Nebivolol and Endothelial Regulation of Fibrinolysis
Phase: PHASE4
Conditions: Prehypertension, Hypertension
Sponsor: University of Colorado, Boulder

CU-Boulder Protocol 
 
Nebivolol and Endothelial Regulation of Fibrinolysis  
08/25/2015 
 
2.  Description of Project 
 
Purpose.  The specific aim and hypothesis associated with the present research protocol are: 
 
Experimental Aim:  To determine, in vivo, the effects of nebivolol on the capacity of the vascular 
endothelium to release t-PA in adult humans with elevated blood pressure. 
 
Hypothesis. We hypothesize that nebivolol will improve endothelial t-PA release in adult humans with 
elevated blood pressure to a greater extent than either metoprolol or placebo.  We further hypothesize 
that the improvement in the capacity of the vascular endothelium to release t-PA with nebivolol is 
mediated, in part, by the compound’s antioxidant properties. 
 
Results supporting this hypothesis will provide important clinical data and novel mechanistic insight into 
the unique antihypertensive and cardioprotective effects of nebivolol.   
 
Background:   
The development of fibrous plaque and the resulting ulceration and subsequent thrombus formation 
within the lumen of an artery is recognized as a precipitating cause of atherothrombotic events such as 
angina, myocardial infarction, thrombotic stroke, and sudden cardiac death (47).  The hemostatic 
mechanism responsible for the proteolytic degradation of intravascular fibrin deposition is the fibrinolytic 
system.  When activated, this enzymatic pathway degrades fibrin within the developing thrombus, thus 
preventing arterial occlusion and interruption of blood flow.  Both clinical and epidemiological data 
indicate that reduced endogenous fibrinolytic activity is a major contributor to the development, 
progression, and severity of atherothrombosis (17, 18, 52, 53, 57, 59, 68).  Endothelial cells are the 
principal site of synthesis and release of tissue-type plasminogen activator (t-PA), the primary 
plasminogen activator in fibrinolysis, and a major producer of its biological inhibitor plasminogen 
activator inhibitor-1 (PAI-1) (40).  t-PA is the key enzyme in initiating an endogenous fibrinolytic 
response due to its ability to preferentially activate plasminogen on the surface of developing thrombi 
(27).  Recent data indicate that it is the capacity of the endothelium to release t-PA rapidly and acutely 
from intracellular storage pools (25, 48) and not circulating plasma fibrinolytic concentrations, that 
determines the efficacy of endogenous fibrinolysis because thrombolysis is much more effective if active 
t-PA is readily available and incorporated during, rather than after, thrombus formation (5, 28).  Thus, the 
capacity of the endothelium to release t-PA is considered to be a major endogenous defense mechanism 
against thrombosis (9, 44, 54).  It has been suggested that impaired fibrinolytic capacity may contribute to 
increased cardiovascular risk with elevated blood pressure (24), identifying an important endothelial 
target for primary and secondary prevention of atherothrombosis in prehypertensive and hypertensive 
individuals.  Very little clinical data are available regarding the effects of antihypertensive agents on 
endothelial control of the fibrinolytic system. 
 
Nebivolol, a third generation beta-blocker with high selectivity for b1-adrenergic receptors, has proven to 
be highly effective in treating hypertension (3, 14, 39).  A distinguishing feature of nebivolol from other 
beta-blockers is its hemodynamic profile, specifically the unique ability to enhance both basal and 
stimulated nitric oxide (NO) release resulting in peripheral vasodilation, improved endothelial function 
and increased myocardial compliance (19, 38, 56, 74).  However, the favorable vascular effects of 
nebivolol that contribute to its blood pressure lowering action, and possible cardioprotectant effects, may 

not be limited to endothelial vasomotor function.  Indeed, there are in vitro data to suggest that nebivolol 
may have favorable effects on the fibrinolytic system, but there is currently no in vivo clinical evidence 
that treatment with nebivolol improves vascular endothelial t-PA release.  This represents a critical void 
in understanding the potential unique antithrombotic effects of nebivolol.  
 
Elevated blood pressure, aside from aging, is the most prevalent cardiovascular disease risk factor.  Sub-
optimal blood pressure (BP>130/80) affects more than 100 million people in the United States and more 
than 1 billion people worldwide (55).  Aggressive reduction of blood pressure can significantly reduce 
cardiovascular risk and acute thrombotic events (16, 63).  Nebivolol is a well tolerated, highly effective 
antihypertensive agent with minimal adverse metabolic effects making it a useful first-line monotherapy 
treatment option for the management of patients with mild to moderate essential hypertension (19, 38, 42, 
56, 74).  The hemodynamic effects of nebivolol, specifically its NO enhancing property, differentiate it 
from traditional beta-blockers (38, 41).  While the importance of the NO system to vasoregulation and 
cardiovascular health is well established, so too is the role of endothelial fibrinolytic regulation in the 
pathogenesis of hypertension-related thrombotic risk (34).  Another potential area of differentiation 
regarding the cardiovascular benefit of nebivolol compared with other beta-blockers may be its effect on 
endothelial fibrinolytic function.  Considering endogenous fibrinolytic potential is determined at the 
vascular wall, understanding the modulatory effects of nebivolol on endothelial release of t-PA would 
provide further insight into unique vascular, and thromboprotective, effects of nebivolol.  The results of 
the proposed study will provide new and clinically important information regarding the effects of 
nebivolol on endogenous fibrinolytic potential in adults with sub-optimal blood pressure. 
 
2.  Methodology: 
 
I.)  Subjects.  Subjects will be men and women of all races and ethnic backgrounds aged 45-65 years.  The 
rationale for studying this age group is based on epidemiological data showing that the prevalence of 
suboptimal blood pressure is highest in this age-range (26).  Subjects will be normotensive defined as 
resting blood pressure <120/80 mm Hg and prehypertensive/hypertensive defined as resting systolic blood 
pressure >125 mmHg and/or diastolic blood pressure >80 mmHg (15).  The rationale for including adults 
with prehypertension is that this is a rapidly expanding segment of the population who are likely to 
develop clinical hypertension (15, 24, 26, 46, 66) and who already exhibit many of the vascular 
abnormalities common with hypertension (75). 
 
All of the women in the study will be postmenopausal and not receiving hormone replacement therapy 
(HRT) currently or in the preceding 3-year period. Our rationale for this approach is as follows.  First, if 
pre-, peri-, and postmenopausal women all were included in this cohort the widely varying circulating 
reproductive hormone levels (which may have profound effects on insulin sensitivity as well as 
endothelial vasomotor function) could introduce substantial variability into this group making it difficult 
to identify main effects of obesity/insulin resistance (if present).  Second, with respect to HRT use, the 
modes of application, the dosage of estrogen and whether or not it is combined with progesterone, the 
number of years of use, and many other factors can vary widely among individual women, adding 
significant inter-subject variability to the data. 
 
To establish blood pressure status, resting blood pressure measurements will be performed in the sitting 
position between 8 a.m. and 10 a.m. on at least 2 separate days one week apart to establish both normal 
and sub-optimal blood pressure (resting systolic blood pressure >125 mmHg and/or diastolic blood 
pressure >80 mmHg).  Subjects will not have ingested caffeine-containing beverages prior to all blood 
pressure measurements.  The recordings will be made under quiet, comfortable ambient (~24°C) 
laboratory conditions.  To avoid any possibility of investigator bias, measurements will be made with a 
semi-automated device (Dinamap, Crtikon, FL) that uses an oscillometric technique over the brachial 

artery.  Recordings will be made in triplicate in the upright sitting position.  All measurements will 
conform strictly to American Heart Association guidelines as established by the Council for High Blood 
Pressure Research (49).   
 
Candidates who smoke (currently or in the past 7 years), report more than low-risk alcohol consumption 
as defined as no more than 14 standard drinks/wk and no more than 4 standard drinks/day for men and 7 
standard drinks/wk and 3 standard drinks/day for women (a standard drink is defined as 12 ounces of 
beer, 5 ounces of wine, 1½ ounces of 80-proof distilled spirits) (71) or who are taking cardiovascular-
acting (i.e. statins) medications will not be eligible.   
 
Subjects will be free of overt chronic diseases as assessed by:  a) medical history; b) physical 
examination; c) electrocardiogram and BP at rest; d) blood chemistries and lipid and lipoprotein 
evaluation; and e) fasting plasma glucose <126 mg/dL (2).  In addition potential candidates with a resting 
heart rate of < 50 beats/minute will be excluded.  All fasting plasma lipid and lipoprotein, glucose and 
insulin concentrations will be determined by the UC-Boulder Clinical and Translational Research Center 
(CTRC) core laboratory using conventional methods (1, 10, 30). 
 
Lastly, candidates will be sedentary as determined from the Stanford Physical Activity Questionnaire 
(<35 kcal/wk; (58)) and will not have engaged in any program of regular physical activity for at least 1 
year prior to the study.  The nature, purpose, and risks of all procedures and protocols will be explained to 
each subject prior to obtaining their written informed consent.   
 
General Experimental Design:   
 
The proposed study will employ a 3-month randomized, double-bind placebo controlled single site trial to 
determine the effects of nebivolol on the capacity of the vascular endothelium to release t-PA in adults 
with suboptimal blood pressure.  An initial telephone screening will be used to ensure that interested 
candidates satisfy the basic criteria of the study (see above).  Potential candidates, after providing written 
informed consent, will be invited to undergo laboratory screening to assess blood pressure status.  
Candidates who successfully meet the blood pressure requirements and complete the screening process 
will be enrolled in the study.  The subjects will then undergo baseline testing and assessment of 
endothelial t-PA (described in detail below under “Measurements and Procedures”).  Drs. DeSouza and 
Stauffer have successfully recruited and studied human subjects across the adult age-range with varying 
levels of blood pressure in the Boulder-Denver metropolitan area since 1995 (20, 22, 23, 32, 33, 60-62, 
64, 73). 
 
Intervention.  Eligible subjects will be randomly assigned (computer generated assignment) to one of 
three treatment groups: nevibolol (5 mg/day), metoprolol (100 mg/day) or placebo (control condition).  
Subjects will participate in their respective treatment for a period of 12 weeks (~3 months).  During this 
12-week period measurements of the key outcome variables will be made at baseline and 12 weeks.  
Before randomization subjects will be reminded that the purpose of the study is to determine the effects of 
nebivolol on ET-1 vasoconstrictor tone and fibrinolytic capacity and that they will be randomly assigned 
to one of the three treatments.   
 
The subjects will be instructed to take their medication (nebivolol, metoprolol or placebo) in the morning, 
except on the morning of a testing session, and not to take any other medications during the course of the 
study. Subjects will return to the UC-Boulder Clinical and Translational Research Center (CTRC) every 
two weeks to have their body weight and blood pressure measured, receive their next allotment of pills 
and to meet with a study research assistant to discuss any problems he/she may be experiencing.  
Adherence to each treatment will be documented by bi-monthly pill counts.  The CTRC pharmacy will 

dispense all pills. 
 
Dr. Stauffer will oversee the randomization of subjects and will not be blinded to treatment group 
assignment of the subjects. 
 
Drug Tapering:  At the completion of the study, or if a subject withdraws from the study at any time, the 
doses of nebivolol and metoprolol will be tapered over a 3-day period.  Nebivolol will be tapered as 
follows:  2.5 mg/day for 3 days then stop.  Metoprolol will be tapered as follows: 50 mg/day for 1 day, 25 
mg/day for 2 days then stop. 
 
General Information for All Protocols.  All protocols will be performed in the outpatient research 
protocol laboratories in the UC-Boulder CTRC providing the highest quality of technical and clinical 
support for the safe and successful performance of the proposed intra-arterial infusion protocols. 
 
Experimental Hypothesis   
 
Nebivolol will improve endothelial t-PA release in adult humans with elevated blood pressure to a 
greater extent than either metoprolol or placebo.  We further hypothesize that the improvement in the 
capacity of the vascular endothelium to release t-PA with nebivolol is mediated, in part, by the 
compound’s antioxidant properties. 
 
Experimental Design and Strategy. To determine the effects of nebivolol on endothelial t-PA release in 
vivo, an isolated human forearm model will be used (11-13).  This approach has the advantages of 
allowing for: (a) stimulation of local and rapid endothelial release of t-PA (by intra-arterial infusion of 
various vasoactive agents) without producing potential confounding systemic responses; (b) direct 
determination of endothelial t-PA release in vivo free of the confounding effects of hepatic clearance and 
shifts between free and complexed (with PAI-1) molecular forms; (c) the study of an intact viable 
vascular bed with preserved innervations, blood flow, and cell-to-cell interaction (7, 36, 37, 65); and (d) a 
peripheral measure of endothelial t-PA release that correlates strongly and positively with t-PA release in 
the coronary circulation (45). 
 
Study Methods.  Subjects: Middle-aged and older male and female adults (see above under “Subjects”).  
Preliminary Measurements (separate day from main protocol): DXA measures of body mass and 
composition, and dietary analysis (see above under “Measurements and Procedures”).  Main Protocols: 
Assessing Endothelial t-PA Release:  The brachial artery will be catheterized as described above under 
“Measurements and Procedures”.  An intravenous catheter will then be placed in an antecubital vein of 
the same arm.  We (32, 33, 72, 73) and others (7, 36, 37, 65) have shown that brachial artery 
catheterization and intravenous catheterization in the antecubital region of the same arm is safe and well 
tolerated by adult human subjects.  The experience of the investigative team and CTRC environment will 
provide the highest level of technical and clinical support for the safe and successful performance of the 
proposed arterial and venous catheterizations.   
 
After arterial and venous catheterization, instrumentation, and establishment of steady-state baseline 
conditions and prior to beginning the infusion protocol, blood will be drawn (5 mL) from the antecubital 
vein for basal fibrinolytic measures.  Following the measurement of baseline FBF and FBF to 
acetylcholine (doses: 4.0, 8.0, 16.0 µg/100 mL tissue/min), and at the end of each dose of bradykinin and 
sodium nitroprusside, blood will be drawn simultaneously from the brachial artery and antecubital vein of 
the experimental arm to measure local release of fibrinolytic factors.  Bradykinin was selected to stimulate 
endothelial t-PA release based on its specificity and effectiveness at eliciting local and rapid endothelial t-
PA release in adult humans (6, 7).  Bradykinin will be infused at 12.5, 25, 50 ng/100 mL tissue/min and 

sodium nitroprusside at 1.0, 2.0, 4.0 µg/100 mL tissue/min for 5 min at each dose.  The concentrations of 
bradykinin have been shown to elicit significant dose-response increases in the net release of t-PA without 
affecting net release of PAI-1 (8).  Fibrinolytic determinations in response to sodium nitroprusside are 
required to establish that any observed differences in local endothelial release of t-PA to bradykinin are 
not due to increased blood flow related shear stress (8, 31, 35).  Each drug will be administered in random 
order after allowing sufficient time for FBF and plasma fibrinolytic concentrations to return to resting 
levels (30 minutes) (32, 33, 72, 73).  Net endothelial release or uptake of t-PA and PAI-1 (both antigen 
and activity levels) at each time point will be calculated as the product of the arteriovenous concentration 
gradient and the infused forearm plasma flow.  Arteriovenous concentration gradients for both t-PA and 
PAI-1 antigen and activity for each subject (at each time point) will be determined by subtraction of the 
values measured in simultaneously collected venous and arterial blood samples.  A positive difference 
indicates a net release and a negative difference, a net uptake.  Forearm plasma flow will be calculated 
from FBF and arterial hematocrit corrected for 1% trapped plasma.  Thus, net release (or uptake) will be 
calculated using the following equation (7, 37). 
 
Net Release = (CV-CA) X (FBF X [101-Hematocrit/100]) 
 
(CV-CA) = arteriovenous concentration gradient   
CV = venous concentration CA= arterial concentration 
 
Intra-Arterial Administration of Vitamin C.  Acute intra-arterial infusion of the potent antioxidant vitamin 
C will be used to assess the vascular effects of oxidative stress on bradykinin-stimulated endothelial t-PA 
release before and after each intervention.  Our rationale for selecting vitamin C over other antioxidants 
such as vitamin E is as follows.  First, vitamin C is the main water-soluble antioxidant in human plasma 
(29).  Second, it effectively scavenges superoxide and other reactive oxygen species and spares other 
endogenous antioxidants from consumption (4, 51).  Third, it plays a key role in the regulation of 
intracellular redox state through its interaction with glutathione (43, 76).  Fourth, vitamin C has been used 
successfully to demonstrate that oxidative stress contributes to endothelial fibrinolytic dysfunction in a 
number of pathologies, including hypertension (72). 
 
After allowing sufficient time (~20 minutes) for FBF to return to baseline following the initial infusions 
of bradykinin, sodium nitroprusside and vitamin C will be infused at a constant rate (12 mg/100 mL 
tissue/min) for 5 minutes.  The vitamin C dose was selected because it has been shown to both protect 
human plasma from free radical-mediated lipid peroxidation (29) and improve endothelium-dependent 
vasodilation and endothelial t-PA release in conditions associated with oxidative stress (50, 67, 69, 70, 
72).  The vitamin C infusion will be maintained at the same rate while the bradykinin and sodium 
nitroprusside dose-response curves are repeated in the same order as performed earlier.  Net endothelial 
release rates of t-PA and PAI-1 (antigen and activity) will be determined at time 0 and 5 minutes of 
vitamin C infusion and after each dose of bradykinin and sodium nitroprusside in the presence of vitamin 
C.  A 15-minute washout period will be provided between each dose response curve (72).  The acute 
vitamin C protocol will be performed at baseline and after each intervention.  
 
 
Statistical Analysis. The proposed statistical approach was chosen to take advantage of the randomized 
study design with repeated measures. Differences in the FBF responses and t-PA and PAI-1 responses to 
each vasoactive agent (bradykinin, sodium nitroprusside, bradykinin+vitamin C, sodium 
nitroprusside+vitamin C) will be determined by a mixed-effects regression model. We chose a mixed-
effect model over a repeated measures ANOVA to accommodate any missing data (due to drop-out) and 
to allow us to model a more realistic covariance structure.  The model will include factors for treatment 

condition (indicator variables for each treatment condition) and any covariates such as baseline blood 
pressure, age, gender that were not balanced under the randomized design (to be tested using one-way 
ANOVA prior to mixed model analysis).  To test for any trends over time, we may also include a term for 
the time of measurement (and interaction term of time with treatment) to determine if there is any impact 
of the time of measurement on measures of FBF. We will initially model the covariance structure of the 
repeatedly measured FBF and fibrinolytic responses assuming an unstructured pattern and test whether 
more restrictive covariance assumptions (i.e. exponential, compound symmetry) are more appropriate.  
Given the design, treatment conditions and assessment protocols, we anticipate the missing data (due to 
subject dropout) to be missing at random (MAR).  In other words, for example, we do not expect the 
initial treatment condition to influence whether a subject opts to continue with the study. Nevertheless, we 
plan to explore this issue by using all available baseline information to determine whether dropout is 
associated with any of the important covariates (baseline blood pressure, medication use and age) using 
logistic regression.  However, as emphasized previously, we do not anticipate encountering missing data 
that are not missing at random (NMAR), given the treatment conditions under study.  Assuming missing 
data that are MAR, an “intent-to-treat analysis” would yield valid inference. However, this approach 
would come at a cost of reduced statistical efficiency.  Therefore, we will also employ Schafer’s 
multivariate normal imputation approach as implemented in SAS v9.1 (SAS Institute Inc.; proc MI - 
MCMC Method and proc MIANALYZE).  According to a recent simulation study, this multiple imputation 
approach performs well and generates more reliable information than complete-case analysis. Results 
from the complete-case analysis will be compared with that of the multiple imputation analysis to ensure 
consistency of the regression parameters. To account for multiple testing, the Bonferroni-correction will 
be used to adjust the significance level appropriately. 
 
Sample Size Calculations.  All power calculations were performed using nQuery Advisor 6.0 statistical 
software (Statistical Solutions, MA). Sample sizes were determined based on data from the P.I.’s 
laboratory and available published results of comparable treatment-related changes in endothelial t-PA 
release (61, 72, 73).  Sample sizes were calculated based on 80% power at an alpha level of 0.05.  This 
power value is equivalent to a probability of <0.20 of committing a type-II error.  Power calculations 
yielded sample size of 18 subjects per treatment group (nebivolol, metoprolol, placebo) based on effect 
sizes of 0.74.  This sample size (N=54; n=18/group) should be sufficient to determine clinically and 
physiologically significant differences between treatment groups if present.  To account for an estimated 
20% dropout rate, we will recruit 22 subjects per group. 
II.)  Recruitment:  Subjects will be recruited from the greater Boulder/Denver area.  It is important to note 
that the investigative team has been conducting cardiovascular research Boulder for over 15 years.  
During this time we have established a recruiting network for middle-aged and older adults.  For example, 
our laboratory is supported by the City of Boulder Aging Services and we have access to senior centers in 
Boulder. Recruiting mechanisms include flyers posted around the community and from senior-emphasis 
organizations (see attached flyer). 
 
III.)  Copies of Surveys, Questionnaires, or Interview Schedules: Copy of Stanford Physical Activity 
Questionnaire will be submitted electronically. 
 
IV.)  Description of Procedures: All procedures will be performed at the CTRC unless otherwise stated.  
Estimated time of each procedure is provided in bold.  The order in which the following procedures will 
be performed, in separate experimental sessions, is provided below under “Sequence of Measurements”. 
 
1)  Telephone Screening/Subject Information (performed by laboratory personnel at the Integrative 
Vascular Biology Laboratory located in Carlson Gymnasium).  When interested candidates voluntarily 
call the laboratory (after seeing our advertisements or through word of mouth) for more information on 

the proposed study and once we have obtained verbal consent according to prescreen consent form they 
will be asked general questions regarding their, age, physical activity status, medical history to determine 
whether they satisfy the basic criteria for the study.  Subjects who meet the basic criteria for the study will 
be invited to come to the CTRC to discuss the study in more detail, have the informed consents explained 
to them, and to provide written informed consent for blood pressure screening, and if appropriate, to 
participate in the study.  Subjects who provide written informed consent will be scheduled to undergo 
screening and testing procedures at the University of Colorado CTRC.  For subjects who do not meet the 
inclusion criteria all clinical screening information will be kept for a period of 10 years.  All other 
information will be destroyed.  Total time: 60 minutes. 
 
2) Physical Examination and Resting ECG.  A physician will perform a standard medical exam (like an 
office visit with a doctor) including vital signs such as heart rate and blood pressure as well as 
examination of ear, nose, throat, eye exam, and listen to your lungs and heart.  Total time: 30 minutes. 
 
3) Body Composition or body fat percentage will be measured non-invasively by skinfold thickness and 
by Dual Energy X-ray Absorptiometry or DXA.  This test involves subjects lying on a padded table while 
a small probe, that emits energy to measure tissue density, passes over their body. This test will be 
performed before and after each intervention.  Total time: 30 minutes. 
 
4) Blood Pressure will be measured non-invasively in the laboratory by the standard cuff technique (i.e., 
as in a Doctor’s office) and using an automated blood pressure monitor. Total time: 20 minutes. 
 
5) Daily Physical Activity will be estimated using a standardized questionnaire.  The questionnaire will be 
administered via personal interview with subject by a trained exercise physiologist associated with the 
CTRC.  Total time: 30 minutes. 
 
6) Dietary Recall Records will be used to determine total daily caloric intake and the amounts of 
carbohydrates, protein, and fat in subject’s diets.  Subjects will be instructed on how to complete their 
food records by the CTRC bionutritionist who will also review all records.  Total time for dietary 
instruction: 45 minutes. 
 
7) Blood Draw to Measure Blood Chemistries and Risk Factors for Cardiovascular Disease in order to 
measure well established risk factors for cardiovascular disease (such as blood cholesterol, glucose and 
insulin levels, oxidative markers, inflammatory cytokines, microparticles and microRNA) and to screen 
for disease subjects will be asked to fast overnight for 12 hours prior to the blood draw.  A small needle 
will be inserted into a vein in the forearm to collect a sample of blood (~ 50 mL) for these risk factor 
determinations.  Total time: 15 minutes. 
 
8) Forearm Blood Flow will be measured by placing a flexible rubber cuff around the forearm while 
periodically inflating and deflating two blood pressure cuffs (one around the upper arm and one around 
the wrist).  Flow will also be measured in response to the local infusion of small safe quantities of 
vasoactive drugs (bradykinin, sodium nitroprusside and vitamin C) into the brachial artery via an 
indwelling catheter. Bradykinin and sodium nitroprusside will cause blood flow to the forearm to 
increase.  Note, a licensed medical person will place all indwelling arterial catheters and administer all 
vasoactive drugs. Total time: 4.0 hours. 
 
9)  Fibrinolytic Factors.  Blood samples for determination of fibrinolytic factors will be collected in 
chilled tubes containing 0.45 mol/L sodium citrate buffer, pH 4.3 (Stabilyte, Biopool AB; final dilution 
volume 1:10) and stored as previously described by the P.I. (31, 32, 61).  Arterial and venous plasma 
concentrations of t-PA and PAI-1 antigen and activity will be determined by enzyme immunoassay (31).  

Reliability and reproducibility of these assays in our laboratory have been reported (21). These analyses 
will be performed in the Department of Integrative Physiology Core Biochemistry Laboratory.  Total 
amount of blood to be collected is ~100 mL. 
 
Study Sessions:  Table 1 provides an overview of the study sessions associated with the study and the 
measurements to be performed at each session. 
 
 
 Session 
Screening and Experimental 
Measurement 
(All measures performed at 
the UC-Boulder CTRC) 
Time 
1 
(Screening) 
 
Blood Pressure  
Resting ECG and Physical 
Examination 
Blood Draw 
20 min 
30 min 
 
15 min 
 
2 
(Experimental) 
 
 
Body Composition 
Daily Physical Activity 
Dietary Recall Records 
 
30 min 
30 min 
45 min 
3 
(Experimental) 
 
Assessment of Endothelial 
Fibrinolytic Capacity 
4.0 hrs 
4 
(Experimental) 
 
Blood Pressure 
Body Weight 
30 min 
5 
(Experimental) 
 
Blood Pressure 
Body Weight 
30 min 
6 
(Experimental) 
 
Blood Pressure 
Body Weight 
30 min 
7 
(Experimental) 
 
Blood Pressure 
Body Weight 
30 min 
8 
(Experimental) 
 
Blood Pressure 
Body Weight 
30 min 
9 
(Experimental) 
 
Blood Pressure 
Body Weight 
30 min 
10 
(Experimental) 
 
 
Body Composition 
Daily Physical Activity 
Dietary Recall Records 
30 min 
30 min 
45 min 
11 
(Experimental) 
 
 
Assessment of Endothelial 
Fibrinolytic Capacity 
4.0 hrs 
 

3.  Benefits and Risks. Indirect Benefits: Subjects will receive the following benefits: 1) medical 
information-- a medical history, physical examination, blood pressure readings, blood levels of glucose 
and insulin, blood fats, and resting electrocardiograms free of charge; 2) physical information-- 
measurement of body fat and bone density levels free of charge; and 3) financial compensation—subjects 
will receive $40.00 for each experimental session completed (maximum $400 for participating in the 
study). 
 
Risks:  The risks associated with the experimental protocols include:  
 
1.  Body Composition: there is a small amount of radiation exposure (0.04 mRem) associated with the 
DEXA scan. This amount of exposure is less than the amount that a subject would receive on daily basis 
living in Colorado. This study will expose a subject to radiation in addition to the natural background 
radiation a subject would receive each day. The more radiation a subject receives over the course of their 
life, the greater the risk of having cancerous tumors or inducing changes in genes. The radiation in this 
study is not expected to greatly increase these risks, but the exact increase in such risks is unclear.  
 
2. Venous Catheter Risks: a hollow needle/plastic tube will be placed in your arm for taking small blood 
samples, giving fluids, or infusing glucose and insulin (intravenous glucose tolerance test).  This will be 
left in for as long as 4 hours.  When the needle goes into a vein, it hurts for a short time.  Also there will 
be the minor discomfort of having the needle/plastic tube taped to the subjects arm.  In about 1 in 10 cases 
a small amount of bleeding under the skin will produce a bruise.  The risk of a blood clot forming in the 
vein is about 1 in 100, while the risk of infection or significant blood loss is 1 in 1,000.  There is a side 
effect/risk of participants fainting as a result of placing the venous catheter and/or drawing blood from the 
vein. 
 
3. Arterial Catheter Risks: placing a catheter into an artery causes some discomfort/pain, however, 
subjects will receive a small amount of numbing medication to lessen this discomfort/pain.  In about 1 in 
10 cases a small amount of bleeding under the skin will produce a bruise.  Very rarely (1 in 4,000) 
damage may occur to the artery which can result in decreased blood flow to the forearm or hand caused 
by a blood clot or artery spasm.  Although rare, these conditions can be serious if they develop and may 
require surgery to correct.  Tests involving arterial catheters will be performed twice – before and after the 
3-month intervention. There is a side effect/risk of participants fainting as a result of placing the arterial 
catheter and/or drawing blood from the artery. 
 
4.  Infusion of Vasoactive Drugs: infusion of bradykinin and sodium nitroprusside cause only local 
changes in forearm blood flow.  Since low doses of these drugs will be used there is little or no effect on 
the rest of the body.  There is no pain or discomfort associated with the drug infusions.  The only 
significant risks associated with the infusion of the drugs are: a small drop or increase in blood pressure 
(+5-7 mm Hg); very slight chance of chest tightness, light-headedness, headache, and fainting; slight 
chance of an allergic reaction. There are no documented cases of serious events such as heart attack or 
stroke with the small doses of the drugs used in this procedure.  The investigators have FDA approval to 
for this research (FDA IND#s are provided in the electronic application).  Drs. DeSouza and Stauffer 
are currently conducting UC-IRB approved research protocols involving the above vasoactive 
drugs at the doses outlined in this protocol. 
 
5.  Vitamin C:  The intra-arterial dose (24 mg/min) used for this study has been shown to be safe and 
without side effects. 
 
6.  Nebivolol: The most common adverse reactions that may occur with nebivolol are headache, nausea, 
diarrhea, tiredness, dizziness, slow heart rate, low blood pressure (hypotension) insomnia and leg 

swelling.  Subjects will be monitored closely throughout the study.  Should they experience any of these 
symptoms or other symptoms that they think might be associated with the treatment they are instructed to 
call the CTRC at 303-735-2304 or after hours at 303-206-6339 (nurse pager).  The nurse may contact Dr. 
Brian Stauffer if necessary.  Drug package insert for nebivolol has been submitted electronically.  
Subjects will be asked to not stop taking their treatment pills without consulting with a CTRC nurse or the 
study investigators. 
 
7. Metoprolol (TOPROLOL-XL [extended release]):  The most common adverse reactions that may occur 
with metoprolol are tiredness, dizziness, slow heart rate, diarrhea, shortness of breath, pruritus (itching), 
rash, depression, and low blood pressure (hypotension).  Subjects will be monitored closely throughout 
the study.  Should they experience any of these symptoms or other symptoms that they think might be 
associated with the treatment they are instructed to call the CTRC at 303-735-2304 or after hours at 303-
206-6339 (nurse pager).  The nurse may contact Dr. Brian Stauffer if necessary.  Drug package insert for 
metoprolol has been submitted electronically. 
 
There are no alternative methods that would provide the same type of accuracy of information as the 
state-of-the-art procedures proposed in this application.  It is important to note that all invasive procedures 
will take place at the University of Colorado CTRC under the supervision of Dr. Stauffer or the CTRC 
medical staff.  Furthermore, emergency medical care will be immediately available during all testing.  
There are no risks to the investigators.  
 
4.  Assurance of Confidentiality. All data collected on individual subjects will only be viewed by the 
investigative team involved in the project (i.e., the Principal Investigator, Research Assistant, and Pre- 
and Post doctoral Research Fellows, and CTRC staff), although these data will be provided to the subject. 
The individual data for each subject will be analyzed and stored by code, and at no time will any 
individual data point be identified with a particular individual subject.  The data will almost exclusively 
be presented as average responses. All data will be stored on hard and compact disk using the 
aforementioned codes. All data and information regarding the study will be stored in a secure (always-
locked) filing cabinet in the Integrative Vascular Biology Laboratory in the Carlson building on the CU-
Boulder campus.  Data will be kept for 10 years after the study is completed then erased.  
 
5.  Consent Form.  See Attached. 
 
6.  Investigator’s Qualifications. The principal investigator for this project is Christopher A. DeSouza, 
Ph.D.  Dr. DeSouza is Professor in the Department of Integrative Physiology.  Dr. DeSouza received his 
doctoral training in the Department of Kinesiology, University of Maryland and Division of Gerontology 
at the Baltimore Veterans Affairs Medical Center in Baltimore Maryland and has been conducting 
research in the area of vascular function and aging in humans for the past 15 years.  Dr. Stauffer is a board 
certified cardiologist and an Associate Professor of Cardiology in the Department of Medicine at the UC- 
Denver.  Dr. Stauffer has extensive experience conducting studies involving vasoactive drugs such as the 
ones proposed herein.  The medical staff at the CTRC are appropriately trained and have experience 
safely administering the procedures outlined in this protocol. 
 
7.  Publications employing the use of bradykinin and sodium nitroprusside in different study populations. 
 
 
 
 
 

Bradykinin and Sodium Nitroprusside References: 
 
Cardillo C, CM Kilcoyne, AA Quyyumi, RO Cannon, and JA Panza. Selective defect in nitric oxide 
synthesis may explain the impaired endothelium-dependent vasodilation in patients with essential 
hypertension. Circulation: 851-856, 1998. 
DeSouza CA, LF Shapiro, CM Clevenger, FA Dinenno, K D Monahan, H Tanaka, and DR Seals. Regular 
aerobic exercise prevents and restores the age-related decline in endothelium-dependent vasodilation in 
healthy men. Circulation 102: 1351-1357, 2000. 
Hambrecht R, E Fiehn, C Weigl, S Gielen, C Hamann, R Kaiser, J Yu, V Adams, J Niebauer, and G 
Schuler. Regular physical exercise corrects endothelial dysfunction and improves exercise capacity in 
patients with chronic heart failure. Circulation 98: 2709-2715, 1998. 
Hambrecht R, A Wolf, S Gielen, A Linke, J Hofer, S Erbs, N Schoene, and G Schuler. Effect of exercise 
on coronary endothelial function in patients with coronary artery disease. New England Journal of 
Medicine 342: 454-460, 2000. 
Higashi Y, S Sasaki, S Kurisu, A Yoshimizu, N Sasaki, H Matsuura, G Kajiyama, and T Oshima. Regular 
aerobic exercise augments endothelium-dependent vascular relaxation in normotensive as well as 
hypertensive subjects.  Role of endothelium-derived nitric oxide. Circulation 100: 1194-1202, 1999. 
Panza JA, PR Casino, CM Kilcoyne, and AA Quyyumi. Impaired endothelium-dependent vasodilation in 
patients with essential hypertension: evidence that the abnormality is not at the muscarinic receptor level. 
Journal of American College of Cardiology 23: 1610-1616, 1994. 
Panza JA, PR Casino, CM Kilcoyne, and AA Quyyumi. Role of endothelial-derived nitric oxide in the 
abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation 
87: 1468-1474, 1993. 
Panza JA, CE Garcia, CM Kilcoyne, AA Quyyumi, and RO Cannon. Impaired endothelium-dependent 
vasodilation in patients with essential hypertension.  evidence that nitric oxide abnormality is not 
localized to a single signal transduction pathway. Circulation 91: 1732-1738, 1995. 
Brown NJ, JV Gainer, LJ Murphy, and DE Vaughan. Bradykinin stimulates tissue plasminogen activator 
release from human forearm vasculature through B2 receptor-dependent, NO synthase-independent, and 
cylcooxygenase-independent pathway. Circulation 102: 2190-2196, 2000. 
Brown NJ, JV Gainer, CM Stein, and DE Vaughan. Bradykinin stimulates tissue plasminogen activator 
release in human vasculature. Hypertension 33: 1431-1435, 1999. 
Brown NJ, JH Nadeau, and DE Vaughan. Selective stimulation of tissue-type plasminogen activator in 
vivo by infusion of bradykinin. Thrombosis and Haemostasis 77: 522-525, 1997. 
Gazis A, DJ White, SR PAge, and JR Cockcroft. Effect of oral vitamin E (a-tocoperol) supplementation 
on vascular endothelial function in Type 2 diabetes mellitus. Diabetes Medicine 16: 304-311, 1999. 
Gilligan DM, V Guetta, JA Panza, CE Garcia, AA Quyyumi, and RO Cannon. Selective loss of 
microvascular endothelial function in human hypercholesterolemia. Circulation 90: 35-41, 1994. 
Hornig MLH, P Smits, PJ Morrison, and TJ Rabelink. Bradykinin-induced vasodilation of human forearm 
resisitance vessels is primarily mediated by endothelium-dependent hyperpolarization. Hypertension 35: 
1314-13-18, 2000. 
Panza JA, CE Garcia, CM Kilcoyne, AA Quyyumi, and RO Cannon. Impaired endothelium-dependent 
vasodilation in patients with essential hypertension.  evidence that nitric oxide abnormality is not 
localized to a single signal transduction pathway. Circulation 91: 1732-1738, 1995. 
 
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