation. 2004; 109:3191-5
18. Treiber et al. Hypertension. 2000; 35:722-5
19. Bourque et al. Hypertension. 2013; 62:753-8
20. Torrens et al. Br J Nutr. 2009; 101:27-33
21. Fox et al. J Am Heart Assoc. 2018; 7:e007863
22. Su et al. Hypertension. 2014; 64:201-7
23. Yammine et al. Psychosom Med. 2014; 76:109-21
24. Cooper et al. Am J Hypertens. 2009; 22:698-704
25. Christian. Neurosci Biobehav Rev. 2020; 117:319-326
26. Martin et al. Natl Vital Stat Rep. 2019; 68:1-47
27. Felitti et al. Am J Prev Med. 1998; 14:245-58
28. Malcarne et al. J Pers Assess. 2006; 86:150-61
29. Spong. JAMA. 2013; 309:2445-6
30. Schreuder et al. Exp Physiol. 2014; 99:1253-64
31. Weber et al. Clin Pharmacol Ther. 1996; 60:124-37
32. Dingemanse et al. Clin Pharmacokinet. 2004; 43:1089-115
Data Collection Sheet 
The following data collection instruments were created using REDCap, and 
PDF copies were included in this proposal: 

Medical History Form

Randomization Form

Protocol Sheet Visit 1

Protocol Sheet Visits 2 and 3

Questionnaire Adverse Childhood Experiences

Questionnaire Scale of Ethnic Experience

2021-06-11 
Funding Sources 
NIH (P20GM104357) 
Department of Physiology & Biophysics 

INCLUSION OF INDIVIDUALS ACROSS THE LIFESPAN 
Healthy Black and White men and women will be recruited between the ages of 18 to 40 years. The focus of 
our research is to examine differences in vascular function associated with race. Therefore, to avoid individuals 
with impaired vascular function due to aging and comorbidities, we will recruit a relatively young cohort of 
participants. 

INCLUSION OF WOMEN AND MINORITIES 
This research proposal is designed to elucidate deleterious alterations in vascular function specifically in 
young Black individuals. Accordingly, Black men and women will be examined and, as traditionally done in 
previous studies in this field, results will be compared with those from White men and women. Subjects will 
qualify for the study if they identify their race and the race of their biological parents as being Non-Hispanic Black 
(i.e., African American) or Non-Hispanic White (i.e., Caucasian American). Individuals whose parents are mixed 
Black and White, or of other races (Hispanic or Latino, Asian, Native American, etc.) will not qualify for the study. 
To avoid confounding factors associated with lifestyle, only individuals born and raised in the U.S. will be 
included. We anticipate to include a total of 60 individuals, with a distribution of 15 Black men, 15 Black women, 
15 White men, and 15 White women. While men and women will be primarily grouped according to race, this 
large and balanced planned sample size will allow for sub-analyses comparing the effects of race on vascular 
responses separately in men and women. 

RECRUITMENT AND RETENTION PLAN 
Individuals will be recruited via UMMC e-mail listserv, UMMC intranet, flyers on campus, and word of mouth. 
Potential participants will contact the study personnel via e-mail or phone, and answer screening questions. If 
they meet the inclusion criteria, a consenting and familiarization visit will be scheduled. Consenting will be 
conducted at the research laboratory by a study person. After signing the Informed Consent Form, participants 
will answer a medical history questionnaire, and be familiarized with the research measurements and 
procedures. 
Study visits will be scheduled in pairs according to each specific aim of the project. In other words, each 
intervention and its respective placebo will be scheduled for consecutive visits. Participants will receive gift cards 
after completing each pair of visits. Furthermore, participants will learn more about the development of 
physiological studies in humans. Particularly for UMMC students, this experience may contribute for deciding to 
pursue a career in human physiology research. 

Fall
2020
Spring
2021
Fall
2021
Spring
2022
Fall
2022
Spring
2023
Fall
2023
Studies – Specific Aim 1a
Studies – Specific Aim 1b
Studies – Specific Aim 1c
Studies – Specific Aim 2
Abstract submissions
Manuscript submissions
IRB – Initial submission
IRB – Amendments and new 
protocols
Funding – COBRE Pilot Grant 
Program submission
Funding – AHA Career 
Development Award submissions
Funding – NIH Research Project 
Grant (R01) submissions

 
PROTECTION OF HUMAN SUBJECTS 
 
Risks to Human Subjects 
a. Human Subjects Involvement, Characteristics, and Design 
Cardiovascular diseases (CVD) are disproportionately more prevalent in Black (i.e., African 
American) men and women than in other racial groups in the U.S., which has been associated with 
deleterious alterations in vascular function starting early in life in Black individuals. Our research is 
designed to examine the impairments in vascular function in young Black individuals compared with 
White individuals, understand the contributions of nitric oxide (NO; Aim 1) and endothelin-1 (ET-1; Aim 
2) driving these racial differences, generate evidence on potential interventions to improve vascular 
function and support future clinical practices to prevent CVD in the Black population. 
Healthy Black and White men and women of age between 18 and 40 years will be recruited via UMMC 
e-mail listserv, UMMC intranet, flyers on campus, and word of mouth. Subjects will qualify for the study 
if they identify their race and the race of their biological parents as being only Black (i.e., African 
American) or only White (i.e., Caucasian American). Individuals whose parents are mixed Black and 
White, or of other races (Hispanic or Latino, Asian, Native American, etc.) will not qualify for the study. 
To avoid confounding factors associated with lifestyle, only individuals born and raised in the U.S. will be 
included. Also, to avoid comorbidities, we will not include individuals with any chronic or ongoing disease, 
on prescribed pharmacological treatment, smoking or using tobacco, and with obesity (BMI > 30 kg / m2). 
We anticipate to include a total of 60 individuals. 
In each visit, we will examine cardiovascular measurements during various stimuli commonly used in 
research (i.e., flow-mediated dilation, passive leg movement, rhythmic handgrip exercise, and knee 
extension exercise). Prior to, or at the beginning of each visit, participants will undergo Placebo-controlled 
interventions [single oral administration of nitrate-rich (~12.8 mmol) beetroot juice; 7-day oral 
administration of L-citrulline (6 g daily); single oral administration of Sildenafil (100 mg); single oral 
administration of Bosentan (125 mg)] to acutely modulate NO and ET-1 signaling. 
b. Sources of Materials 
The following data will be collected from the subjects: Peripheral arterial blood flow (duplex Doppler 
ultrasound); beat-to-beat mean arterial pressure (finger photoplethysmography); heart rate 
(electrocardiogram); plasma concentrations of nitrate, nitrite, L-arginine, L-citrulline, and ET-1. All data 
will be coded, i.e., each study participant will be given a study ID and the data will be associated with this 
ID. Only study personnel will have access to the ID coding system, which will be accessed only if needed. 
All data will be assessed via HIPAA compliant methods, directly collected in or transferred to digital 
format, and stored using REDCap or other UMMC-provided cloud system. Plasma samples will be 
disposed of after conclusion of the study analyses. 
c. Potential Risks 
There are no psychological, financial or legal risks associated with participating in this research. 
Physical risks are only minor and brief discomforts associated with the normal operation of the 
research measurements and procedures, such as local skin irritation during electrocardiogram; 
numbness and tingling during blood pressure measurements and procedures with cuff inflations; muscle 
fatigue after handgrip and knee extension exercise, and minor risks for infection, bruising or occasional 
light-headedness due to venous blood draws.  
Oral administration of beetroot juice, L-citrulline and Sildenafil implicate no known risks. Prolonged 
use of Bosentan has been associated with alterations in liver function, potential harm to an unborn fetus, 
and a possibility of failure in contraception when co-administered with hormonal contraceptives.  
There are no alternative interventions in the study. If a subject chooses not to undergo an intervention, 
he or she will not be included in that portion of the study. 
 
Adequacy of Protection Against Risks 
a. Recruitment and Informed Consent 
Announcements will be made via UMMC e-mail listserv, UMMC intranet, flyers on campus, and word 
of mouth. Potential participants will contact the study personnel via e-mail or phone, and answer 
screening questions. If they meet the inclusion criteria, a consenting and familiarization visit will be 
scheduled. Consenting will be conducted at the research laboratory by a study person. After signing the 

Protection of Human Subjects 
Informed Consent Form, participants will answer a medical history questionnaire, and be familiarized with 
the research measurements and procedures. 
b. Protections Against Risk 
Research personnel will only get involved in this study after approval by the IRB, which will require 
completion of institutional and/or web-based training (e.g., CITI Program) in protection of human subjects. 
Furthermore, all personnel will be extensively trained in the specific research techniques. Study visits will 
be conducted in the Clinical Research and Trials Unit at UMMC, where medical doctors and clinical 
researchers will provide oversight, technical assistance, safety monitoring, and explanation of incidental 
findings (e.g., high blood pressure) to the subjects. The Investigational Drug Services at UMMC will assist 
in the preparation, storage, and dispensing of drugs, supplements, and respective placebo. A portion of 
the procedures and interventions proposed in this study has already been approved by the IRB of the 
Human Research Office at UMMC, and amendments will be submitted as this study progresses. 
Although the physical risks in this study are only minor and brief, the researchers will continuously 
interact with the subjects in the study visits to monitor their level of discomfort. The techniques using cuff 
inflation and arterial occlusion (i.e., blood pressure measurement and flow-mediate dilation) will be 
discontinued at the subject’s request if deemed too uncomfortable. Similarly, exercise trials will be 
discontinued if not tolerated. Also, the risks associated with blood draws will be minimized by being 
performed under sterile conditions only by research personnel with required training and experience with 
this technique. 
Oral administration of beetroot juice, L-citrulline and Sildenafil implicate no known risks. The use of 
Bosentan is well tolerated up to 2400 mg in health individuals and, accordingly, we do not expect any 
adverse events caused by Bosentan in this study. Nevertheless, out of an abundance of caution, we will 
exclude from participation in the study individuals with a history of liver disease; women who are pregnant, 
trying to become pregnant or breastfeeding; and women using hormonal contraceptives, since these 
conditions may be more vulnerable to adverse events with Bosentan. 
 
Potential Benefits of the Proposed Research to Human Subjects and Others 
The benefit for the participants in this study is to learn more about the development of research in 
humans. Particularly for UMMC students, this experience may contribute for deciding to pursue a career 
in human physiology research. As discussed above, the risks to participants are minimal, thus it is our 
opinion that the benefits derived from the proposed studies largely outweigh the risks. 
 
Importance of the Knowledge to be Gained 
Healthy young Black individuals must cope with subclinical impairments in vascular regulation that 
place continuous stress on the cardiovascular system and predispose to vascular remodeling and 
dysfunction, which are predictors of CVD. With this investigation, we strive to provide evidence on the 
roles of NO and ET-1 underlying the racial differences in vascular function and generate preliminary data 
on tailored novel interventions to improve vascular regulation in Black individuals, ultimately contributing 
to reduce mortality and improve cardiovascular health in this underrepresented group of the American 
population. 
 

 
DATA SAFETY MONITORING PLAN 
As approved by the IRB of the Human Research Office at UMMC, a physician and senior faculty of the 
Clinical Research and Trials Unit will serve as an independent Data Safety Monitor for this study. The Principal 
Investigator and study personnel will conduct semi-annual meetings with the Monitor to present the research 
progress and discussion of adverse events observed, if any. In case of a participant’s emergency health care 
that could be associated with the study, the study personnel will notify the Monitor within 24 h. 

 
STATISTICAL DESIGN AND POWER 
Sample size estimation (statulator.com) is 11 individuals per group, based on the femoral artery blood flow 
response to knee extension exercise from our preliminary data, using Student’s t-test for independent samples, 
and considering a two-tailed alpha of 0.05, and power of 0.8 as parameters. This estimated sample size is 
adequate relative to our previous investigations. Due to potential influence of sex and race on vascular function, 
we will duplicate the estimated sample size to recruit an equal number of men and women. To account for 
incompletion and exclusion of data during analyses, additional participants will be recruited, leading to an 
estimate of 30 individuals enrolled in each racial group. We will use a mixed-model ANOVA to compare vascular 
responses according to the effects of drug administration (Placebo vs. Drug), group (Black vs. White), and 
interaction. Subjects’ characteristics will be compared using Student’s t-test for independent samples and, in 
case of significant differences, such characteristics will be used as covariates in the ANOVA. Additionally, while 
men and women will be primarily grouped according to race, the large and balanced sample size will allow for 
sub-analyses comparing the effects of race on vascular responses separately in men and women.