Dietary Restriction in Vascular Surgery
Phase: NA
Conditions: Vascular Diseases, Surgery
Sponsor: Brigham and Women's Hospital

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1 
 
DIETARY RESTRICTION IN VASCULAR SURGERY 
 
Key Unmet Need Addressed 
Vascular surgery continues to offer enormous benefits to selected patients. However, vascular patients 
continue to suffer some of the highest complications rates: cardiac morbidity (e.g. myocardial infarction [MI]), 
stroke, ischemia/reperfusion injury (IR), restenosis, and death peri-operatively, with subsequent high expense 
for our health care systems and enormous toll for the individual patients1-3. With the exception of a few 
evidence-based guidelines that modestly improve perioperative care, there is no uniformly accepted medical 
therapy to reduce complications in these patients4. Rising health care expenditures, the aging population, and 
the increasing incidence of the metabolic syndrome all point to a dire need for accelerated research into 
feasible strategies to enhance outcomes of major operations5. 
 
Project Goals/Specific Aims 
Hydrogen sulfide (H2S) has emerged as a critical gaseous signaling molecule with a role in multiple 
processes including IR injury, angiogenesis, intimal hyperplasia, and anti-inflammatory mechanisms6-11. It even 
appears to hold anti-atherosclerotic properties12. However, the gas is toxic with a half-life of minutes, and it can 
be rapidly oxidized. Furthermore, no clinically useful pharmacologic H2S donors have been developed to date. 
In our 2015 Cell paper, we link substantial upregulation of endogenous H2S to short-term manipulation of 
mammalian dietary intake: simple dietary restriction11. The current project serves as the second phase in the 
accelerated translation of this important discovery toward clinical utility that may substantively impact the 
vascular patient at several levels: protection from IR injury, intimal hyperplasia, peri-procedural events such as 
stroke, cardiac dysfunction and MI, and promotion of angiogenesis7, 8, 12-17.  
Chronic protein and calorie dietary restriction (PCR; reduced food intake without malnutrition) is known for 
extending longevity in multiple species18. PCR is also an established approach to positively impact metabolic 
fitness and resistance to multiple forms of acute stress19-21. Despite these wide-ranging benefits, potential 
clinical applications have never been considered feasible in humans due to the practical difficulties associated 
with voluntary food restriction combined with the assumption that PCR benefits take a long time to accrue. 
Recent preclinical studies by the applicants reveal a rapid onset of PCR benefits against surgically induced 
traumatic inflammation, IR injury, and the fibroproliferative vascular response to injury in rodents14-17, 22, 23. 
Mechanistically, rapid changes in adipose phenotype and associated adipokine profiles appear to underlie 
these benefits16; but the key mediator appears to be endogenous H2S. This field thus stands poised to move to 
clinical utility, and vascular surgery offers an optimal risk/benefit ratio for translation of the PCR/H2S 
hypothesis15.  
For the current project we propose a randomized, controlled trial to evaluate patient compliance and 
biologic mechanisms of a short-term pre-operative PCR diet in comparison to a normal ad libitum diet for 4 
days before elective vascular surgery involving an open major operation. Both Endpoints relate to the long-
term primary scientific objective to test the hypothesis that brief upregulation of endogenous H2S via pre-
operative PCR in elective major surgery improves clinical outcomes in humans. After a successful pilot 
study of the PCR diet conducted inpatient before carotid endarterectomy titled Short-Term Endogenous 
Hydrogen Sulfide Upregulation (PHRC Protocol No. 2016P002507), we now aim to expand the study to at 
home diet among a variety of vascular surgery procedures. The primary endpoints of the study are:  
 
1) Feasibility, and in particular outpatient subject compliance with PCR (measured by direct dietary intake 
data and serum markers including plasma free amino acids, pre-albumin, and insulin-like growth factor in 
comparison to the ad libitum diet). 
 
2) Measurement of H2S and standard biological markers of stress in blood collected at baseline, 
immediately before surgery, and after surgery including stress hormones, oxidative stress, and inflammation. 
We will also collect intra-operative biologic samples of discarded tissues and a gram of adipose tissue (peri-
vascular, subcutaneous) to inform potential mechanistic links between PCR induced H2S upregulation and any 
perturbations in peri-operative stress responses.  
 
A secondary exploratory aim is to compare clinical outcomes and complications between groups. These 
endpoints will include surgical and medical complications such as cardiac, neurologic, infectious, vascular, 

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wound, and any other clinically significant events that occur within 30 days of surgery.  We will also look 
broadly at one-year survival and major cardiovascular events, though it is acknowledged that the project lacks 
fiscal resources to fully power this mechanistic trial for clinical efficacy.   
 
Research Strategy (Background, Significance, Preliminary Data, Approach and Methods) 
New Approaches are Needed to Reduce the Morbidity and Mortality of Vascular Surgery Procedures Elective 
surgery presents an opportunity unique in medicine: the planned stressful event. The need for and submission 
to surgery represents a major disruption in most patients’ family and professional lives (including morbidity and 
mortality). Surgical mortality rates vary up to 1000 fold 24, 25, with vascular surgery carrying one of the highest 
rates of perioperative events. Much effort has been expended in studying methods of assessing and modifying 
the risk of vascular surgery26. Several factors continue to challenge care in these patients. First, the population 
undergoing vascular surgery is getting sicker (e.g. higher prevalence of octogenarians 27 and diabetics28). 
Furthermore, appropriate medical therapy to reduce CV events, particularly β-adrenergic blocking agent 
administration, remains an issue of great contention29. Thus, the aging population, rising incidence of the 
metabolic syndrome, increasing vascular surgery access, and the lack of agreed upon mitigating medical 
therapy creates an environment susceptible for increased CV events with associated morbidity and mortality, 
all at enormous economic cost.  
Compounding these problems, there is now evidence that the rates of cardiac injury after vascular surgery 
are far higher than previously recognized with the focus shift of events from perioperative MI to myocardial 
injury. One in seven (129/782) vascular surgery patients had biomarker evidence of cardiac injury30; 5% of 
vascular subjects had a MI with an associated five-fold increased risk of mortality in the first 30 days31. 
Basic Mechanisms Underlying Adverse Events in Vascular Surgery Have Been Defined Abnormal activation of 
the stress response by surgery has been associated with adverse CV events. Three components of the stress 
response have been studied in relation to postoperative complications: the sympathetic nervous system, 
oxidative stress, and inflammation. Vascular surgery increases catecholamine release32-34; for instance 
norepinephrine and cortisol are increased postoperatively in these patients35. While still evolving, some clinical 
trials have demonstrated that antagonism of this system (β-blockers) reduces the risk of perioperative CV 
events36, 37. 
Oxidative stress participates in the stress response38, 39. Reactive Oxygen Species (ROS) are produced in 
the vascular wall in response to injury40, 41. Patients with atherosclerosis undergoing vascular surgery manifest 
elevated levels of oxidative stress42. In addition to acting as signaling molecules and mediators of the stress 
response, ROS individually and in combination contribute to many abnormalities associated with vascular 
disease, including vascular endothelial dysfunction and attenuated bioavailability of nitric oxide (NO)43. 
Diminished NO bioavailability has been associated with increased rates of cardiac injury during vascular 
surgery44. Patients undergoing both aortic aneurysm repair45 and bypass surgery46 manifest increased levels of 
oxidative stress post-operatively, possibly through an ischemia-reperfusion mechanism.  
Inflammation is now recognized as a causative mechanism underlying atherogenesis47 and acute clinical 
events related to atherosclerosis48-51. Elevated inflammation markers predict both first CV events52 and 
recurrent events53. High sensitivity C reactive protein (hs-CRP) strongly predicts postoperative MI54. Pre-
operative hs-CRP elevation was associated with a 2.5 fold increased rate of postoperative events55. Statins 
reduce markers of inflammation and reduce postoperative cardiac events by 45% in vascular surgery 
patients56, potentially via H2S. Thus any strategy that can modulate inflammation, oxidative stress and 
sympathetic tone might reduce adverse events after vascular surgery. We propose pre-operative PCR as such 
a strategy. 
PCR Appears to Positively Impact the Acute Stress Response via H2S Upregulation An emerging method of 
improving the response to stress is PCR15, 16. Defined as reduced food intake without malnutrition, PCR is best 
known for extending lifespan in model organisms from yeast to non-human primates57, 58. The efficacy of PCR 
against acute stress, including surgical stress, in preclinical models is also well established19, 59-64. Pilot studies 
suggest that humans respond to PCR in beneficial ways with respect to metabolic fitness (including improved 
glucose homeostasis, lipid profiles, and CV performance)65, 66. But humans also have great difficulty complying 
with long-term voluntary food restriction, which is a prime reason that PCR has not been previously exploited in 
the clinical setting despite promising efficacy. However, recent data in model organisms point to a rapid onset 
of PCR benefits and challenges the notion that long-term PCR is required for benefits to accrue. In fruit flies, 
the maximal benefit of PCR on mortality risk occurs within 2-3 days67. In rodents, we have shown that surgical 

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stress resistance also occurs within days19, 68, and the benefits center on upregulation of endogenous H2S11. 
Nonetheless, with the exception of pre-operative overnight fasting (which serves a different purpose69) dietary 
recommendations are largely absent from peri-op management4.  
Pilot data suggest that adverse post-operative outcomes linked to oxidative stress, inflammation and stress 
hormones may be modified by brief PCR in humans as well. In subjects who undertook food restriction for 12 
hours per day for a month, markers of inflammation decreased significantly70. PCR has been shown to reduce 
oxidative stress in both chronic71 and acute72 settings. PCR may decrease stress hormone release73, and it 
also appears to work rapidly even in obese individuals or ill individuals. In gastric bypass surgery, two weeks of 
PCR reduced the risk of complications and procedure difficulty74. With respect to patient compliance, brief (4 
days to 2 weeks) pre-operative dietary interventions have been shown to be feasible and safe in selected 
patients, ranging from obese candidates for laparoscopic surgery74 to living organ donors75. Our own pilot work 
involving humans showed that the proposed PCR diet is feasible and without excess risk for CEA patients 
before surgery.  
Pre-operative PCR Modulates Response to Ischemic Injury The root cause of both MI’s and strokes is 
ischemia resulting in cell death. The artery is clamped during an open vascular surgery. In most cases, 
restoration of blood flow (e.g., with unclamping), or reperfusion, is beneficial for cell survival and return to 
organ function. However, the return of oxygen and nutrients comes at the cost of increased free radical 
generation by inappropriate activation of cellular oxidases such as xanthine oxidase, further increasing 
oxidative damage. Inflammatory mediators further exacerbating tissue injury. While it is well known that long-
term PCR (three months or greater) protects against ischemia-reperfusion injury to multiple organs in 
preclinical models, we have recently shown that robust protection from renal and hepatic IR injury can be 
obtained within 3 days of total food deprivation, with beneficial effects on survival observed in as little as one 
day19. Damage can be mitigated without restriction of food intake per se. Ad libitum diet lacking total protein or 
the essential amino acid tryptophan can still provide protection against renal and hepatic IR injury68. The 
conserved amino acid deprivation sensor Gcn2 is required for the benefits of tryptophan deficiency against IR 
injury, implicating this nutrient sensing pathway for the first time in protection from surgical stress68. The other 
major amino acid sensing pathway in mammals is controlled by the signal transducing kinase mTOR. Inhibition 
of mTOR by rapamycin increases longevity in rodents, possibly by mimicking nutrient deprivation. Our data 
indicate that mTOR also plays a role in stress resistance mediated by PCR. Using a genetic model in which 
mTOR is constitutively activated in the liver by genetic deletion of the mTOR repressor Tsc1, we recently found 
that PCR no longer protects against IR injury. We also recently linked the biologically active gas H2S to the 
protective effects of PCR11. 
Short-Term PCR Attenuates Intimal Hyperplasia Restenosis is a recurrent problem from vascular patients and 
applicable to a range of procedures. While fortunately an infrequent event, restenosis after CEA can lead to 
strokes and repeat interventions for patients76.  In a validated mouse model of the primary driver of restenosis, 
intimal hyperplasia77, we discovered that a short-term pre-operative PCR diet resulted in less intimal area 
(p=0.032) vs controls. Mice on a PCR diet also had significantly larger internal elastic lamina length (p=0.003), 
a remodeling measure. Finally, the PCR cohort serum exhibited significant decreases in certain classes of 
circulating lipids, including a collapse of multiple triglyceride species, suggesting a fundamental shift in 
metabolism toward beta oxidation. In addition, the lowered levels of circulating triglycerides may help reduce 
secondary insulin resistance and inflammation. 
 
Approach and Methods 
Eighty subjects undergoing carotid artery endarterectomy, aortic aneurysm repair (open, and endovascular 
if groin cut down planned), open lower extremity arterial procedures (bypasses, aneurysm repair, arterial and 
bypass graft reconstructions), major amputation of the lower extremity (below knee and above knee 
amputations), or open hemodialysis procedures either symptomatic or asymptomatic disease will be recruited 
and enrolled at BWH. Using a randomized (3:2), parallel design, patients will receive either the PCR diet 
(n=48; ScandiShake [any of 4 flavors] mixed with almond milk, calculated individually for a total daily volume to 
achieve 30% caloric restriction and 70% protein restriction, based on body weight and activity level), or 
continued routine ad libitum diet (n=32). The Mifflin St. Jeor equation will be used to calculate the total 24-hour 
energy needs based on gender, age, height, weight, and activity factor for the PCR diet. Daily physical activity 
will be assessed by questionnaire to determine the activity factor for accurate calorie restriction calculations.  
The general ratio recipe for the ScandiShake is 85 grams ScandiShake powder to 240 cc almond milk. Water 

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intake is ad libitum for both cohorts, and both diets can be consumed throughout the day and night (except on 
the day of surgery). We have selected the ScandiShake as the PCR diet because it is currently the lowest 
protein shake available and there are other trials ongoing with this approach, though none [except our prior 
pilot study] are in vascular surgery patients. We paired the shake with almond milk since it is also low protein. 
Patients will consume their assigned diets for the four days leading up to surgery until midnight the day of 
surgery when both cohorts will be NPO.  
Inclusion Criteria: Patients greater than 18 years old who present for one of the following elective 
procedures at BWH:  
 
Carotid artery endarterectomy 
 
Aortic/iliac aneurysm repair (open, and endovascular if groin cut down planned)  
 
Open lower extremity arterial procedures (bypasses, aneurysm repair, arterial and bypass graft 
reconstructions)  
 
Major amputation of the lower extremity (below knee and above knee amputations). 
 
Open hemodialysis access procedures   
 
Exclusion Criteria: Patient intolerance or allergy to any of the ingredients in the PCR diet; Active infection; 
Pregnancy; Malnutrition, based on abnormally low serum albumin (lower than 3 g/dL); Uncontrolled diabetes 
(HgbA1c greater than 12%); Substance dependency that could interfere with protocol adherence and assent 
as determined by the PI; Active non-cutaneous cancer under treatment with chemotherapeutics or radiation; 
Emergency surgery; Active participation in any another interventional or randomized study; Participation in the 
current study within the past 30 days. 
 
Recruitment 
Subjects will be recruited through the BWH Vascular Surgery outpatient clinics and inpatients wards. 
Physician-Investigators will notify the study coordinators about candidates that fit inclusion/exclusion criteria. 
Study coordinators may discuss the study with potential subjects in detail and answer questions about the 
research being conducted. Patients will be introduced to the study in outpatient clinics or inpatient units and 
given ample time (>12 hours) to consider enrollment. Patients will be provided with the opportunity to discuss 
the trial with family or another physician if they wish to consent at a later time. Consent will only be obtained by 
a licensed Physician Investigator. All patients screened for this study will either have an established 
relationship with the physician-investigator or an established collaboration between a clinician known to them 
and the physician-investigator. Only with the collaborative clinician’s approval may the investigator or study 
coordinators approach the patient about the research trial. Once the patient’s permission has been obtained, 
screening tests and procedures will be performed to determine subject eligibility.  
If the patient consents and remains eligible for the study, then the subject will then be randomized.  
This randomization will occur before the OR event to allow for resources preparation, baseline testing, and 
dietary intervention. 
Foreseeable Risk and Discomforts 
     
Patients randomized to the PCR diet may have a difficult time adhering to the PCR four-day diet, 
however our pilot data shows that it is feasible for vascular patients to adhere to the diet pre-operatively. We 
hope to alleviate this by introducing a variety of flavors that patients may choose from. Furthermore, by 
requiring the patient to log their meals with either the MealLogger app or paper record, we can closely monitor 
and control dietary intake while also allowing the patient to continue their normal lifestyle before surgery.  
Patients randomized to the PCR diet may also experience hunger or discomfort higher than normal 
from limiting their diet for four days. We hope that with additional flavors some discomfort can be alleviated. If 
the patient feels too hungry or uncomfortable they may choose to withdraw from the study. Patients may also 
experience fatigue pre-operatively if randomized to the PCR diet. Based on our pilot data of PCR diet research 
in humans, patients were able to complete the diet safely and without excess risk.  
Due to the high quantity of carbohydrates in the PCR diet, hypoglycemia will not be a major concern for 
subjects. However, at baseline we will screen diabetic patients for glucose control by questionnaire and 
HgbA1c testing. Those with very poor glucose control (HgbA1c >12%) will be excluded. Patients over the age 
of 65 on hypoglycemic agents (ie sulfonylureas) will hold their hypoglycemic agent medication for the four-day 
PCR diet intervention. Patients who have a baseline HgbA1c value less than 7% will decrease their insulin by 

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20%. Patients who report daily episodes of hypoglycemia will be reviewed by our study staff endocrinologist for 
glycemic control. Recommendations will be made to insulin dependent patients to check their blood glucose 
four times a day before meals and fasting while on the PCR diet. Our study staff endocrinologist will be 
available for patient consult in the event of PCR related hypoglycemia to change patients’ insulin regimen. 
We anticipate that there will be no significant adverse effects of the H2S upregulation and PCR diet but 
do expect a 20% dietary compliance failure rate. However, there may be additional risks or discomforts that are 
not known now. 
Adipose biopsies add approximately 30 seconds to 1 minute to total surgery time but pose no other 
known risks.  
Intellectual property and data generated under this project will be administered in accordance with 
Harvard policies. Materials and data will be transferred between BWH and material transfer and use 
agreements are already in place for such transfers. Publication of data shall occur during the project, 
consistent with normal scientific practices. Research data which documents/supports/validates research 
findings will be made available after the main findings have been accepted for publication. 
Inadvertent disclosure of private medical information is a risk for study patients. Data records will be 
maintained in the locked Vascular Surgery Clinical Research office. Electronic files will be maintained on the 
Partners network drive, or securely input into selected professional data management system REDCap with 
access limited to study staff only. 
 
Benefits 
Patients may or may not benefit medically from taking part in this study. Theoretically patients receiving the 
PCR diet may have less chance of complications after surgery, but this is unproven. We do not expect to 
observe any physiological benefit if patients are randomized to the non-dietary restricted arm. 
 
Data Collection 
< 30 Days
4 days
3 days
2 days
1 day
Day of
Recovery
Post-
Post-Op
Post-
Pre-op
Pre-Op
Pre-Op
Pre-Op
Pre-Op
Op
Area
Op
Day #30
Op
(Baseline)
(Pre-Op)
Arrival
Day #1
(+/- 14 days)
1 Year
Control
Continue Usual Dietary
NPO except 
Patient
Enrollment,
Pattern (2:5 randomization)
meds
Resume
Study
Chart Review
Screening
Randomization,
Usual
Participation
for major
(estimated
Education
Diet
Terminates
outcomes
n=600)
(n=80)
Dietary Restriction
Protein-Calorie Restriction
NPO except 
x 4 days (3:5 randomization)
meds
MealLogger app Entry
X
X
X
X
X
X
Post Op Day 14
Blood Draw
X
X
X
Determinations
Physcal Activity Questionnaire
X
24-Hour Food Recall
X
Height, Weight
X
X
X
X
eGFR
X
X
X
Wound Evaluaton
X
X
Tissue Collection*
X
Full Medical History
X
Satisfaction Questionnaire
X
Physical Exam
X
X
Chart Review
X
*discarded artery, vein, peri-vascular or subcutaneous fat, aneurysmal specimens, and amputated tissues
Registry
Time
CBC with diff, Chem 7, lipidomics, cortisol, 
epinephrine, norepinephrine, dopamine, 
hs CRP,  IL-6, leptin, HMW adiponectin, 
resistin, IGF-1, P3, serum pregnancy test 
(as needed); pre-albumin (baseline and 
preop)
Preprocedure 
Inpatient 
Post Procedure
 
Dietary compliance will be measured by way of dietician calculated intake (for data analysis will be the gold 
standard). The subjects will be asked to document their meals using either the MealLogger app or paper 

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record. The MealLogger app allows subjects to take a picture of their meals, either the ScandiShake or their 
routine ad libitum diet. Study dieticians will interpret dietary intake through photos and directly contact subjects 
using the app for clarification of intake, or through paper record if subjects are unable to use the MealLogger 
app.  Compliance with the dietary intervention will also be evaluated through measuring expected changes in 
the levels of plasma amino acids using an LC-MSMS platform, and insulin-like growth factor-1 (IGF-1) by 
ELISA. Because free amino acids are not stored in the body, PCR results in a rapid decline of essential amino 
acids, including methionine and tryptophan, that be monitored easily in blood plasma79, 80. Isolated protein 
restriction also results in reduced plasma IGF-1, even in the absence of overall PCR81. 
H2S and H2S production capacity (two distinct entities, see Cell paper11) will be longitudinally measured, as 
will stress and safety markers in blood samples taken prior to dietary intervention (PCR or control), after the 
dietary intervention but before surgery, and sequentially after vascular surgery. Blood samples will also be 
collected and analyzed by Dr. Ho’s team at Northwestern to assess for changes related to PCR. Adipose 
tissue biopsies from fat depots (subcutaneous, perivascular) will be taken at the time of surgery and analyzed 
for the effects of diet on lipid composition and on inflammatory markers predictive of the response to surgical 
stress. Additionally, discarded specimens (discarded artery, vein, peri-vascular or subcutaneous fat, 
aneurysmal specimens, and amputated tissues) will be collected and stored for microscopic analysis. Enrolled 
patients and their clinical data will be entered into a study-specific database within REDCap.  
 
Statistical Considerations 
The focus of this Mechanistic Trial is to gain insights into practical future trial construction considerations 
such as patient compliance and physiologic impact. We anticipate that the supervised PCR will be completed 
by a majority (~80%) randomized to that arm and that H2S and H2S production capacity will rise on day two 
and be significantly elevated above baseline until re-feeding POD#1. We expect that markers of stress will 
change significantly less in the active treatment group compared to normal diet group. Using an α of 0.05 and 
β of 0.8, based on the differentials observed in rodent work and factoring in non-compliance, via intent-to-treat 
the study is powered to test these two primary scientific markers (H2S and norepinephrine/IL-6). Furthermore, 
we believe that there will be no significant adverse effects of the H2S upregulation and PCR diet but do expect 
a 20% dietary compliance failure rate. Based on our own pilot work, we anticipate that markers of inflammation 
will be elevated (compared to non-atherosclerotic patient’s samples in hand) in the enrolled subjects and 
further amplified by the surgical stress. We anticipate that the blood and adipose phenotypic signatures will 
have high inflammation, but that the subjects compliant with PCR will have a reduced global inflammatory 
activation state, and trends toward less carotid plaque inflammation and fewer postoperative complications. 
Our key endpoint for upregulation of endogenous hydrogen sulfide will be the P3 cell sorting assay.  Based on 
pilot data we anticipate a 4% increase in the PCR diet cohort compared to ad lib diet controls, with an expected 
standard deviation of 8%.  Using a power of 80% and an alpha of 0.05, and 8% specimen loss rate we need a 
sample size of n=80 to reach statistically meaning conclusions (un-paired T-test). Importantly for future trials, 
we anticipate that the unbiased discovery platform will generate novel mechanistic hypotheses founded on 
human data and tissues that will be ripe for future research and translation. 
 
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