Gut Inflammation and Gut-Gut Microbiome Interactions in the Pathogenesis of Hypertension
Conditions: Gut Inflammation, Hypertension
Sponsor: University of Florida

Protocol: 201903360 
 
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Protocol for NCT04497727 
 
1. Project Title: Gut Inflammation and Gut-Gut Microbiome Interactions in the 
Pathogenesis of Hypertension. 
 
2. Investigators: Drs. Carl J. Pepine, Chris Forsmark, Angela Pham, Eyad Alakrad and 
Eileen Handberg. 
 
3. Abstract: Dysbiosis of the gut microbiome has been associated with a variety of 
disorders and disease conditions ranging from mental health diseases to obesity. One 
such disease is hypertension. The initial point of contact between the gut microbiome 
and the host is their gut epithelium. The gut epithelium and the gut microbiota have 
evolved together to exist in balance during health. The epithelium has barrier, immune 
and metabolic functions that support and contain the microbiota. The microbiota in turn 
makes products that support the health of the gut epithelium, such as short chain fatty 
acids. There is emerging evidence to suggest that the gut itself, as well as the gut 
microbiome, is disturbed during hypertension. Little is known about this from animal 
models of hypertension except in studies by our group, and even less is known about 
the health of the gut in people with hypertension. We aim to examine the gut epithelium 
of the colon from colonoscopy biopsies, and we will derive gut organoids from this 
tissue. We will test the hypothesis that gut epithelium is distinct in terms of basic growth 
and differentiation and gene expression, in people with hypertension compared to those 
without cardiovascular disease (CVD) and normal blood pressure (e.g. reference 
subjects) 
 
4. Background: It is estimated that almost half of the adults in the United States have high 
blood pressure1, a leading modifiable risk factor for CVD, stroke and dementia. 
Approximately 20% of these people are termed treatment-resistant hypertensives 
because they do not achieve goal blood pressures even taking three or more 
antihypertensive medications of different classes 1.  
The gut and its microbiome have been shown to be associated with hypertension in both 
animal models and patients2-4. The gut microbiome is distinct in people and animals with 
hypertension from those with normal blood pressure. Both the bacterial populations of 
the gut microbiome and the functions of those populations differ5, 6. Most strikingly, the 
transfer of fecal matter from hypertensive people or animals to animals with normal 
blood pressure increases their blood pressure6-8. In animal models of hypertension, gut 
pathology such as decreased villus length and goblet cell number and increased fibrosis 
and muscularis layer are also observed2. Additionally, the gut barrier has reduced 
strength in hypertensive animals allowing large molecules normally retained in the gut to 
enter (“leak”) into the blood circulation5. In humans with hypertension there is also 
evidence of gut leakiness as there are increased markers of barrier dysfunction and 
substances normally well contained behind the gut barrier appear in the circulation5. 
Furthermore, there are more immune cells with surface markers targeting them to the 
gut in the circulation of hypertensive subjects, another sign that there is a potential 
problem in the gut5. We also have unpublished data in experimental models that gut 
epithelial cells express different proteins when the animal is hypertensive. Altogether, 
this suggests that the gut and its microbiome may be a novel target for blood pressure 
control. 
Gut pathology has not been explored in human hypertension, except by the above-
mentioned circulating markers. This has prompted us to hypothesize that the interaction 

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between the gut and its microbiota is faulty in hypertension, particularly in the light of 
observations of a good balance between the two in health.  
We propose to investigate these interactions in human gut by utilizing colonoscopy 
biopsies for two main purposes. Firstly, to examine the gene expression profile of the gut 
epithelium from individuals with and without hypertension. Secondly, we will use the 
crypts of the gut to culture gut organoids from the stem cells present in the crypts. These 
are three dimensional cultures that grow and differentiate to form an organ that is 
structurally similar to the gut. They have a lumen, a polarized epithelium, good barrier 
function and contain all of the cell types in the correct proportions that are found in the 
gut epithelium in vivo. We will use these to test whether there is a fundamental 
difference in the epithelium of the gut in hypertension compared to normotension. We 
will also test gut organoid growth and differentiation in direct comparison to those from 
normotensive individuals grown under identical culture conditions. In these, we 
hypothesize that there will be no difference between those from people with 
hypertension compared to those without hypertension in terms of cellular composition 
and gene expression patterns. 
 
5. Specific Aims: 
 
We will compare basic properties of gut epithelia of hypertensive and normotensive 
reference subjects. We will determine if there are fundamental differences in the gut 
epithelium in hypertension compared to normotension. We propose the following aims:   
 
Aim 1: To determine if gene expression patterns are distinct in gut epithelium derived 
from hypertensive compared to normotensive reference individuals.  
Aim 2: To determine if gut organoids prepared from gut biopsies have different growth 
rates and patterns, including the proportions of cell populations in the organoids, when 
derived from hypertensive versus normotensive reference subjects. 
 
6. Research Plan: 
 
These studies will include a controlled sampling of human hypertension (n=60, 30 
hypertensive subjects and 30 normotensive reference subjects, equal numbers of males 
and females) recruited at UF. 
 
Aim 1:  From one part of the biopsy, the gut epithelium will be isolated, and immediately 
frozen (-80 degrees) until RNA isolation for RNA sequencing and examination of gene 
expression within the epithelium.  
Aim 2: The remainder of the biopsy will be immediately (within 30 minutes of collection) 
used to prepare gut organoid cultures using methods essentially as developed by 
StemcellTM Technologies. The growth parameters and the proportions of each cell type, 
for example, enterocyte, Paneth, goblet, enteroendocrine, making up the organoid will 
be determined by immunohistochemistry or fluorescence activated cell sorting (FACS) 
analysis. 
 
Recruitment: University of Florida (UF) will recruit participants from the UF GI clinic (with and 
without hypertension) who are scheduled for routine “surveillance” colonoscopy.  Most 
patients undergoing screening colonoscopy have polyps noted (at UF, our adenoma detection 
rate is around 50%).  These folks would have biopsies of other sites (polyps) already, and 
would get additional biopsies for study. The other 50% without polyps would only have 
biopsies as part of study. During screening of upcoming “surveillance” colonoscopies potential 

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participants will be asked by their gastroenterologist about their interest in taking part in this 
study. If the patient agrees to participate or desires more information about the study, the 
study coordinators in  Cardiology Clinical Trials will contact the patient via phone, email, or an 
in-person meeting and provide more details and answer any questions. If they wish to take 
part in the trial, informed consent will be obtained prior to the colonoscopy procedure..  
 
In order to address our hypotheses, colonoscopy biopsies from subjects diagnosed with 
hypertension as well as a reference group will be collected. After which, a member of Dr. 
Raizada or Dr. Pepine’s group will immediately transport biopsy samples to Dr. Mohan 
Raizada’s lab in the McKnight Brain Institute Building L4-135.  
 
A. Specific Procedures: Inclusion/exclusion criteria for hypertension participants  
Inclusion criteria:  
• 
Ages: 18-80 years old with a weight 110 lbs. 
• 
Diagnosis of hypertension or without hypertension for the reference cohort. 2017 
ACC/AHA definition for hypertension (systolic blood pressure ≥130 mmHg and/or 
diastolic BP ≥80 mmHg) will be used.  
• 
Scheduled elective colonoscopy. 
 
Exclusion criteria: 
• 
Hypertensive or reference individuals with history of autoimmune disease or other 
chronic inflammatory conditions. 
• 
Pregnant or have been pregnant in the last six months. 
• 
Antibiotic treatment within two months of study enrollment 
• 
Currently taking a medication(s) (e.g. antibiotic, anti-inflammatory agents, 
glucocorticoids, other immune modulating medications, antacids or proton pump 
inhibitor drugs like Prilosec) known to modify gut microbiota or currently taking a 
medication(s) and stopping the medication(s) to prepare for the colonoscopy then 
restarting after procedure is completed. 
• 
The use of probiotics 
• 
History of intestinal surgery, inflammatory bowel disease, celiac disease, lactose 
intolerance, chronic pancreatitis or other malabsorption disorder. 
• 
History of blood transfusion within 4 weeks. 
• 
Subjects who, in the opinion of the investigator, will be uncooperative or unable to 
comply with study procedures 
 
Subjects will participate until the biopsy procedure is completed.  
 
 
 

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Specific Procedures: 
• Methods/Statistical Analyses: Each study participant will provide, in addition to body weight, 
BMI, BP, PR, and CVD risk factors, a colonoscopy biopsy. 
• Analysis of gene expression in the epithelium of a portion of the biopsy will be examined and 
gut organoids cultured from the remainder.  
• The subjects’ medical records will be reviewed and summarized for the study. All collected 
data will be stored in an encrypted computer data base kept behind UF’s firewall. These data 
will be coded and connected to the biopsy samples by code, not identifiers. Biopsy samples 
will be processed immediately and portions either frozen for storage or the crypts isolated for 
gut organoid culture. The experiments will be performed in Dr. Raizada’s laboratory in the 
McKnight Brain Institute and remaining samples stored until the end of the study. Experiments 
examining gene expression in the colonic epithelium of normotensive and hypertensive rats 
using the same methodology intended for use here, revealed significant differences between 
groups with a sample size of n=4 per group. Hence, we expect a sample size of n=60 to 
provide sufficient power to observe differences in gene expression. Multiple gut organoids can 
be generated from a single biopsy. This allows mean characteristics for each individuals’ 
organoids to be determined, reducing variability. We have no previous data upon which to 
base a formal power analysis. However, we believe these culture conditions, the ability to 
have multiple repetitions of the experiment in each biopsy sample, and 30 subjects per group 
will be adequate to determine whether there are differences worth pursuing in a larger cohort 
of hypertensive and normotensive subjects in a future study.  
Prepped colonoscopy biopsies: Dr. Christopher Forsmark, Dr. Eyad Alakrad, and Dr. Angela 
Pham in the UF GI Division will perform all colonoscopy procedures on subjects (hypertensive 
or healthy reference subjects) to obtain colon biopsies. All subjects will have up to 4 additional 
biopsies collected for research.   Biopsy and blood samples will be coded as quickly as 
possible and securely stored with only a subject number assigned for this study. 
 
Medical Record Abstraction. For participants in this study, their pertinent medical history 
will be abstracted from their records and entered into a RedCap database.   These data will 
be correlated with the research results to determine any relationships or mechanisms in 
hypertension. We will correlate the research results with the type of hypertension (i.e. 
controlled, uncontrolled, treatment-resistant).  
 
Subject Confidentiality. Clinical information will not be released without written permission 
of the subject. The site Investigator must assure that the privacy of subjects, including their 
personal identity and personal medical information, will be maintained at all times. UF site has 
additional privacy obligations to study subjects under the Health Insurance Portability and 
Accountability Act (HIPAA). After a subject has signed an informed consent form, the 
coordinator will review the signed informed consent(s) and the PI will review that portion of 
the subject’s medical record that is directly related to the study including electronic medical 
records. This shall include all study relevant documentation including subject medical history 
to verify eligibility, laboratory test result reports, admission/discharge summaries for hospital 
admissions occurring while the subject is in the study, and autopsy reports for deaths 
occurring during the study (when available). All study files will be kept on site in locked 
drawers. The only personnel with access to this data are the PI and study staff. All data and 
samples will be coded with a study ID.  
 
 
7. Possible Discomforts and Risks: Colonoscopy procedures are only those performed as 
part of a clinically indicated procedure. Possible risks for colonoscopy biopsies include 

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persistent bleeding after biopsy or perforation of the wall of the colon which might require 
surgery. The chance of a serious complication from colonoscopy biopsy is less than 1 in 
10,000. 
 
This study will collect personal data and PHI. There is slight risk of personal information 
becoming public, but measures are in place to prevent that unlikely event. 
 
8. Possible Benefits. There are no direct benefits of participating in this study. 
 
9. Conflict of Interest. Study investigators and staff have no conflicts to report. 
 
10. References 
 
1. Whelton P, Carey R, Aronow W, Casey DJ, Collins K, Dennison Himmelfarb C, DePalma S, 
Gidding S, Jamerson K, Jones D, et al. 2017 
ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the 
prevention, detection, evaluation, and management of high blood pressure in adults: A 
report of the American College of Cardiology/American Heart Association task force on 
clinical practice guidelines. J Am Coll Cardiol 2017 Nov7;pii: S0735-1097(17):41519-1. 
 
2. Santisteban MM, Qi Y, Zubcevic J, Kim S, Yang T, Shenoy V, Cole-Jeffrey CT, Lobaton GO, 
Stewart DC, Rubiano A, et al. Hypertension-linked pathophysiological alterations in the gut. 
Circ Res 2017;120(2):312-23. 
 
3. Wilck N, Matus M, Kearney S, Olesen S, Forslund K, Bartolomaeus H, Haase S, Mähler A, 
Balogh A, Markó L, et al. Salt-responsive gut commensal modulates TH17 axis and disease. 
Nature 2017;551(7682):585-9. 
 
4. Yang T, Santisteban MM, Rodriguez V, Li E, Ahmari N, Carvajal JM, Zadeh M,., Gong M, Qi 
Y, Zubcevic J, et al. Gut dysbiosis is linked to hypertension. Hypertension 2015;65(6):1331-
40. 
 
5. Kim S, Goel R, Kumar A, Qi Y, Lobaton G, Hosaka K, Mohammed M, Handberg E, Richards 
E, Pepine C, et al. Imbalance of gut microbiome and intestinal epithelial barrier dysfunction 
in patients with high blood pressure. Clin Sci (Lond) 2018;132(6):701-18. 
 
6. Li J, Zhao F, Wang Y, Chen J, Tao J, Tian G, Wu S, Liu W, Cui Q, Geng B, et al. Gut 
microbiota dysbiosis contributes to the development of hypertension. Microbiome 
2017;5(1):14. 
 
7. Toral M, Robles-Vera I, de la Visitación N, Romero M, Sánchez M, Gómez-Guzmán M, 
Rodriguez-Nogales A, Yang T, Jiménez R, Algieri F, et al. Role of the immune system in 
vascular function and blood pressure control induced by faecal microbiota transplantation in 
rats. Acta Physiol (Oxf) 2019;Apr 20:e13285. 
 
8. Toral M, Robles-Vera I, de la Visitación N, Romero M, Yang T, Sánchez M, Gómez-Guzmán 
M, Jiménez R, Raizada M, Duarte J. Critical role of the interaction gut microbiota - 
sympathetic nervous system in the regulation of blood pressure. Front Physiol 2019;10:231.