HIV-1, Insufficient Sleep and Vascular Endothelial Dysfunction
Phase: NA
Conditions: HIV-1
Sponsor: University of Colorado, Boulder

 
 
HIV-1, Insufficient Sleep and Vascular Endothelial Function 
 
NCT04956861 
 
Date: 03/08/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

I. 
Research Design and Methods (Be sure to include a statistical analyses plan and sample size 
justification as part of this section)  
Subjects:  Study subjects will be recruited through the Infectious Disease Group 
Practice (IDGP) located at the University of Colorado Hospital, which cares for 
approximately 1,700 HIV-1-infected individuals including more than 300 HIV-
infected women.  Subjects will be men and women of all races and ethnic 
backgrounds aged 40-75 years with documented HIV-1 infection.  Subjects will be 
HIV-1-seropositive individuals on a stable Department of Health and Human 
Services approved Anti-retroviral (ART) regimen for at least 6 months, with 
documented virologic suppression (<50 copies HIV-1 RNA/mL) for at least 3 
months. All subjects must have CD4+ T cell counts >200 cells/mm3 at the time of 
study entry.  Importantly, in 2014, 42% of the patients cared for in the IDGP were 50 
years of age of older, 85% of clinic patients had a viral <50 copies Ml and the mean 
clinic patient CD4+ T cell count was 613 cells/mm3.  Thus, there should be ample 
potential study subjects in the clinic population.  Subjects will be free of overt 
Cardiovascular disease (CVD) as assessed by:  a) medical history; b) physical 
examination; c) electrocardiogram and BP at rest and maximal exercise; d) complete 
blood chemistries, lipid and lipoprotein (35), glucose, insulin and hematological 
evaluation.  Subjects will be free of a recent (<3 months) acute infection or surgery.  
All candidates will be sedentary as determined from the Stanford Physical Activity 
Questionnaire (<35 kcal/wk; (89)) and will not have engaged in any program of 
regular physical activity for at least 6 months prior to the study.  Habitual nightly 
sleep duration and quality will be determined by self-report using the Pittsburgh 
Sleep Quality Index questionnaire (PSQI) (14), Berlin Questionnaire for sleep apnea, 
Insomnia Severity Index (ISI), and Epworth Sleepiness Scale (ESS). A board certified 
sleep clinician will also conduct a clinical assessment of sleep disorder.  Potential 
study candidates will stratified into 2 experimental groups: normal sleep (> 7 
hr/night) or short sleep (< 6.5 hr/night) based on their habitual sleep behavior. 
 
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.  Firstly, estrogen and HRT use can 
markedly influence endothelial t-PA release (48) producing widely varying baseline 
levels within groups of women.  Furthermore, these effects may persist for years 
after the cessation of use (65).  It is also possible that women who receive HRT may 
differ constitutionally from non-users.  Secondly, 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 (continual or intermittent), and many 
other factors can vary greatly among individual women, adding significant inter-
subject variability to the data.  Thirdly, 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 endothelial function) could introduce 
substantial variability into this group making it difficult to identify main effects of 
insufficient sleep (if present).  Taken together, HRT use represents an important and 
avoidable potential confound in the interpretation of the results of the proposed 
studies. We realize that the exclusion of these groups will somewhat limit the 

generalizability of our results.  However, few women in the IDGP are taking HRT and 
therefore, this is not likely to significantly reduce ability to recruit subjects.  
Postmenopausal status will have been confirmed clinically by the subject’s personal 
physician along with demonstration of plasma levels of follicle stimulating hormone 
(FSH) >40 IU/L (92). 
 
Experimental Protocols 
 
General Information for Vascular Protocols.  All subjects will be studied in the 
supine position starting 1 to 2 hours after habitual morning wake time following an 
overnight fast with proper hydration (water drinking only). In addition, 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 
and drug trial.  The UC-Boulder CTRC is located 22 miles northwest of the UC 
medical campus in Denver.  Drs. Connick, DeSouza and Stauffer have conducted 
clinical studies on the Boulder campus for close to a decade involving HIV-1 
populations from the medical campus without difficulties/problems involving 
subjects commuting between campuses. 
 
Experimental Design.  The proposed study design will consist of two separate 
phases to address the stated specific aims.  Phase I is designed to assess endothelial 
vasodilator and fibrinolytic function in HIV-1-seropositive adults who habitually 
sleep more than 7 hours/night (normal sleep) and those who habitually sleep less 
than 6.5 hours night (short sleep) (Specific Aims 1 and 2).  Phase II is designed to 
determine the effects of individualized targeted sleep interventions that increase 
sleep duration and improve sleep on endothelial vasodilator and fibrinolytic 
function in HIV-1 seropositive adults who habitually sleep less than 6.5 hours/night 
(Specific Aims 3).  It is important to note that HIV-1 subjects who habitually sleep 
less than 6.5 hours/night who participate in the acute studies associated with Phase 
I will not necessarily be interested in participating in the intervention trial 
associated with Phase II, although they will be given the option of doing so. Subjects 
interested in Phase II will be consented during or at the end of Phase I. 
 
Example timeline.  Detailed information for each procedure provided in 
following sections. 
 
 
PHASE I Vascular Studies: ~10.5 hours 
 
Visit 1 CTRC: Time approximately 2 hours Physical Examination, Blood Chemistries 
and Risk Markers for Heart Disease, Blood Pressure, Treadmill Stress Test 
Visit 2 CTRC: Time approximately 3.0 hours, Maximal Exercise Test, Body 
Composition, Heart Rate, Dietary Records, Physical Activity Questionnaire, Sleep 
Questionnaires 

 
Ambulatory sleep monitoring: Days between Visits 2 and 3 
  
Each subject will keep a sleep/wake log at home and wear a wrist activity and light 
exposure recorder (Actiwatch Spectrum, Philips Respironics, Bend, OR) between 
Visits 2 and 3 to assess their sleep. Subjects will be given an instruction sheet on 
how to use the Actiwatch. These ambulatory techniques have proven successful in 
examining the timing of sleep and obtaining an objective estimate of sleep duration 
and quality (e.g., awakenings during the night).  We will use these data to examine 
associations between sleep and vascular outcomes. 
 
Visit 3 CTRC: Time approximately 5.5 hours Forearm Blood Flow, Measure of 
Fibrinolytic Capacity 
 
Compensation for Phase I is $125.00 per visit for a total compensation up to $375. 
 
PHASE II Sleep Intervention:  ~12.3 weeks Baseline sleep assessments 
Visit 1 Sleep lab 
• Days 1 
- Initial sleep assessment 
• Days 2-8 
- Ambulatory monitoring 
Visit 2 Sleep lab 
• Day 8 
- Arrive at the sleep laboratory in the afternoon and provide 
saliva samples each hour for 24 hours 
 
Sleep health intervention 
• 8 weeks 
 
 
Follow-up sleep assessments 
Visit 3 Sleep lab  
• Days 1 
– Follow-up sleep assessment (including sleep questionnaires) 
• Days 2-8 
Ambulatory monitoring 
Visit 4 Sleep lab  
• Day 8 
Arrive at the sleep laboratory in the afternoon and provide saliva 
samples each hour for 24 hours 
 
Follow-up vascular assessments 
Visit 5 CTRC: Time approximately 3.0 hours, Maximal Exercise Test, Body 
Composition, Heart Rate, Dietary Records 
 
Visit 6 CTRC: Time approximately 5.5 hours Forearm Blood Flow, Measure of 
Fibrinolytic Capacity 
 
Compensation for Phase II is $125 for participating in each overnight sleep study 
(Visits 1 and 3), $125 per week of ambulatory monitoring (2 weeks x $125 each), 
$125 for each 24h sleep lab visit (Visits 2 and 4), and  

$125 for CTRC Visits 5 and 6.  Maximum compensation for participating in the sleep 
health component of this study is $1000.00.  The 8 week sleep health intervention 
will be provided free of charge and there is no further compensation for the sleep 
health intervention component. 
 
Specific Aims 1 and 2:  To determine the influence of chronic insufficient sleep 
on endothelium-dependent nitric oxide-mediated vasodilation and endothelial t-
PA release in ART-treated HIV-1-seropositive adults. 
 
Experimental Strategy.  Endothelium-dependent, NO-mediated vasodilation and 
endothelial t-PA release will be determined using an isolated human forearm model 
(28, 109).  This model has the advantages of allowing for the: a) measurement of 
whole foream blood flow (FBF;  by plethysmography); b) manipulation (by intra-
arterial infusion of vasoactive drugs) of regional blood flow, via different 
mechanisms, without producing potentially confounding systemic effects; (c) 
stimulation of local and rapid endothelial release of t-PA (by intra-arterial infusion 
of various vasoactive agents) without producing potential confounding systemic 
responses; (d) 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; (e) the study of an intact viable vascular bed with 
preserved innervations, blood flow, and cell-to-cell interaction (12, 53, 54, 96); and 
(f) a peripheral measure of endothelial vasodilator and fibrinolytic function that 
correlates strongly and positively with coronary vascular function (63, 71). 
 
Study Methods: Subjects: HIV-1-seropositive adults receiving ART (see above under 
“Subjects”).  Plasma HIV-1 RNA measurements will be determined by RT PCR by the 
University of Colorado Hospital Virology Laboratory.  CD4+ T cell counts will be 
determined by flow cytometry by the University of Colorado AIDS Research Center 
Flow Cytometry Lab located at the University of Colorado at Denver.  Preliminary 
Measurements: (separate day from main protocol): measures of body mass and 
composition will be determined by basic anthropometric techniques as well as by 
dual energy x-ray absorptiometry (58); graded exercise test to screen for underlying 
coronary artery disease; maximal oxygen consumption will be used as a measure of 
aerobic fitness to document the sedentary status of the subjects.  It will be 
determined using a modified Balke incremental treadmill exercise protocol, as 
previously described by the P.I. (29).  Daily energy expenditure will also be 
estimated using the Stanford Physical Activity Questionnaire (PAQ) (89).  Because 
there is no consensus gold standard plasma marker of oxidative stress, oxidative 
stress will be assessed by a variety of standard measures; plasma total 8-epi-PGF2a 
by gas chromotography (44), plasma oxLDL and MPO by enzyme immunoassay 
(Mercodia AB, Sweden) (112).  Biomarkers of inflammation (CRP, TNF-a, IL-6, IL-
18) and endothelial markers will be determined by enzyme immunoassay as 
previously described by Dr. DeSouza (107).  Experimental Protocol:  Assessing 
Endothelial Vasodilator and Fibrinolytic Function:  The brachial artery of the non-
dominant arm will be catheterized as we have previously described (47, 48, 93, 
109).  An intravenous catheter will then be placed in an antecubital vein of the same 

arm.  We (47, 48, 93, 109) and others (12, 53, 54, 96) 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.  FBF at rest and in 
response to each vasoactive agent will be measured using venous occlusion 
plethysmography as previously described by the P.I. (30).  Endothelium-dependent 
vasodilation will be assessed by changes in FBF in response to intra-arterial 
infusions of the endothelial agonists, acetylcholine and bradykinin.  FBF responses 
to intra-arterial sodium nitroprusside will be used to assess endothelium-
independent vasodilation.  Acetylcholine will be infused at rates of 4.0, 8.0, 16.0 
µg/100 mL of forearm tissue/min, bradykinin at 12.5, 25, 50 ng/100 mL of forearm 
tissue/min, and sodium nitroprusside at 1.0, 2.0, 4.0 µg/100 mL forearm tissue/min 
for 5 min at each dose. To avoid an order effect the sequence in which these agents 
will be administered will be randomized.  To determine the contribution of NO to 
acetylcholine-mediated vasodilation, the acetylcholine dose-response curves will be 
repeated in the presence of the endothelial NO synthase inhibitor L-NMMA.  After 
allowing 15 minutes for FBF to return to baseline, L-NMMA will be infused at 5 
mg/min for 10 minutes intra-arterially.  This dose of L-NMMA has been shown to 
safely and effectively inhibit endogenous NO production in adult humans (94).  
Bradykinin will not be co-infused with L-NMMA because bradykinin stimulated 
forearm vasodilation is largely mediated by endothelium-derived hyperpolarizing 
factor rather than NO (50).  
 
To assess endothelial t-PA release, following the measurement of baseline FBF 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 (11).  Fibrinolytic 
determinations in response to sodium nitroprusside are required to establish that 
any observed differences in local endothelial release of t-PA to bradykinin is not due 
to increased blood flow related shear stress (12, 53). Net endothelial release or 
uptake of t-PA and PAI-1 (both antigen and activity levels) at each dose of 
bradykinin and sodium nitroprusside will be calculated as the product of the 
arteriovenous concentration gradient and the infused forearm plasma flow (12, 54).  
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.  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 (12, 54). 
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:  After allowing sufficient time (45 
minutes) for FBF to return to baseline following the infusions of acetylcholine + L-
NMMA, vitamin C will be infused at a constant rate (12 mg/100 mL tissue/min) for 5 
minutes.  This vitamin C concentration has been shown to both protect human 
plasma from free radical-mediated lipid peroxidation (37) and improve 
endothelium-dependent vasodilation and endothelial t-PA release in conditions 
associated with oxidative stress (79, 97, 101, 106).  Vitamin C infusion will be 
maintained at the same rate while the bradykinin, sodium nitroprusside and 
acetylcholine 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 (106). 
 
Statistical Analysis for Specific Aims 1 and 2.  For Aims 1 and 2 we will deploy 
standard linear regression techniques to evaluate whether chronic insufficient sleep 
is associated with (Aim 1) endothelium-dependent nitric oxide-mediated 
vasodilation and (Aim 2) vascular endothelium capacity to release tissue-type 
plasminogen activator. In both scenarios, the continuous vascular measures from 
Aim 1 and Aim 2 will be modeled as dependent variables in separate linear 
regression procedures. Independent variables for these models will include the 
primary variable of interest - insufficient sleep - as well as other important 
covariates including age, sex, race, BMI, CD4 count, and duration of ART. The 
primary test of the hypothesis will focus on the parameter estimate for insufficient 
sleep for both models under Aims 1 and 2.  
 
Sample Size Calculations For Specific Aims 1 and 2.  Sample sizes were determined 
based on data from Dr. DeSouza’s laboratory and available published results of 
comparable condition-related (e.g. hypertension, obesity) group differences (<6.5h 
versus >7h habitual sleep) in acetylcholine-mediated endothelium dependent 
vasodilation and bradykinin-stimulated endothelial t-PA release (13, 77, 81, 93, 98, 
99, 105, 106, 108, 109).   Sample sizes were calculated based on 90% power at an 
alpha level of 0.05.  This power value is equivalent to a probability of <0.10 of 
committing a type-II error.  Power calculations yielded sample size of 28 subjects 
per sleep group based on effect sizes of 0.74.  This sample size (N=56; n=28/group) 
should be sufficient to determine clinically and physiologically significant 
differences between groups if present.  To account for an estimated 20% dropout 
rate, we will enroll 36 subjects. 
 
Specific Aim 3:  To determine if individualized targeted sleep interventions 
that increase sleep duration and improve sleep quality improve vascular 
endothelium-dependent nitric oxide-mediated vasodilation and endothelial 
tissue-type plasminogen activator release in ART-treated HIV-1-seropositive 
adults. 
 

Experimental Strategy.  Middle-aged and older ART-treated HIV-1-serpositive adults 
who completed Phase I and habitually sleep less than 6.5 hours/night will be offered 
to undergo 8 weeks of an individualized targeted sleep intervention and follow-up 
vascular assessments.  Given the heterogeneity of sleep problems in patients with HIV-
1 infection resulting in habitual short nightly sleep duration, a “one size fits all” 
approach is not appropriate for optimization of sleep health in this population. Thus, 
we have assembled a team of sleep and circadian specialists to provide an 
individualized targeted intervention.  After a comprehensive sleep and circadian 
assessment of each subject, members of the investigative team will meet to create 
an individualized targeted treatment plan.  Expert sleep clinicians will implement 
the plan and use established procedures to monitor compliance. 
 
Study Design and Methods.  Subjects.  Middle-aged and older ART-treated HIV-1-
serpositive adults who habitually sleep less than 6.5 hours/night.  Baseline Sleep 
and Circadian Assessments.  Baseline PSG assessment will occur in the Sleep and 
Chronobiology Laboratory to determine sleep EEG staging using a standard 8-hour 
sleep opportunity at the participant’s preferred bedtime. The baseline PSG 
recording will also be used to confirm absence of sleep apnea, periodic limb 
movements, and other sleep disorders. PSG records will be  interpreted by a board 
certified sleep clinician. Participants’ sleep will then be monitored for a week using 
standardized sleep diaries, wrist actigraphy recorders with concurrent light 
exposure assessment, and call-ins of bed and wake times to a time stamped voice 
recorder.   This will be followed by a second laboratory visit to assess circadian 
melatonin phase. 
 
Individualized Targeted Sleep Intervention.  Older ART-treated HIV-1 infected adults 
experience a variety of sleep problems.  All sleep problems result in sleep deficiency 
and share features that contribute mechanistically to vascular disease.  Our working 
hypothesis is that regardless of the specific sleep problem, improving sleep will 
improve vascular endothelial function.  We will employ an 8-week individualized 
targeted sleep intervention.  Individualized targeted interventions have the 
advantage of improving adherence, reducing attrition, and making the strategy 
personally meaningful.  We chose an 8-week duration for the targeted sleep 
intervention since Cognitive Behavioral Therapy for Insomnia (CBT-I) is a 6-week 
long treatment and endothelial function has been shown to improve following 4-8 
weeks of CPAP treatment for sleep apnea (52, 75, 103). 
 
Participants will be provided all sleep treatments at no cost. 
 
All subjects will be given three brochures published by the American Academy of 
Sleep Medicine and National Sleep Foundation: one on sleep and health, one on 
insomnia, and the other on sleep hygiene (Helping Yourself To a Good Night’s Sleep).  
We will supplement this information with an individualized targeted sleep medicine 
approach to address individual sleep disturbance profiles.  Sleep and circadian 
expert members of the investigative team will use results from baseline assessments 
and clinical assessment of sleep disorders to develop the individualized targeted 

intervention. We will use evidence based sleep medicine best practice strategies and 
combined treatments to optimize sleep and treat sleep disorders.  We will use the 
International Classification of Sleep Disorders 3rd Edition (ICSD-3) diagnostic 
criteria.  The following strategies are not meant to be comprehensive of all possible 
treatment approaches rather details are provided for treatment of disorders most 
likely to be seen in our subject population. 
 
 
Insomnia. We estimate that approximately 66% of patients with HIV studied 
will meet ICSD-3 criteria for insomnia.  Insomnia will be treated using the approach 
of Morin et al. consisting of a 6-week course of CBT-I combined with 5-10mg 
zolpidem nightly (5mg females/5-10mg males per standard guidelines; taken at 
bedtime) and discontinuation of zolpidem at the end of the 6 week treatment 
(titrated reduction). We selected this approach as the addition of medication to CBT-
I has been shown to result in a more rapid improvement in insomnia symptoms and 
since treatment outcome can be maintained long-term with learned CBT-I skills (68, 
69). The CBT-I protocol will be conducted by the behavioral sleep medicine 
specialist in individual sessions for 60 min once a week for the first 4 weeks. The 1st 
stage of CBT-I will involve sleep restriction and stimulus control therapies (31, 32, 
88).  These therapies will strengthen homeostatic sleep drive, consolidate sleep by 
reducing time in bed, establish a regular sleep schedule, and curtail sleep-
incompatible behaviors. The 2nd stage of CBT-I will utilize cognitive therapy to 
attenuate sleep-disruptive and mood-disturbing cognitions that exacerbate 
insomnia. Standard procedures will be used to identify and alter negative cognitive 
activity by recording automatic thoughts, Socratic dialog, constructive worry, and 
behavioral experiments (19, 73).  After the first 4 weeks of treatment, telephone 
calls to each participant, once in each of weeks 5 and 6, will be used to discuss 
strategies to maintain improvements beyond the treatment period. Tapering of 
zolpidem treatment--Those on 10mg will be tapered to 5mg per night for one week, 
5mg every other night during a second week, and then stop medication the third 
week. Those on 5mg per night will go to an every other night regimen for one week 
and then stop medication the second week. Those on existing sleep medication that 
are ineffectively treated will be tapered before beginning treatment. Subjects will 
continue practicing CBT-I until post-treatment assessments.  Dr. Wright has 
conducted multicenter clinical trials on insomnia, as well as circadian rhythm sleep 
disorders that produce insomnia complaints, to provide understanding of the 
pathophysiology, daytime consequences, and effective treatment strategies for such 
sleep difficulties in young and older adults with primary insomnia (86) and patients 
with circadian sleep disorders (120).  
 
Although there have been no clinical trials assessing the effectiveness of CBT-I for 
insomnia in adults with HIV-1, CBT-I has been shown to be effective in the 
treatment of insomnia in patients with other medical comorbidities. Thus we are 
confident that patients with HIV will also respond to CBT-I treatment for insomnia 
when combined with the other individualized treatment strategies.  
 

 
Apnea. We estimate that <50% of participants will show clinically relevant 
sleep apnea. Sleep disordered breathing will be treated with standard of care 
practices. Because patients with HIV are expected to have comorbid conditions (e.g., 
hypertension, depression, cardiovascular disease), we will treat patients with five or 
more predominantly obstructive respiratory events (obstructive and mixed apneas, 
hypopneas, or respiratory effort related arousals [RERAs]) per hour of sleep during 
a PSG). In the absence of comorbidities, treatment is typical if there are > 15 events 
per hour.  Participants will undergo a sleep medicine evaluation at Sleep 
Therapeutics to determine titration level for CPAP treatment of their apnea.  
Established behavioral sleep medicine and monitoring of CPAP usage (goal for a 
minimum of 5 h per night) will be performed to ensure adequate treatment of apnea 
(1, 2).  CPAP adherence will be objectively monitored using Encore Pro SmartCard 
(Philips Respironics, Inc.) data.  Study staff will contact participants twice within the 
first week after starting CPAP and then once every following week to ensure use and 
manage any problems. Alternative therapies, such a dental appliances, will be 
considered for those with <20 events per hour. 
 
 
Insufficient Sleep Syndrome (ISS). We expect <25% of patients to be 
diagnosed with ISS. ISS will be treated with standard of care practices by increasing 
the duration of the sleep opportunity from <6.5 to 7h at a minimum. This includes 
instructing the participant to maintain a consistent sleep schedule.   
 
 
Circadian intervention strategies to promote circadian alignment.  Dr. Wright 
has shown that the severity of sleep onset latency problems are associated with 
sleep being attempted at suboptimal circadian times (see preliminary data). We will 
use circadian science and medicine principles to enhance the strength of 
environmental time cues and thus promote circadian alignment of the sleep 
schedule. Most participants will receive a version of the circadian intervention. 
Consistent bed and wake times. Participants will be instructed to maintain a 
consistent bedtime (plus/minus 30 min) and wake time (plus/minus 15 min) seven 
days per week (with exceptions allowed for special occasions and/or for CBT-I 
treatment).  Maintenance of a consistent sleep schedule has been shown to stably 
align the internal circadian clock and sleep timing, likely through exposure to a 
consistent daily light-dark cycle.  Maintaining a consistent wake time is most 
important as morning sunlight exposure is a strong time cue for synchronizing the 
circadian clock.  Increased flexibility with bedtime improves adherence. We will 
review call-ins daily, download the actiwatch  weekly, and provide participants 
weekly feedback about maintaining a consistent schedule. Light exposure.  We will 
increase the strength of the environmental light-dark cycle to enhance circadian 
alignment (118).  Participants will be instructed to increase exposure to sunlight 
throughout the day and especially in the morning (e.g., morning walk, morning 
coffee outside, open blinds and sit near windows when inside, go outside without 
sunglasses during work/school breaks).  We will give participants a Sunsprite 
(GoodLux Technology), which provides feedback about the amount of light exposure 
obtained that day, to help encourage participants to obtain more sunlight.   
 

We have been successful in modifying sleep duration using similar techniques, thus 
we are confident in increasing exposure to sunlight using our proposed techniques.  
Participants will also be instructed to reduce exposure to electrical light after 
sunset. Investigators will make an initial home visit to measure the light 
environment and instruct participants on how to reduce the intensity of artificial 
light exposure (dim TVs, computers and cell phones [e.g., programs such as Flux], 
home lights—we will provide dimmers and/or fixtures with lower intensity light 
bulbs to be used at night).  When not at home at night, subjects will still be 
instructed to use light reducing methods on their light emitting technologies.  We 
will use the actiwatch information to ensure subjects are being exposed to less light 
at night, and we will give feedback. 
 
 
Other sleep issues. Other individual and co-morbid ICSD-3 sleep disorders and 
factors/physical symptoms commonly associated with HIV that may contribute to 
sleep disturbance (e.g., night sweats, cough, dyspnea, diarrhea, and pain) will be 
treated with best clinical practices.  We recognize factors, such as ART medications, 
may contribute to sleep disruption (40, 74, 80) and will include these as covariates 
in our analysis.  
 
After the 8-week targeted individualized sleep intervention, we will perform follow-
up PSG sleep and circadian assessments to objectively quantify improvements in 
sleep duration and quality and to assess changes in circadian timing relative to 
sleep.  Sleep assessment measures will include: 
 
Sleep Disorder and Fatigue Questionnaires will be used to describe sleep and any 
changes pre- post- treatment: PSQI as an indicator of sleep disturbance; ESS as an 
indicator of excessive daytime sleepiness; ISI as an indicator of the clinical severity 
of insomnia; Berlin Questionnaire to pre-screen apnea.  The cognitive battery of the 
NIH Toolbox will be used to assess mental processes involved in gaining knowledge 
and comprehension, such as thinking, knowing, remembering, judging, and 
problem-solving. The post sleep questionnaire is used after each PSG recorded sleep 
opportunity to assess subjective sleep latency, sleep quality, number of awakenings, 
sleep duration, and factors that may have disturbed sleep and a sleepiness rating; 
the Fatigue Severity Scale (FSS) with modification for HIV patients to quantify self-
reported fatigue.  These questionnaires have been selected for their sensitivity to 
sleep problems and treatment. 
 
Ambulatory sleep, light exposure and activity monitoring.  Wrist actigraphy with 
concurrent light exposure (Actiwatch Spectrum; Philips Respironics) will be 
recorded the baseline week prior to the circadian phase assessment, throughout the 
8 week sleep intervention, and the week prior to follow-up circadian and vascular 
assessments.  Participants will also maintain a daily sleep-wake log. All subjects 
(current and newly enrolled) will receive an instruction sheet on how to use the 
Actiwatch.  Measuring light exposure is critical to assess adherence to circadian light 
exposure treatment strategies. We have successfully used these methods in many 
prior studies (8, 59, 117). Actigraphy is a valid and reliable method for estimating 

sleep timing and total sleep time (TST). Sleep logs will be used to assess subjective 
sleep onset latency (SOL) and TST. Our standardized sleep log also includes 
questions on sleep quality, number of awakenings, daily caffeine and alcohol intake, 
factors that may have disturbed sleep and a morning sleepiness rating. For 
descriptive analyses, the average TST for baseline, treatment and follow-up sleep 
episodes will be calculated based on wrist actigraphy using Actiwatch v.5.57 
software (1-minute epochs; medium sensitivity).  
 
Sleep and Chronobiology Laboratory PSG Research Sleep Recording and Scoring. We 
will use Compumedics Inc Siesta digital sleep recorders to assess PSG data (F3xM2, 
C3xM2, F4xM2, C4xM1, and O2xM1 O1xM2). Sleep will be scored according to 
revised AASM guidelines and used to describe how the sleep treatments influenced 
sleep duration, architecture, arousal number and duration. Oronasal thermal sensor, 
nasal pressure, pulse oxymiter, thoracic and abdominal plethysmography, snoring 
MIC, body position, ECG, and bilateral bipolar anterior tibialis EMG recordings will 
be used to screen for sleep disorders (AHI >5/h, PLM >10/h). Apnea: A thermal 
sensor amplitude drop of >90% from baseline during sleep lasting for at least 10 sec 
will define apnea (inspiratory effort will distinguish between central, obstructive 
and mixed). A nasal pressure drop by >30% for at least 10 sec with >4% 
desaturation will define hypopnea (90% of duration must meet amplitude 
criterion). Periodic limb movements (PLMS): duration of movement 0.5-10 sec, min 
amplitude 8mV increase above resting EMG, min of 4 leg movements in a series; 
length between leg movements 5-90 sec; movements on separate legs within 5 sec 
will be counted as single movement.  Standard precautions will be taken for 
biohazard exposure (e.g., wearing medical gloves when instrumenting participants; 
disinfecting equipment etc.) 
 
Sleep Therapeutics - Sleep Center Test for Suspected Apnea. The same PSG 
measures noted above will be recorded to verify the diagnosis of sleep apnea and 
participants will undergo CPAP titration to determine pressure needed for 
treatment. 
 
Sleep and Chronobiology Laboratory Circadian Melatonin Phase Assessment. Saliva 
samples will be taken every 30 min [at night] and 60 min [daytime] across 24h to 
measure melatonin, and we will determine the Dim Light Melatonin Onset (DLMO) 
and the Dim Light Melatonin Offset (DLMOff) as done in our prior research (linear 
interpolation of 25% of the fitted peak to trough amplitude.  We will also calculate 
the timing of the midpoint between the DLMO and DLMOff.  Light <10 lux, no food 
intake and stable posture 30 min prior to sampling. We will use RIA to assess 
melatonin in saliva (Alpco, Inc). We considered measuring additional circadian 
phase markers, such as core body temperature and cortisol, but decided on 
melatonin as it is the most precise and accurate marker of circadian clock in 
humans.  Standard precautions will be taken for biohazard exposure. 
 
Lastly, to minimize any potential independent influence of weight loss or changes in 
physical activity, body weight will be maintained during the sleep (and active 

control) intervention at +2 kg from baseline in all subjects.  It is very difficult to 
maintain body weight any more stable than within this range.  Body weight will be 
measured and daily physical activity (Stanford Physical Activity questionnaire) will 
be assessed every two weeks during the intervention.  All subjects will return to the 
CTRC bi-weekly to meet with the CTRC’s bionutritionist and exercise physiologist to 
monitor and identify any consistent changes in body weight outside the allowable 
range and changes habitual physical activity.  If necessary CTRC staff will work with 
the subjects to make the necessary adjustments in caloric intake or activity level.  It 
is important to note that the P.I.s have employed this approach to successfully monitor 
changes in weight and physical activity (30, 91, 93). 
 
Statistical Analysis for Specific Aim 3.    The outcome variables FBF responses to 
acetylcholine (in the absence and presence of L-NMMA and vitamin C), bradykinin 
(in the absence and presence of vitamin C) and sodium nitroprusside (in the 
absence and presence of vitamin C) as well t-PA and PAI-1 release in response to 
bradykinin and sodium nitroprusside (both in the absence and presence of vitamin 
C) will be measured at baseline and after 8 weeks.  To accommodate the 
longitudinal collection these continuous outcomes will be modeled using linear 
mixed effects methods as implemented SAS (proc mixed; SAS Institute, Inc., Cary 
NC).  Within-individual difference between measures before and after treatment 
condition (di) can be modeled as the primary outcome measure using standard 
linear regression techniques as described under Aims 1 and 2.  Potential 
confounders such as age, sex, race, family history, BMI, nadir CD4 count, baseline 
CD4 count, duration of ART and HIV-1 RNA prior to ART will be considered.  B-
spline transformations to allow for a non-linear relationship will be considered for 
continuous predictors.  Based on available data, literature and clinical input from co-
investigators, other potential confounders may also be considered.  Potential 
covariates will be identified using a univariate screen with entry criteria of p < 0.25 
and non-significant covariates will be excluded in a backward stepwise approach.  
The primary analysis will be intention to treat, although a secondary as-treated 
analysis will be conducted as well.  Given the design, treatment conditions and 
assessment protocols, we anticipate the missing data (due to subject dropout) to be 
at least missing at random (MAR), if not missing completely at random (MCAR).  
Assuming missing data that are MAR or MCAR, a “complete case analysis” would 
yield valid inference.  However, this approach would come at a cost of reducing 
statistical efficiency.  Therefore, we will also employ Schafer’s multivariate normal 
imputation approach as implemented in SAS v9.3 (SAS Institute Inc.; proc MI - 
MCMC Method and proc MIANALYZE).  According to a recent simulation study, this 
multiple imputation approach generally performed well and “generated more 
reliable information than complete-case analysis” 
(http://www.ncbi.nlm.nih.gov/pubmed/20106935).  Results from the complete-
case analysis will be compared with that of the multiple imputation analysis to 
ensure consistency of the regression parameters. 
 
Sample Size Calculations for Specific Aim 3.  Due to the lack of published data 
regarding the effects of enhancing sleep duration and sleep quality on vascular 

endothelial vasodilator and fibrinolytic function in HIV-1-seropositive adults, 
sample sizes for the targeted sleep intervention were determined based on data 
from the P.I.s laboratory in HIV-1 adults and published studies on comparable 
lifestyle intervention (e.g. exercise) effects on nitric oxide-mediated endothelium-
dependent vasodilation and endothelial t-PA release in middle-aged and older 
adults (30, 93, 109).  The average magnitude of change in FBF response to 
acetylcholine was ~30% and a change in t-PA release of ~35%.  Corresponding 
effect sizes were 0.71-1.25.  Sample sizes were calculated based on 80% power at an 
alpha level of 0.05.  A sample size of 23 subjects is required to detect this 
physiologically meaningful change.  To facilitate equal gender distribution a sample 
size of 24 subjects should be sufficient to show significant changes if present.  This 
sample size also allows for detection of a correlation coefficient of 0.35 with greater 
than 80% power at an alpha level of 0.05 (95).  To account for an estimated ~20% 
dropout rate from the intervention, we will enroll 30 subjects. 
 
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