Coronary (Artery) Disease Clinical Trial
Official title:
Does the Human Gut Microbiome Serve as a Novel Personalized Therapeutic Target for Coronary Atherosclerosis?
Background: The human gastrointestinal system is populated with a variety of symbiotic
microorganisms, namely microbiota. The microbiome is the total genetic data of the
microbiota. The human gut microbiota interacts extensively with the host through metabolic
exchange; thereby contribute to a variety of metabolic and immunologic mechanisms in the
human body. Coronary artery disease (CAD) is major cause of morbidity and mortality
worldwide and is a major field of interest in microbiota research. There have been several
findings that connect the gut microbiota to CAD pathophysiology, but these data relates
solely to the interaction between human gut microbiome and cardiovascular risk factors. As
far as known , data regarding patients who already developed CAD is lacking.
Aims: To investigate gut microbiota of patients with CAD, thereby allowing the adjustment of
personalized treatment by changing the pro-atherosclerotic environment in the gut.
Methods: Study participants will include patients arriving to Rabin Medical Center with
suspected CAD. Patients will provide medical, lifestyle, and nutritional questionnaires.
Vital signs measurements will be taken as well as fecal samples and/or rectal swabs. Blood
samples will be drawn to measure blood chemistry including lipid profile and
trimethylamine-N-oxide (TMAO) levels. Patients will undergo cardiac CT and/or cardiac
catheterization in accordance with the decision of the cardiologist to evaluate and/or treat
CAD. Genomic DNA will be extracted from stool samples for Microbiome analysis.
Innovation: The hypothesis is that there is a unique microbiota pattern in patients with
coronary atherosclerosis, which may contribute to the pathogenesis and/or expression of CAD.
Knowing the unique microbiota in patients with coronary disease, would render it as novel
target for treatment, either primary or secondary prevention.
Collaboration: Between Cardiology department at Rabin Medical Center and the lab of Prof.
Eran Segal located at the Weizmann Institute of Science. The collaboration between these two
groups will combine the clinical expertise of treating cardiac patients with novel
scientific technology and concept.
Introduction:
The human gastrointestinal system is populated with a variety of symbiotic microorganisms,
namely microbiota. Its total weight is approximately 2 kilograms, containing trillions of
microorganisms. The microbiome is the total genetic (metagenomic) data of the microbiota. In
recent years, the development of efficient methods for genome sequencing and bio-informatics
has enabled fast and accurate quantification and qualification of the microbiome, made
microbiome analysis leading method of microbiota research.
Coronary artery disease (CAD) accounted for more than 8 million deaths yearly worldwide. In
particular, acute coronary syndrome (ACS) remains a major cause of morbidity and mortality
and is responsible for more than 1 million hospital admissions in the United States
annually. The pathophysiologic hallmark of ACS is coronary thrombosis caused by
atherosclerotic plaque injury, with two types of injuries being described. The first is
plaque rupture, which remains the most common cause of coronary athero-thrombosis, and the
second is superficial plaque erosion which is recognized with increasing frequency. As
opposed to plaque rupture, lesions that are caused by erosion do not have thin fibrous caps,
abundant inflammatory cells, or a large lipid core, but rather rich in extracellular matrix,
such as proteoglycans and glycosaminoglycans.
Imaging studies such as coronary computed tomographic angiography (CCTA) and diagnostic
coronary catheterizations with or without optical coherence tomography (OCT) are being used
increasingly in clinical practice in order to characterize the mechanism responsible for
unstable/vulnerable atherosclerotic plaque.
The human gut microbiota interacts extensively with the host through metabolic exchange and
co-metabolism of substrates; thereby contribute to a variety of metabolic and immunologic
mechanism in the human body. CAD is a major field of interest in microbiota research, and
there have been several findings that connect the gut microbiota to CAD pathophysiology.
First, microbiota was associated with metabolic syndrome, namely obesity and insulin
resistance. It is hypothesized that gut microbiota may increase short-chain fatty acid, that
eventually increase appetite, thus causing obesity. Another hypothesis is that gut
microbiota endotoxins may translocate into the bloodstream, elicit inflammatory cascade that
eventually promote atherosclerosis. Second, microbiota may also have a role in the
development of atherosclerosis. In patients with symptomatic atherosclerosis, there is a
unique microbiome pattern that may have pro-inflammatory characteristics. Recently, a unique
microbial pattern was found among patients with high cardiovascular risk profile. Third, gut
microbiota metabolize dietary phosphatidylcholine (lecitine) to produce the metabolite
trimethylamine-N-oxide (TMAO), which is associated with increased risk of cardiovascular
events.
The data published so far relates solely to the interaction between human gut microbiome and
cardiovascular risk factors. To the best of the investigator's knowledge and understanding,
the microbiome analysis of patients with an established diagnosis of CAD (including ACS) is
lacking.
Objectives:
The purpose of the current study is to investigate the gut microbiota of patients with
symptomatic CAD, both in stable and during acute phases. The investigators hypothesize that
the study participants would present with a unique microbiome signature that may provide a
novel insight into the pathophysiology of atherosclerotic CAD while affording putative
therapeutic implications.
After establishing the unique microbiome signature in a large cohort of CAD patients, the
investigators would correlate it to TMAO levels to further investigate its part in the
pathophysiology of CAD. In the final stage, the investigators would try to find ways to
adjust personalized treatment options to change the "pro-atherosclerotic" gut microbiota. By
using data from the current study with the investigators' previous 1000 patient cohort with
known microbiome and nutritional profile, it would be able to search for a specific target
for nutritional intervention, such as probiotics. Then, the investigators would monitor the
patients with sequencing the microbiome after the nutritional intervention.
Methods:
Study design and recruitment. Study participants will be patients aged 30-80 arriving to
Rabin Medical Center with suspected CAD and able to provide informed consent. Participants
will provide medical, lifestyle, and nutritional questionnaires. Blood pressure and
heart-rate measurements will be taken during hospitalization as well as blood tests and
fecal samples and/or rectal swabs. In order to evaluate and/or treat suspected
atherosclerotic disease participants will undergo cardiac CT and/or cardiac catheterization
in accordance with the standard of care and based upon the decision of the treating
cardiologist. Diagnostics and treatment options will be based only on participants' medical
condition and regardless of the aforementioned study protocol.
The control group will be selected to represent an age, sex and cardiovascular risk factors
-matched group without current CAD. Further exclusion criteria in the control group will be
antibiotic consumption in the following 3 months, inflammatory bowel disease, or other
significant chronic disease that may influence the microbiota (such as cancer, autoimmune
disease, and chronic immunosuppressive treatment). The control group will undergo cardiac CT
or coronary angiography according to clinical suspicion in order to rule-out CAD and
irrespective of the study protocol.
Blood samples. 10 ml venous blood will be collected into Ethylenediaminetetraacetic acid
(EDTA) and gel with clot activator -containing tubes from the enrolled patients in all study
participants. The concentrations of serum creatinine, troponin, creatine phosphokinase
(CPK), hemoglobin, triglycerides (TG), total cholesterol (TC), high-density lipoproteins
(HDL), low-density lipoproteins (LDL), glucose, c-reactive protein (CRP), b-type natriuretic
peptide (BNP) and hemoglobin A1c (HbA1C) will be measured by automatic biochemistry
analyzer.
In addition, the levels of TMAO will be measured from blood plasma by using Ultra-High
Performance Liquid Chromatography - Mass Spectrometric - Multiple Reaction Monitoring
(UHPLC-MS/MRM) as previously described.
Nutritional profiling. All participants will report their habits of food consumption by
filling up the Food Frequency Questionnaire (FFQ).
Cardiac CT analysis. Selected participants will undergo CT angiography for the evaluation
and quantification of CAD using a 256-slice system (Brilliance iCT, Philips Healthcare,
Cleveland, Ohio). Data will be acquired with a collimation of 96 X 0.625 mm and a gantry
rotation time of 330 ms. Intravenous injection of 60 to 90 ml of nonionic contrast agent at
a flow rate of 5 ml/s will be followed by a 30-ml saline chase bolus (3 ml/s). Acquisition
will be performed during an inspiratory breath hold while the electrocardiogram will be
recorded simultaneously to allow, dependent from the heart rate, retrospective or
prospective gating of the data. All images will be reconstructed with a slice thickness of
0.67 mm and a slice increment of 0.34 mm. The complete dataset will be transmitted to a
dedicated CT workstation with a 3-dimensional reconstruction tool specifically designed for
coronary angiography (Philips Intellispace Portal, version 7.0) to allow for multiplanar
reformations and quantitative plaque analysis. An independent reader will review all
studies. Each vessel containing significant stenosis will be analyzed in curved multiplanar
reformatted images in long-axis and cross-sectional views. Diameters at the site of maximum
stenosis and at proximal and distal references will be measured. Degree of stenosis will be
calculated as the ratio of the difference between the diameter at maximum stenosis and the
mean of diameters at the proximal and distal references divided by the mean of diameters at
proximal and distal references and expressed as percentage. Remodeling index will be
calculated as the outer vessel area at the site of maximum stenosis divided by the mean of
outer vessel areas at proximal and distal references. Positive remodeling will be defined as
a remodeling index ≥ 1.05. Plaque volume will be automatically calculated as the volume of
all voxels segmented between the luminal and outer vessel boundaries on curved multiplanar
reformatted images. Proximal and distal references will be used as the proximal and distal
ends of the plaques. The investigators will report the total volume of plaque and volumes of
plaque subtypes: calcified, non-calcified, and mixed plaques.
Cardiac catheterization and percutaneous coronary intervention (PCI). Patients will be
admitted to the catheterization laboratory according to their clinical presentation, taking
into account the current ESC/AHA clinical guidelines. The cardiac catheterization procedure
will be performed using standard percutaneous techniques via the radial or femoral artery.
Coronary lesions will be evaluated by the operator in terms of stenosis by visual estimation
or other using objective measurements, such as quantitative coronary analysis (QCA).
Coronary intervention, including balloon angioplasty and stent implantation will be
implemented as needed and according to the severity of coronary stenosis i.e. (≥70% diameter
stenosis). Adjunctive coronary imaging (OCT or intravascular ultrasound) will be performed
according to operator's discretion and regardless of the study protocol. All patients will
be treated during the procedure with anticoagulation (mostly unfractionated heparin) using
careful monitoring of activated clotting time between 250-300 seconds. After the angioplasty
procedure, all patients will be treated with dual anti-platelet therapy combining aspirin
and P2Y12 inhibitor (clopidogrel, prasugrel or ticagrelor, according to the clinical
indication) for 6-12 months, unless there will be contra-indication, such as treatment with
oral anti-coagulants.
Genomic DNA Extraction and Filtering. Genomic DNA from stool samples will be purified using
PowerMag Soil DNA isolation kit (MoBio) optimized for Tecan automated platform. For shotgun
sequencing, 100 ng of purified DNA will be sheared with a Covaris E220X sonicator.
Microbiome analysis. Microbiome samples will be processed by an automated robotic pipeline
in 96-well format. Each sample group collected will be processed robotically for both 16S
and metagenomic sequencing.
Generating microbiome-based features. The investigators will employ and further extend a
computational pipeline that was developed for generating a rich set of features from a
metagenomic sample. These features will be the basis for models that identify
microbiome-based signatures.
Bacterial and viral abundances - Mapping the metagenome sample to a reference bacterial
genome database, and then counting the number of reads mapping to each bacteria, resulting
in a vector of relative bacterial abundances for each sample.
Bacterial diversity - Using the relative bacterial abundances derived above, the
investigators will compute several measures of the diversity of bacteria and viruses in a
metagenome sample (e.g., Shannon entropy of the relative abundance vector, number of
bacteria above some minimal abundance level), as sample diversity was shown to be associated
with certain physiological aspects of the host such as overall adiposity and insulin
resistance.
Bacterial growth rates - For each metagenome sample, the investigators will compute a vector
that corresponds to the growth rate of each bacteria in the sample, using a novel method
that was recently developed for this purpose. Briefly, by examining the pattern of
sequencing read coverage (depth) across the length of different bacterial genomes, the
investigators found that many bacteria exhibit a prototypical coverage pattern, consisting
of a single trough and a single peak. Notably, the location of the peak coincides with the
bacteria's known origin of replication, suggesting that the added read coverage near the
peak represents newly replicated DNA. For any given bacteria, the ratio between the peak and
trough coverage varies greatly across samples from different human gut microbiomes, with
high ratios being similar to those obtained during exponential growth phase of bacteria
grown in culture, and low ratios being similar to growth in stationary phase.
Gene abundances - The investigators will compute the relative abundances of genes in
metagenome samples by applying a similar approach to that above for deriving relative
abundances of bacteria. To this end, rather than mapping the reads to the reference
bacterial genome database, the investigators will map them to a reference database of
bacterial genes which was recently extended and that collectively contain over 3 million
distinct bacterial genes. The derived gene abundance vectors are complementary to the
bacteria abundance vectors, with the advantage being mapping to genes whose embedding
bacterial genome is unknown and thus allowing more metagenome sequencing reads to be mapped,
and with the disadvantage of producing a much larger feature vector.
Biological pathway abundances - As another set of features that provide information at the
functional level of the microbiota, the investigators will use the KEGG database of
biological pathways51 and the above vector of gene abundance of each sample to compute an
abundance score for each biological pathway. The key advantage of this set of features is
that its associations provide direct hypotheses regarding the underlying mechanisms through
which the microbiota may be involved with the correlated phenotype.
Sample storage. The samples as well as remaining DNA and frozen serums will be stored in
-80C freezers. Unprocessed stool samples (e.g., not all samples collected annually will be
processed initially) will also be stored in -800C freezers.
Statistical analysis. Clinical data such as vital signs, cardiovascular risk factors (age,
gender, lipid profile, glycemic index, smoking status, previous CAD), chronic
co-morbidities, regular drug use, imaging findings on cardiac CT and/or cardiac
catheterization as well as cardiac enzymes levels will be collected at Rabin Medical Center.
At the Weizmann institute of science, in the computer science department by the Segal Lab,
the investigators will analyze the data. For each parameter an association analyses will be
performed to identify all of the microbiome parameters that are associated with these
clinical parameters.
After analyzing the microbiome signatures in those patients, where known probiotic and/or
nutritional interventions can lead to the desired change the investigators will intervene
and then monitor with sequencing the microbiome after the intervention.
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Observational Model: Cohort, Time Perspective: Cross-Sectional
| Status | Clinical Trial | Phase | |
|---|---|---|---|
| Recruiting |
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